AIX-MARSEILLE UNIVERSITE
ÉCOLE DOCTORALE DES SCIENCES DE LA VIE ET DE LA SANTÉ
FACULTÉ DE MÉDECINE DE MARSEILLE
Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes
(URMITE), UMR 63, CNRS 7278, IRD 198, Inserm 1095
Thèse présentée pour obtenir le grade universitaire de docteur
Discipline : Pathologie Humaine
Spécialité : Maladies Infectieuses
Rezak DRALI
Poux humains : Différenciation, distribution
phylogéographique, Host-Switching et contrôle
Soutenue le 15 Décembre 2014 devant le jury :
Mr le Professeur Pierre MARTY
Président du Jury/Rapporteur
Mr le Docteur
Rapporteur
Arezki IZRI
Mr le Professeur Philippe BROUQUI
Directeur de thèse
Mr le Professeur Didier RAOULT
Co-directeur de thèse
1
À Tassadit, mon épouse.
Merci pour ton soutien indéfectible
À ma mère
À mes enfants Taous, Mokrane et Ania.
3
Sommaire
Avant-propos
7
Résumé
8
Abstract
11
Introduction générale
15
Revue: Taxonomy, lifestyle and current genetic advances of primate’s lice.
23
Chapitre 1: Différenciation pou de tête - pou de corps
59
Article I: Distinguishing Body Lice from Head Lice by Multiplex
63
Real-Time PCR Analysis of the Phum_PHUM540560 Gene.
Article II: Bartonella quintana in body lice from scalp hair
75
of homeless persons, France.
Article III: Detection of Bartonella quintana in African
81
Body and Head Lice.
Chapitre 2: Distribution phylogéographique des poux
91
humains contemporains et anciens.
Article IV: A New Clade of African Body and Head Lice Infected
95
by Bartonella quintana and Yersinia pestis –
Democratic Republic of the Congo.
Article V: Studies of ancient lice reveal unsuspected past
107
migrations of vectors.
Article VI: Evidence of sympatry of Clade A and Clade B head lice
119
in a pre-Columbian Chilean mummy from Camarones.
Chapitre 3: Host-Switching
127
Article VII: Host switching of human lice to New World monkeys
131
in Amazonia
5
Chapitre 4: Détection et monitoring de la résistance moléculaire
161
des poux de corps à la perméthrine
Article VIII: Detection of a knockdown resistance mutation
165
associated with permethrin resistance in the
body louse Pediculus humanus corporis by use of
melting curve analysis genotyping.
Article IX: Effect of permethrin-impregnated underwear on
177
body lice in sheltered homeless persons:
a randomized controlled trial.
Conclusions et perspectives
189
Références
191
Annexes
201
Article X: Matrix-Assisted Laser Desorption Ionization–
203
Time of Flight Mass Spectrometry for
Rapid Identification of Tick Vectors.
Mini review: Typhus in World War I.
213
Remerciements
219
6
Avant-propos
Le format de présentation de cette thèse correspond à une
recommandation
de
la
spécialité
Maladies
Infectieuses
et
Microbiologie, à l’intérieur du Master des Sciences de la Vie et de la
Santé qui dépend de l’Ecole Doctorale des Sciences de la Vie de
Marseille.
Le candidat est amené à respecter des règles qui lui sont imposées et
qui comportent un format de thèse utilisé dans le Nord de l’Europe et
qui permet un meilleur rangement que les thèses traditionnelles. Par
ailleurs, la partie introduction et bibliographie est remplacée par une
revue envoyée dans un journal afin de permettre une évaluation
extérieure de la qualité de la revue et de permettre à l’étudiant de
commencer le plus tôt possible une bibliographie exhaustive sur le
domaine de cette thèse. Par ailleurs, la thèse est présentée sur article
publié, accepté ou soumis associé d’un bref commentaire donnant le
sens général du travail. Cette forme de présentation a paru plus en
adéquation avec les exigences de la compétition internationale et
permet de se concentrer sur des travaux qui bénéficieront d’une
diffusion internationale.
Professeur Didier RAOULT
7
Résumé
Les poux hématophages de primates sont des ectoparasites spécifiques
de leurs hôtes avec qui ils ont Co évolué depuis environ 25 millions
d’années. Ceux infestant l’Homme sont sans doute les mieux étudiés,
particulièrement le pou de tête (Pediculus humanus capitis) et le pou
de corps (Pediculus humanus humanus). Ce sont deux écotypes
indiscernables occupant chacun une niche écologique: les cheveux
pour le pou de tête et les vêtements pour le pou de corps. La
pédiculose due au pou de tête touche chaque année des centaines de
millions d'enfants à travers le monde nonobstant la classe sociale à
laquelle ils appartiennent alors que le pou de corps infeste
spécialement les populations n’ayant pas un accès facile aux
conditions standard d’hygiène, telles les sans-abri, les prisonniers et
les réfugiés de guerre. Le pou de corps représente une menace réelle
pour l’Homme en raison de son rôle de vecteur dans la transmission
de trois maladies graves ayant tué des millions de personnes à travers
l’histoire de l’humanité à savoir : le typhus épidémique, la fièvre des
tranchées et la fièvre récurrente causées par Rickettsia prowazekii,
Bartonella quintana, et Borrelia recurrentis respectivement. Le pou
8
de corps est également soupçonné dans la transmission d'un quatrième
agent pathogène fatidique, Yersinia pestis, l'agent de la peste.
L'analyse de ADN mitochondrial a permis de classer les poux humains
en trois clades A, B et C où seul le clade A distribué mondialement
comprend à la fois des poux de tête et des poux de corps.
Durant cette thèse, nous avons voulu apporter des réponses à un
certain nombre de questions restées posées à travers le traitement de
certaines thématiques qui nous semblaient importantes. Ainsi nous
avons cherché à (i) trouver un moyen pour différencier entre le pou de
tête et le pou de corps, (ii) en savoir d’avantage sur la distribution
phylogéographique mondiale des poux humains, (iii) étudier les poux
anciens pour nous aider à comprendre la circulation des vecteurs, la
circulation des agents pathogènes transmis par ces vecteurs et les flux
migratoires des hôtes de ces vecteurs, (iv) étudier le phénomène de
Host-Switching à travers P. mjobergi le poux de singe du Nouveau
Monde et (v) trouver un moyen efficace de contrôle des poux de corps
infestant les sans-abri à Marseille à travers la détection et le
monitoring de la résistance des poux aux insecticides.
Nous avons obtenu des résultats concrets dans chacune des
thématiques abordées, résultats par ailleurs valorisés par des
9
publications scientifiques. En effet, nous avons (i) mis en place un
outil moléculaire qui permet de différencier pour la première fois entre
le pou de tête et le pou de corps qui a montré efficacité sur le terrain,
(ii) mis en évidence l’existence d’un nouveau clade mitochondrial
(Clade D) renfermant des poux de tête et des poux de corps
susceptible de vectoriser B. quintana et Y. pestis, (iii) retracé les
migrations humaines à travers l'analyse de poux anciens provenant de
différentes périodes et localisations, (iv) démontré pour la première
fois que P. mjobergi est génétiquement proche du pou humain et
confirmé l’hypothèse qu’à l’origine P. mjobergi était un pou humain
qui a été transféré aux singes du Nouveau Monde par les premiers
Hommes à avoir atteint le continent américain il y a des milliers
d’années et (v) mis en place un outil de détection et de contrôle de la
résistance moléculaire des poux à la perméthrine. Cet outil fut
particulièrement utile dans l'étude clinique que nous avons menée
pour déterminer si l'utilisation de sous-vêtements imprégnés
d'insecticide offrait une protection efficace à long terme contre les
poux de corps infestant les personnes sans-abri.
Mots clés : pou de tête, pou de corps, différenciation, distribution
phylogéographique, poux anciens, Host-Switching, contrôle des poux.
10
Abstract
Primate sucking lice are obligate host-specific parasites that have Co
evolved with their hosts for over 25 million years. Lice infesting
humans are probably the best studied, particularly head louse
(Pediculus humanus capitis) and body louse (Pediculus humanus
humanus). These are two indistinguishable ecotypes each occupying
an ecological niche: hair for head louse and clothing for the body
louse. Pediculosis due to head louse affects each year hundreds of
millions of children worldwide despite the social class to which they
belong while body louse infests especially populations that have not
ready access to standard conditions of hygiene, such as the homeless,
prisoners and war refugees. Body louse represents a real threat to
humans because of its role as vector for the transmission of three
deleterious diseases that have killed millions of people, namely
epidemic typhus, trench fever and relapsing fever caused by Rickettsia
prowazekii,
Bartonella
quintana,
and
Borrelia
recurrentis
respectively. The body louse is also suspected in the transmission of a
fourth fateful pathogen, Yersinia pestis, the agent of plague.
Mitochondrial DNA allowed the classification of human lice in three
11
clades designed A, B and C where only clade A that is distributed
worldwide comprises both head and body lice.
During my PhD, some thematic that seemed important have been
addressed. Thus we aimed to (i) find a way to differentiate between
human head and body lice, (ii) learn more about the worldwide
phylogeographic distribution of lice (iii) study of ancient human lice
which can help to understand the circulation of vectors, the flow of
vector-borne pathogens and the migratory flu hosts of these vectors,
(iv) study the host-switching phenomenon through P. mjobergi the
lice that parasite New World monkeys and (v) find an efficient way to
control body lice infesting homeless people in Marseille.
In each of these issues, we obtained concrete results that have led to
scientific publications. Indeed, we (i) implemented a molecular tool to
differentiate for the first time between head and body louse, (ii) we
highlighted the existence of a fourth mitochondrial clade (Clade D)
comprising head and body lice that can vectorize B. quintana and Y.
pestis, (iii) we traced human migration through the analysis of ancient
lice from different periods and different area, (iv) we demonstrated for
the first time that P. mjobergi is genetically close to human louse and
confirmed the hypothesis that initially P. mjobergi was a human louse
12
has been transferred to New World monkeys by the first humans who
have reached the American continent thousands of years ago and (v)
we have implemented a tool for detecting and monitoring the
molecular resistance to permethrin of body lice that parasite sheltered
homeless persons in Marseille. This tool was particularly useful in the
clinical study we conducted to determine whether the use of longlasting insecticide–treated underwear provides effective long-term
protection against body lice in homeless persons.
Keywords: head louse, body louse, differentiation, phylogeographic
distribution, ancient lice, host-switching, lice control.
13
Introduction générale
Les poux (Insecta: Phthiraptera) sont des ectoparasites obligatoires des
oiseaux et des mammifères. A ce jour, environ 4.900 espèces ont été
répertoriées et réparties dans quatre sous-ordres: d’une part les poux
mâcheurs (chewing / biting lice) comprenant les Rhynchophthirina,
les Ischnocera et les Amblycera et d’autre part les anoploures
(Anoplura) suceurs de sang [1]. Les données morphologiques [2]
combinées aux données moléculaires [3] ont montré qu'il y avait une
relation para phylétique entre les poux mâcheurs et les anoploures. La
récente découverte de deux fossiles de poux, le premier vieux de 44
millions d’années parasitait les oiseaux [4] et le second 100 millions
d’années appartenaient à la famille Liposcelididae [5] a permis
d’estimer l’âge des poux à plusieurs millions d’années. Les méthodes
de datation moléculaire ont démontré que les poux ont survécu à la
grande extinction des espèces du Crétacé-Paléogène (K-Pg) survenue
il y’a 65 millions d’années [6].
Aptères et ne possédant pas d’hôte intermédiaire, les poux ont dû Co
évoluer et s’adapter au microenvironnement de leur hôte. De ce fait, la
transmission des poux s’effectue lors des contacts directes entre hôtes
conspécifiques [7].
15
Le processus de cospeciation hôtes-parasites est exemplifié à travers la
relation des poux avec leurs hôtes [8], cependant ce processus n’est
pas toujours parfait comme énoncé dans la règle de Fahrenholz qui
édicte que les phylogénies des parasites et de leurs hôtes doivent se
refléter l’une et l’autre [9]. Parfois, cette association est marquée par
une série d'événements historiques tels le changement d'hôte “hostswitching” ou encore la duplication du parasite [10,11]. A juste titre,
le phénomène de host-switching a été rapporté deux fois chez les
primates. D’abord en 1938, lorsque Ewing a suggéré que Pediculus
mjobergi le pou des singes du Nouveau Monde était à l'origine un pou
humain (P. humanus) transmis par les premiers Hommes ayant
traversé le détroit de Béring il y'a quelques milliers d'années [12].
Puis, en 2007, lorsque Reed et al. suggérèrent que l’Homme avait
hérité de Pthirus pubis, le pou du pubis, à travers les contacts directs
ayant eu lieu entre gorilles et hominidés archaïques il y’a environ 3
millions d’années [13].
Les poux du genre Pediculus infestant l’Homme sont les mieux
étudiés en raison notamment de l’intérêt médical qu’ils suscitent. Ce
genre fut initialement établit par Linnaeus en 1758. Deux décennies
plus tard en 1778, De Geer proposa les nomenclatures Pediculus
16
capitis de Geer 1778 et Pediculus corporis de Geer 1778 pour
différencier le pou de tête du pou de corps [14]. Hopkins en 1952 a
proposé une nomenclature qu’il souhaitait finale eu égard au
foisonnement d’appellations données aux poux: Pediculus humanus
humanus Linnaeus, 1758 pour le pou de corps et Pediculus humanus
capitis de Geer 1778 pour le pou de tête [15].
Le pou de tête vit et se multiplie sur les cheveux alors que le pou de
corps occupe une niche écologique différente, à savoir les plis et les
coutures de vêtements [14]. La pédiculose due au pou de corps est très
répandue parmi les populations précaires telles les pauvres, les sansabri, les prisonniers et les réfugiés de guerre [16], tandis que les poux
de tête affectent des centaines de millions d'écoliers à travers le monde
indépendamment des conditions d'hygiène causant du prurit et une
perte de sommeil dans certains cas [17]. Plusieurs études comparatives
basées sur des critères morphologiques ont été réalisées sans pour
autant avoir réussi à différencier entre le pou de tête et le pou de corps.
Dès 1919, Nuttall concluait déjà que les deux écotypes appartenaient à
la même espèce puisque les deux écotypes ne présentaient aucune
différence concernant les points essentiels [18].
17
Durant un laps de temps, la pigmentation des poux fut en débat. En
1917, Hindle à partir de ses expériences de croisement/sélection avait
conclu que la pigmentation des poux était un caractère héréditaire [19].
Hypothèse réfutée par Nuttall en 1919 puis Ewing en 1926 qui
considéraient que la pigmentation des poux adultes était due à la
couleur du fond (background) utilisée durant les différents stade de
développement larvaire [20,21]. Plus récemment, une étude comparant
les données phénotypiques et génotypiques des poux d’origine
Africaine avait montré qu’il n’existait aucune concordance entre la
couleur et le génotype des poux [22].
L’analyse moléculaire des gènes mitochondriaux a permis d’inférer
une classification phylogéographique robuste de Pediculus humanus.
En effet, les poux humains du genre Pediculus ont été classés dans
trois clades A, B et C où seul le Clade A distribué à travers le monde
comprend aussi bien des poux de tête que des poux de corps [23]. Le
Clade B comprenant des poux de tête retrouvés jusqu’ici sur le
continent américain, en Europe de l’ouest, en Australie et en Algérie.
Le Clade C a été retrouvé au Népal, en Ethiopie et au Sénégal [24].
L’analyse moléculaire de gènes mitochondriaux des poux récupérés
sur des momies précolombiennes a montré que la présence des Clades
18
A et B sur le continent Américain était antérieure à l'arrivée des colons
européens [25,26]. Ce qui renforce l’hypothèse selon laquelle le Clade
B a une origine Américaine qui par la suite a été redistribué dans le
vieux monde par les colons ayant regagné l’Europe [24].
La taille prédictive du génome de P. humanus (108 Mb) faisait de lui
un excellent candidat au séquençage du génome [27]. En 2010,
Kirkness et al. [28] publiaient une première version du génome du pou
de corps et de son endosymbiont Candidatus Riesia pediculicola. Ce
génome renferme 10.773 gènes codants parmi lesquels 163 étaient
spécifiques à P. humanus [28].
La comparaison du profil transcriptionnel des poux de tête et de corps
avait révélé que les deux écotypes avaient le même nombre de gènes
excepté le gène phum_PHUM540560 absent chez le pou de tête [29] .
A ce jour, seul le pou de corps est reconnu comme vecteur de trois
maladies délétères ayant tué des millions de personnes à travers
l’histoire de l’humanité qui sont le typhus épidémique, la fièvre des
tranchées et la fièvre récurrente causées par Rickettsia prowazekii,
Bartonella quintana et Borrelia recurrentis respectivement [30]. Le
pou de corps est également soupçonné dans la transmission d'un
quatrième agent pathogène mortel, Yersinia pestis, l'agent de la peste
19
[31,32]. Au cours des dernières années, l'ADN de B. quintana a été
détecté chez les poux de tête de Clade A [16,32–34] et de Clade C
[35,36], l'ADN de B. recurrentis chez les poux de tête de Clade C [37].
Avant l'avènement des pédiculicides, le contrôle des poux était basé
sur les moyens physiques tel le peignage, l’épouillage à la main, le
rasage et le blanchiment de vêtements [38]. Au cours des dernières
décennies, différentes molécules chimiques ont été développées pour
lutter contre la pédiculose. Malheureusement les poux ont pu
développer une résistance contre la plupart de ces molécules [39].
L’ivermectine semble donner de bons résultats dans le traitement des
poux de tête [40] bien qu’une résistance potentielle à cette molécule
eut été démontrée dans des conditions de laboratoire [41].
Durant cette thèse nous avons voulu apporter notre contribution dans
le domaine de la recherche sur les poux humains. Ainsi, nous avons
commencé par rédiger une revue de littérature intitulée « The lice of
primates »
pour
donner
un
aperçu
sur
le
positionnement
phylogénétique des poux humains au sein des anoploures.
Nous avons organisé ce manuscrit de thèse autour de 4 chapitres
couvrant chacun une thématique différente à savoir (1) la
différenciation entre le pou de tête et le pou de corps, (2) la
20
distribution phylogéographique des poux humains contemporains et
anciens, (3) étude du phénomène de Host-Switching et (4) la détection
et le monitoring de la résistance moléculaire des poux de corps à la
perméthrine.
21
Revue de littérature:
Taxonomy, Lifestyle and Current Genetic
Advances of Primate’s Lice
Revue proposée au journal Annual Review of Entomology
23
Taxonomy, Lifestyle and Current Genetic Advances of Primate’s Lice
1 2 Rezak Drali1 and Didier Raoult1
3 4 5 6 1
Aix Marseille Université, URMITE, UM63, CNRS 7278, IRD 198, Inserm 1095,
27 Boulevard Jean Moulin 13005 Marseille, France
7 8 Corresponding author: Raoult, D. (mailto:didier.raoult@gmail.com)
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Keywords: lice, primates, co evolution, host-switching
1 25
25 Abstract
26 The relationship between lice (Insecta: Phthiraptera) and their host, birds and mammals, is
27 very old, it dates back to tens of millions of years. Thus, their status as obligate parasite
28 makes lice remarkable markers of the evolution of their hosts. On the other hand, among the
29 4,900 species of lice identified, only the human body louse Pediculus humanus humanus, is
30 known to be a vector of three serious diseases that killed millions of people throughout the
31 history of mankind. Here we reviewed the various studies that targeted lice parasitizing
32 primates, with particular attention to human lice of the genus Pediculus. We also provided an
33 overview on the current taxonomy of primates.
34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 2 26
50 Lice
51 Lice (Phthiraptera) are permanent obligate parasitic insects that infest birds and mammals.
52 They are about 4,900 known species distributed into four suborders: chewing or biting lice
53 including Rhynchophthirina, Ischnocera and Amblycera; sucking lice grouped under the name
54 of Anoplura (Johnson et al., 2004). The morphological data (Lyal, 1985a) supported by
55 molecular evidences (Barker et al., 2003) showed that there was a paraphyletic relation
56 between the chewing lice and Anoplura . The discovery of two rare fossils allowed estimating
57 the age of lice to about tens millions of years. Indeed, the first fossil that parasitized birds was
58 44 million years and the second was 100 million years and belongs to family Liposcelididae,
59 commonly called book-louse, a close relatives to parasitic lice (Grimaldi and Engel, 2006).
60 Molecular dating methods showed that the radiation of the louse suborders started before the
61 mass extinctions of species 65 million years ago (Ma), corresponding to Cretaceous-
62 Paleogene (K-Pg) boundary (Smith et al., 2011).
63 Because they are wingless and have no intermediate hosts, lice coevolved with their host to
64 adapt to its microenvironment and the transmission of lice occurs mostly through direct
65 contact between conspecific hosts (Barker, 1991). Lice are probably the most successful
66 pattern of host-parasite cospeciation (Hafner and Nadler, 1988), however, the cospeciation
67 process is not always perfect as stated in Farenholz's rule which enacts that host and parasite
68 phylogenies should mirror each other (Brooks, 1979). Occasionally this association is marked
69 by a range of historical events such as host switching, parasite duplication, sorting event and
70 failure of the parasite to speciate in response to host speciation (Johnson et al., 2003;Page and
71 Charleston, 1998).
72 Rightly in primates, the host-switching phenomenon has been described at least twice. Firstly
73 in 1938 when Ewing suggested that Pediculus mjobergi the louse parasitizing New World
74 monkeys was originally a human louse (P. humanus) transmitted to the monkey by the first
3 27
75 humans who crossed the Bering Strait it there's thousands of years (Ewing, 1938). Hypothesis
76 we have also confirmed thanks to the molecular analysis of howler monkey lice from
77 Argentina – South America (Drali, unpublished data). Then in 2007 when Reed et al. based
78 on molecular and phylogenetic analyzes suggested that the presence of the genus Pthirus in
79 humans would be the result of host-switching from archaic gorillas to archaic hominids
80 roughly 3 Mya throughout direct contact between them (Reed et al., 2007).
81 Characteristics
82 Lice
83 Hemimetabolous, they pass through three nymphal stages before becoming adults (Lyal,
84 1985a). The mouthparts consist of mandibles for chewing lice allowing them to feed upon the
85 skin (feathers, fur, and squamae) and sometimes the blood of their hosts, and stylet (piercing
86 mouthparts) for sucking lice (Johnson and Clayton, 2003;Light et al., 2010). Initially, the
87 sucking lice ancestors lived free in burrows and nests of vertebrates and have chewing
88 mouthparts (Light et al., 2010;Lyal, 1985a). The development of mouthparts allowing some
89 of these lice to take blood meals has created a dependence on their hosts and thus have
90 become obligate parasites (Light et al., 2010). The tibiotarsal claws of sucking lice have
91 adapted for grasping host hairs (Durden, 2001).
92 Unlike the chewing lice, that have large heavily sclerotized heads that are as wide as the
93 prothorax, sometimes wider, the sucking lice have heads that are narrower than their
94 prothorax. Yet, Rhynchophthirina belonging to chewing lice group constitute an exception
95 with their mandibles that are worn at the end of a long snout which enables them to penetrate
96 the thick skin of their host to feed (Johnson and Clayton, 2003).
97 Classification
98 Phthiraptera would be derived from original Pscopteran-like ancestor is divided into two
99 groups the chewing lice and the sucking lice (Johnson et al., 2004). About 4400 species
are
wingless,
dorsoventrally flattened
insects
(Price
and
Graham,
1997).
4 28
100 compose the group of chewing lice which includes three major families: Amblycera (1182
101 species of bird lice and 162 species of mammal lice), Ischnocera (2683 species of bird lice
102 and 377 species of mammal lice) and Rhynchophthirina (3 species of mammal lice confined
103 to elephants and pigs) (Cruickshank et al., 2001). The Anoplura group comprises about 540
104 species that parasitize mammals exclusively, two-third are parasites of rodents (Durden and
105 Musser, 1994b). Concerning the primates (Mammalia), of the 78 genera recognized, including
106 our own species (Finstermeier et al., 2013), 61.7% are parasitized by lice. Among these,
107 53.3% are parasitized by sucking lice, 13.3% by chewing lice and 5% by both sucking and
108 chewing lice (Light, 2005).
109 Behaviors
110
Lice usually do not leave their hosts without which they can only survive a short period (two
111 days) (Barker, 1994;Johnson and Clayton, 2003). The females lay eggs on the fur, hair or
112 feathers of their hosts at a suitable temperature until hatching for avoiding desiccation and
113 other physical factors (Price and Graham, 1997). Lice move to conspecific host when the
114 latter is in close contact, but exceptionally they can cling to the abdomen of haematophagous
115 flying insects to reach to another host (phoresis) (Durden, 1990;Harbison and Clayton, 2011).
116 Usually, chewing lice parasitize only a single species of hosts at a time (Johnson and Clayton,
117 2003).
118 Ecology
119 Of the 4400 species of chewing lice identified, 550 are hosted by mammals (Cruickshank et
120 al., 2001). The primates are parasitized by four genera of chewing lice among which three
121 belong to Ischnocera and the fourth belongs to Amblycera (Table 1).
122 Sucking lice are about 540 known species distributed in 50 genera and 15 families that spread
123 worldwide (Light et al., 2010).
5 29
124 Six genera of sucking lice were found in primates. Three belong to the family Polyplacidae
125 and the three other genera, each belonging to a different family, namely Pediculidae,
126 Pedicinidae and Pthiridae (Table 1) (Durden and Musser, 1994b;Price and Graham, 1997).
127 Among the 29 orders of mammals that are distributed in 153 families, 1,200 genera and 5,400
128 species (Wilson and Reeder, 2005), eight orders are not known to harbor sucking lice
129 including Monotremata, Marsupialia, Xenarthra, Pholidota, Chiroptera, Cetacea, Proboscidea,
130 and Sirenia (Kim, 2006).
131 Phylogeny of lice
132 The molecular phylogeny of lice was inferred after the analysis of 18S rRNA sequences in 33
133 species (Barker et al., 2003). This analysis showed that Amblycera is the sister-group to all
134 other lice but the Rhynchophthirina is sister to the Anoplura. Ischnocera is sister to the
135 Rhynchophthirina and Anoplura, which gives the following configuration (Amblycera
136 (Ischnocera (Anoplura, Rhynchophthirina))) (Barker et al., 2003) (Figure 1).
137 Primates (Mammalia)
138 Primates, term meaning "prime, first rank", represent an order of mammals, including
139 humans, apes, monkeys and prosimians (Wilson and Reeder, 2005). Arboricoles for the most
140 part, primates live in tropical forests. There are nevertheless partially terrestrial species such
141 as baboons or fully terrestrial species such as geladas and humans (Reed and Fleagle, 1995).
142 Until recently, the taxonomy of primates has undergone many changes. Today based on
143 mitogenomic phylogeny, primates including our own species comprise 480 species distributed
144 in 78 genera originated from a common ancestor during the Cretaceous/Paleocene boundary
145 approximately 80 to 90 Mya (Finstermeier et al., 2013;Perelman et al., 2011). Three suborders
146 compose primate’s order: Strepsirrhini, Tarsiiformes and Simiiformes (Perelman et al., 2011).
147 148 6 30
149 Strepsirrhini
150 Strepsirrhini include Lorisiformes (galagos, pottos, and lorises), Chiromyiformes (Malagasy
151 aye-aye) and Lemuriformes (Malagasy lemurs) (Roos et al., 2004). Insectivores, Strepsirrhini
152 are provided with a tail covered with fur, they have an elongated hairless and wet nose and
153 round eyes adapted to nocturnal activities. They have large mobile ears, sensitive tactile hairs
154 and a strong sense of smell (Mittermeier et al., 1999). Strepsirrhini characterizes mainly from
155 other primates by the presence of a toothcomb to the front of their teeth that consists of four
156 incisors and two canines, all elongated and facing forward. The dental comb is used to
157 retrieve the gum trees on which they feed, but also for grooming/delousing (Seiffert et al.,
158 2003). The habitat of Strepsirrhini overlaps two continents, Africa and Asia. Lorises are found
159 in equatorial Africa and Southeast Asia, the galagos in sub-Saharan African forests and the
160 Lemurs are endemic in Madagascar (Mittermeier et al., 1999). However, based on the results
161 obtained by the analysis of short interspersed elements (SINEs) combined with complete
162 mitochondrial cytochrome b sequences from all recognized strepsirrhine genera, Roos and
163 collaborators conclude that strepsirrhines originated from Africa and that Madagascar and
164 Asia were colonized by respective single immigration events (Roos et al., 2004).
165 Tarsiiformes (tarsiers)
166 Tarsiiformes are small nocturnal primates, including three genera (Tarsius, Cephalopachus
167 and Carlito) allopatrically distributed in the islands of Southeast Asia (Groves and Shekelle,
168 2010). Their weight varies between 80 and 150 g, they have round heads with very large eyes
169 that are directed forward and very mobile ears (Ankel-Simons, 2000).
170 For a long time, the position of Tarsiiformes relative to Strepsirrhini and Anthropoidea in the
171 phylogeny of primates was unclear (Yoder, 2003). Finally, the mitochondrial genome analysis
172 allowed to resolve the issue by showing the subdivision of primates into infra-orders
7 31
173 Strepsirrhini and Haplorhini, with tarsiers as sister group of Anthropoidea (Finstermeier et al.,
174 2013).
175 Simiiformes
176 Composed of Platyrrhini (New World monkeys) and Catarrhini that include Cercopithecoidea
177 (Old World monkeys) and Hominoidea (great apes and gibbons) (Perelman et al., 2011).
178 Platyrrhini
179 Platyrrhini comprises 5 families, 6 subfamilies, 19 genera, and 199 species that occupy large
180 areas in Central and South America (Rylands and Mittermeier, 2009). Callitrichidae, Cebidae
181 (Cebinae and Saimiriinae), Aotidae, Pitheciidae (Pitheciinae and Callicebinae) and Atelidae
182 (Alouattinae and Atelinae) compose the five families (Rylands and Mittermeier, 2009).
183 The shape of their nose earned them the name of Platyrrhini meaning "flatted nose" (Laitman,
184 2011). Their body size is extremely variable (about a factor of 100): 120 grams for the pygmy
185 marmoset (Cebuella pygmaea) and 10-12 kg for the muriqui (Brachyteles arachnoides) and
186 gray woolly monkey (Lagothrix cana) (Di Fione and Campbell, 2007). Platyrrhini are
187 provided with a long prehensile tail that can be controlled and used to hold onto branches
188 (Schmitt et al., 2005). According to performed studies, Platyrrhini separated from Catarrhini
189 during the Eocene about 45 million years ago (Finstermeier et al., 2013;Perelman et al.,
190 2011). The arrival of Platyrrhini the in the American continent remains ambiguous however,
191 the phylogeny resolves the relative divergence pattern among families members from a
192 common ancestor about 20 million years (Hodgson et al., 2009).
193 Catarrhini
194 Catarrhini includes Cercopithecoidea (Old World monkeys) and Hominoidea (great apes and
195 gibbons) (Perelman et al., 2011).
196 197 8 32
198 Cercopithecoidea
199 Named Old World monkeys in opposition to the New World monkeys, Cercopithecoidea are
200 distributed mainly in Africa and Asia (Rowe et al., 1996). As for Platyrrhini, they owe their
201 name to the form of their nose whose openings and directed downwardly (Groves, 1993).
202 Cercopithecidae includes two extant subfamilies, Colobinae (the leaf-eating monkeys) and
203 Cercopithecinae (the cheek-pouch monkeys) (Groves, 1993). Colobines are arboreal and
204 possess a long nonprehensile tails while cercopithecines have well-developed thumbs and tails
205 of varying lengths (Rowe et al., 1996). The body size of cercopithecines can varies from 1 kg
206 for talapoin (Miopithecus talapoin) to over 37 kg for the large olive baboon (Papio
207 hamadryas anubis) (Rowe et al., 1996).
208 Cercopithecidae comprises 21 genera. 10 belong to subfamily Colobinae in which 7 have an
209 Asian distribution (Nasalis, Simias, Presbytis, Semnopithecus, Trachypithecus, Pygathrix and
210 Rhinopithecus) and 3 have an African distribution (Colobus, Piliocolobus and Procolobus).
211 11 genera belong to subfamily Cercopithecinae that are endemic to Africa with exception of
212 the genus Macaca which is now mainly found in Asia (Allenopithecus, Cercocebus,
213 Cercopithecus, Chlorocebus, Erythrocebus, Lophocebus, Macaca, Mandrillus, Miopithecus,
214 Papio and Theropithecus) (Wilson and Reeder, 2005).
215 Old World monkeys separated from hominoids about 32 Ma and diverged into the subfamilies
216 Cercopithecinae and Colobinae in the Early Miocene (Finstermeier et al., 2013;Perelman et
217 al., 2011).
218 Hominoidea
219 Hominoidea includes two families: Hominidae (great apes) and Hylobatidae (gibbons, small
220 apes) (Perelman et al., 2011). Four extant genera compose the family Hominidae:
221 chimpanzees (Pan), gorillas (Gorilla), orangutans (Pongo), and humans (Homo) (Hirai et al.,
222 2012). Orangutans live in Asia, chimpanzees and gorillas in Africa and humans are distributed
9 33
223 worldwide (Rowe et al., 1996). Bipedalism is certainly the most characteristic feature of the
224 Hominidae, though not widely used by African apes which prefer to move on knuckles
225 (Richmond and Strait, 2000).
226 In 1990s, the analysis of whole mitochondrial sequences has allowed showing that humans
227 have a sister relationship with chimpanzees among Hominidae and therefrom, the phylogeny
228 of the Hominidae has been established (Horai et al., 1995).
229 The family Hylobatidae is composed from four genera (Hylobates, Hoolock, Nomascus, and
230 Symphalangus) (Groves, 2005). They live in Southeast Asia (India, China, Malay Peninsula,
231 Java, Borneo, and Sumatra) (Rowe et al., 1996). Given their lower size relative to the other
232 Hominoidea, the name lesser apes was given to the gibbons (Hirai et al., 2012). The four
233 genera of family Hylobatidae are phylogenetically divergent. Indeed, each genus of gibbons is
234 monophyletic (Roos and Geissmann, 2001).
235 Hominoids separated into gibbons (Hylobatidae) and great apes and humans (Hominidae) in
236 the Early Miocene (23 to 5 Mya). Within Hylobatidae, Nomascus separated first (7.8 Mya),
237 followed by the divergence of Symphalangus and Hylobates 6.2 Mya.
238 In Hominidae, orangutans (Pongo) diverged 15.2 MYA from the African great apes and
239 humans, while Gorilla separated from the Homo + Pan Clade 8.4 MYA. Finally, chimpanzees
240 and humans separated in the Latest Miocene, about 5.9 MYA (Finstermeier et al.,
241 2013;Perelman et al., 2011).
242 Lice of primates
243 Sucking lice
244 Genus Pediculus
245 Pediculus includes three known species that parasite primates: P. schaeffi the louse of
246 chimpanzees, P. mjobergi the louse of New World monkeys and P. humanus the louse of
247 humans (Durden and Musser, 1994b).
10 34
248 P. schaeffi was described for the first time in 1910 by Fahrenholz thanks to specimens found
249 in monkeys from a zoo in Hamburg, Germany (Durden and Musser, 1994b). P. schaeffi can
250 be found on any part of the body of their host but with a preference for head, groin and
251 armpits (Allen et al., 2013). By using a molecular clock analysis of mtDNA performed studies
252 showed that P. schaeffi diverged from its sister taxon P. humanus about 5.5 million years ago
253 (Kittler et al., 2003;Reed et al., 2004).
254 New World monkeys also harbored louse of the Pediculidae family (Ferris, 1916). Ferris gave
255 the name P. mjobergi to this species in reference to Mjoberg who was the first to describe it in
256 1910 (Ferris, 1951). In a study we have just completed, we reported the case of transfer of
257 human lice to the New World monkeys. Indeed, the morphological examinations and genetic
258 analyzes performed on the P. mjobergi lice collected from the howler monkeys Alouatta
259 caraya have supported these findings (Drali, unpublished Data).
260 Humans, chimpanzees and New World monkeys are not the only ones to harbor lice of the
261 genus Pediculus, in 1951, Ferris inventoried the siamang (Hylobates syndactylus) among the
262 hosts of Pediculus humanus (Ferris, 1951).
263 Pediculus humanus
264 Of lice infesting primates, those parasitizing humans are the best studied particularly head and
265 body lice, Pediculus humanus capitis and Pediculus humanus humanus respectively (Durden
266 and Musser, 1994b).
267 Taxonomy
268 Linnaeus in 1758 was the first who established the genus Pediculus. In 1778, De Geer
269 proposed the nomenclatures P. capitis de Geer 1778 and P. corporis de Geer 1778 to
270 discriminate between head and body louse (De Geer, 1778). Different terminologies have
271 been proposed to name the two variants: P. cervicalis Latreille 1803 and P. consobrinus
272 Piaget 1880 for head louse, P. vestimenti Nitzsch 1818 and P. tabescentium Alt 1824 for body
11 35
273 louse (Nuttall, 1917). In 1952 Hopkins suggested to give the final names: Pediculus humanus
274 humanus Linnaeus, 1758 to the body louse and Pediculus humanus capitis de Geer, 1778 to
275 the head louse (Hopkins, 1952).
276 Head and body lice ecotypes
277 The classification and nomenclature of human lice genus Pediculus derived from their
278 ecology. Indeed, the head louse lives, breeds and lays its eggs at the base of the hair of the
279 head while the body louse occupies a different ecological niche namely garments where it
280 lays its eggs in the seams and folds (De Geer, 1778).
281 Several comparative studies based on morphological criteria were conducted without reaching
282 differentiates between head lice and body lice. Early as 1919, Nuttall concluded that the two
283 ecotypes represent the extremes in the variation of the same species since they are
284 undistinguishable in all essential point of structure (Nuttall, 1919c).
285 For a while, pigmentation of lice was under debate. In 1917, Hindle starting from its breeding
286 experiments concluded that the pigmentation in lice is an inherited character (Hindle, 1917).
287 Assumption refuted by Nuttall in 1919 relayed later by Ewing in 1926 who considered that
288 the pigmentation of adult lice is due to the color of background used during nymphal
289 development (Ewing, 1938;Nuttall, 1919b). More recently, a study comparing the phenotypic
290 and genotypic data of human lice from Africa showed that no congruence between louse color
291 and genotype has been identified (Veracx et al., 2012).
292 Pediculosis due to the body louse is prevalent in precarious populations such as poor
293 populations, homeless, prisoners and war refugees (Sangare et al., 2014), while head lice
294 affects hundreds of millions of schoolchildren worldwide regardless hygienic conditions with
295 symptoms that include itching and in some cases loss of sleep (Chosidow et al., 1994).
296 297 12 36
298 Mitochondrial clades
299 Thanks to mitochondrial genes, a robust phylogeographic classification of Pediculus humanus
300 was inferred. Indeed, human lice are distributed into 3 clades designed Clade A, B and C
301 where only Clade A that is spread worldwide includes both head and body lice (Reed et al.,
302 2004). Clade B comprises head lice found in the Americas, Western Europe, Australia and
303 North Africa and Clade C including head lice was found in Nepal, Ethiopia and Senegal
304 (Boutellis et al., 2014). Analysis of mitochondrial gene of Pre-Columbian mummies lice
305 showed that Clades A and B were previously present on the Americas before the coming of
306 European settlers (Boutellis et al., 2013a;Raoult et al., 2008). This supports an American
307 origin for Clade B and a dispersal in the Old World with European colonists returning to
308 Europe (Boutellis et al., 2014). Recently, we described a fourth clade, Clade D comprising
309 head and body lice in Democratic Republic of the Congo (Figure 2).
310 Genome of Pediculus humanus
311 Until very recently (Kelley et al., 2014), Pediculus humanus had the smallest genome sizes
312 known in insects (ca. 108 Mb) at that time (Johnston et al., 2007) what made him an excellent
313 candidate for sequencing. In 2010, Kirkness et al. (Kirkness et al., 2010) published a first
314 version of the body louse genome, and its primary bacterial endosymbiont Candidatus Riesia
315 pediculicola. This genome contained 10,773 predicted protein-coding genes, 163 of which are
316 specific to it (Kirkness et al., 2010). It contains 58 microRNAs genes (Kirkness et al.,
317 2010;Olds et al., 2012). Transposable elements represent only 1% of the genome and GC
318 percentage is very low, about 28%, which would explain the small size of this genome
319 (Kirkness et al., 2010). Thirty seven P450s were found in louse, far of the number of P450
320 found in other insects, an advantage to explore the insecticide resistance in human lice (Lee et
321 al., 2010;Yoon et al., 2011). P. humanus is equipped with a complete genetic repertoire
322 enable him to carry out its essential biological functions, but compared to Drosophila
13 37
323 melanogaster, some genes associated with sensing and responding to the environment are in
324 reduced number (Kirkness et al., 2010).
325 Mitochondrial genome
326 Unlike bilateral animals from which the 37 mitochondrial (mt) genes are arranged on a single
327 circular chromosome, the head and body lice have their mt genes on 20 minichromosomes
328 respectively. Each minichromosome has a size of three to four kilobases and contains one to
329 three genes in addition to a large non coding region that includes a control region (Shao et al.,
330 2012). This organization would be due to the lack of the mitochondrial single-stranded
331 binding protein (mtSSB) involved in mitochondrial genome replication (Kirkness et al.,
332 2010).
333 Genome of Candidatus Riesia pediculicola endosymbiont
334 The genome of Candidatus Riesa pediculicola comprises less than 600 genes distributed
335 between a linear chromosome and plasmid that includes specifically genes required for the
336 synthesis of pantothenate, an indispensable vitamin deficient in the louse diet (Kirkness et al.,
337 2010).
338 Recently, the sequencing of the genome of the P. schaeffi endosymbiont namely Candidatus
339 Riesa pediculischaeffi and its comparison to the genome of Candidatus Riesia pediculicola
340 showed that the two species diverged roughly 5.4 Mya ago (Boyd et al., 2014).
341 Head and body louse transcriptome
342 The comparison of the body and head louse transcriptome revealed that the two ecotypes had
343 the same numbers of genes except one gene missing in the head louse (PHUM540560) (Olds
344 et al., 2012) . The refined analysis of partial sequence of this gene in both body and head lice
345 showed that the gene was also present in the head louse but with a rearranged sequence.
346 Based on the difference between the two sequences, a multiplex RT-PCR was developed to
347 differentiate between the two ecotypes (Drali et al., 2013). The tool developed was
14 38
348 particularly effective in field since it able to determine that lice populations collected on the
349 head and clothing of the same homeless in Marseille had a body louse genotype (Drali et al.,
350 2014).
351 Vector competence of human lice
352 So far, body louse is the only known that can transmit at least three deleterious diseases that
353 have killed millions of people, namely epidemic typhus, trench fever and relapsing fever
354 caused by Rickettsia prowazekii, Bartonella quintana, and Borrelia recurrentis respectively
355 (Raoult and Roux, 1999). It is also suspected in the transmission of a fourth lethal pathogen,
356 Yersinia pestis, the agent of plague (Houhamdi et al., 2006;Piarroux et al., 2013). In recent
357 years, the DNA of B. quintana was found in head lice belonging to Clade A (Bonilla et al.,
358 2009;Boutellis et al., 2012;Piarroux et al., 2013;Sangare et al., 2014) and Clade C (Angelakis
359 et al., 2011;Sasaki et al., 2006). DNA of B. recurrentis found in head lice Clade C (Boutellis
360 et al., 2013b) and DNA of Y. pestis was detected in head lice of Clade A and D (Figure 2).
361 Treatment
362 Before the advent of pediculicides, the louse control was mainly by physical means such
363 combing, louse and nit removal, shaving and laundering of clothing (Mumcuoglu and Zias,
364 1988).
365 In recent decades, different chemical molecules with various mechanism of action have been
366 developed for the treatment of pediculosis. Unfortunately lice developed a resistance against
367 most of these molecules namely organochloride (DDT and Lindane), synthetic pyrethroids
368 (Permethrin), organophosphate (Malathion), and Carbamate (Carbaryl), (Clark et al., 2013).
369 Macrocyclic lactone (Ivermectin) seems to be working for the treatment of head lice (Pariser
370 et al., 2013) although a potential resistance to this molecule has been demonstrated under
371 laboratory conditions (Yoon et al., 2011).
15 39
372 Today, the availability of louse genome opens new perspectives in understanding its biology
373 vector competence which will help to consider new ways to fight against lice infestation.
374 Genus Pedicinus
375 Genus Pedicinus includes 16 species that parasitize 41 species of Old World monkeys
376 distributed among Colobinae and Cercopithecinae (Durden, 2001;Price and Graham, 1997).
377 More than one species of Pedicinus can parasitize different hosts but the opposite is rare
378 (Durden and Musser, 1994a). However, erratic cases where two different species of Pedicinus
379 that parasite the same host species have been reported in Colobinae; in Africa where two non-
380 interbreeding populations of lice were found on both groups of red colobus (Scholl et al.,
381 2012) and in Southeast-Asia on douc langurs (Pygathrix spp.) (Mey, 2010). In both cases, the
382 authors believe that this is due to host-switching that can occur during direct contact between
383 the respective hosts of both species of Pedicinus (Mey, 2010;Scholl et al., 2012).
384 Kuhn in 1967, cited by Price in 1997, reported cases of infestation of White-handed gibbon
385 (Hylobates lar) by Pedicinus eurygaster, the only case reported in literature (Kuhn and
386 Ludwig, 1967;Price and Graham, 1997).
387 Other sucking lice
388 The group of lemurs of Madagascar are hosting three genera of lice (Lemurpediculus,
389 Lemurphthirus and Phthirpediculus) all belonging to the family Polyplacidae (Durden and
390 Musser, 1994a). Comprising 190 species distributed in 20 genera, Polyplacidae is the largest
391 family of Anoplura (Durden and Musser, 1994b). Members of this family parasitize mainly
392 rodents and insectivores (Price and Graham, 1997).
393 Genus Pthirus
394 Pthirus pubis usually known as the crab or pubic louse is the third type of louse that
395 parasitizes humans (Durden and Musser, 1994a). Pt. pubis is prevalent worldwide and is
396 present in all categories of the population. It is sexually transmitted and has often been found
16 40
397 in combination with sexually transmitted infections (Anderson and Chaney, 2009). Crab louse
398 lives in pubic hair but he can be found also on the eyelashes and / or eyebrows of the children
399 (Chosidow, 2000). The second louse belonging to the family Pthiridae, Pthirus gorillae is the
400 parasite of gorillas (Durden and Musser, 1994a). Then, the legitimate question facing
401 scientists was: why humans and gorillas shared the same genus of lice? The molecular
402 analyzes of both species showed that P. pubis and P. gorillae were sister taxa that diverged 3
403 to 4 million years ago (Reed et al., 2007), more freshly than the gorilla/human-chimp split
404 which is estimated at about 9 million years ago (Wilkinson et al., 2011). The proposed
405 scenario assumes that the Pthirus lineage from archaic gorillas switched to the archaic human
406 who transmitted it to his descendants (Reed et al., 2007).
407 Chewing lice
408 Genus Trichophilopterus
409 This genus contains only one species namely Trichophilopterus babakotophilus for whom
410 Mjöberg (Nuttall, 1919a) established the monotypic family Trichophilopteridae that parasites
411 only the lemurs of Madagascar (Price and Graham, 1997). Trichophilopterus has been
412 classified for a time in the family Philopteridae (parasites of birds) by Ferris (1933), finally
413 taxonomists have found that it was quite different to constitute a family itself (Price and
414 Graham, 1997).
415 Genus Felicola
416 Genus Felicola is composed of 55 species of which 54 are parasites of carnivores. The fifty
417 fifth species is found in Lorisidae, the Asian-African family of primates (Lyal, 1985b;Perez
418 and Palma, 2001).
419 Genus Cebidicola
420 Belonging to the family of Trichodectidae, genus Cebidicola comprises three species found in
421 New World monkeys living in Brazil: C. amiatus found in Ring-tailed monkey (Alouatta
17 41
422 ursina) and Woolly spider monkey (Brachyteles arachnoïdes); C. extrarius found in Red
423 howler monkey; C. semiarmatus found in Rufous-handed howler monkey (Alouatta belzebul),
424 Black howler (Boero and Boerhinger, 1963), Brown howler (Alouatta guariba) and Ring-
425 tailed monkey (Price and Graham, 1997).
426 Genus Aotiella
427 Two species of Aotiella a parasite of South American night monkeys (Aotidae) have been
428 described: A. aotophilus hosted by (Aotus azarai) and A. hershkovitzi found in gray-necked
429 night monkey (Aotus trivirgatus) (Price and Timm, 1995).
430 Co evolution and Host-Switching
431 The partnership hosts - lice parasites old of 65 million years is marked by a succession of
432 events like cospeciation, coevolution, host-switching and adaptation (Barker, 1991;Barker,
433 1994;Hafner and Page, 1995;Smith et al., 2011). For lice, Host-switching is not accidental but
434 rather a vital necessity. This is also the case for blood sucking insects that can feed in
435 fortuitous host when its favorite host becomes scarce or is not accessible (Takken and
436 Verhulst, 2013). Physiological factors like famine and/or abundance of accessible new hosts
437 may favor to host-switching (Takken and Verhulst, 2013).
438 Host-switching phenomenon seems marked by strict rules. Indeed, according to Clayton, lice
439 cannot establish viable populations on novel hosts that differ in size from the native host
440 (Clayton et al., 2003). In real life, this rule does not always respected. Indeed, in 1940s, a
441 colony of human body lice (Orlando strain) was adapted to take blood meals on rabbits
442 (Culpepper, 1944). Cases of infestation with Pediculus and Pedicinus were reported in
443 gibbons we thought no parasitized by lice just like orangutans (Kuhn and Ludwig, 1967;Price
444 and Graham, 1997). New World monkeys have inherited human lice (Ewing, 1938). Indeed,
445 P. mjobergi has well adapted to its new host. It is even likely that recombination events can
446 take place between the two populations of lice on the occasion of their meeting (Drali,
18 42
447 unpublished data). Humans and gorillas share the louse of genus Pthirus as a result of host-
448 switching from archaic gorillas to archaic hominids roughly 3 Mya throughout direct contact
449 between them (Reed et al., 2007).
450 Concluding remarks
451 The study of lice is essential for understanding complex processes such as coevolution,
452 cospeciation, adaptation and host-switching. Through carrying out this literature review, we
453 realized that little is known about lice parasitizing primates, apart from the human louse of the
454 genus Pediculus. Thus, some primates can harbor lice belonging to different sub-orders. This
455 is the case of Lemuridae, Indridae and Cebidae (Table 1). Whether for humans an hypothesis
456 was proposed to explain his hosting of two genera of sucking lice belonging to two different
457 families (Reed et al., 2004), we do not yet know how certain primates can harbor lice
458 belonging to different sub-orders. Is this the result of a rehosting followed by an adaptation as
459 in P. mjobergi? Further investigations and larger collection of specimens in the field will be
460 necessary to try to answer this question. The sequencing of the genomes could be a real
461 breakthrough in understanding biology, epidemiology and phylogenetic relationship of the
462 different species of lice.
463 464 465 466 467 468 469 470 471 19 43
472 References Cited
473 474 1. Allen, J. M., C. O. Worman, J. E. Lihgt, and D. L. Reed, 2013, Parasitic Lice Help to
475 Fill in the Gaps of Early Hominid History., in JF Brinkworth and K Pechenkina eds.,
476 Primates, Pathogens, and Evolution: New York, Springer, p. 161-186.
477 2. Anderson, A. L., and E. Chaney, 2009, Pubic lice (Pthirus pubis): history, biology and
478 treatment vs. knowledge and beliefs of US college students: Int.J.Environ.Res.Public
479 Health, v. 6, no. 2, p. 592-600.
480 3. Angelakis, E., G. Diatta, A. Abdissa, J. F. Trape, O. Mediannikov, H. Richet, and D.
481 Raoult, 2011, Altitude-dependent Bartonella quintana genotype C in head lice,
482 Ethiopia: Emerg.Infect.Dis., v. 17, no. 12, p. 2357-2359.
483 4. Ankel-Simons, F., 2000, Primate anatomy: an introduction. Academic Press.
484 5. Barker, S. C., 1991, Evolution of host-parasite associations among species of lice and
485 rock-wallabies: coevolution? (J. F. A. Sprent Prize lecture, August 1990):
486 Int.J.Parasitol., v. 21, no. 5, p. 497-501.
6. Barker, S. C., 1994, Phylogeny and classification, origins, and evolution of host
487 associations of lice: Int.J.Parasitol., v. 24, no. 8, p. 1285-1291.
488 489 7. Barker, S. C., M. Whiting, K. P. Johnson, and A. Murrell, 2003, Phylogeny of the lice
490 (Insecta, Phthiraptera) inferred from small subunit rRNA.: Zoologica Scripta, v. 32,
491 no. 5, p. 407-414.
492 8. Boero, J. J., and I. K. Boerhinger, 1963, Reflexiones sobre un nuevo caso de Pediculus
493 mjöbergi en el mono aullador Alouatta caraya.: Rev.Fac.Agr.Vet., v. 15, no. 3, p. 87-
494 98.
20 44
495 9. Bonilla, D. L., H. Kabeya, J. Henn, V. L. Kramer, and M. Y. Kosoy, 2009, Bartonella
496 quintana in body lice and head lice from homeless persons, San Francisco, California,
497 USA: Emerg.Infect.Dis., v. 15, no. 6, p. 912-915.
10. Boutellis, A., L. Abi-Rached, and D. Raoult, 2014, The origin and distribution of
498 human lice in the world: Infect.Genet.Evol., v. 23, p. 209-217.
499 500 11. Boutellis, A., R. Drali, M. A. Rivera, K. Y. Mumcuoglu, and D. Raoult, 2013a,
501 Evidence of sympatry of clade a and clade B head lice in a pre-Columbian Chilean
502 mummy from Camarones: PLoS.One., v. 8, no. 10, p. e76818.
503 12. Boutellis, A., O. Mediannikov, K. D. Bilcha, J. Ali, D. Campelo, S. C. Barker, and D.
504 Raoult, 2013b, Borrelia recurrentis in head lice, Ethiopia: Emerg.Infect.Dis., v. 19, no.
505 5, p. 796-798.
13. Boutellis, A., A. Veracx, E. Angelakis, G. Diatta, O. Mediannikov, J. F. Trape, and D.
506 507 Raoult,
2012,
Bartonella
quintana
in
508 Vector.Borne.Zoonotic.Dis., v. 12, no. 7, p. 564-567.
head
lice
from
Senegal:
509 14. Boyd, B. M., J. M. Allen, V. de Crecy-Lagard, and D. L. Reed, 2014, Genome
510 Sequence of Candidatus Riesia pediculischaeffi, Endosymbiont of Chimpanzee Lice,
511 and Genomic Comparison of Recently Acquired Endosymbionts from Human and
512 Chimpanzee Lice: G3.(Bethesda.).
15. Brooks, D. R., 1979, Testing the context and extent of host-parasite coevolution.:
513 SysT.Zool., v. 28, p. 229-307.
514 515 16. Chosidow, O., 2000, Scabies and pediculosis: Lancet, v. 355, no. 9206, p. 819-826.
516 17. Chosidow, O., C. Chastang, C. Brue, E. Bouvet, M. Izri, N. Monteny, S. Bastuji-
517 Garin, J. J. Rousset, and J. Revuz, 1994, Controlled study of malathion and d-
518 phenothrin lotions for Pediculus humanus var capitis-infested schoolchildren: Lancet,
519 v. 344, no. 8939-8940, p. 1724-1727.
21 45
520 18. Clark, J. M., K. S. Yoon, S. H. Lee, and B. R. Pittendrigh, 2013, Human lice: Past,
521 present and future control.: Pesticide Biochemistry and Physiology, v. 106, p. 162-
522 171.
523 19. Clayton, D. H., S. E. Bush, B. M. Goates, and K. P. Johnson, 2003, Host defense
524 reinforces host-parasite cospeciation: Proc.Natl.Acad.Sci.U S A, v. 100, no. 26, p.
525 15694-15699.
526 20. Cruickshank, R. H., K. P. Johnson, V. S. Smith, R. J. Adams, D. H. Clayton, and R. D.
527 Page, 2001, Phylogenetic analysis of partial sequences of elongation factor 1alpha
528 identifies major groups of lice (Insecta: Phthiraptera): Mol.Phylogenet.Evol., v. 19, no.
529 2, p. 202-215.
530 21. Culpepper, G. H., 1944, The rearing and maintenance of a laboratory colony of the
531 body louse.: The American journal of tropical medicine and hygiene, v. 1, no. 5, p.
532 327-329.
22. De Geer,C. Mémoires pour servir à l'histoire des Insectes. [7], 62-68. 1778. Stokholm,
533 Hesselberg.
534 535 Ref Type: Serial (Book,Monograph)
536 23. Di Fione, A., and C. J. Campbell, 2007, The Atelines: Variation in ecology, behavior,
537 and social organization., in CJ Campbell, A Fuentes, KC MacKinnon, M Panger, and
538 SK Bearder eds., Primates in Perspective: New York, Oxford University Press, p. 155-
539 185.
540 24. Drali, R., A. Boutellis, D. Raoult, J. M. Rolain, and P. Brouqui, 2013, Distinguishing
541 body lice from head lice by multiplex real-time PCR analysis of the
542 Phum_PHUM540560 gene: PLoS.One., v. 8, no. 2, p. e58088.
543 25. Drali, R., A. K. Sangare, A. Boutellis, E. Angelakis, A. Veracx, C. Socolovschi, P.
544 Brouqui, and D. Raoult, 2014, Bartonella quintana in body lice from scalp hair of
545 homeless persons, France: Emerg.Infect.Dis., v. 20, no. 5, p. 907-908.
22 46
546 26. Durden, L. A., 1990, Phoretic relationships between sucking lice (Anoplura) and flies
547 (Diptera) associated with humans and livestock.: Entomologist, v. 109, no. 3, p. 191-
548 192.
549 27. Durden, L. A., 2001, Lice (Phthiraptera)., in WM Samuel, MJ Pybus, and AA Kocan
550 eds., Parasitic diseases of wild mammals.: Ames: Iowa State University Press, p. 3-17.
551 28. Durden, L. A., and G. G. Musser, 1994a, The Mammalian Hosts of The Sucking Lice
552 (Anoploura) of the World: A Host-Parasite List.: Bull.Soc.Vector Ecol., v. 19, p. 130-
553 168.
554 29. Durden, L. A., and G. G. Musser, 1994b, The sucking lice (Insecta, Anoplura) of the
555 world: a taxonomic checklist with records of mammalian hosts and geographical
556 distributions.: Bulletin of the American Museum of Natural History, v. 218, p. 1-90.
30. Ewing, H. E., 1938, The Sucking Lice of American Monkeys.: The Journal of
557 Parasitology, v. 24, no. 1, p. 13-33.
558 31. Ferris, G. F., 1916, A catalogue and lost list of the Anoplura. San Francisco, The
559 Academy.
560 561 32. Ferris, G. F., 1951, The sucking lice: Mem.Pacific Coast Entomol.Soc., v. 1, p. 1-320.
562 33. Finstermeier, K., D. Zinner, M. Brameier, M. Meyer, E. Kreuz, M. Hofreiter, and C.
563 Roos, 2013, A mitogenomic phylogeny of living primates: PLoS.One., v. 8, no. 7, p.
564 e69504.
34. Grimaldi, D., and M. S. Engel, 2006, Fossil Liposcelididae and the lice ages (Insecta:
565 Psocodea): Proc.Biol Sci., v. 273, no. 1586, p. 625-633.
566 567 35. Groves, C., 1993, Order Primates., in DE Wilson and DM Reeder eds., Mammal
568 Species of the World: A Taxonomic and Geographic Reference: Washington, D.C.,
569 Smithsonian Institution, p. 243-278.
23 47
570 36. Groves, C., 2005, Order Primates., in DE Wilson and DM Reeder eds., Mammal
571 Species of the World: A Taxonomic and Geographic Reference: Washington, D.C.,
572 Smithsonian Institution, p. 243-278.
37. Groves, C., and M. Shekelle, 2010, The genera and species of Tarsiidae.: International
573 Journal of Primatology, v. 31, no. 6, p. 1071-1082.
574 38. Hafner, M. S., and S. A. Nadler, 1988, Phylogenetic trees support the coevolution of
575 parasites and their hosts: Nature, v. 332, no. 6161, p. 258-259.
576 577 39. Hafner, M. S., and R. D. Page, 1995, Molecular phylogenies and host-parasite
578 cospeciation: gophers and lice as a model system: Philos.Trans.R Soc Lond B Biol
579 Sci., v. 349, no. 1327, p. 77-83.
40. Harbison, C. W., and D. H. Clayton, 2011, Community interactions govern host-
580 581 switching
with
implications
for
host-parasite
582 Proc.Natl.Acad.Sci.U S A, v. 108, no. 23, p. 9525-9529.
coevolutionary
history:
41. Hindle, E., 1917, Notes on the biology of Pediculus humanus L.: Parasitology, v. 9, p.
583 259-265.
584 42. Hirai, H., H. Imai, and Y. Go, 2012, Post-Genome Biology of Primates. Tokyo,
585 Springer.
586 587 43. Hodgson, J. A., K. N. Sterner, L. J. Matthews, A. S. Burrell, R. A. Jani, R. L. Raaum,
588 C. B. Stewart, and T. R. Disotell, 2009, Successive radiations, not stasis, in the South
589 American primate fauna: Proc.Natl.Acad.Sci.U S A, v. 106, no. 14, p. 5534-5539.
590 44. Hopkins,GHE. The correct names of the body and head lice of man. [85], 91-92. 1952.
The Entomologist.
591 592 Ref Type: Serial (Book,Monograph)
593 45. Horai, S., K. Hayasaka, R. Kondo, K. Tsugane, and N. Takahata, 1995, The recent
594 African origin of modern humans revealed by complete sequences of hominoid
24 48
595 mitochondrial DNAs.: Proceedings of the National Academy of Sciences., v. 92, no. 2,
596 p. 532-536.
597 46. Houhamdi, L., H. Lepidi, M. Drancourt, and D. Raoult, 2006, Experimental model to
598 evaluate the human body louse as a vector of plague: J.Infect.Dis., v. 194, no. 11, p.
599 1589-1596.
47. Johnson, K. P., R. J. Adams, R. D. Page, and D. H. Clayton, 2003, When do parasites
600 fail to speciate in response to host speciation?: Syst.Biol, v. 52, no. 1, p. 37-47.
601 602 48. Johnson, K. P., and D. H. Clayton, 2003, The biology, ecology, and evolution of
603 chewing lice., in RD Price, RA Hellenthal, RL Palma, KP Johnson, and DH Clayton
604 eds., The chewing lice: world checklist and biological overview.: Illinois Natural
605 History Survey Special Publication, p. 449-476.
49. Johnson, K. P., K. Yoshizawa, and V. S. Smith, 2004, Multiple origins of parasitism in
606 lice: Proc.Biol Sci., v. 271, no. 1550, p. 1771-1776.
607 608 50. Johnston, J. S., K. S. Yoon, J. P. Strycharz, B. R. Pittendrigh, and J. M. Clark, 2007,
609 Body lice and head lice (Anoplura: Pediculidae) have the smallest genomes of any
610 hemimetabolous insect reported to date: J.Med.Entomol., v. 44, no. 6, p. 1009-1012.
611 51. Kelley, J. L., J. T. Peyton, A. S. Fiston-Lavier, N. M. Teets, M. C. Yee, J. S. Johnston,
612 C. D. Bustamante, R. E. Lee, and D. L. Denlinger, 2014, Compact genome of the
613 Antarctic midge is likely an adaptation to an extreme environment: Nat.Commun., v.
614 5, p. 4611.
615 52. Kim, K. C., 2006, Blood-sucking lice (Anoplura) of small mammals: True parasites.,
616 in S Morand, BR Krasnov, and R Poulin eds., Micromammals and Macroparasites.:
617 Springer Japan, p. 141-160.
618 53. Kirkness, E. F. et al., 2010, Genome sequences of the human body louse and its
619 primary endosymbiont provide insights into the permanent parasitic lifestyle:
620 Proc.Natl.Acad.Sci.U.S.A, v. 107, no. 27, p. 12168-12173.
25 49
54. Kittler, R., M. Kayser, and M. Stoneking, 2003, Molecular evolution of Pediculus
621 humanus and the origin of clothing: Curr.Biol., v. 13, no. 16, p. 1414-1417.
622 623 55. Kuhn, H. J., and H. W. Ludwig, 1967, Die Affenlause der Gattung Pedicinus.:
624 Zeitschrift fur zoologische Systematik und Evolutionsforschung, v. 5, p. 144-297.
625 56. Laitman, J. T., 2011, A (New World monkey) tree grows in Brooklyn:
Anat.Rec.(Hoboken.), v. 294, no. 12, p. 1953-1954.
626 627 57. Lee, S. H. et al., 2010, Decreased detoxification genes and genome size make the
628 human body louse an efficient model to study xenobiotic metabolism: Insect Mol.Biol,
629 v. 19, no. 5, p. 599-615.
630 58. Light, J. E., 2005, Host-parasite cophylogeny and rates of evolution in two rodent-
631 louse assemblages (Doctoral dissertation, Faculty of the Louisiana State University
632 and Agricultural and Mechanical College in partial fulfillment of the requirements for
633 the degree of Doctor of Philosophy in The Department of Biological Sciences by
634 Jessica E. Light BS, University of Michigan).,
635 59. Light, J. E., V. S. Smith, J. M. Allen, L. A. Durden, and D. L. Reed, 2010,
636 Evolutionary history of mammalian sucking lice (Phthiraptera: Anoplura):
637 BMC.Evol.Biol, v. 10, p. 292.
638 60. Lyal, C. H., 1985a, Phylogeny and classification of the Psocodea, with particular
639 reference to the lice (Psocodea: Phthiraptera).: Systematic Entomology, v. 10, no. 2, p.
640 145-165.
61. Lyal, C. H. C., 1985b, A cladistic analysis and classification of trichodectid mammal
641 lice (Phthiraptera: Ischnocera): Bull.Brit.Mus.(Nat.Hist.) Entomol., v. 51, p. 187-346.
642 62. Mey, E., 2010, The Pedicinus species (Insecta, Phthiraptera, Anoplura, Pedicinidae)
643 on douc langurs (Pygathrix spp.).: Vietnamese Journal of Primatology, v. 4, p. 57-68.
644 26 50
645 63. Mittermeier, R. A., A. B. Rylands, and W. R. Konstant, 1999, Primates of the world:
646 An introduction, in RM Nowak ed., Walker's Mammals of the World (6th ed.): Johns
647 Hopkins University Press, p. 1-52.
64. Mumcuoglu, K. Y., and J. Zias, 1988, Head lice, Pediculus humanus capitis
648 (Anoplura, Pediculidae) from hair combs excavated in Israel and dated from
649 650 the first century B.C. to the eighth century A.D.: J.Med.Entomol., v. 25, p. 545-547.
65. Nuttall, G., 1919a, The biology of Pediculus humanus.: Parasitology, v. 10, p. 201-
651 220.
652 66. Nuttall, G. H., 1917, Studies on Pediculus. I. The copulatory apparatus and the process
653 of copulation in Pediculus humanus.: Parasitology, v. 9, no. 2, p. 293-324.
654 67. Nuttall, G. H., 1919b, The biology of Pediculus humanus L. (Supplementary notes).:
655 Parasitology, v. 11, p. 201-220.
656 68. Nuttall, G. H., 1919c, The systematic position, synonymy and iconography of
657 Pediculus humanus and Phthirus pubis.: Parasitology, v. 11, no. 3-4, p. 329-346.
658 69. Olds, B. P. et al., 2012, Comparison of the transcriptional profiles of head and body
659 lice: Insect Mol.Biol, v. 21, no. 2, p. 257-268.
660 70. Page, R. D., and M. A. Charleston, 1998, Trees within trees: phylogeny and historical
661 associations: Trends Ecol.Evol., v. 13, no. 9, p. 356-359.
662 71. Pariser, D. M., T. L. Meinking, and W. G. Ryan, 2013, Topical ivermectin lotion for
663 head lice: N.Engl.J.Med., v. 368, no. 10, p. 967.
664 72. Perelman, P. et al., 2011, A molecular phylogeny of living primates: PLoS.Genet., v.
665 7, no. 3, p. e1001342.
666 27 51
667 73. Perez, J. M., and R. L. Palma, 2001, A new species of Felicola (Phthiraptera:
668 Trichodectidae) from the endangered Iberian lynx: another reason to ensure its
669 survival.: Biodiversity and Conservation, v. 10, p. 929-937.
670 74. Piarroux, R., A. A. Abedi, J. C. Shako, B. Kebela, S. Karhemere, G. Diatta, B.
671 Davoust, D. Raoult, and M. Drancourt, 2013, Plague epidemics and lice, Democratic
672 Republic of the Congo: Emerg.Infect.Dis., v. 19, no. 3, p. 505-506.
75. Price, R. D., and O. H. Graham, 1997, Chewing and Sucking Lice as Parasites of
673 Mammals and Birds.: Technical Bulletin, no. 1849.
674 675 76. Price, R. D., and R. M. Timm, 1995, The chewing louse genus Aotiella (Phthiraptera:
676 Gyropidae) from the South American night monkeys, Aotus (Primates: Cebidae).:
677 Proceedings of the Entomological Society of Washington, v. 97, no. 3, p. 659-665.
678 77. Raoult, D., D. L. Reed, K. Dittmar, J. J. Kirchman, J. M. Rolain, S. Guillen, and J. E.
679 Light, 2008, Molecular identification of lice from pre-Columbian mummies:
680 J.Infect.Dis., v. 197, no. 4, p. 535-543.
78. Raoult, D., and V. Roux, 1999, The body louse as a vector of reemerging human
681 diseases: Clin.Infect.Dis., v. 29, no. 4, p. 888-911.
682 683 79. Reed, D. L., J. E. Light, J. M. Allen, and J. J. Kirchman, 2007, Pair of lice lost or
684 parasites regained: the evolutionary history of anthropoid primate lice: BMC.Biol., v.
685 5, p. 7.
686 80. Reed, D. L., V. S. Smith, S. L. Hammond, A. R. Rogers, and D. H. Clayton, 2004,
687 Genetic analysis of lice supports direct contact between modern and archaic humans:
688 PLoS.Biol, v. 2, no. 11, p. e340.
81. Reed, K. E., and J. G. Fleagle, 1995, Geographic and climatic control of primate
689 diversity: Proc.Natl.Acad.Sci.U S A, v. 92, no. 17, p. 7874-7876.
690 28 52
82. Richmond, B. G., and D. S. Strait, 2000, Evidence that humans evolved from a
691 knuckle-walking ancestor: Nature, v. 404, no. 6776, p. 382-385.
692 83. Roos, C., and T. Geissmann, 2001, Molecular phylogeny of the major hylobatid
693 divisions: Mol.Phylogenet.Evol., v. 19, no. 3, p. 486-494.
694 84. Roos, C., J. Schmitz, and H. Zischler, 2004, Primate jumping genes elucidate
695 strepsirrhine phylogeny: Proc.Natl.Acad.Sci.U S A, v. 101, no. 29, p. 10650-10654.
696 85. Rowe, N., J. Goodall, and R. A. Mittermeier, 1996, The Pictorial Guide to the Living
697 Primates. East Hampton, New York, Pogonias Press.
698 699 86. Rylands, A. B., and R. A. Mittermeier, 2009, The Diversity of the New World
700 Primates (Platyrrhini): An Annotated Taxonomy, in PA Garber, A Estrada, JC Bicca-
701 Marques, EW Heymann, and KB Strier eds., South American Primates. Comparative
702 Perspectives in the Study of Behavior, Ecology, and Conservation.: Springer, p. 23-54.
703 87. Sangare, A. K. et al., 2014, Detection of Bartonella quintana in African Body and
Head Lice: Am.J.Trop.Med.Hyg., v. 91, no. 2, p. 294-301.
704 705 88. Sasaki, T., S. K. S. Poudel, H. Isawa, T. Hayashi, S. Seki, T. Tomita, K. Sawabe, and
706 M. Kobayashi, 2006, First Molecular Evidence of Bartonella quintana in Pediculus
707 humanus capitis (Phthiraptera: Pediculidae), Collected from Nepalese Children:
708 J.Med.Entomol., v. 43, no. 1, p. 110-112.
89. Schmitt, D., M. D. Rose, J. E. Turnquist, and P. Lemelin, 2005, Role of the prehensile
709 710 tail
during
ateline
locomotion:
experimental
711 Am.J.Phys.Anthropol., v. 126, no. 4, p. 435-446.
and
osteological
evidence:
712 90. Scholl, K., J. M. Allen, F. H. Leendertz, C. A. Chapman, and D. L. Reed, 2012,
713 Variable microsatellite loci for population genetic analysis of Old World monkey lice
714 (Pedicinus sp.): J.Parasitol., v. 98, no. 5, p. 930-937.
29 53
91. Seiffert, E. R., E. L. Simons, and Y. Attia, 2003, Fossil evidence for an ancient
715 divergence of lorises and galagos: Nature, v. 422, no. 6930, p. 421-424.
716 717 92. Shao, R., X. Q. Zhu, S. C. Barker, and K. Herd, 2012, Evolution of extensively
718 fragmented mitochondrial genomes in the lice of humans: Genome Biol Evol., v. 4,
719 no. 11, p. 1088-1101.
720 93. Smith, V. S., T. Ford, K. P. Johnson, P. C. Johnson, K. Yoshizawa, and J. E. Light,
721 2011, Multiple lineages of lice pass through the K-Pg boundary: Biol Lett., v. 7, no. 5,
722 p. 782-785.
94. Takken, W., and N. O. Verhulst, 2013, Host preferences of blood-feeding mosquitoes:
723 Annu.Rev.Entomol., v. 58, p. 433-453.
724 725 95. Veracx, A., A. Boutellis, V. Merhej, G. Diatta, and D. Raoult, 2012, Evidence for an
726 African cluster of human head and body lice with variable colors and interbreeding of
727 lice between continents: PLoS.One., v. 7, no. 5, p. e37804.
728 96. Wilkinson, R. D., M. E. Steiper, C. Soligo, R. D. Martin, Z. Yang, and S. Tavare,
729 2011, Dating primate divergences through an integrated analysis of palaeontological
730 and molecular data: Syst.Biol, v. 60, no. 1, p. 16-31.
97. Wilson, D. E., and D. M. Reeder, 2005, Mammal species of the world: a taxonomic
731 and geographic reference. JHU Press.
732 733 98. Yoder, A. D., 2003, The phylogenetic position of genus Tarsius: whose side are
734 you on?, in PC Wright, EL Simons, and S Gursky eds., Tarsiers: past, present, and
735 future: New Jersey, Rutgers University Press, p. 161-175.
736 99. Yoon, K. S., J. P. Strycharz, J. H. Baek, W. Sun, J. H. Kim, J. S. Kang, B. R.
737 Pittendrigh, S. H. Lee, and J. M. Clark, 2011, Brief exposures of human body lice to
738 sublethal amounts of ivermectin over-transcribes detoxification genes involved in
739 tolerance: Insect Mol.Biol., v. 20, no. 6, p. 687-699.
740 30 54
Suborder
Philopteridae
Family
Table 1. The lice of primates
Ischnocera
Trichodectidae
Lemuridae
Indri and Propithecus
Lemur and Eulemur
Primate Genus
Indridae
Nycticebus
Primate Family
Lorisidae
Alouatta and Brachyteles
Genus
Felicola
Cebidae
Trichophilopterus
Cebidicola
Lemuridae
Cheirogaleidae
Lepilemur
Eulemur
Cheirogaleus and Microcebus
Aotus
Megaladapidae
Avahi and Propithecus
Aotiella
Indridae
Galago, Galagoides and Otolemur
Gyropidae
Galagonidae
Pan and Homo
Amblycera
Phthirpediculus
Hominidae
Alouatta, Cebus, Cacajao, Pithecia and Ateles
Lemurpediculus
Lemurphthirus
Cebidae
Hylobates
Polyplacidae
Pediculus
Hylobatidae
Gorilla and Homo
Presbytis, Procolobus, Trachypithecus and Semnopithecus
Lophocebus, Chlorocebus, Miopithecus, Colobus, Nasalis,
Cercocebus, Cercopithecus, Erythrocebus, Macaca, Papio,
Pedicinus
Pthirus
Hominidae
Cercopithecidae
Anoplura
Pediculidae
Pedicinidae
Pthiridae
55
Figure 1. Maximum-likelihood (ML) phylogram of cox1 mt gene in primates.
The symbols represent the different genera of lice that parasitize different primate families.
56
Figure 2. Maximum-likelihood (ML) phylogram of cytb mt gene.
ML bootstrap that support values greater than 75 are located above the nodes. Mitochondrial clade
memberships are indicated to the right of each tree. GenBank accession numbers, manuscript lead author,
locality are indicated for each louse specimen. Localities are abbreviated as follows: California (CA),
Florida (FL), Georgia (GA), Maryland (MD), Massachusetts (MA), Papua New Guinea (PNG), Republic
Democratic of the Congo (RDC), United Kingdom (UK), and Utah (UT).
57
Chapitre 1 :
Différenciation pou de tête - pou de corps
59
Préambule
Le pou de tête et le pou de corps appartiennent-ils à la même espèce?
Une question restée en débat pendant plus de deux siècles. En effet,
une fois détachés de leurs niches écologiques respectives, ils ne sont
plus différenciables [42]. La comparaison de différents critères
morphologiques chez les deux types de poux tels la forme, la taille ou
encore la pigmentation n’a pas réussi à les séparer [43]. Certains
auteurs soutiennent que les différences morphologiques observées
entre les deux écotypes seraient dues à une adaptation à leur
environnement. À juste titre, des poux de tête
élevés sous les
conditions habituelles des poux de corps deviennent progressivement
indiscernables de ces derniers [44,45].
C’est finalement le séquençage du génome du pou de corps [28] qui a
permis de faire une percée dans le traitement de la question. En effet,
dans une étude visant à comparer le profil transcriptionnel du pou de
tête et du pou de corps, Olds et al. avaient soutenu que seul le gène
phum_PHUM540560 séparait les deux types de poux. Pour les
auteurs, ce gène était présent et transcrit chez le pou de corps, mais
absent chez le pou de tête [29].
61
Dans la foulée, nous avons ciblé et réussi à amplifier une séquence
partielle de 187 pb de ce gène aussi bien chez des poux de tête que des
poux de corps appartenant au Clade A provenant de 13 pays, 5
continents. L’alignement des séquences obtenues a révélé l’existence
de plusieurs polymorphismes entre les séquences des deux types de
poux. Nous nous sommes appuyés sur ces différences pour mettre en
place un outil moléculaire basé sur de la RT PCR multiplexe capable
de différencier rapidement entre le pou de tête et le pou de corps [42].
Cet outil s’est révélé particulièrement utile sur le terrain pour
connaître le statut génotypique des poux infectés par B. quintana
[16,46].
62
Article I:
Distinguishing Body Lice from Head Lice by Multiplex Real-Time
PCR Analysis of the Phum_PHUM540560 Gene
PLoS One 8: e58088
63
Distinguishing Body Lice from Head Lice by Multiplex
Real-Time PCR Analysis of the Phum_PHUM540560 Gene
Rezak Drali1,2, Amina Boutellis1, Didier Raoult1, Jean Marc Rolain1, Philippe Brouqui1*
1 Aix Marseille Université, URMITE, UM63, CNRS 7278, IRD 198, Inserm 1095, Marseille, France, 2 Service des Entérobactéries et Hygiène de l’Environnement, Institut
Pasteur d’Algérie, Algiers, Algeria
Abstract
Background: Body louse or head louse? Once removed from their environment, body and head lice are indistinguishable.
Neither the morphological criteria used since the mid-18th century nor the various genetic studies conducted since the
advent of molecular biology tools have allowed body lice and head lice to be differentiated. In this work, using a portion of
the Phum_PHUM540560 gene from the body louse, we aimed to develop a multiplex real-time polymerase chain reaction
(PCR) assay to differentiate between body and head lice in a single reaction.
Materials and Methods: A total of 142 human lice were collected from mono-infested hosts from 13 countries on five
continents. We first identified the louse clade using a cytochrome b (CYTB) PCR sequence alignment. We then aligned a
fragment of the Phum_PHUM540560 gene amplified from head and body lice to design-specific TaqMan� FAM- and VIClabeled probes.
Results: All the analyzed lice were Clade A lice. A total of 22 polymorphisms between the body and head lice were
characterized. The multiplex real-time PCR analysis enabled the body and head lice to be distinguished in two hours. This
method is simple, with 100% specificity and sensitivity.
Conclusions: We confirmed that the Phum_PHUM540560 gene is a useful genetic marker for the study of lice.
Citation: Drali R, Boutellis A, Raoult D, Rolain JM, Brouqui P (2013) Distinguishing Body Lice from Head Lice by Multiplex Real-Time PCR Analysis of the
Phum_PHUM540560 Gene. PLoS ONE 8(2): e58088. doi:10.1371/journal.pone.0058088
Editor: Ramy K. Aziz, Cairo University, Egypt
Received August 29, 2012; Accepted January 30, 2013; Published February 28, 2013
Copyright: � 2013 Drali et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors have no support or funding to report.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: philippe.brouqui@univmed.fr
Introduction
18S ribosomal RNA has enabled the sub-Saharan African
phylogenetic group of lice to be distinguished from a second
group that encompasses the remainder of the lice worldwide [8,9].
An analyses of the mitochondrial cytochrome b (CYTB) and
cytochrome oxidase I (COI) genes, have allowed the differentiation of three clades of lice. Clade A contains both body and head
lice that are distributed worldwide. Clade B contains head lice
encountered in America, Europe and Australia, whereas Clade C
contains head lice found in Ethiopia, Nepal and Senegal [10–14].
Recently, a method targeting intergenic spacers that utilizes four
highly polymorphic markers has revealed associations between the
sources and genotypic distributions of lice [15,16]. Nevertheless,
none of the above genetic studies were able to differentiate
between body and head lice. In 2010, the sequencing of the entire
genome of P. humanus corporis provided new perspectives for
understanding the relationship between the biology and genetics of
the louse [17]. More recently, a study comparing the transcriptional profiles of body and head lice reported that the two types of
lice had a single, 752-base pair (bp) difference in the Phum_PHUM540560 gene, which encodes a hypothetical, 69-amino
acids protein of unknown function [18]. Based on the alignment of
a portion of the two Phum_PHUM540560 gene sequences, we
have designed a novel multiplex real-time PCR assay to efficiently
differentiate, for the first time, between body and head lice
Body and head lice are hematophagous ectoparasites that are
specific to humans [1] and have different ecologies. The body
louse, Pediculus humanus corporis, lives and multiplies in clothing,
whereas the head louse, Pediculus humanus capitis, lives and lays its
eggs on hair [2,3]. The body louse is known as a vector of three
life-threatening infectious diseases: epidemic typhus, caused by
Rickettsia prowazekii; relapsing fever, caused by Borrelia recurrentis; and
trench fever, caused by Bartonella quintana [4,5].
Distinguishing body from head lice has always been a challenge.
Once a louse leaves its biotope (head or clothes), it becomes
indistinguishable from other lice, which has presented a critical
problem in historical and paleobiological studies of lice.
Since the mid-18th century, morphological criteria such as size,
shape and color gradation have been used to differentiate body
and head lice into two distinct species [6]. In 1978, the use of
microscopes to observe body and head lice collected from
Ethiopians with double infestations allowed a researcher to
conclude that the lice represented two distinct species, Pediculus
humanus Linnaeus and Pediculus capitis De Geer. He based his assertion
on the length of the tibia of the louse’s middle leg [7].
The advent of molecular biology and gene sequencing has led to
the development of genetic studies to address issues concerning
louse phylogeny. The investigation of the gene that encodes the
PLOS ONE | www.plosone.org
1
65
February 2013 | Volume 8 | Issue 2 | e58088
Differentiating Body from Head Louse
collected from a mono-infested host. This assay has been tested by
analyzing a large collection of worldwide specimens belonging to
Clade A, the only clade known to contain both body and head lice.
Research, Waltham, MA, USA). The final reaction volume was
20 ml, with 0.4 U of Phusion polymerase (Finnzymes, Thermo
Scientific, Vantaa, Finland), 4 ml of 5x Phusion buffer, 0.5 mM of
each primer, 0.16 mM dNTP mix and 30–50 ng of genomic
DNA. The following cycling conditions were used for the
amplifications: an initial 30-s denaturation at 98uC; 35 cycles of
denaturation for 5 s at 98uC and annealing for 30 s at 56uC (for
CYTB gene) or 59uC (for the Phum_PHUM540560 gene); and a
final 15 min extension at 72uC. The amplification was completed
by a 5-min extension at 72uC. Subsequently, the PCR products
were subjected to electrophoresis on 1.5% agarose gels with
ethidium bromide staining and were then purified using NucleoFast 96 PCR Plates (Macherey-Nagel EURL, Hoerdt, France)
according to the manufacturer’s instructions.
Bidirectional DNA sequencing of the targeted PCR products
was performed using the 3130XL genetic analyzer (Applied
Biosystems, Courtaboeuf, France) with the BigDye Terminator
v1.1 cycle (Applied Biosystems). The electropherograms obtained
for each sequence were analyzed using Chromas Pro software
(Technelysium PTY, Australia).
Materials and Methods
Ethics statement
Lice from foreign countries were obtained from the private
frozen collection of our laboratory (The URMITE/WHO
Collaborative Research Center). The lice in that collection were
required for various epidemiological and entomological studies or
to perform diagnoses abroad and were sent to our laboratory as a
WHO reference facility. The specimens were collected according
to the ethics laws of each country; however, because lice are not
part of the human body, lice removed from individuals are not
considered to be human samples in most countries. The body lice
were collected from clothing, and the head lice were removed from
hair, with the verbal consent of the infested individuals. Written
consent was not obtainable in the majority of cases because most
of the subjects were illiterate. However, in most instances, the
investigator, local authorities and/or village chief approved and
were present when it was performed.
The lice collected in France were obtained from homeless
individuals during a registered epidemiological study (French
Bioethics laws nu 2011–814). Informed consent was obtained from
these subjects, and the study was approved by the ‘‘Comité de
Protection des Personnes Sud Mediterranée I’’ on January, 12,
2011 (ID RCB: 2010-A01406-33).
The anonymity of the individuals who provided the lice used in
the present genetic analysis was preserved.
Phylogenetic analysis
The DNA sequences were aligned using the multi-sequence
alignment software CLUSTAL X, version 2.0.11. The partial
CYTB gene sequences were aligned with sequences available from
GenBank. The percentages of similarity were determined using the
MEGA 5 software package (Molecular Evolution Genetic Analysis, The Biodesign Institute, AZ, USA) [20]. The PhyML
phylogeny software was used to create an unrooted phylogenetic
tree based on the DNA sequences using maximum likelihood (ML)
100 bootstrap replicates [21].
Sampling
A total of 142 lice, including 88 body lice and 54 head lice, were
collected from mono-infested human hosts. The head lice were
collected exclusively from the hair, and the body lice were
collected exclusively from clothing. No lice were collected from the
neck or the beard; the purpose of this precaution was the
avoidance hybrid lice, as previously reported [7]. The strain
information, geographic origin and anatomical sources (body or
head) of the analyzed lice are provided in Table 1.
Real-time PCR and PCR products sequencing
TaqMan� FAM- and VIC-labeled probes (Table 2) specific to
body and head lice, respectively, were designed for the sequences
obtained in this study. Both probes contained a TAMRA
quencher dye at the 39 end. The probes were synthesized by
Applied Biosystems (Courtaboeuf, France).
Monoplex and multiplex real-time PCRs were performed in the
CFX96 thermal cycler (Bio-Rad Laboratories, Foster City, CA,
USA). The final reaction volume of 20 ml contained 5–20 ng of the
DNA template, 10 ml of 2x QuantiTect Probe PCR Master Mix
(Qiagen), 0.5 mM of each primer and 0.2 mM of the FAM- or
VIC-labeled probes. A monoplex protocol designed to optimize
the conditions for the multiplex real-time PCR was used: a
denaturation step at 95uC for 15 min; and 40 cycles of 95uC for
15 s and 60uC for 45 s. The multiplex real-time PCR was
performed using the optimized conditions that were determined in
the monoplex real-time PCR assay. Each reaction contained 10 ml
of 2x QuantiTect Probe PCR Master Mix (Qiagen), 0.5 mM of
each primer, 0.2 mM of each fluorogenic probe, 5–20 ng of the
DNA template, adjusted to a final volume of 20 ml with the
addition of nuclease-free dH2O. The cycling parameters consisted
of 95uC for 15 min and 40 cycles of 95uC for 15 s and 60uC for
1 min. To evaluate the specificity (the ability of the test to identify
negative results) and sensitivity (the ability of the test to identify
positive results) of the developed method, all the products of the
multiplex real-time PCR amplifications were sequenced, and these
sequences were used as the gold standard reference.
DNA preparation
Prior to DNA isolation, each louse was immersed in 70%
ethanol for 15 min and was then rinsed twice in sterile water.
Total genomic DNA was extracted using the QIAamp Tissue Kit
(QIAGEN, Hilden, Germany) according to the manufacturer’s
instructions. The extracted DNA was assessed for quantity and
quality using a NanoDrop instrument (Thermo Scientific,
Wilmington, United Kingdom) before being stored at 220uC [19].
Conventional PCR and sequencing
Two conventional PCR experiments were performed in this
study. The first was performed to identify the Clades of the
collected lice by amplifying and sequencing a 347-bp fragment of
the mitochondrial cytochrome b (CYTB) gene [7]. The second
PCR targeted a 187-bp fragment of the Phum_ PHUM540560
gene using a pair of primers designed in this study and based on
the Phum_PHUM540560 gene sequence available from GenBank
(Pediculus humanus corporis strain USDA 1103172108290, GenBank
accession no. NW_002987859.1 GI: 242022583). The obtained
PCR products from three body lice and three head lice were
sequenced to enable comparison of the body and head lice DNA
sequences. All the PCRs were performed using the primers
outlined in Table 2 and a PTC-200 automated thermal cycler (MJ
PLOS ONE | www.plosone.org
Results
The concentration of the genomic DNA extracted from the 142
lice analyzed in this study ranged from 5 to 20 ng/ml.
2
66
February 2013 | Volume 8 | Issue 2 | e58088
Differentiating Body from Head Louse
Table 1. The Clade A lice examined in this study and the results of the real-time PCR assay.
Country
Town/province
Analysis channel results
Number
FAM-positive
VIC-positive
0
Body lice
France
Marseille
15
15
Hungary
Budapest
10
10
0
Nepal
Pokava
9
9
0
China
Inner Mongolia Province
5
5
0
Tiligi
7
7
0
Japan
Tokyo
10
10
0
Madagascar
Borenty village
9
9
0
Kenya
Nairobi
10
10
0
USA
Orlando
13
13
0
Head lice
USA
Washington
6
0
6
Brazil
Sao Cristovao
6
0
6
Amazonia
8
0
8
Madagascar
Bedaro village
12
0
12
Senegal
Dakar
3
0
3
Australia
Brisbane
5
0
5
Papua New Guinea
Highlands
5
0
5
New Zeland
Auckland
9
0
9
doi:10.1371/journal.pone.0058088.t001
Genotypic distribution of the lice based on the
mitochondrial CYTB gene
acid and the I19N (ATT.AAT) mutation that would replace
isoleucine with asparagine.
The remainder of the polymorphisms were spread throughout
the first intron and included the insertion of nucleotides at two
different locations: approximately nucleotide (nt) 96+11 ins.G and
nt 96+80, (ins.CT). This triplex insertion resulted in the
amplification of a 190-bp fragment from the head lice and a
187-bp fragment from the body lice (Figure 1).
A 347-bp DNA fragment was successfully amplified from the
CYTB gene in all 142 lice. Direct sequencing and multiple
alignments of the obtained sequences revealed that all the lice
belonged to Clade A (data not shown).
Characterization of the partial Phum_PHUM540560 gene
in body and head lice
Real-time PCR and PCR product sequencing
The multiple alignments of the partial Phum_PHUM54056
gene sequences obtained from the six analyzed lice revealed 22
polymorphisms between body and head lice (Figure 1). The first
exon contained two point mutations: a silent (CCA.CCC)
transversion affecting codon 18 that would not change the amino
The monoplex real-time PCR results demonstrated that the
FAM-labeled probe was specific to the body lice and that the VIClabeled probe was specific to the head lice. This assay was
optimized by testing louse specimens from known anatomical
locations.
Table 2. The oligonucleotide primers and probes used in this study.
Name
Purpose
Sequence 59R39
Cytb_F
Forward sequencing primer partial
cytochrome b gene
GAGCGACTGTAATTACTAATC
Cytb_R
Reverse sequencing primer
partial cytochrome b gene
GGACCCGGATAATTTTGTTG
Phum540560_F
Forward sequencing primer
partial Phum_PHUM540560 gene
GTCACGTTCGACAAATGTT
Phum540560_R
Reverse sequencing primer
partial Phum_PHUM540560 gene
TTTCTATAACCACGACACGATAAAT
BL probe
Specific to the body lice
FAM-CGATCACTCGAGTGAATTGCCA-TAMRA
HL probe
Specific to the head lice
VIC-CTCTTGAATCGACGACCATTCGCT-TAMRA
doi:10.1371/journal.pone.0058088.t002
PLOS ONE | www.plosone.org
3
67
February 2013 | Volume 8 | Issue 2 | e58088
Differentiating Body from Head Louse
Figure 1. Primer and probe alignments with partial Phum_PHUM540560 gene sequences from body and head lice [35]. A portion of
the Phum_PHUM540560 gene sequences from body and head lice were aligned with the primers and probes designed for the multiplex RT-PCR
assay. Part of the first exon spanings nucleotides 1 to 64 was analyzed. The forward and reverse primer sequences are boxed in black. The FAM- and
VIC-labeled probe sequences are boxed in purple and green, respectively. The nucleotides in blue represent single-nucleotide polymorphisms that
are specific to head lice. The nucleotides in black represent single-nucleotide polymorphisms that are specific to body lice. BL: body louse; HL: head
louse; NW_002987859.1: Pediculus humanus corporis strain USDA 1103172108290 Phum_PHUM540560 (gene sequence available in GenBank).
doi:10.1371/journal.pone.0058088.g001
The multiplex real-time PCR assay clearly identified and
simultaneously differentiated among the 142 lice included in this
work. Specifically, the signal emitted by the FAM-labeled probe
was detected only in the body louse samples, whereas the signal
emitted by the VIC-labeled probe was detected only in the head
louse samples (Figure 2). No signals were detected in the nontemplate controls (NTCs). The Ct values obtained in this assay are
outlined in Table S1. The sequencing of the 142 PCR products
has confirmed our results. In addition, 100% of the samples that
were positive for the FAM-labeled probe contained sequences
specific to body lice, and 100% of the samples that were positive
for the VIC-labeled probe contained sequences specific to head
lice (100% sensitivity and 100% specificity; data not shown).
Discussion
Presently, comparisons of the body and head lice genomes are
not possible because the head louse genome is not yet available.
Recently published comparative transcriptional profiles of both
body and head lice demonstrated that among the nine genes with
differential expression, only one gene was absent in the head louse
but present in the body louse [18]. We considered this difference
to be a possible opportunity for distinguishing body lice from head
Figure 2. Amplification curves from multiplex real-time PCR assays. Figure 1A. Real-time PCR amplification curves for body lice using a
partial Phum_PHUM540560 gene in the FAM channel (495–520). Figure 1B. Amplification curves for head licee louse using a partial
Phum_PHUM540560 gene in the VIC channel (522–544).
doi:10.1371/journal.pone.0058088.g002
PLOS ONE | www.plosone.org
4
68
February 2013 | Volume 8 | Issue 2 | e58088
Differentiating Body from Head Louse
lice. Unexpectedly, our first PCR amplification of the 187- bp
fragment of the Phum_PHUM540560 gene produced a PCR
product from both head and body louse samples, suggesting that at
least a portion of the gene was present in both types of lice. The
sequencing of the PCR products revealed significant differences
between the sequences from the head and body lice, which may
explain why the head louse sequence failed to amplify in Old’s
experiment [18].
In this study, we exploited this sequence variation in the partial
Phum_PHUM540560 gene to discriminate between body and
head lice from a global collection of lice collected from monoinfested hosts originating from five different continents. Lice from
Clade A were used because Clade A is the only currently
recognized clade that includes both head and body lice [10,13,14],
the two types of lice that our assay was developed to distinguish.
Finally, our choice of specimens was based on the commonly
recognized definitions of body and head lice. Under these
conditions, we developed a multiplex real-time PCR assay that is
rapid (two hours) and simple and has 100% specificity and
sensitivity.
The purpose of this study was to distinguish between body and
head lice, a long-standing challenge. Resolving this challenge has
become even more important because both head and body lice
have been reported to harbor Bartonella quintana, the trench fever
agent, raising the question of whether head lice, similar to body
lice, can transmit the agent [22,23]. Currently, B. quintana DNA
has been detected only in head lice collected from impoverished
people in situations where co-infestations with body lice are
possible [24–27]. In fact, co-infestations have been recently
reported in the same homeless population [28]. One study of
head lice collected from schoolchildren in France failed to detect
B. quintana [29]. The ability to distinguish body lice from head lice
will help advance our understanding of the role of head louse in
the transmission of B. quintana. Moreover, 22% of the homeless
people who frequent shelters in Marseille, France are infested with
lice, and some people can harbor more than 10,000 lice in their
clothing. In such a situation, finding lice on the head challenges
the ‘‘head louse definition’’, making an identification tool useful.
Recent studies have suggested that head and body lice can be
mixed in people infested with both types of lice [28]. Although
head and body lice do not interbreed in the wild [7], fertile hybrids
with an intermediate morphology [30] have been reported under
laboratory conditions [31,32]. Moreover, several observational
studies have also suggested that head lice may become body lice
when raised under the appropriate conditions [33,34]. Our
technique can be used to identify heterozygous specimens, which
may prove valuable for studies on the population dynamics of lice.
This work confirmed that the Phum_PHUM540560 gene may
be a useful genetic marker for the study of lice. However, the
genetic differences between head and body lice do not put back
into question whether head and body lice are conspecific [11]. The
ability to distinguish between head and body lice may facilitate
future research into the behavior of Clade A body and head lice.
Supporting Information
Table S1 Ct values obtained in multiplex real-time PCR for
differentiating between body and head louse.
(DOCX)
Acknowledgments
The text was edited by American Journal Experts under Certificate
Verification Key: 6B5B-1E56-39B6-59E4-BE7F. We thank Idir BITAM,
Institut Pasteur d’Algérie, Algiers, Algeria; and Christophe ROGIER,
Institut Pasteur de Madagascar, Antananarivo.
Author Contributions
Conceived and designed the experiments: PB DR JMR. Performed the
experiments: RD AB. Analyzed the data: RD AB DR JMR PB. Wrote the
paper: RD PB.
References
14. Reed DL, Smith VS, Hammond SL, Rogers AR, Clayton DH (2004) Genetic
analysis of lice supports direct contact between modern and archaic humans.
PLoS Biol 2: e340. 10.1371/journal.pbio.0020340 [doi].
15. Li W, Ortiz G, Fournier PE, Gimenez G, Reed DL, et al. (2010) Genotyping of
human lice suggests multiple emergencies of body lice from local head louse
populations. PLoS Negl Trop Dis 4: e641. 10.1371/journal.pntd.0000641 [doi].
16. Veracx A, Boutellis A, Merhej V, Diatta G, Raoult D (2012) Evidence for an
African Cluster of Human Head and Body Lice with Variable Colors and
Interbreeding of Lice between Continents. PLoS One 7: e37804. 10.1371/
journal.pone.0037804 [doi]; PONE-D-12-05586 [pii].
17. Kirkness EF, Haas BJ, Sun W, Braig HR, Perotti MA, et al. (2010) Genome
sequences of the human body louse and its primary endosymbiont provide
insights into the permanent parasitic lifestyle. Proc Natl Acad Sci U S A 107:
12168–12173. 1003379107 [pii];10.1073/pnas.1003379107 [doi].
18. Olds BP, Coates BS, Steele LD, Sun W, Agunbiade TA, et al. (2012)
Comparison of the transcriptional profiles of head and body lice. Insect Mol Biol
21: 257–268. 10.1111/j.1365-2583.2012.01132.x [doi].
19. Drali R, Benkouiten S, Badiaga S, Bitam I, Rolain JM, et al. (2012) Detection of
a knockdown resistance (kdr) mutation associated with permethrin resistance in
the body louse Pediculus humanus corporis using melting curve analysis
genotyping. J Clin Microbiol. JCM.00808-12 [pii];10.1128/JCM.00808-12
[doi].
20. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, et al. (2011) MEGA5:
molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28: 2731–2739.
msr121 [pii];10.1093/molbev/msr121 [doi].
21. Guindon S, Gascuel O (2003) A simple, fast, and accurate algorithm to estimate
large phylogenies by maximum likelihood. Syst Biol 52: 696–704.
54QHX07WB5K5XCX4 [pii].
22. Angelakis E, Rolain JM, Raoult D, Brouqui P (2011) Bartonella quintana in
head louse nits. FEMS Immunol Med Microbiol 62: 244–246. 10.1111/j.1574695X.2011.00804.x [doi].
1. Durden LA (2002) Lice (Phthiraptera). In: Mullen G, Durden LA, editors.
Medical and veterinary entomology. 45–65.
2. Burgess IF (2004) Human lice and their control. Annu Rev Entomol 49: 457–
481. 10.1146/annurev.ento.49.061802.123253 [doi].
3. Light JE, Toups MA, Reed DL (2008) What’s in a name: the taxonomic status of
human head and body lice. Mol Phylogenet Evol 47: 1203–1216.
4. Brouqui P, Stein A, Dupont HT, Gallian P, Badiaga S, et al. (2005)
Ectoparasitism and vector-borne diseases in 930 homeless people from
Marseilles. Medicine (Baltimore) 84: 61–68.
5. Raoult D, Roux V (1999) The body louse as a vector of reemerging human
diseases. Clin Infect Dis 29: 888–911.
6. Veracx A, Raoult D (2012) Biology and genetics of human head and body lice.
Trends Parasitol. S1471-4922(12)00163-8 [pii];10.1016/j.pt.2012.09.003 [doi].
7. Busvine JR (1978) Evidence from double infestations for the specific status of
human head lice and body lice (Anoplura). Systematic Entomology 3: 1–8.
8. Leo NP, Barker SC (2005) Unravelling the evolution of the head lice and body
lice of humans. Parasitol Res 98: 44–47. 10.1007/s00436-005-0013-y [doi].
9. Yong Z, Fournier PE, Rydkina E, Raoult D (2003) The geographical segregation
of human lice preceded that of Pediculus humanus capitis and Pediculus
humamus humanus. C R Biol 326.
10. Kittler R, Kayser M, Stoneking M (2003) Molecular evolution of Pediculus
humanus and the origin of clothing. Curr Biol 13: 1414–1417.
S0960982203005074 [pii].
11. Leo NP, Campbell NJ, Yang X, Mumcuoglu K, Barker SC (2002) Evidence
from mitochondrial DNA that head lice and body lice of humans (Phthiraptera:
Pediculidae) are conspecific. J Med Entomol 39: 662–666.
12. Light JE, Allen JM, Long LM, Carter TE, Barrow L, et al. (2008) Geographic
distributions and origins of human head lice (Pediculus humanus capitis) based
on mitochondrial data. J Parasitol 94: 1275–1281. GE-1618 [pii];10.1645/GE1618.1 [doi].
13. Raoult D, Reed DL, Dittmar K, Kirchman JJ, Rolain JM, et al. (2008)
Molecular identification of lice from pre-Columbian mummies. J Infect Dis 197:
535–543.
PLOS ONE | www.plosone.org
5
69
February 2013 | Volume 8 | Issue 2 | e58088
Differentiating Body from Head Louse
23. Brouqui P, Raoult D (2006) Arthropod-borne diseases in homeless. Ann N Y Acad
Sci 1078: 223–235.
24. Bonilla DL, Kabeya H, Henn J, Kramer VL, Kosoy MY (2009) Bartonella
quintana in body lice and head lice from homeless persons, San Francisco,
California, USA. Emerg Infect Dis 15: 912–915.
25. Boutellis A, Veracx A, Angelakis E, Diatta G, Mediannikov O, et al. (2012)
Bartonella quintana in head lice from Senegal. Vector Borne Zoonotic Dis 12:
564–567. 10.1089/vbz.2011.0845 [doi].
26. Sasaki T, Poudel SK, Isawa H, Hayashi T, Seki N, et al. (2006) First molecular
evidence of Bartonella quintana in Pediculus humanus capitis (Phthiraptera:
Pediculidae), collected from Nepalese children. J Med Entomol 43: 110–112.
27. Angelakis E, Diatta G, Abdissa A, Trape JF, Mediannikov O, et al. (2011)
Altitude-dependent Bartonella quintana genotype C in head lice, Ethiopia.
Emerg Infect Dis 17: 2357–2359. 10.3201/eid1712.110453 [doi].
28. Veracx A, Rivet R, McCoy KD, Brouqui P, Raoult D (2012) Evidence that head
and body lice on homeless persons have the same genotype. PLoS One 7:
e45903. 10.1371/journal.pone.0045903 [doi];PONE-D-12-20556 [pii].
PLOS ONE | www.plosone.org
29. Bouvresse S, Socolovshi C, Berdjane Z, Durand R, Izri A, et al. (2011) No
evidence of Bartonella quintana but detection of Acinetobacter baumannii in
head lice from elementary schoolchildren in Paris. Comp Immunol Microbiol
Infect Dis 34: 475–477. S0147-9571(11)00076-2 [pii];10.1016/j.cimid.2011.08.007 [doi].
30. Busvine JR (1948) The head and body races of Pediculus humanus L.
Parasitology 39: 1–16.
31. Bacot AW (1917) A contribution to the bionomics of Pediculus humanus (vestimenti)
and Pediculus capitis. Parasitology 9: 228–258.
32. Mullen G, Durden LA (2009) Medical and Veterinary Entomology. Academic
Press, San Francisco.
33. Alpatov V, Nastukova OA (1955) Transformation of the head form of Pediculus
humanus into the body form under changed conditions of existence. Bull Moscow
Nat Hist Res Soc 60: 92.
34. Nuttall G (1919) The biology of Pediculus humanus. Supplementary notes.
Parasitology 11: 201–221.
35. Corpet F (1988) Multiple sequence alignment with hierarchical clustering.
Nucleic Acids Res 16: 10881–10890.
6
70
February 2013 | Volume 8 | Issue 2 | e58088
Table S1. Ct values obtained in multiplex real-time PCR for differentiating between body and
head louse
Louse
Body louse_1003/4
Body louse_1160/1
Body louse_1200/2
Body louse_1229/6
Body louse_1237/9
Body louse_1237/10
Body louse_1237/11
Body louse_1237/16
Body louse_1237/17
Body louse_1237/19
Body louse_1240/1
Body louse_1245/4
Body louse_1246/1
Body louse_2002/2
Body louse_2215
Body louse_104
Body louse_105
Body louse_106
Body louse_107
Body louse_108
Body louse_109
Body louse_110
Body louse_111
Body louse_112
Body louse_113
Body louse_80
Body louse_82
Body louse_83
Body louse_83
Body louse_86
Body louse_87
Body louse_88
Body louse_89
Body louse_90
Body louse_91
Body louse_92
Body louse_93
Body louse_94
Body louse_95
Body louse_96
Body louse_97
Body louse_98
Ct values
FAM
VIC
21,56
NA
20,84
NA
20,02
NA
18,33
NA
19,04
NA
20,19
NA
20,11
NA
20,24
NA
20,09
NA
20,51
NA
20,87
NA
20,72
NA
18,74
NA
19,04
NA
20,10
NA
22,02
NA
25,10
NA
26,51
NA
23,90
NA
24,17
NA
22,10
NA
27,59
NA
22,74
NA
23,19
NA
25,00
NA
23,08
NA
21,58
NA
23,50
NA
30,84
NA
31,98
NA
20,68
NA
22,17
NA
26,41
NA
20,49
NA
22,43
NA
21,94
NA
22,84
NA
21,74
NA
21,28
NA
20,08
NA
20,89
NA
21,43
NA
71
Country
Town/Province
France
Marseille
Hungary
Budapest
Inner Mongolia
Province
China
Tiligi
Japan
Tokyo
Body louse_99
Body louse_100
Body louse_101
Body louse_102
Body louse_103
Body louse_70
Body louse_71
Body louse_72
Body louse_73
Body louse_74
Body louse_75
Body louse_76
Body louse_78
Body louse_79
Body louse_223
Body louse_224
Body louse_226
Body louse_227
Body louse_228
Body louse_229
Body louse_230
Body louse_231
Body louse_232
Body louse_233
Body louse_175
Body louse_176
Body louse_177
Body louse_178
Body louse_179
Body louse_180
Body louse_181
Body louse_182
Body louse_183
Body louse_PDL1
Body louse_PDL3
Body louse_PDL4
Body louse_PDL6
Body louse_PDL11
Body louse_PDL13
Body louse_PDL14
Body louse_PDL7
Body louse_PDL2
Body louse_PDL9
Body louse_PDL10
Body louse_PDL4
Body louse_PDL5
Head louse_13
23,07
21,86
22,04
22,07
22,42
21,66
21,71
23,48
23,08
22,29
23,22
26,08
24,11
23,88
30,34
35,20
33,64
30,26
36,03
30,68
31,23
30,21
32,72
35,21
20,46
21,04
20,10
20,12
22,11
21,87
23,76
23,45
23,02
19,28
18,88
18,66
18,62
18,90
18,57
19,13
18,92
18,30
17,53
19,01
21,18
21,03
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
31,52
72
Nepal
Pokava
Kenya
Nairobi
Madagascar
Borenty village
USA
Laboratory colony
USA
Washington
Head louse_14
Head louse_15
Head louse_16
Head louse_17
Head louse_19
Head louse_9
Head louse_10
Head louse_11
Head louse_12
Head louse_14
Head louse_172
Head louse_173
Head louse_175
Head louse_20
Head louse_21
Head louse_23
Head louse_24
Head louse_25
Head louse_29
Head louse_30
Head louse_31
Head louse_34
Head louse_36
Head louse_37
Head louse_50
Head louse_51
Head louse_54
Head louse_55
Head louse_56
Head louse_60
Head louse_61
Head louse_62
Head louse_63
Head louse_64
Head louse_66
Head louse_67
Head louse_68
Head louse_69
Head louse_160
Head louse_161
Head louse_162
Head louse_163
Head louse_165
Head louse_166
Head louse_168
Head louse_170
Head louse_171
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
32,36
32,04
35,44
37,34
36,01
34,43
35,87
33,92
39,17
35,69
37,51
36,56
37,32
35,47
37,84
35,22
39,33
38,18
23,20
36,56
35,45
38,16
37,01
37,88
36,43
36,33
36,26
36,21
37,64
35,71
35,94
37,74
36,43
38,07
37,20
36,89
37,45
37,16
24,45
24,79
24,54
36,13
24,09
28,02
26,78
26,23
36,16
73
Amazonia
Brazil
Sao Cristovao
Australia
Brisbane
Papua New Guinea
Highlands
New Zeeland
Auckland
Madagascar
Bedaro village
Head louse_172
Head louse_173
Head louse_174
Head louse_48
Head louse_95
Head louse_96
NTC
NA
NA
NA
NA
NA
NA
NA
28,20
29,55
27,44
27,54
28,17
24,03
NA
NA: signal not detected; NTC: non template control
74
Senegal
Dakar
Article II:
Bartonella quintana in Body Lice from
Scalp Hair of Homeless Persons, France
Emerging Infectious Diseases 20: 907-908.
75
LETTERS
Address for correspondence: Pietro E.
Varaldo, Unità di Microbiologia, Dipartimento
di Scienze Biomediche e Sanità Pubblica,
Università Politecnica delle Marche, Via Tronto
10/A, 60126 Ancona, Italy; email: pe.varaldo@
univpm.it
Bartonella
quintana in Body
Lice from Scalp
Hair of Homeless
Persons, France
To the Editor: Bartonella quintana is a body louse–borne human
pathogen that can cause trench fever,
bacillary angiomatosis, endocarditis, chronic bacteremia, and chronic
lymphadenopathy (1). Recently, B.
quintana DNA was detected in lice
collected from the heads of poor and
homeless persons from the United
States, Nepal, Senegal, Ethiopia, and
the Democratic Republic of the Congo
and in nits in France (2,3). The head
louse, Pediculus humanus capitis, and
the body louse, Pediculus humanus
humanus, are obligatory ectoparasites
that feed exclusively on human blood
(4). Outside of their habitats, the 2
ecotypes are morphologically indistinguishable (1). Sequence variation
in the PHUM540560 gene discriminates between head and body lice by
determining the genotype of the lice
(5). While surveying for trench fever
among homeless persons in shelters
in Marseille, France during October
2012–March 2013, we investigated
the presence of B. quintana DNA in
nits, larvae, and adult lice collected
from mono-infested and dually infested persons and determined the genotypes of the specimens.
The persons included in this study
received long-lasting insecticide-treated underwear; lice were collected by
removing them from clothing, including underwear, pants, and shirts. Because body lice reside in the clothing of
infested persons except when feeding,
they are sometimes called clothing lice.
A total of 989 specimens were
tested, including 149 (83 from clothing and 66 from hair) first–instar larvae hatched in the laboratory from
eggs collected from 7 dually infested
persons, and 840 adult body lice collected from the clothing of 80 monoinfested patients. We included DNA
isolated from 3 nits collected from the
hair of a mono-infested person who
had previously been confirmed as positive for B. quintana 6) (Table).
Total DNA was extracted by using an EZ1 automated extractor (QIAGEN, Courtaboeuf, France) and subjected twice to real-time PCR specific
for B. quintana. The first PCR targeted
the 16S-23S intergenic spacer region.
Positive samples were confirmed by
using a second real-time PCR targeting the yopP gene (6). Samples that
tested positive for B. quintana DNA
were analyzed by multiplex real-time
PCR that targeted the PHUM540560
gene (5). We used head and body lice
that had known genotypes positive
controls. Negative controls were included in each assay.
Of the hatched larvae, 5 (6%) of
the 83 recovered from clothing and
7 (11%) of 66 from the hair (Table)
of 4 of the 7 dually infested persons
were positive for B. quintana DNA
(online Technical Appendix Table 1
wwwnc.cdc.gov/EID/article/20/5/131242-Techapp1.pdf). Of the 840 adult
body lice, 174 (21%) collected from
42 (53%) of 80 of the mono-infested
persons contained B. quintana DNA
(Table, online Technical Appendix 2).
The multiplex real-time PCR that targeted the PHUM540560 gene clearly
identified all nits, larvae, and adult lice
as belonging to the body lice lineage.
Negative controls remained negative
in all PCR-based experiments.
For 2 decades, B. quintana DNA
has been regularly detected in lice collected from the heads of persons living
in poverty, but it had not been detected
in head lice that infest schoolchildren
(7,8). All of the lice collected during
this study that tested positive for B.
quintana from homeless persons were
body lice, including some that were
recovered from hair. This observation
supports our assertion that body lice
are not confined to the body. The 3 eggs
that were removed from the hair of a
mono-infested homeless person whose
samples tested positive for B. quintana
were also body lice. During the clinical
examination, no adult head lice or adult
body lice were found on that person, confirming that the patient had been heavily infested with body lice in the past,
not head lice. The nits were most likely
laid by body lice that migrated toward
the patient’s head. When a member of
Table.DistributionofBartonella quintana DNAinnits,larvae,andadultbodylicecollectedfromhairandclothingofhomelesspersons
inshelters,Marseille,France,October2012–March2013*
No.persons
No.(%)licepositivefor
B. quintana DNA
Reference
Location
Duallyinfested,n=7
Monoinfested,n=80
Hair
Nits
0
3
3(100)
(6)
Thisstudy
Hatchedlarvae
66
0
7(10.60)
Clothing
Hatchedlarvae
83
0
5(6.00)
Thisstudy
Adults
0
840
174(20.70)
Thisstudy
*Alllicewereidentifiedasbodylice.Studyparticipantswereprovidedwithlong-lastinginsecticide-treatedunderwear,andkilledbodylicewerecollected
fromtheclothingofinfestedpersons.
77
EmergingInfectiousDiseases•www.cdc.gov/eid•Vol.20,No.5,May2014
907
LETTERS
this research team (DR) collected the
eggs from the hair shaft, they were
found ≈3 3.5 cm from the hair follicle. Because hair grows ≈1.25 cm per
month, the louse infestation occurred
≈3 months before egg collection (6).
Homeless persons that we have
monitored for many years are often
heavily infested by body lice but are
also occasionally infested with head
lice. Before genetic tools that differentiate the head and body louse lineages
were available (5), it was speculated
that body lice may have originated
from head lice (9). From our study, it is
clear that under conditions of massive
infestation, body lice can migrate and
colonize hair; the opposite may also be
true. However, there is no evidence that
body lice are capable of causing an outbreak of lice living on the head, as happens among schoolchildren that have
been found to be infested only by head
lice. This suggests that body lice cannot thrive in the environment of head
lice, which infest millions of children
worldwide (10), further suggesting
that outbreaks of trench fever are most
likely not linked to head lice in industrialized countries. In conclusion, by
analyzing lice harvested from the heads
and clothing of homeless persons, we
have shown that the 2 ecotypes belong
to the same body lice population.
The text has been edited by American
Journal Experts under certificate verification key 51F8-8A17-1F51-90DD-705C.
Rezak Drali,
Abdoul Karim Sangaré,
Amina Boutellis,
Emmanouil Angelakis,
Aurélie Veracx,
Cristina Socolovschi,
Philippe Brouqui,
and Didier Raoult
Authoraffiliations:AixMarseilleUniversité,
Marseille, France; and Institut HospitaloUniversitaire
Méditerranée
Infection,
13005,Marseille
DOI:http://dx.doi.org/10.3201/eid2005.131242
908
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
Brouqui P. Arthropod-borne diseases
associated with political and social disorder. Annu Rev Entomol. 2011;56:357–74.
http://dx.doi.org/10.1146/annurev-ento120709-144739
Boutellis A, Mediannikov O, Bilcha
KD, Ali J, Campelo D, Barker SC, et al.
Borrelia recurrentis in head lice, Ethiopia.
Emerg Infect Dis. 2013;19:796–8.
Piarroux R, Abedi AA, Shako JC,
Kebela B, Karhemere S, Diatta G, et al.
Plague epidemics and lice, Democratic
Republic of the Congo. Emerg Infect Dis.
2013;19:505–6. http://dx.doi.org/10.3201/
eid1903.121542
Gratz NG. Emerging and resurging
vector-borne diseases. Annu Rev Entomol.
1999;44:51–75. http://dx.doi.org/10.1146/
annurev.ento.44.1.51
Drali R, Boutellis A, Raoult D,
Rolain JM, Brouqui P. Distinguishing body
lice from head lice by multiplex real-time
PCR analysis of the Phum_PHUM540560
gene. PLoS ONE. 2013;8:e58088. http://
dx.doi.org/10.1371/journal.pone.0058088
Angelakis E, Rolain JM, Raoult D,
Brouqui P. Bartonella quintana in
head louse nits. FEMS Immunol Med
Microbiol. 2011;62:244–6. http://dx.doi.
org/10.1111/j.1574-695X.2011.00804.x
Fournier PE, Ndihokubwayo JB, Guidran J,
Kelly PJ, Raoult D. Human pathogens
in body and head lice. Emerg Infect Dis.
2002;8:1515–8. http://dx.doi.org/10.3201/
eid0812.020111
Bouvresse S, Socolovshi C, Berdjane Z,
Durand R, Izri A, Raoult D, et al. No
evidence of Bartonella quintana but
detection of Acinetobacter baumannii
in head lice from elementary schoolchildren in Paris. Comp Immunol Microbiol
Infect Dis. 2011;34:475–7. http://dx.doi.
org/10.1016/j.cimid.2011.08.007
Li W, Ortiz G, Fournier PE, Gimenez G,
Reed DL, Pittendrigh B, et al. Genotyping of human lice suggests multiple
emergencies of body lice from local head
louse populations. PLoS Negl Trop Dis.
2010;4:e641. http://dx.doi.org/10.1371/
journal.pntd.0000641
Chosidow O, Chastang C, Brue C,
Bouvet E, Izri M, Monteny N, et al.
Controlled study of malathion and
d-phenothrin lotions for Pediculus humanus var capitis-infested schoolchildren.
Lancet. 1994;344:1724–7. http://dx.doi.
org/10.1016/S0140-6736(94)92884-3
Address for correspondence: Didier Raoult,
Institut Hospitalo-Universitaire Méditerranée
Infection, 27 boulevard Jean Moulin, 13385
Marseille CEDEX 5, France; email: didier.
raoult@gmail.com
Myasthenia Gravis
Associated with
Acute Hepatitis E
Infection in
Immunocompetent
Woman
To the Editor: Hepatitis E virus (HEV) is a common cause of
acute hepatitis in developing countries. The course of acute hepatitis E
is usually benign, except in pregnant
women and in immunocompromised
patients, who are prone to a lethal or
chronic outcome of the disease. Since
2001, hepatitis E has been emerging in industrialized countries, and
neurologic manifestations such as
Guillain-Barré syndrome, brachial
neuritis, transverse myelitis, and cranial nerve palsies have been reported
in patients with acute or chronic forms
of the disease (1–6). Most cases with
neurologic manifestations have been
characterized by infection with genotype 3 HEV. Data are not available to
indicate whether this association between HEV infection and neurologic
manifestations is related to a specific
antigenic stimulus provided by HEV
or is linked to the more comprehensive assessment for such neurologic
conditions that is available in industrialized countries or to a reporting bias.
We report a case of HEV infection in
an immunocompetent woman who
had muscle-specific kinase (MuSK)
antibody–positive myasthenia gravis
associated with HEV replication.
A 33-year-old woman was hospitalized in France for subacute asthenia and intermittent symptoms
including dysarthria, dysphagia,
muscle weakness, and diplopia. She
had no family history of autoimmune
disease and no notable personal
medical history; she had not received
any recent vaccinations and had not
traveled outside France during the
previous year. Physical examination
showed no pyramidal, vestibular, or
78
EmergingInfectiousDiseases•www.cdc.gov/eid•Vol.20,No.5,May2014
Article DOI: http://dx.doi.org/10.3201/eid2005.131242
Bartonella quintana in Body Lice from Scalp
Hair of Homeless Persons, France
Technical Appendix
Detailed distribution of B. quintana DNA among lice from monoinfested and dually infested homeless persons, France
Technical Appendix Table 1: Distribution of B. quintana DNA among lice from dually infested homeless persons, France, October
2012–March 2013
Lice collected from body
Lice collected from head
Lice tested
B. quintana no. (%)
Lice tested
B. quintana no. (%)
ID code homeless persons
32
5
0
4
0
33
5
2 (40.00)
2
1 (50.00)
40
5
1 (20.00)
4
0
89
29
0
6
0
5 (16.60)
B
17
0
30
D
11
2 (18.20)
10
1 (10.00)
Nov
11
0
10
0
Total
83
5 (6.00)
66
7 (10.60)
Technical Appendix Table 2: Detail of distribution of B. quintana DNA among lice from mono-infested homeless persons, France,
October 2012–March 2013
ID no. homeless persons
Body lice
B. quintana
1
3
0
2
3
1
3
3
3
4
3
0
5
3
0
6
3
0
7
3
3
8
3
3
1001
69
13
1002
17
7
1003
18
6
1005
3
0
1022
10
0
1023
3
3
1029
15
9
1034
3
1
1037
8
2
1038
101
3
1040
5
1
1051
2
0
1052
74
15
1053
19
2
1054
3
1
1059
19
0
1060
3
3
1065
4
2
1066
2
1
1070
7
3
1087
23
4
1101
2
2
1105
1
0
79
ID no. homeless persons
1151
1152
1153
1155
1158
1159
1160
1161
1163
1200
1201
1202
1203
1204
1205
1206
1208
1209
1210
1211
1212
1213
1222
1223
1224
1225
1227
1228
1229
1230
1231
1232
1234
1236
1237
1238
1239
1240
1241
1242
1244
1245
1246
2002
2016
2040
2101
2103
Total (%)
Body lice
10
17
1
6
4
5
3
4
1
9
24
1
1
2
1
5
21
2
23
16
5
4
46
28
2
2
2
2
18
2
6
2
10
4
44
2
2
11
1
5
2
3
7
1
34
1
1
2
840
B. quintana
5
2
0
2
0
4
0
0
1
4
0
0
0
0
0
0
2
2
13
10
1
0
1
5
0
0
0
0
3
0
1
0
1
0
19
0
0
5
1
4
0
0
0
0
0
0
0
0
174 (20.7)
80
Article III:
Detection of Bartonella quintana in African
Body and Head Lice
American Journal of Tropical Medicine and Hygiene 91: 294-301.
81
Am. J. Trop. Med. Hyg., 91(2), 2014, pp. 294–301
doi:10.4269/ajtmh.13-0707
Copyright © 2014 by The American Society of Tropical Medicine and Hygiene
Detection of Bartonella quintana in African Body and Head Lice
Abdoul Karim Sangaré, Amina Boutellis, Rezak Drali, Cristina Socolovschi, Stephen C. Barker, Georges Diatta,
Christophe Rogier, Marie-Marie Olive, Ogobara K. Doumbo, and Didier Raoult*
Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes (URMITE), Unité Mixte de Recherche (UMR)63, 7278 Centre National
de la Recherche Scientifique (CNRS), Institut de Recherche pour le Développement (IRD) 198, Institut National de la Santé et de la Recherche
Médicale (INSERM) 1095, University of Aix, Marseille, France; IRD, Campus Commun Université Cheikh Anta Diop (UCAD)-IRD of Hann,
Dakar, Senegal; Parasitology Section, School of Chemistry and Molecular Biosciences (SCMB), University of Queensland, Brisbane,
Queensland, Australia; Pasteur Institute of Madagascar, Ambohitrakely, Madagascar; University of Bamako, Malaria Research
and Training Center (MRTC)/Département d’Epidemiologie des Affections Parasitaires (DEAP)/Faculté de Médecine
de Pharmacie et d’Odontostomatologie (FMPOS)-Unité Mixte Internationale (UMI)3189, Bamako, Mali
Abstract. Currently, the body louse is the only recognized vector of Bartonella quintana, an organism that causes
trench fever. In this work, we investigated the prevalence of this bacterium in human lice in different African countries.
We tested 616 head lice and 424 body lice from nine African countries using real-time polymerase chain reaction
targeting intergenic spacer region 2 and specific B. quintana genes. Overall, B. quintana DNA was found in 54% and
2% of body and head lice, respectively. Our results also show that there are more body lice positive for B. quintana
in poor countries, which was determined by the gross domestic product, than in wealthy areas (228/403 versus 0/21,
P < 0.001). A similar finding was obtained for head lice (8/226 versus 2/390, P = 0.007). Our findings suggest that head lice
in Africa may be infected by B. quintana when patients live in poor economic conditions and are also exposed to body lice.
INTRODUCTION
Sucking lice (Anoplura) are hematophagous wingless
insects that can infest birds and mammals.1 Among these
hosts, humans constitute the preferred host for only two species: Pediculus humanus and Pthirus pubis (pubic lice).
P. humanus includes two morphotypes: P. humanus morphotype capitis (head lice) and P. humanus morphotype humanus
(body lice).2 Each louse has a specific ecotype; head lice live
and lay eggs in the hair and are prevalent in all countries and
all levels of society, whereas body lice live in clothing and
multiply when cold, promiscuity, and lack of hygiene are present.3 Louse coloration was described at the beginning of 20th
century. The variability in louse color on a single host may be
affected by not only the color of the skin but also, the color of
the hair and clothing.4,5
Many genetic studies have been conducted on human lice—
first based on 18S ribosomal RNA6,7 and then, the mitochondrial genes (cytochrome oxidase subunit 1 [Cox1] and cytochrome b [Cytb]). These studies allowed scientists to classify
lice into three different clades: Clade A, the most common
clade found worldwide and comprised of both head and body
lice; Clade B, composed of only head lice and found in Central and North America, Europe, and Australia8; and Clade C,
including black head lice found in Nepal,9 Ethiopia,10
and Senegal.11
The body louse is linked to poverty. Its transmission occurs
in crowded environments, such as homeless shelters, refugee
camps, and jails, especially when hygienic standards are lacking.12 The body louse is the main vector of three pathogenic
bacteria: Bartonella quintana, the agent of trench fever;
Borrelia recurrentis, the agent of louse-borne relapsing fever;
and Rickettsia prowazekii, the agent of epidemic typhus.13,14
B. quintana is a Gram-negative bacterium that causes trench
fever, bacillary angiomatosis, endocarditis, chronic bacteremia,
and chronic lymphadenopathy.15 It has typically been trans-
*Address correspondence to Didier Raoult, URMITE, UM63, 7278
CNRS, IRD 198, Inserm 1095, University of Aix, 27 Bd Jean Moulin,
13005 Marseille, France. E-mail: didier.raoult@gmail.com
mitted by body lice, but recently, DNA of B. quintana has also
been found in head lice collected from homeless individuals in
Nepal,9 the United States,16 France,17 Senegal,11
and Ethiopia.10,18
The detection of B. quintana in African lice remains limited
to only a small number of countries. Therefore, the objectives
of this study were to investigate the presence of B. quintana in
head and body lice in different areas of African countries
suffering from poverty, social instability, or war and identify
the relationship between louse phenotypes and genotypes.
MATERIALS AND METHODS
Ethics statement. Lice from African countries were obtained
from the private frozen collection of our laboratory (The Unité
de Rcherche sur les Maladies Infectieuses et Tropicales
Emergentes [URMITE]/World Health Organization [WHO]
Collaborative Research Center). The lice in that collection
were required for various epidemiological and entomological
studies or to perform diagnoses abroad, and they were sent to
our laboratory as a WHO reference facility. Body lice were
collected from clothing and head lice were removed from hair
with the verbal consent of the infested individuals. Written
consent was not obtainable in the majority of cases, because
most of the subjects were illiterate. However, in most instances,
the investigator, local authorities/Institutional Review Boards
(IRBs), and/or village/family chief approved and were present
when collection was performed (with individual consent).
Sampling/country. Human lice samples were collected
from patients in various regions of Africa (Figure 1). Nine
countries were investigated. In total, 1,040 lice were collected: 616 head lice and 424 body lice. Exactly 381 head lice
were collected in Senegal (2011), 75 head lice and 22 body
lice were collected in Madagascar (2009, 2011, and 2012),
14 head and body lice were collected in Ethiopia (2011),
92 head lice were collected in Mali (2010), 37 body lice were
collected in Kenya (1999), 166 body lice were collected in
Rwanda (2011), 10 head and body lice were collected in
Burundi (2008), 35 head lice and 154 body lice were collected
in Congo (2010), and 9 head lice and 21 body lice were
29483
BARTONELLA QUINTANA IN AFRICAN LICE
Figure 1.
Map of Africa showing the prevalence of B. quintana in head and body lice collected in the various study areas during 1999–2012.
collected in Algeria (2000). In our laboratory, each sample
was photographed on dorsal and ventral sides with a camera
(Olympus DP71, Rungis, France). Lice were preserved in
70% alcohol, rinsed two times with sterile distilled water for
2 minutes, and dried with filter paper. Samples were then cut
Figure 2.
295
in half on the longitudinal plane, and one-half was stored at
−20 °C for subsequent analysis.
Distribution of regions. We classified these countries into
two regions: poor (with the gross domestic product [GDP] <
$1,844 per capita: Madagascar, Ethiopia, Mali, Kenya,
Phenotypes of head and body lice collected from African countries during 1999–2012 (Head lice: A, B and C; Body lice: D, E, F and G).
84
296
SANGARÉ AND OTHERS
Rwanda, Burundi, and Congo) and wealthy (with GDP ³
$1,844 per capita: Algeria and Senegal) areas. GDP being an
economic indicator of the wealth produced annually in a particular country for our classification, we estimated the average per
capita GDP of all regions.19 The average GDP corresponding
to the total of GDP per capita from nine countries divided by
nine ($16,600/9 = $1,844) was then compared with the infection
rate of B. quintana in lice.
DNA preparation and detection of B. quintana in lice.
After incubation at 56 °C in a dry bath overnight, the lice were
extracted on an automaton EZ1 device using the QIAamp
Tissue Kit (Qiagen, Hilden, Germany) according the manufacturer’s recommendations. The DNA was used as a template in a real-time polymerase chain reaction (PCR) assay
targeting a portion of the Bartonella 16S–23S intergenic
spacer (ITS) region and a specific B. quintana gene (yopP)
Figure 3.
that encodes a hypothetical intracellular effector.17 Each realtime PCR assay was performed using a CFX96 TM REALTime System C1000 Thermal Cycler (Bio-Rad Laboratories,
Foster City, CA). For each PCR assay, two negative and
positive controls were used. The identification of B. quintana
was confirmed by amplification of both ITS2 and yopP genes.
Genotypic status of lice. All samples that tested positive for
B. quintana DNA were analyzed by multiplex real-time PCR
that targeted a portion of the Phum PHUM540560 gene. This
assay allowed for the discrimination of body lice from head
lice, which has described previously.20 As a positive control,
we used a head louse and body louse with genotypic statuses
that were known (VIC-positive for the head louse and FAMpositive for the body louse). Monoplex and multiplex realtime PCRs were performed in a CFX96 Thermal Cycler
(Bio-Rad Laboratories, Foster City, CA).
Phylogenetic tree of lice based on the Cytb (using the ML method with 100 bootstrap replicates).
85
297
BARTONELLA QUINTANA IN AFRICAN LICE
Standard PCR and sequencing. Lice genotype was identified by the analysis of partial (347-bp) mitochondrial Cytb
DNA. The primers used for the Cytb gene were CytbF1 (5¢GAGCGACTGTAATTACTAATC-3¢) and CytbR1 (5¢-GGA
CCCGGATAATTTTGTTG-3¢). Overall, 10 lice belonging to
each region were tested with a standard PCR assay targeting
the Cytb gene as described previously.8 Each amplification
reaction was performed on a PTC-200 thermocycler machine
(MJ Research, Waltham, MA). The PCR reaction contained
9.9 mL water, 4 mL buffer 5 Fusion HF Buffer, 0.4 mL 10 mM
deoxynucleoside triphosphates (dNTPs), 1.25 mL each primer,
0.2 mL Phusion polymerase (Finnzymes, Thermo Scientific,
Vantaa, Finland), and 3 mL DNA template to obtain a total
volume of 20 mL. The cycling conditions were 98 °C for 30 seconds, 35 cycles of 98 °C for 5 seconds, 56 °C for 30 seconds,
and 72 °C for 15 seconds, and a final extension time of
5 minutes at 72 °C. The success of the PCR amplification was
then detected by the migration of the PCR product on 2%
agarose gel (Electrophoresis grade; Invitrogen, Carlsbad, CA)
prepared with 0.5% Tris Borate ethylenediaminetetraacetic
(EDTA) (TBE; Euromodex, Lake Placid, NY) and charged
with a solution of 0.5% ethidium bromide (Invitrogen,
Carlsbad, CA). Purification of PCR-amplified products was
performed using distilled RNase-DNase free water on
NucleoFast 96 PCR plates (Macherey-Nagel EURL, Hoerdt,
France). Sequencing of positive samples was performed
using the ABI Prism Big Dye Terminator Cycle Sequencing
Kit, version 1.1 (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions.
Data analysis. Microsoft Excel was used for data management. Descriptive statistics, such as percentages and means,
were computed to summarize the proportions of infestations
+
with lice. Statistical analysis was performed with Epi Info 6
(www.cdc.gov/epiinfo/Epi6/EI6dnjp.htm), and a P value of
< 0.05 was considered significant.
Phylogenetic analysis. Each DNA sequence was aligned
using multisequence alignment software (CLUSTALX, version 2.0.11). The ChromasPro program (Technelysium PTY,
Australia) was used to analyze, assemble, and correct sequences.
The sequence similarities were determined using MEGA 5,
and phylogenetic trees were obtained using the maximum
likelihood (ML) method with 100 bootstrap replicates.21
We compared our sequences with other sequences that are
present in GenBank.
RESULTS
Morphological analysis. Phenotypical examination of the
lice showed that all head lice from Senegal, Madagascar, and
Ethiopia have a black color (Figure 2A –C). The color of all
body lice in Madagascar and Rwanda is black; in Kenya, all
body lice are brown, whereas in Ethiopia, all body lice are gray
(Figure 2D–G). During our study, the phenotypes of lice from
Congo, Burundi, Mali, and Algeria were not investigated,
because they had been destroyed previously for other studies.
Phylogenetic analysis. On the basis of phylogenetic study
of lice Clade A based on ITSs (ultispacer typing method
[MST])22 and through the analysis of Cytb in this work
(Figure 3), our results show that head lice from Algeria,
Madagascar, Burundi, and Senegal belong to Clade A2.
Clade C was found in head lice from Ethiopia, Mali, and
Senegal. Clade B was found in head lice from Algeria. Based
on Cytb analysis, all head and body lice that are positive for
B. quintana DNA belong to Clade A2 (Table 1).
Table 1
Distribution of B. quintana DNA in head lice (N = 616) and body lice (N = 424) collected from African countries during 1999–2012
Lice analyzed
Country/area
Senegal
Yeumbeul
Malika
Dielmo village
Ndiop village
Keur Massar
Total
Madagascar
Borenty village
Tsiroanomandidy
Anjozorobe
Bedaro village
Total
Ethiopia
Gondar
Mali
Diankabou
Kenya
Nairobi
Rwanda
Kigali
Burundi
Bujumbura
Congo
RDC
Algeria
Batna
Total
Head lice
Positive B. quintana DNA (%)
Body lice
Head lice
Body lice
Cytb
Head lice
Body lice
101
69
71
77
63
381
0
0
0
0
0
0
0
0
2 (2.8)
0 (0)
0
2 (0.52)
Clade A2
Clade A2
Clade A2
Clade C
Clade A2
−
−
−
−
−
0
21
39
15
75
9
13
0
0
22
−
0
2 (5.1)
0
2 (2.66)
1 (11.1)
0
−
−
1 (4.54)
−
Clade A2
Clade A2
Clade A2
Clade A2
Clade A2
−
−
14
14
0
0
Clade C
Clade A2
92
0
0
Clade C
−
0
37
−
27 (72.9)
−
Clade A2
0
166
−
149 (89.7)
−
Clade A2
10
10
35
154
9
616
21
424
−
−
−
−
−
−
0
6 (17.1)
0
10 (1.6)
86
1 (10)
Clade A2
Clade A
50 (32.5)
Clade A2
Clade A2
Clades A and B
Clade A2
0
228 (53.7)
298
SANGARÉ AND OTHERS
Molecular detection of B. quintana in lice. The DNA of
B. quintana was detected in 10 of 616 (1.6%) head lice (the
mean cycle thresholds [ct] value ± SD: 30.64 ± 6.34). Specifically, B. quintana DNA was found in 2 of 71 (2.8%) head lice
from Senegal (Dielmo village), 2 of 39 (5.1%) head lice
from Madagascar (Anjozorobe), and 6 of 35 (17.1%) head
lice from Congo.23 No B. quintana DNA was detected in
Senegal (Yeumbeul, Malika, Ndiop village, and Keur Massar),
Madagascar (Tsiroanomandidy and Bedaro village), Ethiopia,
Mali, Burundi, or Algeria (Table 1).
The DNA of B. quintana was detected in 228 of 424 (54%)
body lice (the mean ct value ± SD: 33.19 ± 3.79). Among 228
B. Quintana-positive body lice, 149 of 166 (89.7%) body lice
are from Rwanda, 27 of 37 (72.9%) body lice are from Kenya,
50 of 154 (32.5%) body lice are from the Democratic Republic of Congo,23 1 (11.1%) body louse is from Madagascar, and
1 (10%) body louse is from Burundi. No B. quintana DNA
was detected in the Madagascar area of Tsiroanomandidy,
Ethiopia, or Algeria. There are significantly more body lice
with B. quintana DNA than head lice (54% versus 1.6%, P <
0.001) (Table 1).
Genotypic status of positive lice to B. quintana DNA.
Among the African Clade A lice positive for B. quintana
DNA, all head lice had the head louse genotype, and all body
lice had the body louse genotype. No signal was detected in
the negative controls.
Table 2
Prevalence of infections of B. quintana in body lice (this study and the literature)
Country/source
Algeria
Schoolchildren
Homeless in Batna
Madagascar
Local population
Ethiopia
Bahir Dar
Poor regions (Jimma)
Poor regions (Jimma)
Kenya
Local population
Rwanda
Jail
Jail
Burundi
Refugee camp
During typhus outbreak
Refugee camp
After outbreak in camp
During typhus outbreak
Refugee camp
After typhus outbreak
Refugee camp
Refugee camp
Refugee camp
Congo
Local population
Refugee camp
Refugee camp
Local population
France
SDF (homeless)
SDF (homeless)
SDF (homeless)
SDF (homeless)
Tunisia
Homeless in Sousse
Zimbabwe
Homeless in Harare
The Netherlands
Homeless in Utrecht
United States
SDF (homeless)
Nepal
Street children and children in slums
Russia
Homeless in Moscow
Australia
Homeless
Peru
Andean rural population
Andean rural population
Year of collection
Percent (no. B. quintana/no. tested)
Source
0 (0/21)
0 (0/33)
This study
2009/2011/2012
4.5 (1/22)
This study
2011
2010
2010
0 (0/14)
3 (1/33)
18 (76/424)
This study
2000
2001
30
29
10
1999
72.9 (27/37)
This study
2011
2001
89.7 (149/166)
2.3 (6/262)
This study
2008
1997
1997
1998
1997
1997
1998
1998
2000
2001
10 (1/10)
0 (0/10)
9.5 (6/63)
14.3 (13/91)
0 (0/10)
9.5 (6/63)
14.3 (13/91)
21 (8/38)
90 (100/111)
93.9 (31/33)
This study
2010
1998
1998
2010
32.5 (50/154)
0 (0/7)
0 (0/7)
32.5 (50/154)
This study
1997
1998
2000
1998–2001
20 (3/15)
4 (3/75)
26 (42/161)
9.8 (32/324)
2000
0 (0/3)
30
32
32
32
30
30
30
30
30
30
32
30
23
34
32
33
30
30
30–32
1998
16.7 (2/12)
2001
36 (9/25)
30
33.3 (11/33)
16
2002
20 (4/20)
9
1998
12.3 (33/268)
32
2001
0 (0/2)
30
NA
NA
1.4 (1/73)
0 (0/10)
32
2007–2008
NA = not available.
87
30
299
BARTONELLA QUINTANA IN AFRICAN LICE
Relationship between B. quintana in body or head lice and
socioeconomic level. We compared our results with the life
conditions of the regions tested and found that the body lice
that were B. quintana-positive were more likely to be found in
poor countries than wealthy countries (228/403 versus 0/21,
P < 0.001). The same finding was made for head lice (8/226
versus 2/390, P = 0.007).
We correlated the GDP to the level of B. quintana infection. Our results indicate that the higher the GDP in a region,
the less prevalent that B. quintana DNA was and vice versa
(correlation r = −0.178).
DISCUSSION
This study of 1,040 human lice of the genus Pediculus collected from nine African countries has enabled us to better
specify some characteristics of human lice. The phenotypic
study allowed us to confirm several previously described
observations, including the black phenotype of head lice in
Senegal and Ethiopia.10,11,24 For the first time, we have
established the phenotype of lice collected in Madagascar
and Kenya. Body lice from Madagascar and Rwanda are
black; body lice collected in Kenya are brown. The head lice
from Madagascar had the same black color as body lice col-
lected in this country (Figure 2). In addition, it seems that the
color of lice is more complex than indicated in the first findings provided in 1926 by Ewing,25 and as suggested by a recent
study,10 lice color may be independent of the host’s skin color.
On the basis of phylogenetic study of lice Clade A, based
on ITSs (MST)22 and through the analysis of Cytb in this work
(Figure 3), we confirmed that head lice in Senegal are Clades
A2 and C11 and that head lice in Ethiopia are Clade C.10 In
addition, we have found that head lice in Madagascar,
Burundi, Congo, and Algeria belong to Clade A2 and that
head lice in Mali are Clade C (Figure 3). The body lice of
Algeria, Ethiopia, Madagascar, Kenya, Burundi, and Rwanda
belong to Clade A2; it is the main clade in sub-Saharan
Africa.26 Therefore, the geographical distribution of lice
seems to be complex and independent of the phenotype.
B. quintana is a re-emerging pathogen that is responsible
for a range of clinical manifestations in humans.27 It has long
been established that trench fever can be transmitted by the
body louse.28 However, the role played by the head louse as a
reservoir or vector of B. quintana remains unclear.29 In total,
B. quintana was found in 54% and 2% of body and head lice
collected in Africa, respectively. Here, we find that there are
more B. quintana in body lice than head lice. This finding
could probably be explained through the role played by
Table 3
Prevalence of infections of B. quintana in head lice (this study and the literature)
Country/source
Algeria
Schoolchildren
Schoolchildren
Burundi
Schoolchildren
Schoolchildren
Senegal
Rural community
Rural community
Ethiopia
Poor region
Poor regions (Jimma)
Poor regions (Jimma)
Congo
Local population
Local population
Madagascar
Local population
Mali
Schoolchildren
Portugal
Schoolchildren
China
Schoolchildren
Thailand
Schoolchildren
Australia
Schoolchildren
United States
SDF (homeless)
Nepal
Street children and children in slums
Russia
Schoolchildren
France
Schoolchildren
Schoolchildren
SDF (homeless)
Year of collection
Percent (no. B. quintana/no. tested)
Source
2000
NA
0 (0/9)
0 (0/18)
This study
2008
NA
0 (0/10)
0 (0/20)
This study
2011
NA
0.52 (2/381)
6.9 (19/274)
This study
2011
2010
2010
0 (0/14)
7 (19/271)
9.2 (6/65)
This study
2010
2010
30
30
11
10
29
17.1 (6/35)
17.1 (6/35)
This study
2.6 (2/75)
This study
2010
0 (0/92)
This study
NA
0 (0/20)
30
NA
0 (0/23)
30
NA
0 (0/29)
30
NA
0 (0/3)
30
2007–2008
25 (3/12)
16
2002
9.5 (2/21)
9
NA
0 (0/10)
30
2010–2011
NA
2008–2009
2008
0 (0/20)
0 (0/288)
100 (3/3)
NA = not available.
88
23
30
31
17
300
SANGARÉ AND OTHERS
the body louse in transmission of diseases. With the new
method of multiplex real-time PCR assay with the Phum_
PHUM540560 gene, all head lice were genotyped as head lice
by the signal emitted by the VIC-labeled probe specific to
head lice, and all body lice were genotyped as body lice by
the signal emitted by the FAM-labeled probe specific to body
lice. In our study, the infection rate was higher in Rwanda (149/
166 versus 6/262, P < 0.001) (Table 2) and lower in Burundi (1/
10 versus 158/346, P = 0.025) than the rate reported in 2002 by
Fournier and others.30 These differences could be explained by
the living conditions in jail in Rwanda and probably, the sample size in this study in Burundi. We have confirmed the presence of B. quintana in body lice in Rwanda and Burundi.30
B. quintana has also been detected in body lice from homeless
people in Zimbabwe (16.7%, 2/12).30 This bacterium has not
been found in body lice in Tunisia.30 We detected B. quintana
for the first time in body lice collected in Madagascar (4.54%,
1/22) and Kenya (72.9%, 27/37). The high rate of B. quintana in
body lice in some African countries (Kenya, Rwanda, and
Congo) may reflect the low socioeconomic level of the study
population. We tested this hypothesis by comparing the average GDPs of nine different countries with the level of
B. quintana infection. We found that the higher the GDP
increases, the lower the level of B. quintana decreases and vice
versa (correlation r = −0.178). For example, for countries with
high GDP, this hypothesis could be justified through the previous work by Fournier and others30 in 2002, which reported 0%
B. quintana in head lice in Portugal, China, Thailand, Australia,
Algeria, and France.30 This percentage of infection with
B. quintana remains stable (0%) in body lice in Australia,
Algeria, and Peru. In France, Bouvresse and others31 also
found the same result (0%) in head lice. Roux and Raoult32
reported a rate of 1.4% in body lice in Peru. For countries with
low GDP, this hypothesis could be confirmed by the work of
Piarroux and others23 in 2010, which reported 32.5% B. quintna
in body lice in Congo, and the work of Fournier and others,30
which found a higher prevalence of 93.9% B. quintana in body
lice in Burundi (a refugee camp in 2001). Elsewhere, we compared the infection rate in B. quintana body lice in people in
Africa with the infection rate in B. quintana body lice in homeless people in Marseille, France,30,32,33 and the result was statistically significant (228/424 versus 77/560, P < 0.001) (Table 2).
In this study, we show a relationship between B. quintana presence in body lice and socioeconomic level, and we found that
the body lice that were B. quintana-positive were more likely
to be found in poor countries (GDP < $1,844 per capita) than
wealthy countries (GDP > $1,844 per capita; 228/403 versus
0/21, P < 0.001).
Head lice can also be infected with B. quintana. In our
study, we detected the DNA of B. quintana in 1.6% (10/616)
of head lice collected: in Senegal, 0.52% (2/381); in Madagascar
for the first time, 2.66% (2/75); in the Congo, 17.1% (6/35).
B. quintana was detected in 7% (19/274) of head lice collected
in Dakar, Senegal11 and 9.2% (6/65) of head lice pools and
7% (19/271) of head lice on persons living in the poorest
areas of Jimma, Ethiopia.10,29 No B. quintana DNA was
detected in head lice from schoolchildren in Marseille,
France,30,31 contrary to the work by Angelakis and others,10
which have reported this result in the homeless population.
In Russia, Portugal, Algeria, Burundi, China, Thailand, and
Australia, no B. quintana was found in schoolchildren30
(Table 3). In this study, we also found the head lice that were
B. quintana-positive were more likely to be found in poor
countries (GDP < $1,844 per capita) than wealthy countries
(GDP > $1,844 per capita; 8/226 versus 2/390, P < 0.007).
Head lice are prevalent around the world and in all levels
of society. Thus, head lice may be infected with B. quintana
when the host is coinfected with body lice in a precarious
environment in which proper hygiene is lacking.
In conclusion, it has been estimated that trench fever
affected several million people, especially in Russia and on
the Eastern, Central, and Western European fronts during
World War II.12,15 Our study on the prevalence of B. quintana
in lice from various geographical and socioecological situations associated with clinical information will help to evaluate
the role of head lice in the transmission of this disease.
Received December 5, 2013. Accepted for publication February 14,
2014.
Published online June 16, 2014.
Acknowledgments: The authors thank Mr. Jean-Michel Berenger
(entomologist) and the molecular biology team of URMITE Marseille
for their technical support.
Authors’ addresses: Abdoul Karim Sangaré, Amina Boutellis, Rezak
Drali, Cristina Socolovschi, Georges Diatta, and Didier Raoult,
URMITE, UM63, 7278 CNRS, IRD 198, Inserm 1095, University of
Aix, Marseille, France, and IRD, Campus Commun UCAD-IRD of
Hann, Dakar, Senegal, E-mails: sangareak@icermali.org, amina.
boutell@yahoo.fr, rezakdrali@hotmail.com, cr_socolovschi@yahoo.
com, georges.diatta@ird.fr, and didier.raoult@gmail.com. Stephen C.
Barker, Parasitology Section, School of Chemistry and Molecular Biosciences (SCMB), University of Queensland, Brisbane, Queensland,
Australia, E-mail: s.barker@uq.edu.au. Christophe Rogier and MarieMarie Olive, Pasteur Institute of Madagascar, Ambohitrakely,
Madagascar, E-mails: crogier@pasteur.mg and mmolive@pasteur.mg.
Ogobara K. Doumbo, University of Bamako, MRTC/DEAP/FMPOSUMI3189, Bamako, Mali, E-mail: okd@icermali.org.
REFERENCES
1. Barker SC, 1994. Phylogeny and classification, origins, and evolution of host associations of lice. Int J Parasitol 24: 1285–1291.
2. Light JE, Toups MA, Reed DL, 2008. What’s in a name: the
taxonomic status of human head and body lice. Mol Phylogenet
Evol 47: 1203–1216.
3. Badiaga S, Brouqui P, 2012. Human louse-transmitted infectious
diseases. Clin Microbiol Infect 18: 332–337.
4. Nuttall GHF, 1917. The biology of Pediculus humanus. Parasitology 10: 80–185.
5. Busvine JR, 1946. On the pigmentation of the body louse
pediculus humanus L. In Proceedings of the Royal Entomological Society of London. Series A, General Entomology
(Vol. 21, No. 10–12, pp. 98–103). Blackwell Publishing Ltd.
6. Yong Z, Fournier PE, Rydkina E, Raoult D, 2003. The geographical segregation of human lice preceded that of Pediculus
humanus capitis and Pediculus humanus humanus. C R Biol
326: 565–574.
7. Leo NP, Barker SC, 2005. Unravelling the evolution of the head
lice and body lice of humans. Parasitol Res 98: 44–47.
8. Raoult D, Reed DL, Dittmar K, Kirchman JJ, Rolain JM,
Guillen S, Light JE, 2008. Molecular identification of lice from
pre-Columbian mummies. J Infect Dis 197: 535–543.
9. Sasaki T, Poudel SK, Isawa H, Hayashi T, Seki N, Tomita T,
Sawabe K, Kobayashi M, 2006. First molecular evidence
of Bartonella quintana in Pediculus humanus capitis
(Phthiraptera: Pediculidae), collected from Nepalese children.
J Med Entomol 43: 110–112.
10. Angelakis E, Diatta G, Abdissa A, Trape JF, Mediannikov O,
Richet H, Raoult D, 2011. Altitude-dependent Bartonella
quintana genotype C in head lice, Ethiopia. Emerg Infect Dis
17: 2357–2359.
89
BARTONELLA QUINTANA IN AFRICAN LICE
11. Boutellis A, Veracx A, Angelakis E, Diatta G, Mediannikov O,
Trape JF, Raoult D, 2012. Bartonella quintana in head lice
from Senegal. Vector Borne Zoonotic Dis 12: 564–567.
12. Brouqui P, 2011. Arthropod-borne diseases associated with political and social disorder. Annu Rev Entomol 56: 357–374.
13. Cutler SJ, 2010. Relapsing fever–a forgotten disease revealed.
J Appl Microbiol 108: 1115–1122.
14. Raoult D, Ndihokubwayo JB, Tissot-Dupont H, Roux V,
Faugere B, Abegbinni R, Birtles RJ, 1998. Outbreak of epidemic typhus associated with trench fever in Burundi. Lancet
352: 353–358.
15. Raoult D, Roux V, 1999. The body louse as a vector of
reemerging human diseases. Clin Infect Dis 29: 888–911.
16. Bonilla DL, Kabeya H, Henn J, Kramer VL, Kosoy MY, 2009.
Bartonella quintana in body lice and head lice from homeless
persons, San Francisco, California, USA. Emerg Infect Dis 15:
912–915.
17. Angelakis E, Rolain JM, Raoult D, Brouqui P, 2011. Bartonella
quintana in head louse nits. FEMS Immunol Med Microbiol 62:
244–246.
18. Boutellis A, Mediannikov O, Bilcha KD, Ali J, Campelo D,
Barker SC, Raoult D, 2013. Borrelia recurrentis in head lice,
Ethiopia. Emerg Infect Dis 19: 796–798.
19. CIA Factbook, 2012. The World Factbook. Central Intelligence
Agency. Available at: https://www.cia.gov/library/publications/
the-world-factbook/index.html.
20. Drali R, Boutellis A, Raoult D, Rolain JM, Brouqui P, 2013.
Distinguishing body lice from head lice by multiplex realtime PCR analysis of the Phum_PHUM540560 gene. PLoS
ONE 8: e58088.
21. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S,
2011. MEGA5: molecular evolutionary genetics analysis using
maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28: 2731–2739.
22. Li W, Ortiz G, Fournier PE, Gimenez G, Reed DL, Pittendrigh
B, Raoult D, 2010. Genotyping of human lice suggests multiple
emergencies of body lice from local head louse populations.
PLoS Negl Trop Dis 4: e641.
23. Piarroux R, Abedi AA, Shako JC, Kebela B, Karhemere S,
Diatta G, Davoust B, Raoult D, Drancourt M, 2013. Plague
90
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
301
epidemics and lice, Democratic Republic of the Congo. Emerg
Infect Dis 19: 505–506.
Veracx A, Boutellis A, Merhej V, Diatta G, Raoult D, 2012.
Evidence for an African cluster of human head and body lice
with variable colors and interbreeding of lice between continents. PLoS ONE 7: e37804.
Ewing HE, 1926. A revision of the American lice of the genus
Pediculus, together with aconsideration of the significance of
their geographical and host distribution. Proc US Natl Museum
68: 1–30.
Boutellis A, Drali R, Rivera MA, Mumcuoglu KY, Raoult D,
2013. Evidence of sympatry of clade A and clade B head lice
in a pre-Columbian Chilean mummy from Camarones. PLoS
ONE 8: e76818.
Foucault C, Brouqui P, Raoult D, 2006. Bartonella quintana
characteristics and clinical management. Emerg Infect Dis 12:
217–223.
Bacot A, 1921. On the probable identity of Rickettsia pediculi
with Rickettsia quintana. BMJ 1: 156–157.
Cutler S, Abdissa A, Adamu H, Tolosa T, Gashaw A, 2012.
Bartonella quintana in Ethiopian lice. Comp Immunol Microbiol
Infect Dis 35: 17–21.
Fournier PE, Ndihokubwayo JB, Guidran J, Kelly PJ, Raoult D,
2002. Human pathogens in body and head lice. Emerg Infect
Dis 8: 1515–1518.
Bouvresse S, Socolovshi C, Berdjane Z, Durand R, Izri A, Raoult
D, Chosidow O, Brouqui P, 2011. No evidence of Bartonella
quintana but detection of Acinetobacter baumannii in head lice
from elementary schoolchildren in Paris. Comp Immunol
Microbiol Infect Dis 34: 475–477.
Roux V, Raoult D, 1999. Body lice as tools for diagnosis
and surveillance of reemerging diseases. J Clin Microbiol 37:
596–599.
La SB, Fournier PE, Brouqui P, Raoult D, 2001. Detection and
culture of Bartonella quintana, Serratia marcescens, and
Acinetobacter spp. from decontaminated human body lice. J
Clin Microbiol 39: 1707–1709.
Brouqui P, La Scola B, Roux V, Raoult D, 1999. Chronic
Bartonella quintana bacteremia in homeless patients. N Engl
J Med 340: 184–189.
Chapitre 2 :
Distribution phylogéographique des poux
humains contemporains et anciens
91
Préambule
Les poux comptent parmi les plus anciens parasites obligatoires de
l'Homme, ce qui fait d’eux un excellent marqueur de l'évolution et de
la migration des espèces du genre Homo au fil du temps [23]. Les
nombreuses études basées sur les analyses de phylogénie des gènes
mitochondriaux ont montré que les poux humains étaient (i) distribués
à l’intérieur de trois clades, les Clades A, B et C et (ii) une relation
phylogéographique existait entre ces trois clades [47,48]. Ainsi, si le
Clade A composé de poux de tête et de poux de corps avait une
distribution géographique mondiale, les deux autres clades avaient une
distribution géographique plus restreinte. le Clade B d’origine
américaine [26] est retrouvé maintenant en Europe, en Australie et en
Algérie alors que le Clade C est distribué en Afrique et au Népal [49].
Dans ce chapitre nous présentons les résultats que nous avons obtenus
grâce à l’analyse de poux humains rares et précieux, ce qui nous a
valu quelques surprises.
Dans le premier papier, nous rapportons pour la première fois
l’existence d’un quatrième clade mitochondrial (Clade D) en
République Démocratique du Congo. Comme le Clade A, le Clade D
93
renferme des poux de tête et des poux de corps chez qui on a détecté
de l’ADN de B. quintana et de Y. pestis.
Dans le deuxième papier, l'analyse d’anciennes lentes de poux de tête
provenant d'Israël datant de 2 périodes différentes, chalcolithique
(4.000 avant JC) et période islamique (650-810 AD), a permis
d'affirmer qu'elles appartenaient probablement à des personnes
originaires de l'Afrique de l'Ouest en raison de leur appartenance à un
sous clade mitochondrial spécifique à cette région.
Dans le troisième papier, nous avons confirmé l’origine américaine du
Clade B à travers l’analyse des lentes de poux de momies
précolombienne datant de 4.000 ans [25].
94
Article IV:
A New Clade of African Body and Head Lice Infected by
Bartonella quintana and Yersinia pestis –
Democratic Republic of the Congo
Accepté avec révisions mineures dans
American Journal of Tropical Medicine and Hygiene
95
American Journal of Tropical Medicine & Hygiene
�
�
�
�
�
�
������������������������������������������������������
�������������������������
���������������������������������������������������
�
�
Fo
��������� ������������������������������������������������
��������������� ��������������
rP
����������������� �������������
������������������������������ ������������
rR
ee
�������������������������� ����������������������������������������������������������������
����������������������������������������������������������������������
�����������
������������������������������������������������������������������
��������������������������������������������������������������������������
�����������������������������������������������������������������������
���������
�������������������������������������
������������������������������������������������������������������
ev
����������� ����������������������������
��
w
ie
�
�
97
Page 1 of 9
1
����������������������
2
�������������������������������������������
3
������������������������������������������������������������������������������
4
���������������������������������������������������
5
�����������������������������������������������������������������������������������
6
������������������������������������������������������������������������������������
7
����������������������������������������������������������������������������������������
8
�����������������������������������������������������������������������������������������
9
�������������������������������������������������������������������������������������
10
��������������������������������������������������������������������������������������������
11
�������������������������������������������������������������������������������������
ee
rP
Fo
���������
12
13
����������������������������� �����������������������������������������������������������������
14
�������������������������������������������������������������������������������������������
15
��������������������������������������������������������������������������������������
16
�������������������������������������������������
17
�
18
�������������������������������������������������������������������������������������������
19
�����������������������������������������������������������������������������������
20
����������������������������������������������������������
21
�
22
��������������������������������������
23
�
24
�
w
ie
ev
rR
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
American Journal of Tropical Medicine & Hygiene
1
98
American Journal of Tropical Medicine & Hygiene
25
��������������������������������������������������������������������������������
26
���������������������������������������������������������������������������������������
27
�������������������������������������������������������������������������������������������
28
����������������������������������������������������������������������������������������������
29
������������������������������������������������������������������������������������
30
��������������������������������������������������������������������������������������������
31
����������������������������������������������������������������������������������������������
32
����������������������������������������������������������������������������������������
33
����������������������������������������������������������������������������������������
34
����������������������������������������������������������������������������������������������
35
������������������������������������������������������������������������������������������������
36
�����������������������������������������������������������������������������������������������
37
����������������������������������������������������������������������������������� �����������
38
������������������������������������������������������������������������������������������������
39
����������������������������������������������������������������������������������������������
40
�����������������������������������������������������������������������������������������
41
��������������������������������������������������������������������������������������������
42
����������������������������������������������������������������������� ������������������������
43
��������������������������������������������������������������������������������������������
44
������������������������������������������������������� ��������������������������������������
45
������������������������������������������������������������������������������������������������
46
��������������������������������������
w
ie
ev
rR
ee
48
rP
47
Fo
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Page 2 of 9
�����������������������������������������������������������������������������������
��������������������������������������������������������������������������������������������������
2
99
Page 3 of 9
49
��������������������������������������������������������������������������������������������
50
����������
51
�����������������������������������������������������������������������������������������
52
��������������������������������� ������������������������������������������������������������
53
����������������������������������������������������������������������� ��������������������
54
������������������������������������������������������������������������������������������������
55
������������� ��������������������������������������������������������������������������
56
57
���������������������������������������������������������������������������������������������������
Fo
����������������������������������������������������������������� �������������������
58
������������������������������������������������������������������������������������������
59
�����������������������������������������������������������������������������������������
60
���������������������������������������������������������������������������������������
61
��������������������������������������������������������������������������������������
62
����������������������������������������������������������������������������������
63
����������������������������������������������������������������������
ev
rR
ee
rP
64
����������������������������������������������������������������������������������������
65
������������������������������������������������������������������������������������������
66
��������������������������������������������������������������������������������� �����
67
��������������������������������������������������������������������
68
w
ie
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
American Journal of Tropical Medicine & Hygiene
�������������������������������������������������������������������������������
69
������������������������������������������������������������������������������������
70
������������������������������������������������������������������������������������������������
71
��������������������������������������������������������������������������������������
72
������������������������������������������������������������������������������������������
73
���������������������������������������������������������������������������������������
3
100
American Journal of Tropical Medicine & Hygiene
74
���������������������������������������������������������������������������������������
75
������������������������������������������������������������������������������������������
76
��������������������������������������
77
�������������������������������������������������������������������������������������
78
��������������������������������������������������������������������������������������������
79
���������������������������������������������������������������������������������������������
80
������������������������������������������������������������������������������������������������
81
�������������������������������������������������������������������������������
Fo
82
������������������������������������������������������������������������������
83
�����������������������������������������������������������������������������������������������
84
���������������������������������������������������������������������������������������������
85
�����������������������������������������������������������������������������������������������
86
������������������������������������������������������������������������������������������������
87
������������������������������������������������������������������
rR
ee
88
rP
����������������������������������������������������������������������������������
ev
89
�����������������������������������������������������������������������������������������
90
���������������������������������������������������������������������������������������������
91
��������������������������������������������������������������������������������������������
92
��������������������������������������������������������������������������������������������
93
��������������������������������������������������������������������������������������������
94
������������������������������������������������������������������������������������������������
95
�����������������������������������������������������������������������������������������
96
����������������������������������������������������������������������������������������
97
�������������������������������������������������������������������������������������������
98
������������������������
w
ie
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Page 4 of 9
4
101
Page 5 of 9
���������������������������������������������������������������������������������������
99
100
����������������������������������������������������������������������������������������������
101
���������������������������������������������������������������������������������������������
102
�������������������������
�������������������������������������������������������������������������������
103
104
���������������������������������������������������������������������������������������
105
��������������������������������������������������������������������������� �����������
106
����������
107
����������������
108
���������������������������������������������������������������������������������������
109
������������������������������������������������������������������������
110
����������������������
111
�������������������������������������������������������
112
������������������������������������������������������������������������������������������
113
�����������������������������������������������������������������������������������
114
����������������������������������������������������������������������������������������
115
�������������������������������������������������������������������������������������������
116
��������������������������������������������������������������������������������������
117
�������������������������������������������������������������������������������������������
118
�����������������������������������������������������������������������������������
119
������������������������������������������������������������������������������������������
120
����������������������������������������������������������������������������
121
�
122
�
123
�
w
ie
ev
rR
ee
rP
Fo
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
American Journal of Tropical Medicine & Hygiene
5
102
American Journal of Tropical Medicine & Hygiene
124
��������������
125
���������������������������������������������������������������������������������
126
���������������������������������������������������� �����������������������������������
127
����������������������������������������������������������������������������������������
128
�����������������������������������������������������������������������������������������������
129
��������������������������������������������������������������������������������������
130
���������������������� ������������������������ ����������������������������������������������
131
��������������������������������������������������������������������������������������
132
��������������������������������������������������������������������������������
133
��������������������������������������������
134
��������������������������������������������������������������������������������������������
135
�����������������������
136
�
137
�
138
�
139
�
140
�
141
�
142
�
143
�
144
�
145
�
146
�
147
�
148
�
w
ie
ev
rR
ee
rP
Fo
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Page 6 of 9
6
103
Page 7 of 9
149
�����������
150
��������������������������������������������������������������������������������������
151
152
153
���������������������������������������������������������������������������������
������������������������������������������������������������������������������������������
�������������������������������������������������
154
������������������������������������������������������������������������������������������
155
��������������������������������������������������������������������������������
156
��������������������
Fo
157
�������������������������������������������������������������������������������������������
158
����������������������������������������������������������������������������������������
159
��������������������������������������������������������������������
161
����������������������������������������������������������������������������������������������
������������������������������
rR
162
ee
160
rP
�������������������������������������������������������������������������������������������
163
������������������������������������������������������������������������������������
164
������������������������������������������
166
167
168
169
170
�������������������������������������������������������������������������������������������
w
ie
165
ev
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
American Journal of Tropical Medicine & Hygiene
������������������������
��������������������������������������������������������������������������������������������
������������������������������������������������������������������
��������������������������������������������������������������������������������
��������������������������������������������������������
171
����������������������������������������������������������������������������������������
172
�����������������������������������������������������������������������������������
173
������������������������������
7
104
American Journal of Tropical Medicine & Hygiene
174
��������������������������������������������������������������������������������������
������������������������������������������������������������������������������������
175
176
���������������������������������������������������������������������������������
177
��������������������������������������������������������������������������������
178
��������������������������������
179
�����������������������������������������������������������������������������������������
��������������������������������������������������������
180
181
�������������������������������������������������������������������������������������������
182
���������������������������������������������������������������������������������������
183
�������������������
rP
Fo
184
���������������������������������������������������������������������������������
185
����������������������������������������������������������������������������
186
���������������������������������������������������������������������������
rR
187
ee
�����������������������������������������������������������������������������������
�������������������������������������������������������������������������
188
�
190
�
191
�
w
ie
189
ev
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Page 8 of 9
8
105
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
Page 9 of 9
Fo
Head louse Body louse Head louse iew
Head louse Head louse ev
rR
ee
rP
Body louse Yersinia pestis Borrelia recurrentis Bartonella quintana Rickettsia prowazekii (B)
106
Figure: Maximum-likelihood (ML) phylogram of the mitochondrial cytochrome b (cytb) gene. (A) American Journal of Tropical Medicine & Hygiene
Article V:
Studies of Ancient Lice Reveal Unsuspected
Past Migrations of Vectors
Accepté avec révisions mineures dans
American Journal of Tropical Medicine and Hygiene
107
American Journal of Tropical Medicine & Hygiene
�����������������������������������������������������������
�����������
��������� ������������������������������������������������
Fo
��������������� ��������������
����������������� �������������
rP
������������������������������ ������������
rR
ee
�������������������������� ����������������������������������������������������������������
�������������������������������������������������������������������
������������������������������������������������������������������������
�������������������������������������������������������������������������
�����������������������
�������������������������������������������������������������������
������������������������������������������������������������������������
�������������������������������������������������������������������������
�����������������������
������������������������������������������������������������������
����������� �������������������������������������������������������������������
w
ie
ev
109
Page 1 of 9
1
LRH: DRALI AND OTHERS
2
RRH: PAST MIGRATIONS OF VECTORS
Studies of ancient lice reveal unsuspected past migrations of vectors
3
4
5
Rezak Drali, Kosta Y. Mumcuoglu, Gonca Yesilyurt, Didier Raoult
6
������������������������������������������������������������������������������������
7
����������������������������������������������������������������������������������������
8
�����������������������������������������������������������������������������������������
9
�������������������������������������������������������������������������������������
10
���������������������������������������������������������������������������������������
11
����������������������������������������������������������������������������������������
12
�������������������������������������������������������������������
ee
rP
Fo
13
Abstract��The analysis of ancient head louse eggs recovered from Israel dating from
14
Chalcolithic period (four millennium B.C.) and early Islamic period (650 – 810 A.D.) allowed
15
to affirm that they probably belonged to people originating from West Africa because of their
16
belonging to the mitochondrial sub clade specific to that region.
ev
rR
17
w
ie
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
American Journal of Tropical Medicine & Hygiene
18
19
20
Address correspondence to: Didier Raoult, Unité de Recherche sur les Maladies Infectieuses
21
et Tropicales Emergentes (URMITE), Faculté de Médecine, 27 Blvd Jean Moulin, 13385
22
Marseille cedex 5, France. Email: didier.raoult@gmail.com
23
24
�
25
���������������������������, ancient lice, paleo-entomology, past migration of vectors�
1
110
American Journal of Tropical Medicine & Hygiene
26
Head lice (�������������������������) and body lice (�������������������������)
27
are strict bloodsucking ectoparasites of humans, and each species lives in a specific ecological
28
niche: hair for head lice, clothing for body lice.1 Hundreds millions of children worldwide are
29
continually infested by head lice unrelated to hygienic conditions, resulting in insomnia and
30
itching.2 Body lice exclusively infest populations exposed to stressful life conditions, such as
31
the homeless, prisoners and war refugees, and can serve as a vector for three serious humans
32
diseases, namely, epidemic typhus, trench fever and relapsing fever caused by ����������
33
����������, ���������� �������� and ��������������������, respectively.1 Body lice are also
34
suspected to be able to host and transmit ���������������, the agent of plague. 3 Genetically,
35
human lice are distributed into three mitochondrial clades (A, B and C), whereby only Clade
36
A comprises head and body lice.4 Clade A is found on all continents, whereas Clade B, with
37
an assignment of an American origin, is present in Europe and Australia and was recently
38
characterized in North Africa. Lastly, Clade C is to date limited to Africa and Asia. 5
rR
ee
rP
Fo
39
Lice are among the oldest parasites of humans, thus representing an excellent marker
40
of the evolution and migration of the Homo species over time. 4 For centuries, archaeologists
41
excavating soil in different locations around the world have found nits, lice and/or combs on
42
mummies or human remains with ages varying between 300 and 10,000 years (see 6 for a
43
review). The most ancient specimens (nits from hair, 10,000 years old) were found in Brazil,
44
South America.7 However, few molecular data on ancient lice are available. In 2008, Raoult
45
et al. showed that the most prevalent and well-distributed clade of lice (A) had a pre-
46
Columbian presence on the American continent.8 In 2013, Boutellis et al� confirmed this
47
result and demonstrated that Clade B also had a presence of at least 4,000 years in America
48
and that Clades A and B could live in sympatry.9
w
ie
ev
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Page 2 of 9
2
111
Page 3 of 9
49
In the present work, we obtained and analyzed ancient head louse eggs recovered from
50
Israel dating from two different periods, namely, the Chalcolithic (four millennium B.C.) and
51
early Islamic periods (650 – 810 A.D.).
52
In 2014, two lots of ancient head louse nits recovered from human remains in Israel
53
were sent to our laboratory for molecular analysis. A total of seven samples were examined.
54
Five operculated nits were found on hair remains in the Cave of the Treasure located at Nahal
55
Mishmar, in the Judean Desert, which date to the Chalcolithic period. 10 The two other nit
56
samples were found on human hair remains belonging to a population who lived in Nahal
57
Omer in the Arava Valley (between Dead Sea and Red Sea) during the Early Islamic Period
58
(650 – 810 A.D.). The site appears to have been a way-station on the north-south Arava route
59
and on the Spice Route between Petra and Gaza.11,12 The operculum on the eggs was most
60
likely detached during the centuries, but the embryos were still present inside the eggs.
ee
rP
61
Fo
The nits were photographed using an Axio Zoom V16 (Carl Zeiss AG, Germany).
rR
62
DNA extraction was performed as previously described.9 To determine the clade of the
63
specimens, an 89-bp fragment of the mitochondrial cytochrome b gene (����) was targeted. A
64
sensitive tool based on the real-time PCR with a hydrolysis probe that was useful to detect
65
and amplify ancient DNA was developed. A pair of primers (the reverse primer was
66
degenerate) that can amplify the different known clades of lice was used: cytbF (5’-
67
AGTGTGAGGAGGGTTTTCAG-3’) and cytbR (5’-CAAACCCCAAYAAVAYAAACGG-
68
3’). A TaqMan© FAM-labeled probe that contained non-fluorescent quencher conjugated to a
69
minor groove binder (MGB) at the 3’ was designed: FAM-
70
CTACTTTAGAGCGGTTGTTTACTC-MGB (Applied Biosystems, Courtaboeuf, France).
w
ie
71
ev
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
American Journal of Tropical Medicine & Hygiene
Real-time PCR was performed using the CFX96 thermal cycler (Bio-Rad
72
Laboratories, Foster City, CA, USA). The final reaction volume of 20 µL contained 3 µL of
73
the DNA template, 10 µL of 2x QuantiTect™ Probe PCR Master Mix (Qiagen), 0.5 µM of
3
112
American Journal of Tropical Medicine & Hygiene
74
each primer and 0.2 µM of the FAM-labeled probe. The thermocycling parameters consisted
75
of 95°C for 15 min and 40 cycles of 95°C for 10 s and 60°C for 30 s. No-template controls
76
(NTCs) were included in RT-PCR assay. The products of the real-time PCR amplifications
77
were sequenced as previously described.9 The nucleotide sequences obtained were aligned,
78
and phylogenetic analyses were performed as previously described.9
79
The appearance of nits under the microscope shows that they were well preserved
80
despite the many centuries spent underground. As shown in Figure 1, we can distinguish the
81
eyes, legs and claws of embryos still inside the operculated nits.
82
RT-PCR was positive for the seven samples of ancient DNA tested (32 < Ct < 34). The Ct
83
value was 20 in the positive control; no fluorescence was detected in the negative control.
84
A Maximum-Likelihood phylogenetic analysis performed for the cytb gene showed that the
85
ancient hair nits belonging to the Chalcolithic and early Islamic periods are part of Clade C.
86
Within Clade C, these nits form a sub-clade with lice found in Senegal (Figure 2).
87
Nonetheless, the ancient DNA sequences are quite unique and are different compared to
88
known sequences of contemporary lice.
ev
rR
ee
rP
89
Fo
In this work, the first molecular data on lice infesting the ancient inhabitants of the
90
Near East were compiled. The tool implemented here based on the real-time PCR was
91
effective in preventing the difficulties associated with the analysis of ancient DNA, which is
92
often damaged or in a very low concentration.13
93
w
ie
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Page 4 of 9
Interestingly, this study reveals the presence of a mitochondrial genotype, Clade C, in
94
a Near East region that has thus far only been found in Africa (Ethiopia and Senegal) and Asia
95
(Nepal).5 Specifically, these old nits are included in a subgroup in Clade C comprising
96
contemporary lice that are characterized only in West Africa. The presence of this genotype in
97
the Near East is necessarily linked to migration flows of humans through the ages. The slave
98
trade practiced by Arab tribes to supply the Near and Middle East in manpower existed for a
4
113
Page 5 of 9
99
long time.14 The genetic information available today suggests that significant gene flow most
100
likely occurred within the past ∼2,500 years between sub-Saharan African and Near East
101
populations.15
102
Thus, the study of ancient lice provides a wealth of interesting information for
103
understanding and elucidating the migration of our species throughout history and also
104
information about the circulation of pathogens transmitted by lice. Indeed, lice have proven to
105
be an excellent recorder, allowing explanations of what occurred when discovering certain
106
mass graves. In 2006, Raoult et al. concluded that louse-borne infectious diseases that
107
affected nearly one-third of Napoleon’s soldiers buried in Vilnius might have been a major
108
factor of mortality in the French retreat from Russia. 13
rP
109
Fo
In conclusion, we assert that the study of ancient lice is very useful to understand the
ee
110
circulation of vectors, the flow of vector-borne pathogens and the migratory flu hosts of these
111
vectors.
112
�����������������
113
We thank Jean-Michel BERENGER (Entomologist) and Abdul Karim SANGARÉ for their
114
technical support.
115
����������������������
116
The authors declare they have no conflict of interest.
117
�����������������: Rezak Drali and Didier Raoult, Unité de Recherche sur les Maladies
118
Infectieuses et Tropicales Emergentes (URMITE), Centre National de la Recherche
119
Scientifique No. 7278 (CNRS7278), Institut de Recherche pour le Développement No. 198
120
(IRD198), Institut National de la Santé et de la Recherche Médicale Unité No. 1095
121
(InsermU1095), Institut Hospitalo-Universitaire Méditerranée-Infection, Aix-Marseille
122
Université, Marseille, France, Emails: rezakdrali@hotmail.com, didier.raoult@gmail.com.
123
Kosta Y. Mumcuoglu and Gonca Yesilyurt, Department of Microbiology and Molecular
w
ie
ev
rR
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
American Journal of Tropical Medicine & Hygiene
5
114
American Journal of Tropical Medicine & Hygiene
124
Genetics, The Kuvin Center for the Study of Infectious and Tropical Diseases, Hadassah
125
Medical School, The Hebrew University, Jerusalem, Israel, Emails: kostasm@ekmd.huji.ac.il,
126
emine.gonca.yesilyurt@gmail.com.
127
References
128
129
130
131
132
1. Raoult D, Roux V, 1999. The body louse as a vector of reemerging human diseases. �����
���������� ��: 888-911.
Fo
2. Chosidow O, Chastang C, Brue C, Bouvet E, Izri M, Monteny N, Bastuji-Garin S,
133
Rousset JJ, Revuz J, 1994. Controlled study of malathion and d-phenothrin lotions
134
for ����������������� var �������-infested schoolchildren. ����������: 1724-1727.
135
3. Houhamdi L, Lepidi H, Drancourt M, Raoult D, 2006. Experimental model to evaluate
ee
136
rP
the human body louse as a vector of plague. ����������������� 1589-1596.
137
4. Reed DL, Smith VS, Hammond SL, Rogers AR, Clayton DH, 2004. Genetic analysis of
138
lice supports direct contact between modern and archaic humans. ������������ e340.
139
5. Boutellis A, Abi-Rached L, Raoult D, 2014. The origin and distribution of human lice in
the world. ��������������������� 209-217.
w
ie
141
ev
140
rR
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Page 6 of 9
6. Mumcuoglu KY, 2008. Human lice: Pediculus and Pthirus. In: Raoult, Drancourt,
142
editors. Paleomicrobiology: Past Human Infections. Berlin: Springer-Verlag. pp.
143
215-222.
144
145
7. Araujo A, Ferreira LF, Guidon N, Maues Da Serra FN, Reinhard KJ, Dittmar K, 2000.
Ten thousand years of head lice infection. ������������������� 269.
146
8. Raoult D, Reed DL, Dittmar K, Kirchman JJ, Rolain JM, Guillen S, Light JE, 2008.
147
Molecular identification of lice from pre-Columbian mummies. �����������������
148
535-543.
149
9. Boutellis A, Drali R, Rivera MA, Mumcuoglu KY, Raoult D, 2013. Evidence of
150
sympatry of clade A and clade B head lice in a pre-Columbian Chilean mummy from
151
Camarones. ����������� e76818.
6
115
Page 7 of 9
152
153
154
155
156
157
10. Bar-Adon P, 1980. The cave of the treasure: The finds from the caves in Nahal
Mishmar. ������������������������������������.
11. Baginski A, Shamir O, 1995. Early Islamic textiles, basketry and cordage from Nahal
Omer, Israel. ���������� 21-42.
12. Negev A, 1966. The Date of the Petra-Gaza Road. �����������������������������������
89-98.
158
13. Raoult D, Dutour O, Houhamdi L, Jankauskas R, Fournier PE, Ardagna Y, Drancourt
159
M, Signoli M, La VD, Macia Y, Aboudharam G, 2006. Evidence for louse-
160
transmitted diseases in soldiers of Napoleon's Grand Army in Vilnius. �������������
161
���� 112-120.
163
University Press.
15. Richards M, Rengo C, Cruciani F, Gratrix F, Wilson JF, Scozzari R, Macaulay V,
ee
164
14. Lewis B, 1992. Race and slavry in the Middle East: an historical enquiry. Oxford
rP
162
Fo
165
Torroni A, 2003. Extensive female-mediated gene flow from sub-Saharan Africa
166
into near eastern Arab populations. ������������������ 1058-1064.
w
ie
ev
rR
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
American Journal of Tropical Medicine & Hygiene
7
116
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
iew
ev
rR
ee
rP
117
Figure 1: Operculated head louse egg recovered from Israel dating from Chalcolithic period (four millennium B.C.)
Fo
American Journal of Tropical Medicine & Hygiene
Page 8 of 9
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
Page 9 of 9
92
50
Clade-B36
Clade C the right of each tree.
118
ML bootstrap that support values greater than 50 are located above the nodes. Mitochondrial clade memberships are indicated to
Figure 2. Maximum-likelihood (ML) phylogram of cytochrome b gene.
P.schaeffi
Chalcolithic-27
iew
Chalcolithic-30
Clade-C39_Senegal
Islamic-11
Chalcolithic-29
Chalcolithic-5
Chalcolithic-36
Islamic-9
Clade-C42_Ethiopia
Clade-C44_Nepal
ev
90
Clade A Clade B Clade-A12
Clade-A5
Clade-B33
55
Clade-A17
rR
ee
rP
0.05
Fo
62
56
American Journal of Tropical Medicine & Hygiene
Article VI:
Evidence of Sympatry of Clade A and Clade B Head Lice in
A Pre-Columbian Chilean Mummy from Camarones
PLoS One 8: e76818.
119
Evidence of Sympatry of Clade A and Clade B Head Lice
in a Pre-Columbian Chilean Mummy from Camarones
Amina Boutellis1, Rezak Drali1, Mario A. Rivera2, Kosta Y. Mumcuoglu3, Didier Raoult1*
1 Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes: URMITE, Aix Marseille Université, UMR CNRS 7278, IRD 198, INSERM 1095. Faculté de
Médecine, 27 Bd Jean Moulin, Marseille, France, 2 Programa Identidad del Fin del Mundo. Universidad de Magallanes-Mineduc, Punta Arenas, Chile, 3 Department of
Microbiology and Molecular Genetics, The Kuvin Center for the Study of Infectious and Tropical Diseases, Hadassah Medical School, The Hebrew University, Jerusalem,
Israel
Abstract
Three different lineages of head lice are known to parasitize humans. Clade A, which is currently worldwide in distribution,
was previously demonstrated to be present in the Americas before the time of Columbus. The two other types of head lice
are geographically restricted to America and Australia for clade B and to Africa and Asia for clade C. In this study, we tested
two operculated nits from a 4,000-year-old Chilean mummy of Camarones for the presence of the partial Cytb mitochondrial
gene (270 bp). Our finding shows that clade B head lice were present in America before the arrival of the European
colonists.
Citation: Boutellis A, Drali R, Rivera MA, Mumcuoglu KY, Raoult D (2013) Evidence of Sympatry of Clade A and Clade B Head Lice in a Pre-Columbian Chilean
Mummy from Camarones. PLoS ONE 8(10): e76818. doi:10.1371/journal.pone.0076818
Editor: Michael Knapp, Bangor University, United Kingdom
Received April 22, 2013; Accepted August 29, 2013; Published October 30, 2013
Copyright: � 2013 Boutellis et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: These authors have no support or funding to report.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: didier.raoult@gmail.com
Introduction
have contributed to populating the Americas. In the current study,
two operculated nits from a mummy found in Camarones, Chile,
were tested to identify the mitochondrial phylotypes of the lice.
Pediculus humanus capitis is an ancient human parasite most likely
associated with humans since our pre-hominid ancestor and
dispersed throughout the world by early human migrants [1].
Louse infestation in ancient human populations has been recorded
in different geographic regions of the world [2] and even affected
wealthy social classes, such as the 15th-century King of Naples,
Ferdinand II of Aragon [3]. The oldest head louse nit was found
on a hair from an archaeological site in northeastern Brazil and
was dated to 8,000 B.C. [4]. The oldest such finding in Asia is
9,000 years old, obtained from a hair sample from an individual
who lived in the Nahal Hemar cave in Israel [5]. Head lice have
also been found at archaeological sites in the southwestern USA,
the Aleutian Islands, Peru, Greenland and Mexico and on
mummies that were Incan sacrifices [4]. Recently, another
discovery of lice was reported for a Maitas Chiribaya mummy
from Arica, in northern Chile, dating to 670–990 A.D. (calibrated)
[6]. The evidence for the presence of ectoparasites on ancient
Americans indicates that head lice most likely arrived with the first
human colonists who entered the Americas [7]. However, by
amplifying mitochondrial DNA (mtDNA) of part of two genes
(Cytb and Cox1) belonging to 10,000-year-old lice collected from
Peruvian mummies, Raoult and colleagues demonstrated that the
worldwide clade A louse was in the Americas before the time of
Columbus [8]. Two other clades of head lice have been reported
and have a specific geographical distribution: clade C is specifically
restricted to head lice in Ethiopia, Nepal and Senegal [9], whereas
the clade B head lice are found in North and Central America,
Australia and certain European countries [7]. However, the origin
of clade B head lice remains unknown because no lice with this
phylotype have been reported in Asia or in any region suspected to
PLOS ONE | www.plosone.org
Materials and Methods
Hair samples from seven mummies from Camarones 15-D,
Chile, carbon-dated to ca. 4,000 B.C., were examined for the
presence of head lice (Figure 1), and in six hair samples, the nits of
head lice were found. No body louse was found on these
mummies. The material (nits fixed on hair) of one mummy was
stored in 70% ethyl alcohol for the last 10 years and was sent to
our laboratory in Marseilles in September 2012.
A first screening of the quality of the nits was performed using
our ZEISS zoom microscope with a fixed camera (ZEISS AXIO
ZOOM.V16. France), and then two operculated nits were selected
for our study (Figure 2). The two nits were rinsed twice in sterile
water then crushed with a scalpel. The total genomic DNA was
extracted and eluted in a 50 ml volume with a QIAamp Tissue Kit
(Qiagen, Courtaboeuf, France) with an EZ1 apparatus, as
described by the manufacturer. The extracted genomic DNA
concentrations were 1.8 ng/ml and 1.3 ng/ml for the two nits, and
the DNA samples were stored at 220uC under sterile conditions to
avoid cross-contamination until further processing. The DNA of
the two nits was amplified using a suicide nested polymerase chain
reaction (PCR) protocol (re-amplification without positive control)
with a partial Cytb gene (270 bp) primers, as previously described
[11]. The same primers were used for both amplifications. PCR
reactions were prepared on ice and contained 3 ml of the DNA
template, 4 ml of 5X HF Phusion Buffer, 250 mM of each
nucleotide, 0.5 mM of each primer, 0.2 ml of Phusion DNA
Polymerase (Thermo Scientific, Lithuania) and water (DNase and
1
121
October 2013 | Volume 8 | Issue 10 | e76818
Head Lice Nits in Pre-Columbian Mummy
RNase-Free) to a final reaction mixture volume of 20 ml. The
PCRs were performed in a PTC-200 automated thermal cycler
(MJ research, Waltham, MA, USA). The cycling conditions were
98uC for 30 sec; 40 cycles of 5 sec at 98uC, 30 sec at 56uC, 15 sec
at 72uC; and a final extension time of 5 min at 72uC. All of the
experiments were performed in a location free of louse DNA,
under a hood with air capture and with sterilized instruments that
were used only once. The negative controls remained negative.
The success of the PCR amplification was then verified by
migration of the PCR product on a 2% agarose gel. The
NucleoFast 96 PCR Plates (Macherey-Nagel EURL, France) and
BigDye Terminator version 1.1 cycle sequencing-ready reaction
mix (Applied Biosystems, Foster City, CA) were then used to purify
the PCR products to be sequenced directly in both directions with
the same primers used in the PCR amplification. The ABI 3100
automated sequencer (Applied Biosystems) resolved the sequenced
products. The program Chromas Pro software (Technelysium
PTY, Australia) was used to analyze, assemble and correct the
sequences.
In addition to our newly obtained data, 18 samples belonging to
the worldwide clade A [8], 19 samples belonging to clade B from
USA, UK [11] and Honduras [7] and 28 samples belonging to
clade C from Senegal [9] and Ethiopia [12] were used.
The Phylogeny Reconstruction was performed from the DNA
sequences using the Maximum Likelihood (ML) with 100
Bootstrap Replications within the MEGA 5 software with
complete deletion, Tamura-Nei model (nucleotide) of substitution
model was used automatically [13].
Ethics statement
Figure 1. Picture of the Mummy. Mummy 23 from Camarones 15-D,
Northern Chile � Mario A Rivera.
doi:10.1371/journal.pone.0076818.g001
Mummies from Camarones were excavated in 1990 by a team
of investigators under the direction of Mario A. Rivera, they have
permit from Consejos Monumentos Nacionales, authorization
Figure 2. Picture of the Pre Columbian nit. A pre-Columbian nit isolated from a Chilean mummy (No. 1) (h: hair; o: operculum; c: cementum)
(picture taken with a ZEISS AXIO ZOOM.V16).
doi:10.1371/journal.pone.0076818.g002
PLOS ONE | www.plosone.org
2
122
October 2013 | Volume 8 | Issue 10 | e76818
Head Lice Nits in Pre-Columbian Mummy
Figure 3. Cytb phylogenic analysis. The phylogenic tree based on ML method of the two pre-Columbian Chilean nits based on the partial Cytb
gene (270 bp). HL: head louse, BL: body louse. The numbers on the branches are bootstrap values.
doi:10.1371/journal.pone.0076818.g003
phylogenetic tree, it was found that one nit belonged to the
worldwide clade A (Genbank accession nuKF498963), whereas the
second nit belonged to the clade B (Genbank accession
nuKF498962) (figure 3). One base distinguished chilean mummy
clade A head lice from other sequences found in GenBank:
position 7 (G in mummy nit lice versus a gap in others) (Figure 4).
There is no difference between the Chilean mummy clade B nit
and other sequences present in GenBank (Figure 4).
number 355, November 12th, 1987. Samples were donated by
Museo Arqueologia San Miguel Azapa, Universidad de Tarapaca,
Arica, Chile and permission was obtained from the said Museum
to access the collections [10].
Results
The DNA of the two nits (848 mm and 912 mm long,
respectively) was amplified and sequenced for the Cytb gene of
270 bp. After assembling the sequences and analyzing the
PLOS ONE | www.plosone.org
3
123
October 2013 | Volume 8 | Issue 10 | e76818
Head Lice Nits in Pre-Columbian Mummy
Figure 4. Sequences alignment. Alignment of the clade A and clade B sequences (Chilean mummy’s nit and other sequences present in GenBank)
P: Philippines, G: Germany, I: Iran, U: United State of America, T: Taiwan, C: Canada, S: Senegal, Pa: Papua New Guinea, H: Honduras.
doi:10.1371/journal.pone.0076818.g004
Discussion
been reported only in Africa; and A3, which is specific to
American lice [14]. Clade B may have developed in North and
Central America before Columbus and is now spreading
throughout the world. This clade’s origin predates modern Homo
sapiens by an order of magnitude (ca. 1.18 million years) [1]. In
contrast, Clade C is mostly confined to Africa and Asia.
The present work confirms that the origin of clade B was
America before the arrival of Columbus, but it will be interesting
to test other mummies from Asia, which is reputed to have peopled
the Americas.
We report the first identification of both the clade A and the
clade B genotypes existing in sympatry in two nits isolated from
human remains from pre-Columbian Chile. Lice with clade A and
clade B mtDNA are not uncommon and were reported to be
present on a single human head in both the USA and Honduras
on several occasions [8]. Clade B was first identified in America
and the United Kingdom and was later found in other European
countries and Australia [7]. Until now, the origin of clade B was
unknown, but according to the clade’s current distribution, it was
speculated that this louse phylotype was imported into Europe by
Europeans returning from America [1]. Currently, the most likely
theory is that the clade A louse issued from Africa and was
distributed worldwide, given the clade’s three different chromosomal signatures: A1, which is found worldwide; A2, which has
PLOS ONE | www.plosone.org
Author Contributions
Conceived and designed the experiments: DR KM. Performed the
experiments: AB RD. Analyzed the data: AB RD DR. Wrote the paper:
AB RD KM MR DR. Collected samples: MR KM.
4
124
October 2013 | Volume 8 | Issue 10 | e76818
Head Lice Nits in Pre-Columbian Mummy
References
8. Raoult D, Reed DL, Dittmar K, Kirchman JJ, Rolain JM, et al. (2008)
Molecular identification of lice from pre-Columbian mummies. J Infect Dis 197:
535–543. 10.1086/526520 [doi].
9. Boutellis A, Veracx A, Angelakis E, Diatta G, Mediannikov O, et al. (2012)
Bartonella quintana in head lice from Senegal. Vector Borne Zoonotic Dis 12:
564–567. 10.1089/vbz.2011.0845 [doi].
10. Rivera MA, Mumcuoglu K, Mathney RT, Matheny DG (2008) Head lice eggs,
Anthropophthirus capitis, from mummies of the Chinchorro tradition, Camarones
15-D, Northern Chile. Chungara, Revista de Antropologia Chilena 40: 31–39.
11. Li W, Ortiz G, Fournier PE, Gimenez G, Reed DL, et al. (2010) Genotyping of
human lice suggests multiple emergencies of body lice from local head louse
populations. PLoS Negl Trop Dis 4: e641. 10.1371/journal.pntd.0000641 [doi].
12. Angelakis E, Diatta G, Abdissa A, Trape JF, Mediannikov O, et al. (2011).
Altitude-dependent Bartonella quintana genotype C in head lice, Ethiopia. Emerg
Infect Dis; 17: 2357–2359. 10.3201/eid1712.110453 [doi].
13. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, et al. (2011) MEGA5.
Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol; 28: 2731–39.
msr121 [pii];10.1093/molbev/msr121 [doi].
14. Boutellis A, Veracx A, Jonatas A, Raoult D (2013) Amazonian head lice-specific
genotypes are putatively pre-Columbian. Am J Trop Med Hyg 88(6):1180–4.
10.4269/ajtmh.12–0766 [doi].
1. Reed DL, Smith VS, Hammond SL, Rogers AR, Clayton DH (2004) Genetic
analysis of lice supports direct contact between modern and archaic humans.
PLoS Biol 2: e340. 10.1371/journal.pbio.0020340 [doi].
2. Rick FM, Rocha GC, Dittmar K, Coimbra CE, Reinhard K, et al. (2002) Crab
louse infestation in pre-Columbian America. J Parasitol 88: 1266–1267.
10.1645/0022–3395 (2002) 088 [1266: CLIIPC]2.0.CO;2 [doi].
3. Fornaciari G, Giuffra V, Marinozzi S, Picchi MS, Masetti M (2009) ‘Royal’
pediculosis in Renaissance Italy: lice in the mummy of the King of Naples
Ferdinand II of Aragon (1467–1496). Mem Inst Oswaldo Cruz 104: 671–672.
S0074–02762009000400026 [pii].
4. Araujo A, Ferreira LF, Guidon N, Maues Da Serra FN, Reinhard KJ, et al.
(2000) Ten thousand years of head lice infection. Parasitol Today 16: 269.
S0169–4758 (00) 01694-X [pii].
5. Mumcuoglu K (2008) Pediculus and Pthirus. In Paleomicrobiology – Past
Human Infections. Raoult, D. & M. Drancourt (eds). Springer, Berlin, 215–222.
6. Arriaza B, Orellana NC, Barbosa HS, Menna-Barreto RF, Araujo A, et al.
(2012) Severe head lice infestation in an Andean mummy of Arica, Chile.
J Parasitol 98: 433–436. 10.1645/GE-2903.1 [doi].
7. Light JE, Allen JM, Long LM, Carter TE, Barrow L, et al. (2008) Geographic
distributions and origins of human head lice (Pediculus humanus capitis) based
on mitochondrial data. J Parasitol 94: 1275–1281. GE-1618 [pii];10.1645/GE1618.1 [doi].
PLOS ONE | www.plosone.org
5
125
October 2013 | Volume 8 | Issue 10 | e76818
Chapitre 3:
Host-Switching
127
Préambule
L’association étroite hôte-parasite peut conduire à une cospeciation.
Ce phénomène est bien illustré par la relation poux-hôtes. En effet, les
poux sont des ectoparasites obligatoires des oiseaux et des
mammifères avec qui le partenariat a commencé il y’a plus de 65
millions d’années [6,7,50].
Parfois, cette association est marquée par une série d'événements
historiques tels le changement d'hôte “host-switching” ou encore la
duplication de parasite [10,11].
Depuis un siècle, la relation pou – hôte entre Pediculus mjobergi et les
primates du Nouveau Monde est restée une énigme du fait de la
ressemblance morphologique frappante de ce dernier avec les poux
humains.
Pour investiguer ces observations centenaires, nous avons recueilli des
poux de singes hurleurs d’Argentine et nous les avons comparés à des
poux humains que nous avons recueillis dans un village d’Amazonie
isolé qui a échappé à la mondialisation.
Cette étude a permis de lever l'ambiguïté existante grâce aux
premières analyses moléculaires et génétiques effectuées sur P.
mjobergi.
129
En effet, les résultats montrent que P. mjobergi a été transmis aux
singes par les premiers humains arrivés sur le continent américain il
y’a des milliers d'années. Ce résultat remet en cause le paradigme de
coévolution stricte entre le pou et son hôte.
130
Article VII:
Host switching of Human Lice to New World
Monkeys in Amazonia
Soumis comme Research Reports dans
Proceedings of the National Academy of Sciences
131
Host switching of human lice to new world monkeys in Amazonia
1
2
3
Rezak Dralia, Laurent Abi-Rachedb, Amina Boutellisa, Félix Djossouc, Stephen C. Barkerd and
4
Didier Raoulta
5
6
a
7
Marseille, France.
8
b
9
Mixte de Recherche 7373, Equipe ATIP, Aix-Marseille Université, 13331 Marseille, France.
Aix Marseille Université, URMITE, UM63, CNRS 7278, IRD 198, Inserm 1095, 13005
Centre National de la Recherche Scientifique, Institut de Mathématiques de Marseille - Unité
10
c
11
Cedex. Guyane française.
12
d
13
of Queensland, Brisbane, Queensland, Australia.
Service de maladies infectieuses et tropicales, Centre hospitalier de Cayenne, 97306 Cayenne
Department of Parasitology, School of Chemistry and Molecular Biosciences, The University
14
15
Corresponding author: Didier Raoult
16
mailto:didier.raoult@gmail.com
17
18
PNAS Research Reports
19
20
21
22
23
24
Key words: Pediculus mjobergi, Clade B, Amazonian head louse, host-switching,
25
cospeciation
1
133
26
Abstract (204/250)
27
The coevolution between a host and its obligate parasite is exemplified in the sucking lice that
28
infest primates (1-4). In the context of close lice-host partnerships and cospeciation, Pediculus
29
mjobergi, the louse of New World primates, has long been puzzling because its morphology
30
resembles that of human lice (5,6). To investigate the possibility that P. mjobergi was
31
transmitted to monkeys from the first humans who set foot on the American continent
32
thousands of years ago, we obtained and compared P. mjobergi lice collected from howler
33
monkeys from Argentina to human lice gathered from a remote and isolated village in
34
Amazonia that has escaped globalization (7). Morphological examinations were first
35
conducted and verified the similarity between the monkey and human lice. The molecular
36
characterization of several nuclear and mitochondrial genetic markers in the two types of lice
37
revealed that one of the P. mjobergi specimens had a unique haplotype that clustered with the
38
haplotypes of Amazonian head lice that are prevalent in tropical regions in the Americas, a
39
natural habitat of New World monkeys. Because this phylogenetic group forms a separate
40
branch within a human clade of sequences that is of American origin, this finding indicates
41
that human lice have transferred to New World monkeys.
42
43
44
45
46
47
48
49
50
2
134
51
Significance Statement (116/120)
52
Lice are permanent obligate parasitic insects that infest birds and mammals. Because they are
53
wingless and have no intermediate hosts, lice coevolved with their hosts and acclimated to
54
their microenvironments. The transmission of lice mostly occurs through direct contact
55
between conspecific hosts. In exceptional cases, lice may migrate to another host and adapt to
56
their new ecological niches. These events are known as the host-switching phenomenon. In
57
this study, we report a case of human lice transfer to New World monkeys. This event
58
happened thousands of years ago when the first men reached the American continent. The
59
morphological examinations and genetic analyses performed on Pediculus mjobergi lice
60
collected from howler monkeys, Alouatta caraya, support these findings.
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
3
135
76
Introduction
77
A close host-parasite association can lead to cospeciation. This phenomenon is exemplified in
78
lice (Insecta: Phthiraptera), the obligate ectoparasites of birds and mammals, as their
79
partnership with their hosts has taken place for over 65 million years (1-4).
80
Humans can be infested by two types of sucking lice (Anoplura), Pediculus humanus and
81
Phthirus pubis, the crab louse (8). P. h. capitis (head louse) and P. h. humanus (body louse)
82
are ecotypes that have spread worldwide as modern humans have moved out of Africa over
83
the past 100,000 years (9). The molecular analysis of the mitochondrial (mt) genes
84
cytochrome oxidase subunit 1 (cox1) and cytochrome b (cytb) has allowed for their
85
classification into three haplogroups, which are designated A, B and C (10). Of these groups,
86
only haplogroup A is distributed worldwide and comprises both head and body lice (11,12).
87
Haplogroup B consists of head lice found in the Americas, Western Europe, Australia and
88
North Africa (13). Haplogroup C consists of head lice found in Nepal, Ethiopia and Senegal
89
(12,14,15). In addition to the inter-haplogroup diversity, human lice also demonstrate intra-
90
haplogroup diversity, which is illustrated by the many distinct A and B haplotypes (16-18).
91
The molecular analysis of lice collected from Pre-Columbian mummies showed that
92
haplogroups A and B were already present in the New World before the arrival of European
93
settlers (19,20). This finding supports an American origin for haplogroup B, followed by a
94
dispersal into the Old World by European colonists returning to Europe (13).
95
Interestingly, humans are not the only species in the Americas that harbor lice of the genus
96
Pediculus. In 1916, Ferris reported that New World monkeys harbored a sucking louse of the
97
Pediculidae family, suborder Anoplura (21). The first description of this louse was reported in
98
1910 by Mjoberg, who named it Pediculus affinis (22). Ferris suggested replacing the name
99
affinis with mjobergi (23). P. mjobergi has been found in three of the five families of
4
136
100
monkeys in the New World, the Cebidae (capuchin monkeys), the Atelidae (howler and spider
101
monkeys) and the Pitheciidae (titi monkeys) (22,24,25).
102
To investigate the relationship between P. humanus and P. mjobergi lice, we obtained and
103
compared P. mjobergi lice with lice recovered from a remote and isolated village in
104
Amazonia that has escaped globalization.
105
Results
106
P. mjobergi specimens highly resemble human lice
107
A comparison between the six adult P. mjobergi specimens from two wild howler monkeys
108
and 19 Amazonian human head lice showed that the P. mjobergi specimens are
109
morphologically similar to human lice (Figure 2). In particular, P. mjobergi were the same
110
gray color as the contemporary head lice recovered in the USA and were not the darker color
111
of the Amazonian head lice (Figure 2).
112
P. mjobergi mtDNA sequences belong to the mtDNA clades of human lice
113
To determine if the morphological similarities between the P. mjobergi specimens and human
114
lice are the result of convergent evolution or a recent common ancestor, we evaluated four
115
mtDNA markers (cytb, cox1, 16S rRNA, and nad2). Although each marker was targeted for
116
amplification from each of the six P. mjobergi specimens, only one marker was amplified
117
from all six individuals (cytb), and the remaining three markers (cox1, 16S rRNA, and nad2)
118
were only amplified from P. mjobergi individual #4 (Figure 3). Because the positive and
119
negative PCR amplification controls worked well in all of the amplification experiments, this
120
finding suggests that for P. mjobergi individuals #1-3 and #5-6, either the DNA extracted was
121
not of sufficient quality or the targeted genes had a sequence at the primer sites that was too
122
divergent.
123
The targets that could be amplified by PCR were sequenced, and the sequences generated
124
were aligned with the publicly available sequences. The P. mjobergi sequences obtained were
5
137
125
highly similar to the sequences obtained from human lice. To investigate their precise
126
relationship, we performed phylogenetic analyses. Maximum-likelihood (ML) and Neighbor-
127
Joining (NJ) phylogenetic analyses were performed for each of the four mtDNA genes and
128
showed that, in all four analyses, the human and P. mjobergi sequences split into three well-
129
supported haplogroups that corresponded to haplogroups A, B and C (Figure 4). In all four
130
analyses, the sequences of the monkey louse P. mjobergi #4 clustered with the Amazonian
131
head lice in haplogroup B. Similarly, the analysis of the cytb gene revealed that the remaining
132
monkey lice (P. mjobergi #1-3 and #5-6) have the widespread haplotype A5 of haplogroup A
133
(Figure 4A).
134
To investigate the geographical distribution of the haplotypes more precisely, we assembled a
135
dataset of 707 cytb sequences of body and head lice (424 from GenBank and 283 from our
136
laboratory). These 707 sequences span 36 worldwide geographic locations on five continents
137
and represent 32 distinct haplotypes, 21 from haplogroup A (66%), five from haplogroup B
138
(16%) and six from haplogroup C (SI Appendix). While P. mjobergi #1-3 and #5-6 have
139
haplotype A5, which is common worldwide (80% of locations and 49% of the 707 analyzed
140
human lice), the P. mjobergi #4 haplotype is closely related to haplotype B54 (Figure 5A),
141
which is unique to Amazonian head lice; these two haplotypes only differ in five nucleotides
142
that result in only a single amino acid difference (Lys129Ile) (SI Appendix).
143
We performed a similar analysis for the cox1 gene. Even though the sequences included in
144
this analysis were primarily collected from across the America (16), we were also able to
145
include 79 sequences from head and body lice from other parts of the world as well as the 33
146
lice sequences obtained in this study (SI Appendix). The 562 sequences collected represent 37
147
haplotypes, fifteen from haplogroup A, fifteen from haplogroup B and two from haplogroup
148
C. As for cytb, P. mjobergi #4 has a unique haplotype that is part of a subgroup of haplogroup
6
138
149
B that contains haplotype B18 from Argentina and Mexico and haplotypes B29-B30 from
150
Amazonia (Figure 5B).
151
This analysis result indicates that the P. mjobergi mtDNA sequences belong to the mtDNA
152
clades of human lice. In particular, specimen P. mjobergi #4 had, for all four markers,
153
sequences belonging to the B haplogroup, a haplogroup that most likely originated in the
154
Americas. Therefore, in addition to the morphological similarities, P. mjobergi lice and
155
human lice also share highly related mtDNA genomes, which suggests that their
156
morphological similarities are not the result of convergent evolution but are the result of their
157
close genetic relationship.
158
P. mjobergi nuclear sequences are related to the sequences of human lice
159
To further investigate whether P. mjobergi and human lice are closely related, we extended
160
our analysis to include nuclear markers, three genes and four intergenic spacers (Figure 3).
161
All three nuclear genes were amplified from P. mjobergi #4 as well as one gene from P.
162
mjobergi individual #3 (18S rRNA), but no PCR product was obtained for specimens #1-2
163
and #5-6 (Figure 3). A comparison of the sequences obtained in these amplifications with
164
publicly available human lice sequences showed that the P. mjobergi sequences are highly
165
related to the human lice sequences but are unique sequences in all cases. In particular, the
166
18S rRNA sequences from P. mjobergi individual #3 and P. mjobergi #4 are both unique (SI
167
Appendix).
168
The intergenic spacers PM1 and PM2 could be amplified from P. mjobergi #4, and PM1 was
169
also amplified from P. mjobergi #3 (Figure 3). Both spacers were also amplified from the 19
170
Amazonian head lice included in this study (see Methods). For PM1, 15 of the 19 Amazonian
171
head lice shared genotype 13 (G13) with human lice from Africa and Europe belonging to a
172
group that also includes P. mjobergi #3 (Figure 6A). The P. mjobergi #4 PM1 sequence,
173
however, belongs to genotype 21 (G21), which was previously characterized in Mexico. The
7
139
174
remaining Amazonian head lice belong to either genotype 36 (G36), which was already
175
characterized in Amazonia, or a new genotype that is highly related to G36 (Figure 6A). For
176
the PM2 marker, P. mjobergi #4 and Amazonian head lice share genotype 47 (G47) with
177
human lice from Africa, Oceania and America (Figure 6B).
178
This analysis of nuclear markers supports the results from the mtDNA analysis and shows that
179
the P. mjobergi specimens are closely related to human lice.
180
Discussion
181
In this study, we report for the first time molecular data for Pediculus mjobergi, which is
182
known as the louse of New World monkeys. The six individual lice analyzed were
183
morphologically similar to Pediculus humanus, the human lice (Figure 2). This observation
184
supports two previous descriptions reported in 1938 and 1983 (5,6).
185
The mtDNA analysis revealed that the New World monkey lice analyzed in this study have
186
cytb haplogroup A and B haplotypes, whereas the Amazonian head lice only have haplogroup
187
B haplotypes. Interestingly, while five P. mjobergi had a haplogroup A haplotype (A5) that is
188
prevalent (49% of analyzed human lice) and well distributed (80% of locations), P. mjobergi
189
#4 had a novel haplogroup B haplotype. This novel haplotype belongs in a group with
190
Amazonian head lice (Figure 5) that are prevalent in tropical areas in the Americas (Mexico,
191
Amazonia and northeast Argentina), a natural habitat of New World monkeys.
192
Because of the genetic and geographic proximity of the lice evaluated in this study, this
193
finding suggests that an exchange of genetic material has taken place between human lice and
194
New World monkey lice during their casual encounters. Consistent with this model, contact
195
between monkeys and humans has never been interrupted in South America. For example,
196
when monkeys are not hunted for their meat, they can become pets (26).
197
In blood sucking insects, including mosquitoes, host preference demonstrates a high degree of
198
flexibility when the favorite host species becomes scarce or is not accessible (27). Under these
8
140
199
conditions, physiological factors (hunger) and the physical abundance of available new hosts
200
may contribute to host switching (27). In this study, we show that this type of host switching
201
has occurred with human lice transmitted to monkeys. Moreover, the evidence suggesting that
202
two different mitochondrial genotypes (Clade A and Clade B) are prevalent, leads us to
203
conclude that the P. humanus-P. mjobergi shift is not a unique event because it appears to
204
have occurred at least twice. Among closely related mammals, louse-host cospeciation
205
primarily results from allopatry (28,29). Conversely, the sympatric life of related hosts can
206
result in an exchange, and potentially a recombination, of their respective lice, which would
207
overrule the strict louse-host coevolution paradigm.
208
Materials and Methods
209
Lice samples. For P. mjobergi, six adult specimens were collected from two wild howler
210
monkeys, Alouatta caraya, located in northeast Argentina. The first four lice were collected
211
from monkey #B2188 from the National Park Iguaza, Province of Misiones. The other two
212
lice were found on monkey B1395 from the Province of Corientes (Figure 1).
213
For the human lice in the Americas, 19 Amazonian human head lice were included in this
214
study. We recovered these specimens in 2013 from members of the Wayapi community (7)
215
living in “Trois-Sauts”, a remote and isolated village on the Oyapock River along the border
216
between French Guyana and Brazil (Figure 1).
217
Human head and body lice collected in France were included as positive controls.
218
Lice were photographed on their dorsal and ventral sides using a fixed camera (Olympus
219
DP71, Rungis, France).
220
DNA samples. Genomic DNA was extracted using the QIAamp DNA tissue extraction kit
221
(Qiagen, Hilden, Germany) in an EZ1 apparatus following the manufacturer's instructions.
222
DNA from each louse was eluted in 100 µl of TE buffer and stored at -20 °C.
9
141
223
PCR amplifications. Seven genes, three nuclear genes [18S rRNA, glycerol-3-phosphate
224
dehydrogenase (GPD) and RNA polymerase II largest subunit (RPII)] and four mtDNA
225
[(cytb), cytochrome oxidase subunit 1 (cox1), 16S ribosomal RNA, and NADH
226
dehydrogenase subunit 2 (nad2)] were investigated. We also targeted intergenic regions using
227
two highly polymorphic microsatellites (PM1 and PM2) as previously described (30).
228
All primers used for these experiments are described in the SI Appendix.
229
PCR amplifications were conducted in a Peltier PTC-200 model thermal cycler (MJ Research
230
Inc., Watertown, Mass. USA). PCR reactions were prepared on ice and contained 3 μl of
231
DNA template, 4 μl of Phusion HF Buffer, 250 μM of each nucleotide, 0.5 μM of each
232
primer, 0.2 μl of high fidelity Phusion DNA Polymerase (Finnzymes, Thermo Scientific,
233
Vantaa, Finland) and water to a final reaction mixture volume of 20 μl. The cycling
234
conditions were 98°C for 30 sec; 35 cycles of 5 sec at 98°C, 30 sec at Tm (SI Appendix), 15
235
sec at 72°C; and a final extension time of 5 min at 72°C. PCR positive and negative controls
236
were included in each assay. The success of PCR amplification was then verified by
237
electrophoresis of the PCR product on a 1.5% agarose gels.
238
Sequencing. NucleoFast 96 PCR Plates (Macherey-Nagel EURL, France) were used to purify
239
the PCR products before sequencing. The purified PCR products were then sequenced in both
240
directions (with the PCR primers) using a BigDye Terminator version 1.1 cycle sequencing-
241
ready reaction mix (Applied Biosystems, Foster City, CA) and an ABI 3100 automated
242
sequencer (Applied Biosystems). The program Chromas Pro software (Technelysium PTY,
243
Australia) was used to analyze, assemble and correct the sequences.
244
The sequences obtained in this study and those representing the haplotypes determined from
245
the set of used sequences have been deposited in GenBank (KM579408 - KM579584).
246
Sequence analysis. For each of the target genes, the nucleotide sequences obtained in this
247
study were aligned with the sequences available on public databases (GenBank) using
10
142
248
CLUSTALX 2.0.11 (31). MEGA 6 was used for phylogenetic analyses and tree
249
reconstruction (32). NJ phylogenetic analysis was performed using the Maximum Composite
250
Likelihood method with 200 replicates. ML analyses were performed using the Jukes-Cantor
251
model with 200 replicates.
252
To determine the phylogeographic relationships among the lice, median-joining networks
253
were assembled for the lice haplotypes of two mitochondrial markers, cytb and cox 1, using
254
the method of Bandelt with the program Network version 4.6.1.1 (33). The sequence between
255
nucleotide positions 370-642 of cytb and 748-1026 of cox1 was determined for all the
256
specimens. Partial gene sequences were aligned with the sequences available in GenBank.
257
The percent similarities were determined using MEGA6 (32).
258
Ancestral sequences were reconstructed for each node of the cytb phylogenetic tree using the
259
marginal reconstruction approach with BASEML of the PAML4 software package (34).
260
261
262
263
264
265
266
267
268
269
270
271
272
11
143
273
Reference List
274
275
1. Barker,S.C. (1991) Evolution of host-parasite associations among species of lice and
276
rock-wallabies: coevolution? (J. F. A. Sprent Prize lecture, August 1990). Int. J.
277
Parasitol., 21, 497-501.
278
279
280
2. Barker,S.C. (1994) Phylogeny and classification, origins, and evolution of host
associations of lice. Int. J. Parasitol., 24, 1285-1291.
3. Hafner,M.S. and Page,R.D. (1995) Molecular phylogenies and host-parasite
281
cospeciation: gophers and lice as a model system. Philos. Trans. R Soc Lond B Biol
282
Sci., 349, 77-83.
283
284
285
286
4. Smith,V.S., Ford,T., Johnson,K.P., Johnson,P.C., Yoshizawa,K. and Light,J.E. (2011)
Multiple lineages of lice pass through the K-Pg boundary. Biol Lett., 7, 782-785.
5. Ewing,H.E. (1938) The Sucking Lice of American Monkeys. The Journal of
Parasitology., 24, 13-33.
287
6. Maunder,J.W. (1983) The Appreciation of lice. Proc. R. Inst. Great Britain., 55, 1-31.
288
7. Woerther,P.L., Angebault,C., Lescat,M., Ruppe,E., Skurnik,D., Mniai,A.E.,
289
Clermont,O., Jacquier,H., Costa,A.D., Renard,M. et al. (2010) Emergence and
290
dissemination of extended-spectrum beta-lactamase-producing Escherichia coli in the
291
community: lessons from the study of a remote and controlled population. J. Infect.
292
Dis., 202, 515-523.
293
8. Durden,L.A. (2001) Lice (Phthiraptera). In Samuel,W.M., Pybus,M.J. and Kocan,A.A.
294
(eds.), Parasitic diseases of wild mammals. Ames: Iowa State University Press, pp. 3-
295
17.
296
9. Ascunce,M.S., Toups,M.A., Kassu,G., Fane,J., Scholl,K. and Reed,D.L. (2013)
297
Nuclear genetic diversity in human lice (Pediculus humanus) reveals continental
298
differences and high inbreeding among worldwide populations. PLoS. One., 8,
299
e57619.
12
144
300
10. Reed,D.L., Smith,V.S., Hammond,S.L., Rogers,A.R. and Clayton,D.H. (2004) Genetic
301
analysis of lice supports direct contact between modern and archaic humans. PLoS.
302
Biol, 2, e340.
303
11. Reed,D.L., Light,J.E., Allen,J.M. and Kirchman,J.J. (2007) Pair of lice lost or
304
parasites regained: the evolutionary history of anthropoid primate lice. BMC. Biol., 5,
305
7.
306
12. Xiong, H., Campelo, D., Pollack, R. J., Raoult, D., Shao, R., Alem, M., Ali, J., Bilcha,
307
K., Barker, S. C. (2014). Second�generation sequencing of entire mitochondrial
308
coding�regions (� 15.4 kb) holds promise for study of the phylogeny and taxonomy of
309
human body lice and head lice. Medical and veterinary entomology, 28(S1), 40-50.
310
311
312
313
13. Boutellis,A., Abi-Rached,L. and Raoult,D. (2014) The origin and distribution of
human lice in the world. Infect. Genet. Evol., 23, 209-217.
14. Light,J.E., Toups,M.A. and Reed,D.L. (2008) What's in a name: the taxonomic status
of human head and body lice. Mol. Phylogenet. Evol., 47, 1203-1216.
314
15. Veracx,A., Boutellis,A. and Raoult,D. (2013) Genetic recombination events between
315
sympatric Clade A and Clade C lice in Africa. J. Med. Entomol., 50, 1165-1168.
316
16. Ascunce,M.S., Fane,J., Kassu,G., Toloza,A.C., Picollo,M.I., Gonzalez-Oliver,A. and
317
Reed,D.L. (2013) Mitochondrial diversity in human head louse populations across the
318
Americas. Am. J. Phys. Anthropol., 152, 118-129.
319
17. Leo,N.P., Campbell,N.J., Yang,X., Mumcuoglu,K. and Barker,S.C. (2002) Evidence
320
from mitochondrial DNA that head lice and body lice of humans (Phthiraptera:
321
Pediculidae) are conspecific. J. Med. Entomol., 39, 662-666.
322
18. Light,J.E., Allen,J.M., Long,L.M., Carter,T.E., Barrow,L., Suren,G., Raoult,D. and
323
Reed,D.L. (2008) Geographic distributions and origins of human head lice (Pediculus
324
humanus capitis) based on mitochondrial data. J. Parasitol., 94, 1275-1281.
325
19. Boutellis,A., Drali,R., Rivera,M.A., Mumcuoglu,K.Y. and Raoult,D. (2013) Evidence
326
of sympatry of clade a and clade B head lice in a pre-Columbian Chilean mummy
327
from Camarones. PLoS. One., 8, e76818.
13
145
328
20. Raoult,D., Reed,D.L., Dittmar,K., Kirchman,J.J., Rolain,J.M., Guillen,S. and
329
Light,J.E. (2008) Molecular identification of lice from pre-Columbian mummies. J.
330
Infect. Dis., 197, 535-543.
331
332
333
334
21. Ferris,G.F. (1916) A catalogue and lost list of the Anoplura. The Academy, San
Francisco.
22. Durden,L.A. and Musser,G.G. (1994) The Mammalian Hosts of The Sucking Lice
(Anoploura) of the World: A Host-Parasite List. Bull. Soc. Vector Ecol., 19, 130-168.
335
23. Ferris,G.F. (1951) The sucking lice. Mem. Pacif. Coast Entomol. Soc..
336
24. Finstermeier,K., Zinner,D., Brameier,M., Meyer,M., Kreuz,E., Hofreiter,M. and
337
338
339
340
341
342
343
344
345
346
347
348
Roos,C. (2013) A mitogenomic phylogeny of living primates. PLoS. One., 8, e69504.
25. Price,M.A. and Graham,O.H. (1997) Chewing and sucking lice as parasites of
mammals and birds. Technical Bulletin., 1849, 1-309.
26. Horwich,R.H. (1998) Effective solutions for howler conservation. International
Journal of Primatology, 19, 579-598.
27. Takken,W. and Verhulst,N.O. (2013) Host preferences of blood-feeding mosquitoes.
Annu. Rev. Entomol., 58, 433-453.
28. Mayr,E. (1944) Systematics, and the Origin of Species. Columbia University Press.,
New York, NY.
29. Mayr,E. (1963) Animal Species, and Evolution. Harvard university Press., Cambridge,
MA.
30. Li,W., Ortiz,G., Fournier,P.E., Gimenez,G., Reed,D.L., Pittendrigh,B. and Raoult,D.
349
(2010) Genotyping of human lice suggests multiple emergencies of body lice from
350
local head louse populations. PLoS. Negl. Trop. Dis., 4, e641.
351
31. Larkin,M.A., Blackshields,G., Brown,N.P., Chenna,R., McGettigan,P.A.,
352
McWilliam,H., Valentin,F., Wallace,I.M., Wilm,A., Lopez,R. et al. (2007) Clustal W
353
and Clustal X version 2.0. Bioinformatics., 23, 2947-2948.
14
146
354
32. Tamura,K., Stecher,G., Peterson,D., Filipski,A. and Kumar,S. (2013) MEGA6:
355
Molecular Evolutionary Genetics Analysis version 6.0. Mol. Biol. Evol., 30, 2725-
356
2729.
357
358
359
360
33. Bandelt,H.J., Forster,P. and Rohl,A. (1999) Median-joining networks for inferring
intraspecific phylogenies. Mol. Biol. Evol., 16, 37-48.
34. Yang,Z. (2007) PAML 4: phylogenetic analysis by maximum likelihood. Molecular
biology and evolution., 24, 1586-1591.
361
362
363
364
365
366
367
368
369
370
15
147
148
Figure 1: Geographical localization of lice sampling
New World monkey lice: Argentina
Human head lice: french Guyana
Province of Corientes, Argentina
Province of Misiones, Argentina
National Park Iguaza,
Trois-Sauts village, French Guyana
149
Male;
Amazonia.
female
(1, 2) human head lice from USA; (3, 4) P. mjobergi from Argentina; (5, 6) human head lice from
Figure 2: Morphological comparisons show that P. mjobergi lice highly resemble human lice
150
Lice # (1-4) were collected from monkey [B2188]. Lice # (5 and 6) were collected from monkey [B1395].
Figure 3: PCR results of genetic markers in monkey lice P. mjobergi.
Not amplified
Amplified
151
Haplogroup C
Haplogroup B
Haplogroup C
Haplogroup B
Haplogroup A
Haplogroup A
(D)
(B)
Haplogroup C
Haplogroup B
Haplogroup A
Haplogroup C
Haplogroup B
Haplogroup A
includes sequences of P.mjobergi
#1-3 and #5-6.
Head & body lice
Head louse
Body louse
Bootstrap support values (greater than 80) are located above and below the nodes, respectively. Mt clade memberships are indicated to the right of each tree.
Maximum-likelihood (ML) and neighbor-joining phylograms resulting from analysis of mt genes: (A) cytb, (B) cox1, (C) 16S rRNA and (D) Nad2.
Figure 4. P. mjobergi sequences highly related to sequences obtained from human lice.
(C)
(A)
152
[A]
P. mjobergi
Oceania
America
Europe
Africa
Asia
Figure 5A. Statistical parsimony network for the cytb gene haplotypes found in haplogroup A, haplogroup B and haplogroup C. Each
connecting branch represents a single mutational step. Sizes are scaled and represent relative frequencies.
[C]
[B]
153
[C]
[B]
Africa
Asia
Europe
America
Oceania
P. mjobergi
Figure 5B. Statistical parsimony network for the cox1 gene haplotypes found in haplogroup A, haplogroup B and haplogroup C. Each
connecting branch represents a single mutational step. Sizes are scaled and represent relative frequencies.
[A]
154
P. mjobergi #3
KM579409 G13
Amazonia
Amazonia
France, Portugal Senegal
EU928786 G6
Burundi
EU928790 G10 Burundi
EU928785 G5 Burundi, Ethiopia
EU928793 G13
KM579426 G13 Amazonia
KM579427 G13 Amazonia
KM579425 G13 Amazonia
KM579423 G13 Amazonia
KM579422 G13 Amazonia
KM579421 G13 Amazonia
KM579419 G13 Amazonia
KM579418 G13
KM579417 G13 Amazonia
KM579416 G13 Amazonia
KM579415 G13
KM579414 G13 Amazonia
KM579412 G13 Amazonia
Amazonia
KM579411 G13
Amazonia
JX178753 G36
EU928802 G22 Burundi
0.005
(B)
EU928831 G28 Russia
EU928841 G38 France
EU928843 G40 Portugal
EU928830 G27 Russia
EU928847 G44 Mexico
EU928862 G59 France
KM579435 G39 Orlando strain
EU928805 G2 France
EU928806 G3
Russia
EU928809 G6 Russia
EU928810 G7 France
KM579434 G6 France
EU928851 G48 Russia
EU928856 G53 Russia
EU928857 G54 Russia
EU928833 G30 Russia
EU928834 G31 Russia
EU928835 G32 Russia
KM579428 G33 Nepal
EU928837 G34 France
EU928817 G14 Burundi
KM579443 P. mjobergi #4
KM579444 - KM579454 G47 Amazonia
KM579442 G47 Amazonia
KM579441 G47 Australia
KM579436 - KM579439 G47 Algeria
KM579433 G47 Australia
EU928861 G58 Mexico
EU928853 G50 France
KM579430 G61 Nepal
EU928846 G43 Russia
EU928840 G37 Mexico
EU928839 G36 Russia
EU928855 G52 Mexico
EU928854 G51 France
EU928852 G49 France
KM579429 G60 Nepal
EU928821 G18 Rwanda
EU928829 G26 Rwanda
EU928828 G25 Burundi
EU928811 G8 Rwanda
KM579431 G62 Ethiopia
KM579440 G64 Ethiopia
EU928826 G23 Rwanda
EU928823 G20 Rwanda
EU928848 G45 Russia
EU928825 G22 Burundi
EU928824 G21 Rwanda
EU928822 G19 Burundi
KM579432 G63 Ethiopia
EU928814 G11 Rwanda
EU928813 G10 Burundi
EU928820 G17 Burundi
EU928819 G16 Burundi
EU928860 G57 France
EU928859 G56 Rwanda
Africa
Asia
Europe
America
Oceania
P. mjobergi
Figure 6. Phylogenetic organization of human lice and P. mjobergi based on two nuclear intergenic spacers, PM1, PM2, using the maximum likelihood method.
EU928789 G9 Burundi
EU928782 G2 Burundi
JQ652455 G35 Burundi
Amazonia
EU928791 G11 Burundi
Amazonia
KM579410 G36
EU928792 G12 Burundi
EU928799 G19 Russia
Amazonia
KM579413
KM579420 G36
EU928796 G16 Russia
EU928795 G15 Russia
EU928781 G1 Burundi
EU928787 G7 Burundi
0.002
EU928803 G23 Russia
P. mjobergi #4
EU928798 G18 France Russia Mexico Ethiopia, Rwanda
KM579408 G21
EU928794 G14 Russia
EU928801 G21 Mexico
EU928783 G3 Burundi, Rwanda
(A)
155
G C A T A G T A A T G A - T A G A C A - A T A A T T C
G C A T A G T A A T G A - T A G A C A - A T A A T T C
G
G
G
G
G
T
T
T
T
T
A
A
A
A
A
A
A
A
A
A
T
T
T
T
T
G
G
G
G
G
A
A
A
A
A
-
C
C
C
C
C
A
A
A
A
A
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
A
A
A
A
A
T
T
T
T
T
T
T
T
T
T
T
T
T
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
-
-
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
T
T
T
T
T
C
C
C
C
C
C
C
C
C
C
C
C
C
BL: body louse; HL: head louse
(A) 18S rRNA gene; (B) RPII gene; (C) GPD gene
Supplementary file 1: Alignment of variable nucleotide positions
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
AY236413 Burundi HL
AY236415 Rwanda HL
T
T
T
T
T
G
G
G
G
G
G
G
G
G
G
G
G
G
T
T
T
T
T
G C A C G G T A A T G A - C A G A C A - A T A A G T C
A
A
A
A
A
C
C
C
C
C
C
C
C
C
C
C
C
C
G
G
G
G
G
AF139482 Zimbabwe BL
C
C
C
C
C
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
C
C
C
C
C
FJ267399 Burundi BL
AY236412 Rwanda BL
AF139486 Burundi BL
KM579472 Ethiopia BL
KM579473 Ethiopia BL
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
G C A - - G T T T C G A C G A G T C A - A C A A G T C
G C A - - G T T T C G A C G A G T C A - A C A A G T C
A
A
A
A
A
A
A
A
A
A
A
A
A
G
G
G
G
G
G T A - - A T A T T C G T A C G T T C - G C C A G T C
A
A
A
A
A
A
A
A
A
A
A
A
A
-
KM579479 Nepal HL
KM579480 USA BL
T
T
T
T
T
T
T
T
T
T
T
T
T
C
C
C
C
C
FJ267395 USA HL
G
G
G
G
G
G
G
G
G
G
G
G
G
T
T
T
T
T
G C A - - G T A A C G A C G A G T C A - A C C A G T C
G C A - - G T A A C G A C G A G T C A - A C C A G T C
-
T
T
T
T
T
AY589939 Nepal HL
AF139481 Peru BL
-
G
G
G
G
G
A C A - - G T A A C G A C G A G T C A - A C A A G T C
A
A
A
A
A
A
A
A
A
A
A
A
A
-
AY236411 Algeria BL
C
C
C
C
C
C
C
C
C
C
C
C
C
-
G
G
G
G
G
G
G
G
G
G
G
G
G
-
KM579469 Nepal BL
AF139479 Russia BL
AF139480 USA BL
AY236416 Netherlands BL
KM579470 Nepal BL
AY589938 Nepal BL
AY589941 Iran HL
FJ267398 Canada BL
AY589940 Iran HL
AY589942 Nepal BL
AF139488 Tunisia BL
AF139478 France BL
KM579471 France BL
-
G T A - - G T A A C G A - - - G T T C - G C C A G T C
-
EF570919 Indonesia HL
-
KM579468 Australia HL
KM579474 Amazonia HL
AY077775 Australia HL
AY236414 HL Portugal
AY236417 HL China
-
G
G
G
G
G
KM579475 Algeria HL
KM579476 Algeria HL
KM579477 Algeria HL
A
A
A
A
A
G T A - - A A A - - - - - - - G T T C - G C C A G T C
G T A - - A A A - - - - - - - G T T C - G C C A G T C
G T A - - A A A - - - - - - - G T T C - G C C A G T C
AY236418 Thailand HL
A
A
A
A
A
G T A - - A A A - - - - - - - G T T C A G C C A G T C
FJ267396 USA HL
FJ267397 HL UT USA
A
A
A
A
A
G T A - - A A A - - - - - - - A T T C - G C C A G T C
G T A - - A A A - - - - - - - A T T C - G C C A G T C
FJ267394 USA HL
-
G T A - - A A A - - - - - - - G T T C - G C C A G T C
AY236410 France HL
-
G C T - - A A A - - - - - - - G T T C G A C C A G T C
AF139484 Russia HL
T
T
T
T
T
G C A - - A A A - - - - - - - G T C A - A C A A G T C
KM579467 P. mjoberg i #4
T
T
T
T
T
G T A - - A A A - - - - - - - G T T C - G C C G G C C
G T A - - A A A - - - - - - - G T T C - G C C A G T A
KM579466 P. mjobergi #3
(A)
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
AY316903 Papua New Guinea HL
FJ267456 USA HL
FJ267457 USA HL
FJ267459 Canada BL
AY316879 Panama HL
AY316880 Panama HL
AY316883 Ethiopia HL
AY316885 Ethiopia HL
AY316898 Ethiopia HL
AY316884 Ethiopia HL
AY316892 Ethiopia HL
FJ267458 USA HL
AY316889 Nepal HL
AY316894 Ethiopia HL
AY316897 Ethiopia HL
AY316893 Ethiopia HL
AY316895 Ethiopia HL
AY316896 Ethiopia HL
AY316899 Nepal HL
AY316902 Papua New Guinea HL
AY316904 Papua New Guinea HL
AY316908 Germany HL
AY316910 Germany HL
A
AY316900 Nepal HL
A
A
AY316891 Ethiopia HL
FJ267455 USA HL
A
AY316890 Nepal HL
A
A
AY316888 Germany HL
AY316911 UK HL
A
AY316887 Germany HL
A
A
AY316886 UK HL
AY316909 Germany HL
A
AY316882 Germany HL
A
A
AY316881 Germany HL
AY316907 Germany HL
A
AY316878 Ecuador HL
A
A
AY316877 Laos HL
AY316906 Ecuador HL
A
AY316876 UK HL
A
T
KM579481 USA BL
AY316905 Ecuador HL
T
KM579491 Ethiopia HL
A
T
KM579482 France BL
AY316901 Papua New Guinea
T
T
KM579490 Amazonia HL
KM579492 P. mjobergi #4
(B)
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
G
G
G
G
G
T
T
T
T
T
T
T
C
T
T
T
T
T
T
C
C
C
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
C
C
C
C
C
C
T
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
T
T
T
T
T
C
C
C
T
T
T
C
C
C
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
C
T
T
T
T
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
G
G
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
G
T
T
C
C
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
G
G
G
G
G
A
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
A
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
A
G
G
G
T
T
C
C
T
T
T
T
T
T
T
C
C
C
T
T
T
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
T
C
C
C
C
G
G
G
G
G
C
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
C
C
T
T
T
C
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
C
T
T
T
T
T
T
T
T
T
T
C
T
C
C
C
T
T
T
C
C
C
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
156
A T T T T A T - T T T C A A A A T A C T A G A C T G G G A A A T T G C - G - C G A
A T T T T A T - T T T T A A A A T A C T A G A C T G G T A A A T T G C - G - C G A
A - - - T A T T T T T T A A A A A A C C A T G A A A C T G A A T T A T - A T C G A
A - - - T A T T T T T T A A A A A A C C A T G A A A C T G A A T T A T - A T C G A
G - - - A A T - T T T T A A A A A A C C A T A A A A C T A A A T T A C - A T C G A
G - - - A A T - T T T T A A A A A A C C A T A A A A C T A A A T T A C - A T C G A
G T A T G T G A A A A T A A T A A G A C A T A A A A C T A A A C T A T T A A C G A
G G A T G T G C A A A T G A A A T A C C A T A C A A C T A G T C T A T T A A T T G
KM579582 Nepal BL
KM579583 Nepal BL
KM579575 Algeria HL
KM579576 Algeria HL
KM579580 Nepal HL
KM579578 Ethiopia HL
KM579577 Amazonia HL
KM579579 Ethiopia BL
BL: body louse; HL: head louse
(A) 18S rRNA gene; (B) RPII gene; (C) GPD gene
Supplementary file 1: Alignment of variable nucleotide positions
A T T T T A T T T T T T A A A A A A A C A T G A A A C T G A A T T A T A A - T C G
KM579584 P. mjobergi 4#
(C)
157
B
Supplementary file 2.
A . Chronogram for the lice haplotypes resulting from analysis cytb gene.
B. Nucleotide positions shared between the different haplotypes
Body louse
Head louse
Head & body lice
A
Haplogroups A & C
Haplogroups B & C
Haplogroup B
Haplogroup A
* Non synonymous nucleotide position
Haplogroups A & B
Haplogroup C
Haplotype 39
Haplotype 54
Burundi
Haplotype
France
Yemen
Philippines
Mongolia
Nepal
Laos
Rwanda
Algeria
158
3
28
1
19
20
8
2
7
1
1
4 1
8
1
16
2
20
8
17
7
1
51
26 18 85 7 18 13 11 6 2 10 1 6 83 14 31 1 1 44 9 10 4 32 57 16 18 50 10 7 2 1 8 10 1 11 84
5
Taiwan
1
Hungary
1
Germany
9
4 10 6
United Kingdom
1
Australia
1
New Zeeland
9
Amazonia
5
Papua-New-Guinea
20
Canada
1
Senegal
6
China
6
Japan
10
Chille
4
1 1 3 1 1 9 34
Ecuador
2
Brazil
38 10 17 1 10
Mexico
1
Portugal
1 44 4 10 4
Honduras
1 1 45 5 27
Panama
1
Iran
2
1
Peru
24
Ethiopia
1
USA
18 21 7 17 13
3
Kenya
2
Madagascar
1
Russia
A1
A2
A3
A4
A5
A6
A7
A8
A9
A10
A11
A12
A13
A14
A16
A17
A18
A19
A45
A52
A53
B32
B33
B34
B36
B54
C39
C40
C41
C42
C43
C44
1
1
1
4
346
3
1
3
1
20
10
16
11
1
18
37
2
1
1
20
8
1
21
2
76
19
5
1
17
7
1
51
707
KM579538
KM579539
KM579540
KM579541
KM579542
KM579543
KM579544
KM579545
KM579546
KM579547
KM579548
KM579549
KM579550
KM579551
KM579552
KM579553
KM579554
KM579555
KM579566
KM579567
KM579568
KM579556
KM579557
KM579558
KM579559
KM579569
KM579560
KM579561
KM579562
KM579563
KM579564
KM579565
Acc. No.
Supplementary file 3: cytb gene haplotype frequency of human lice per location worldwide
Total
Mongolia
Ethiopia
Burundi
Algeria
Haplotype
159
3
1
Nepal
4 14
2
1
1
2 30
1
24
3
1
9 235
10
1
2
1
1
25
4
2 17 29
15
1
1
1
1
1
3
4
1
42
17
1
2
2
10
92
2
1
2
5
4
2 177 14
2
Panama
2
2 4
Yemen
3
Philippines
1
1
France
1
Norway
3
Australia
7
United Kingdom
2
Cook Islands
1
Argentina
3
Papua-New-Guinea
2
Peru
2
Colombia
2
Ecuador
18
9 195 2
6
2
2
1
Amazonia
1
Honduras
5
2
USA
2
Mexico
1
18 KM579493
239 KM579500
113 KM579501
2 KM579502
2 KM579503
1 KM579504
2 KM579505
2 KM579506
3 KM579507
7 KM579494
2 KM579495
1 KM579496
1 KM579497
1 KM579498
1 KM579499
75 KM579508
42 KM579509
12 KM579510
3 KM579511
4 KM579512
1 KM579513
2 KM579514
1 KM579515
1 KM579516
1 KM579517
2 KM579518
1 KM579519
1 KM579520
15 KM579521
1 KM579522
2 KM579523
2 KM579524
7 561
7
Canada
A1
A2
A3
A4
A5
A6
A7
A8
A9
A10
A11
A12
A13
A14
A15
B16
B17
B18
B19
B20
B21
B22
B23
B24
B25
B26
B27
B28
B29
B30
C31
C32
Acc. No.
Supplementary file 4: cox1 gene haplotype frequency of human lice per location worldwide
Total
Africa
Asia
Europe
America
Oceania
Chapitre 4 :
Détection et monitoring de la résistance moléculaire
des poux de corps à la perméthrine
161
Préambule
La prévalence des poux de corps chez les personnes fréquentant les
foyers pour sans-abri à Marseille atteint 22% [51]. Il était donc
indispensable de mettre en œuvre des stratégies de lutte contre le pou
de corps vecteur de trois maladies ayant tué des millions de personnes
par le passé [30]. Comme on l’avait constaté, les poux de corps sont
extrêmement contagieux et peuvent se propager par contact direct
entre les personnes ou par le biais de la literie dans les foyers. Les
mesures thérapeutiques classiques telles le changement fréquent de
vêtements ou le lavage des couvertures à 50 °C restent difficiles à
appliquer auprès de ces personnes qui vivent dans des conditions
précaires. L'ivermectine par voie orale réduit le prurit et la prévalence
des infestations par les poux de corps chez ces patients mais l'effet
reste
transitoire [52,53]. Ces résultats montrent que l'éradication
complète de ces ectoparasites chez les personnes sans-abri demeure un
défi permanent.
Un essai clinique ayant pour objectif l’éradication des poux de corps
par le port de sous-vêtements imprégnés de perméthrine dans les
populations défavorisées de Marseille avait été mis en place. Lors de
cette étude, il était important de vérifier la sélection possible de la
163
résistance moléculaire à la perméthrine par évaluation de la prévalence
des mutations conférant cette résistance avant, pendant et après
l’étude. Pour cela, il fallait développer une méthode rapide, fiable et
permettant la réalisation de plusieurs centaines de tests avant le début
de l’essai clinique.
Nous avions opté pour une méthode de génotypage par PCR en temps
réel avec sondes d’hybridation qui s’était révélée sensible et
spécifique [54].
Les résultats de l’essai clinique avaient suggéré d’éviter l’usage de la
perméthrine en raison de l’augmentation de la résistance kdr chez les
populations de poux de corps ciblées [55].
164
Article VIII:
Detection of a Knockdown Resistance Mutation Associated
with Permethrin Resistance in the Body Louse
Pediculus humanus corporis by Use of
Melting Curve Analysis Genotyping
Journal of Clinical Microbiology 50:2229-2233.
165
Detection of a Knockdown Resistance
Mutation Associated with Permethrin
Resistance in the Body Louse Pediculus
humanus corporis by Use of Melting Curve
Analysis Genotyping
Updated information and services can be found at:
http://jcm.asm.org/content/50/7/2229
These include:
SUPPLEMENTAL MATERIAL
REFERENCES
CONTENT ALERTS
http://jcm.asm.org/content/suppl/2012/06/12/50.7.2229.DC1.ht
ml
This article cites 30 articles, 7 of which can be accessed free at:
http://jcm.asm.org/content/50/7/2229#ref-list-1
Receive: RSS Feeds, eTOCs, free email alerts (when new
articles cite this article), more»
Information about commercial reprint orders: http://journals.asm.org/site/misc/reprints.xhtml
To subscribe to to another ASM Journal go to: http://journals.asm.org/site/subscriptions/
167
Downloaded from http://jcm.asm.org/ on July 5, 2012 by INIST-CNRS BiblioVie
Rezak Drali, Samir Benkouiten, Sékéné Badiaga, Idir Bitam,
Jean Marc Rolain and Philippe Brouqui
J. Clin. Microbiol. 2012, 50(7):2229. DOI:
10.1128/JCM.00808-12.
Published Ahead of Print 9 May 2012.
Detection of a Knockdown Resistance Mutation Associated with
Permethrin Resistance in the Body Louse Pediculus humanus corporis
by Use of Melting Curve Analysis Genotyping
Rezak Drali,a,b Samir Benkouiten,a Sékéné Badiaga,a Idir Bitam,c Jean Marc Rolain,a and Philippe Brouquia
Aix-Marseille Université, URMITE CNRS-IRD, UMR 6236/198 IHU Méditerranée Infection, Faculté de Médecine et de Pharmacie, Marseille, Francea; Service des
Entérobactéries et Hygiène de l’Environnement, Institut Pasteur d’Algérie, Algiers, Algeriab; and Service d’Ecologie des Systèmes Vectoriels, IRD 198, Institut Pasteur
d’Algérie, Algiers, Algeriac
T
he human body louse Pediculus humanus corporis (P. h. corporis) is a hematophagous ectoparasite that lives and multiplies
in clothing. Body lice are vectors of 3 major infectious diseases:
epidemic typhus caused by Rickettsia prowazekii, relapsing fever
caused by Borrelia recurrentis, and trench fever caused by Bartonella quintana (24). The poor living conditions and crowded situations in homeless, war refugee, or natural disaster victim populations provide ideal conditions for the spread of lice (23). Body
louse infestation has been observed in 22% of the sheltered homeless population in Marseille, France, causing trench fever, epidemic typhus, and relapsing fever (4), and coinfestation with head
lice is often noted. Consequently, all measures that can be used to
decrease the burden of these ectoparasites in homeless people are
warranted to avoid the spread and/or outbreak of these diseases.
Clothing change as well as the use of ivermectin to eradicate the
body lice in a cohort of homeless individuals in Marseille was
unsuccessful (10), suggesting that the complete eradication of this
ectoparasite is a true challenge (1, 5). It was therefore essential for
us to investigate other strategies to reach this goal.
During the 1980s, a study conducted by the U.S. Army demonstrated that the use of uniforms impregnated with 0.125 mg/
cm2 permethrin effectively eradicated body lice (28).
The resistance to permethrin, known as knockdown resistance
(kdr), was first identified in the housefly Musca domestica (8) and
is due to the presence of mutations in the gene encoding the �
subunit of the sodium channel that is responsible for the depolarization of nerve cells (9). To date, permethrin resistance has never
been studied in P. h. corporis. However, in the head louse Pediculus
humanus capitis (P. h. capitis), resistance to permethrin was first
reported in France in 1994 and throughout the world thereafter
July 2012 Volume 50 Number 7
(11). This resistance is generated by three point mutations, resulting in the amino acid substitutions M815I, T917I, and L920F in
the paraorthologous voltage-sensitive sodium channel (VSSC) �
subunit (17). According to various authors, these mutations are
often found en bloc as a resistant haplotype (7). In Marseille, we
wished to conduct a pilot prospective study to eradicate body
louse infestations in the homeless individuals frequenting the
shelters by providing them with underwear impregnated with a
solution of 0.4% permethrin. For this purpose, it was critical to be
able to evaluate permethrin resistance before and during the study
using a rapid and specific molecular method.
There are currently several different methods available for detecting the mutations responsible for kdr in head lice, including
PCR-restriction fragment length polymorphism (PCR-RFLP)
(15) and quantitative sequencing (QS), real-time PCR amplification of a specific allele (rtPASA), and serial invasive signal amplification reaction (SISAR) (6). Nevertheless, these methods are
time-consuming and not applicable to the investigation of large
quantities of arthropods. We report here a rapid and reliable sin-
Received 26 March 2012 Returned for modification 17 April 2012
Accepted 27 April 2012
Published ahead of print 9 May 2012
Address correspondence to Jean Marc Rolain, jean-marc.rolain@univmed.fr, or
Philippe Brouqui, philippe.brouqui@univmed.fr.
Supplemental material for this article may be found at http://jcm.asm.org/.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
doi:10.1128/JCM.00808-12
Journal of Clinical
168Microbiology
p. 2229 –2233
jcm.asm.org
2229
Downloaded from http://jcm.asm.org/ on July 5, 2012 by INIST-CNRS BiblioVie
Louse-borne diseases are prevalent in the homeless, and body louse eradication has thus far been unsuccessful in this population. We aim to develop a rapid and robust genotyping method usable in large field-based clinical studies to monitor permethrin
resistance in the human body louse Pediculus humanus corporis. We assessed a melting curve analysis genotyping method based
on real-time PCR using hybridization probes to detect the M815I-T917I-L920F knockdown resistance (kdr) mutation in the
paraorthologous voltage-sensitive sodium channel (VSSC) � subunit gene, which is associated with permethrin resistance. The
908-bp DNA fragment of the VSSC gene, encoding the � subunit of the sodium channel and encompassing the three mutation
sites, was PCR sequenced from 65 lice collected from a homeless population. We noted a high prevalence of the 3 indicated mutations in the body lice collected from homeless people (100% for the M815I and L920F mutations and 56.73% for the T917I mutation). These results were confirmed by melting curve analysis genotyping, which had a calculated sensitivity of 100% for the
M815I and T917I mutations and of 98% for the L920F mutation. The specificity was 100% for M815I and L920F and 96% for
T917I. Melting curve analysis genotyping is a fast, sensitive, and specific tool that is fully compatible with the analysis of a large
number of samples in epidemiological surveys, allowing the simultaneous genotyping of 96 samples in just over an hour (75
min). Thus, it is perfectly suited for the epidemiological monitoring of permethrin resistance in human body lice in large-scale
clinical studies.
Drali et al.
gle-step method to allow the monitoring of kdr in human body
lice.
MATERIALS AND METHODS
2230 jcm.asm.org
RESULTS
PCR amplification and sequencing of the 908-bp fragment of
the VSSC gene. A total of 65 lice, including 52 body lice and 1 head
louse from homeless persons, 11 body lice from the laboratory
colony, and 1 head louse from a schoolchild in Algeria, were used
for this assay. The concentration of genomic DNA extracted from
the lice varied from 10 to 50 ng/�l.
The 908-bp DNA fragment of the VSSC gene encoding the �
subunit of the sodium channel was successfully amplified in all 65
lice tested. Direct sequencing and multiple alignments of these
fragments allowed us to deduce the complete reference sequence.
Four introns of various sizes (87, 86, 88, and 85 bp) and three
exons (141, 174, and 163 bp) were found (see Fig. S1 in the supplemental material). The genotyping results of the 3 mutation
sites in the 65 lice tested are summarized in Table 1, with several
different profiles identified. Of the specimens genotyped using the
Sanger sequencing method and designated RG (reference genotype), 31% exhibited a resistant haplotype (RRR), 29% showed an
RHR haplotype, 22% showed an RSR haplotype, and 18% showed
a wild-type haplotype (SSS). An alignment of the 65 obtained
sequences indicated that all of the lice tested, which contained at
least one of the 3 target mutations, also had a single nucleotide
polymorphism in the second intron, at approximately nucleotide
(nt) 2484 � 4, A� T (data not shown).
Melting curve analysis genotyping. The real-time PCR technique was first improved by testing the known genotype clones.
Each of the three mutations was targeted individually. The results
obtained with the melting curve analysis and sequencing were
superimposable for the three mutation sites. As expected, the
wild-type allele showed a peak with a higher Tm than that of
the mutated allele because the detection probe complementary to
the mutated allele was detached earlier in the presence of a mismatch during the melting step. In addition, the Tm of the duplex
probe/mutated codon is lower than that of the duplex probe/wildtype codon. A double peak is characteristic of heterozygotes (see
Fig. S2 in the supplemental material).
Thus, the G�T transversion responsible for the replacement of
a methionine with an isoleucine at position 815 and the C�T
169
Journal of Clinical Microbiology
Downloaded from http://jcm.asm.org/ on July 5, 2012 by INIST-CNRS BiblioVie
Body louse populations. Body lice were collected from volunteers at 2
homeless shelters in Marseille before the beginning of a permethrin clinical trial. The research complied with all relevant federal guidelines and
institutional policies (ID RCB: 2010-A01406-33). Alternatively, homeless
individuals were given new clothes, and their louse-infested clothing was
removed and brought to the laboratory for louse removal. A colony of
in-house human body lice maintained on rabbits and never exposed to
permethrin has been used as a wild-type control. Two head lice collected
in France and Algeria and stored in our laboratory were also used as
possibly resistant specimens.
Genomic DNA extraction. Prior to DNA extraction, the collected lice
were immersed in 70% ethanol for 15 min and then rinsed with distilled
water. Each louse was cut longitudinally into two parts. DNA extraction
was performed using a Qiagen tissue kit (Hilden, United Kingdom), according to the manufacturer’s instructions. The extracted DNA was assessed for quantity and quality using a NanoDrop instrument (Thermo
Scientific, Wilmington, United Kingdom) before being stored at �20°C.
PCR amplification and sequencing of the 908-bp fragment of the
VSSC gene. By analogy with head lice, Lee et al. (17) hypothesized that
acquired resistance to permethrin in body lice was associated with the
same mutations. Consequently, we chose to use the already-reported specific primers 5=HL-QS (5=-ATTTTGCGTTTGGGACTGCTGTT-3=) and
3=HL-QS (5=-CCATCTGGGAAGTTCTTTATCCA-3=) to amplify the
908-bp DNA fragment of the VSSC gene (16). The Phusion high-fidelity
DNA polymerase (Finnzymes, Thermo Scientific, Vantaa, Finland) was
used. The reactions were performed in a final volume of 50 �l with 1 U
Phusion polymerase, 10 �l 5� Phusion buffer, 0.5 mM (each) primer,
0.16 mM deoxynucleoside triphosphates (dNTPs), and 30 to 50 ng of
DNA. The amplification consisted of 35 cycles (98°C for 5 s, 56°C for 30 s,
and 72°C for 15 s), preceded by an initial phase at 98°C for 30 s and
followed by a termination phase at 72°C for 5 min. The PCRs were performed using the Mastercycler Thermocycler (Eppendorf, Hamburg,
Germany). After amplification, all of the PCR products were analyzed by
electrophoresis on 1.5% agarose gels using ethidium bromide staining.
Bidirectional DNA sequencing of the targeted 908-bp PCR products
was performed using the 3130XL genetic analyzer (Applied Biosystems,
Courtaboeuf, France) with the BigDye Terminator v1.1 cycle (Applied
Biosystems). The electropherograms obtained for each sequence were analyzed using ChromasPro software. A multiple alignment was performed
by the ClustalW method using the Basic Local Alignment Search Tool
(BLASTn for nucleotide comparisons) available at http://blast.ncbi.nlm
.nih.gov/.
Cloning of the 908-bp DNA fragments. As a high-quality source of
target DNA to optimize the test, we amplified DNA fragments from two
body lice that had previously been sequenced and shown to be homozygous for the three mutations and cloned them into the pGEM-T Easy
vector systems (Promega, Madison, WI) according to the manufacturer’s
recommendations. The first louse exhibited a wild-type susceptible genotype (SSS), and the second displayed a resistant genotype (RRR). To obtain a heterozygous genotype (HHH), we mixed the two cloned DNA
fragments in equal proportions (1:1). R, S, and H represented homozygous resistant, homozygous wild-type, and heterozygous genotypes, respectively. One clone per louse was chosen to conduct our experiments.
The plasmids containing the cloned fragments were extracted using the
alkaline lysis method (3).
Melting curve analysis genotyping. The melting curve analysis genotyping method is based on real-time PCR with hybridization probes, using
fluorescent resonance energy transfer (FRET) technology. Two sets of
primers were designed to amplify specific regions: (i) a 144-bp genomic
fragment of the first exon to characterize the M815I (ATG�ATT) mutation and (ii) a 203-bp fragment of the third exon to detect the 2 other
mutations, T917I (ACA�ATA) and L920F (CTT�TTT). In addition to
the two primer pairs, two hybridization probes, an anchor probe and a
reporter probe, were designed by Tib Molbiol (DNA Synthesis Service,
Berlin, Germany) to detect each of the three targeted mutations (see Table
S1 in the supplemental material). These probes are alternatively labeled
with fluorescein and LC Red 640 fluorochromes. The probes hybridize
head to tail to the region containing the mutation site to genotype, allowing the transfer of the fluorescence energy of fluorescein to that of Light
Cycler Red 640 (Fig. 1). The emitted fluorescence is measured continuously during the melting phase, during which the temperature is gradually
increased, allowing the determination of the melting temperature (Tm) of
the reporter probe. The reactions were performed using a LightCycler 480
(Roche Diagnostics Corp.) in 96-well plates (LightCycler Multiwell Plate
96). In a final volume of 20 �l, 10 �l 2� QuantiTect Probe PCR Master
Mix (Qiagen), 0.5 mM (each) primer, 0.2 mM anchor probe, 0.2 mM
probe reporter, and between 5 and 20 ng DNA were mixed. Two different
programs were used for the detection of three mutations in this study (see
Table S2 in the supplemental material).
Data analysis. The sensitivity and specificity of the melting curve analysis genotyping method were tested using a contingency table representing the findings of the method test compared to the gold standard of
sequencing. The statistical analyses were performed using SPSS for Windows, version 17.2.
Knockdown Resistance in Body Louse
(ACA�ATA) and L920F (CTT�TTT) in the third exon, 2 amplicons (a to c) spanning the region containing the polymorphic sites are amplified with 2 sets of
primers (primers Seq1 F [forward] and Seq1 A [reverse] [a] and primers Seq2 S [forward] and Seq2 A [reverse] [b and c]).
TABLE 1 Validation of the method of genotyping by comparing the
results of melting curve analysis (predicted genotype) and Sanger
sequencing (reference genotype)a
No. of lice in exon with mutation,
determined by method
Louse type (no.
of lice tested)
First exon,
M815I,
ATG�ATT
Phenotype
PG
RG
Third exon
T917I,
ACA�ATA
L920F,
CTT�TTT
PG
RG
PG
RG
c
Body lice from
homeless (52)
S
H
R
0
0
52
0
0
52
13
18
21b
13
19
20
1
0
51
0
0
52
Head louse from
homeless (1)
S
H
R
0
0
1
0
0
1
0
1
0
0
1
0
0
0
1
0
0
1
Human body
lice bred on
rabbits (11)
S
H
R
11
0
0
11
0
0
11
0
0
11
0
0
11
0
0
11
0
0
Head louse from
Algeria (1)
S
H
R
1
0
0
1
0
0
1
0
0
1
0
0
1
0
0
1
0
0
a
DISCUSSION
Abbreviations: PG, predicted genotype obtained by melting curve analysis genotyping
method; RG, reference genotype obtained by the Sanger sequencing method; R,
resistant homozygote in which both alleles are mutated; S, susceptible homozygote in
which neither allele is mutated; H, heterozygote in which only one of the two alleles is
mutated.
b
False-positive result.
c
False-negative result.
July 2012 Volume 50 Number 7
transitions causing the replacements of a threonine with an isoleucine at position 917 and a leucine with an phenylalanine at
position 920 each shift the Tm by 7 to 8°C.
To ensure that the results reflected the targeted fragments, we
have verified the PCR products by sequencing after genotyping
(data not shown).
We then focused on the 65 lice for which the sequence of interest was previously determined using the Sanger sequencing
method. The genotypes for each louse obtained by the melting
curve analysis genotyping, designated PG (predicted genotype),
are reported in Table 1.
Sensitivity and specificity of the real-time PCR with hybridization probe technique. Of the 195 tests performed, only two
results were discordant with those obtained by the reference
method: a false positive for the T917I mutation and a false negative for the L920F mutation (Table 1). The calculated sensitivity
was 100% for the M815I and T917I mutations and 98% for the
L920F mutation; the specificity was 100% for M815I and L920F
and 96% for T917I.
In contrast to head lice, for which the kdr frequency was sometimes as high as 0.90, the permethrin resistance of the body lice
was still unknown. Consequently, to promote the eradication of
body lice in the homeless population, we have planned a clinical
study using permethrin-impregnated underwear. Monitoring the
resistance of body lice to permethrin in the homeless population
before and during the clinical trial was determined to be mandatory. To achieve this goal, we opted to use a robust and simple
170
jcm.asm.org 2231
Downloaded from http://jcm.asm.org/ on July 5, 2012 by INIST-CNRS BiblioVie
FIG 1 Schematic diagram showing the hybridization of the FRET probes. For the detection of the kdr mutations, M815I (ATG�ATT) in the first exon and T917I
Drali et al.
2232 jcm.asm.org
tive, and specific tool that is fully compatible with the analysis of a
large number of samples in epidemiological surveys, allowing the
simultaneous genotyping of 96 samples in just over an hour (75 min).
Therefore, this method is perfectly suited to the epidemiological
monitoring of permethrin resistance in large-scale clinical studies.
ACKNOWLEDGMENTS
This study was funded in part by PHRC 2010 from the French Ministry of
Health.
The text has been edited by American Journal Experts under certificate
verification key 7DCD-BB1D-3851-ACC7-FB0D.
We gratefully thank Didier Raoult from URMITE Marseille for suggestions and help with this study and Amina Boutellis, Jean Christophe
Lagier, Elisabeth Botelho-Nevers, Mathieu Million, Djamel Thiberville,
Nadim Cassir, and Aurélie Veracx for their assistance.
REFERENCES
1. Badiaga S, et al. 2008. The effect of a single dose of oral ivermectin on
pruritus in the homeless. J. Antimicrob. Chemother. 62:404 – 409.
2. Bass C, et al. 2007. Detection of knockdown resistance (kdr) mutations in
Anopheles gambiae: a comparison of two new high-throughput assays
with existing methods. Malar. J. 6:111.
3. Birnboim HC, Doly J. 1979. A rapid alkaline extraction procedure for
screening recombinant plasmid DNA. Nucleic Acids Res. 7:1513–1523.
4. Brouqui P, et al. 2005. Ectoparasitism and vector-borne diseases in 930
homeless people from Marseilles. Medicine (Baltimore) 84:61– 68.
5. Chosidow O. 2000. Scabies and pediculosis. Lancet 355:819 – 826.
6. Clark JM. 2009. Determination, mechanism and monitoring of knockdown resistance in permethrin-resistant human head lice, Pediculus humanus capitis. J. Asia Pac. Entomol. 12:1–7.
7. Clark JM. 2010. Permethrin resistance due to knockdown gene mutations
is prevalent in human head louse populations. Open Dermatol. J. 4:63– 68.
8. Davies TG, Field LM, Usherwood PN, Williamson MS. 2007. DDT, pyrethrins, pyrethroids and insect sodium channels. IUBMB Life 59:151–162.
9. Dong K. 2007. Insect sodium channels and insecticide resistance. Invert.
Neurosci. 7:17–30.
10. Foucault C, et al. 2006. Oral ivermectin in the treatment of body lice. J.
Infect. Dis. 193:474 – 476.
11. Hemingway J, Ranson H. 2000. Insecticide resistance in insect vectors of
human disease. Annu. Rev. Entomol. 45:371–391.
12. Hodgdon HE, et al. 2010. Determination of knockdown resistance allele
frequencies in global human head louse populations using the serial invasive signal amplification reaction. Pest Manag. Sci. 66:1031–1040.
13. Hurlbut HS, Peffly RL, Salah AA. 1954. DDT resistance in Egyptian body
lice. Am. J. Trop. Med. Hyg. 3:922–929.
14. Kim HJ, Symington SB, Lee SH, Clark JM. 2004. Serial invasive signal
amplification reaction for genotyping permethrin-resistant (kdr-like) human head lice, Pediculus capitis. Pestic. Biochem. Physiol. 80:173–182.
15. Kristensen M. 2005. Identification of sodium channel mutations in human head louse (Anoplura: Pediculidae) from Denmark. J. Med. Entomol. 42:826 – 829.
16. Kwon DH, Yoon KS, Strycharz JP, Clark JM, Lee SH. 2008. Determination of permethrin resistance allele frequency of human head louse
populations by quantitative sequencing. J. Med. Entomol. 45:912–920.
17. Lee SH, et al. 2003. Sodium channel mutations associated with knockdown resistance in the human head louse, Pediculus capitis (De Geer).
Pestic. Biochem. Physiol. 75:79 –91.
18. Lee SH, et al. 2000. Molecular analysis of kdr-like resistance in permethrin-resistant strains of head lice, Pediculus capitis. Pestic. Biochem.
Physiol. 66:130 –143.
19. Lyon E, Wittwer CT. 2009. LightCycler technology in molecular diagnostics. J. Mol. Diagn. 11:93–101.
20. McLintock J, Zeini A, Djanbakhsh B. 1958. Development of insecticide
resistance in body lice in villages of North-Eastern Iran. Bull. World
Health Organ. 18:678 – 680.
21. Motovska Z, et al. 2010. Platelet gene polymorphisms and risk of bleeding
in patients undergoing elective coronary angiography: a genetic substudy
of the PRAGUE-8 trial. Atherosclerosis 212:548 –552.
22. Randegger CC, Hachler H. 2001. Real-time PCR and melting curve analysis for reliable and rapid detection of SHV extended-spectrum betalactamases. Antimicrob. Agents Chemother. 45:1730 –1736.
171
Journal of Clinical Microbiology
Downloaded from http://jcm.asm.org/ on July 5, 2012 by INIST-CNRS BiblioVie
single-step melting curve analysis genotyping method. Since its
establishment in 1997 (19), this method has been particularly useful in detecting known mutations causing human diseases such as
cancer (27) and other disorders (30). The melting curve analysis
genotyping technique is also used in other fields, such as molecular haplotyping (21), bacteriology (22), parasitology (26), and virology (25). Using this method allowed us to determine the genotype of human lice at each mutation site. Furthermore, its ability
to detect heterozygotes makes it particularly useful for following
the dynamics of the kdr mutation in a targeted population of lice.
Despite the two inaccurate results, a false positive and a false negative for the T917I and L920F mutations, respectively, in our total
of 195 tests, the melting curve analysis genotyping method proved
to have great sensitivity (98% to 100%) and excellent specificity
(96% to 100%) in the detection and characterization of allelic
resistance to permethrin in human lice. The false-positive result
could be due to differences in the strength of probe binding (2) or
polymerization errors during the insertion of nucleotides by the
Taq polymerase during real-time PCR. The resequencing of this
sample confirmed its initial genotypic status, which was heterozygous. Conversely, the false-negative result could have been due to
a manipulation error during the distribution of the DNA samples
or the reagent mixtures into the 96-well plates. Several tools described in the literature are available to monitor both phenotypic
and genotypic resistance traits in head lice. In addition to being
faster than conventional phenotypic bioassays, which are tedious
and not adapted to such large-scale purposes (18), these techniques can simultaneously detect the allele frequencies and point
mutations associated with resistance to permethrin without gene
sequencing (14).
The eradication of body lice in the homeless is a major challenge. We implemented several different clinical trials during the
past 10 years to fight louse infestations without success (4). Although resistance to DDT was reported at the end of the 1940s and
in the early 1950s in the United States, southeast Asia, Egypt, and
Iran (13, 20), to the best of our knowledge, no data are available
for permethrin resistance in P. h. corporis. In this work, we noted a
high prevalence of the 3 indicated mutations in the body lice collected from homeless people (100% for the M815I and L920F
mutations and 56.73% for the T917I mutation). As suggested for
head lice, under the selective pressure of permethrin, the mutations M815I and L920F are the first to take place. This event would
be followed by the onset of the T917I mutation (12), which is
considered the main cause of this resistance, while the M815I and
L920F mutations would reduce permethrin sensitivity (29). These
findings suggest that the population of body lice infesting homeless people in Marseille has been subjected to selective pressure.
Overuse of synthetic pyrethroids to eradicate the various insect
pests, notably the increasing infestation with bedbugs in the last 4
decades, has been well documented and probably accounts for the
level of resistance reported here.
The presence of a single nucleotide polymorphism (SNP), at
approximately nt 2484 � 4, A�T, at the beginning of the second
intron of the 908-bp target sequence of the VSSC gene, was an
unexpected finding. This polymorphism was found exclusively in
those lice that displayed at least one of the 3 tested mutations and
could represent an informative SNP for the presence or absence of
these mutations; however, further investigations are required to
address this possibility.
In conclusion, melting curve analysis genotyping is a fast, sensi-
Knockdown Resistance in Body Louse
23. Raoult D, Foucault C, Brouqui P. 2001. Infections in the homeless.
Lancet Infect. Dis. 1:77– 84.
24. Raoult D, Roux V. 1999. The body louse as a vector of reemerging human
diseases. Clin. Infect. Dis. 29:888 –911.
25. Ratcliff RM, Chang G, Kok T, Sloots TP. 2007. Molecular diagnosis of
medical viruses. Curr. Issues Mol. Biol. 9:87–102.
26. Safeukui I, et al. 2008. Evaluation of FRET real-time PCR assay for rapid
detection and differentiation of Plasmodium species in returning travellers and migrants. Malar. J. 7:70.
27. Schnittger S, et al. 2006. KIT-D816 mutations in AML1-ETO-positive
AML are associated with impaired event-free and overall survival. Blood
107:1791–1799.
28. Sholdt LL, Rogers EJ, Jr, Gerberg EJ, Schreck CE. 1989. Effectiveness of
permethrin-treated military uniform fabric against human body lice. Mil.
Med. 154:90 –93.
29. SupYoon KS, Symington SB, Lee SH, Soderlund DM, Clark JM. 2008.
Three mutations identified in the voltage-sensitive sodium channel alpha
subunit gene of permethrin-resistant human head lice reduce the permethrin sensitivity of houde fly Vssc1 sodium channels expressed in Xenopus
oocytes. Insect. Biochem. Mol. Biol. 38:296 –308.
30. Wee L, Vefring H, Jonsson G, Jugessur A, Lie RT. 2010. Rapid genotyping of the human renin (REN) gene by the LightCycler instrument:
identification of unexpected nucleotide substitutions within the selected
hybridization probe area. Dis. Markers 29:243–249.
Downloaded from http://jcm.asm.org/ on July 5, 2012 by INIST-CNRS BiblioVie
July 2012 Volume 50 Number 7
172
jcm.asm.org 2233
87 bp
Intron 1
86 bp
Intron 2
815
H
D
M
D
K
CAC GAC ATG GAT AAA
+
+
I
+
+
*** *** **T *** ***
141 bp
1st exon
174 bp
2nd exon
88 bp
163 bp
3rd exon
85 bp
Intron 4
3’ HL-QS
917
920
L
T
F
V
L
C
TTA ACA TTC GTC CTT TGC Wild type
+
I
+
+
F
+
*** *T* *** *** T** *** Mutated
Intron 3
173
type sequence in the region of interest obtained from a body louse that was raised on a rabbit in the laboratory.
corporis, containing the three mutations, M815I (ATG / ATT), T917I (ACA / ATA) and L920F (CTT / TTT). (b) Electropherograms of the wild-
Supplementary figure 1. (a) A diagram of the 908 bp fragment of the VSSC gene encoding the α-subunit of a sodium channel in P. humanus
(b)
(a)
5’ HL-QS
Genotyping of the mutation site
the same position.
one mutated (CT). Bottom, the mutated allele showed a T at
C., Middle, heterozygote with two alleles, one wild-type and
mutation site in 3 samples. Top, the wild-type allele bearing a
the melting curves. (c) Electropherograms of the (T917I)
negative control. (b) Derivative melting peaks obtained from
in red, the compound heterozygote (CT); and in azure the
green, the homozygous mutant (TT); in blue, wild-type (CC);
(T917I) using melting curve analysis. (a) Melting curves: in
Supplementary figure 2.
(b)
(a)
174
Negative control
TT
Homozygote
CT
Heterozygote
CC
Wild Type
(c)
Supplementary Table 1. DNA sequences of the primers and probes used to detect kdr
mutations in body lice.
Primer or
probe
Sequence
Seq1 F (forward)
ACCCATTCGTCGAATTATTCATAACT
Seq1 A (reverse)
CCCCGCATTAAAATTAAATTTTTAC
Seq1 mut
TCTGTCCATGTCTTTATCCATGTC-FLb
Anchor Seq1
LC-640-TGATGATCCAAAGCCATAAATAGTGTGTT-P
Seq2 S (forward)
TTTTTTTCTTTTTATGACGAAAC
Seq2 A (reverse)
CCCGTGTAATTTTTTCCA
Anchor 1
AATTTCAATTATGGGTCGAACTGTTGG-FL
Sensor 1 C
LC-640-GCTTTGGGTAATTTAACATTCGTC-P
Sensor 2 C
ATTCGTCCTTTGCATTATCATATTCAT-FL
Anchor 2
LC-640-TTTGCCGTTATGGGAATGCAACTT-P
Exon
and a.a.
positiona
exon 1
M815I
exon 3
T917I
and
L920F
LC-Red 640 and fluorescein are fluorophores. The 3’ end of the one of the two probes in each
pair was phosphorylated to prevent probe elongation by Taq polymerase during the PCR.
Bold, codons; underlined, mutated bases.
a
The amino acids are numbered is according to GenBank accession no. AY191155
b
FL, fluorescein; LC-Red 640, LightCycler-Red 640; P, phosphorylated.
175
Supplementary Table 2. Programs for real-time PCR to detect kdr mutations using the Light
Cycler apparatus 480.
Mutations
M815I
Primers
Seq1F/Seq1A
Size
Denaturation
144 bp
95 °C; 15 min
T917I
and or
LightCycler© 480 Program
Seq2S/Seq2A
203 bp
L920F
176
Amplification
Melting
95 °C; 01 sec
95 °C; 1 sec
60 °C; 10 sec
40 °C; 30 sec
72 °C; 06 sec
80 °C;
95 °C; 01 sec
continuous
51 °C; 15 sec
transition rate:
72 °C; 10 sec
0.1 °C/sec
Article IX:
Effect of Permethrin-Impregnated Underwear on Body Lice
in Sheltered Homeless Persons: a Randomized Controlled Trial
JAMA Dermatology 150:273-279.
177
Research
Original Investigation
Effect of Permethrin–Impregnated Underwear
on Body Lice in Sheltered Homeless Persons
A Randomized Controlled Trial
Samir Benkouiten, MPH; Rezak Drali, MSc; Sékéné Badiaga, MD, PhD; Aurélie Veracx, PhD;
Roch Giorgi, MD, PhD; Didier Raoult, MD, PhD; Philippe Brouqui, MD, PhD
IMPORTANCE The control of body lice in homeless persons remains a challenge.
Supplemental content at
jamadermatology.com
OBJECTIVE To determine whether the use of long-lasting insecticide–treated underwear
provides effective long-term protection against body lice in homeless persons.
DESIGN, SETTING, AND PARTICIPANTS A randomized, double-blind, placebo-controlled trial
was conducted in February and December 2011 in 2 homeless shelters (Madrague Ville and
Forbin) in Marseille, France. Of the 125 homeless persons screened for eligibility, 73 body
lice–infested homeless persons, 18 years or older, were enrolled.
INTERVENTIONS Body lice–infested homeless persons were randomly assigned to receive
0.4% permethrin–impregnated underwear or an identical-appearing placebo for 45 days, in a
1:1 ratio, with a permuted block size of 10. Visits were scheduled at days 14 and 45. Data
regarding the presence or absence of live body lice were collected.
MAIN OUTCOMES AND MEASURES The primary and secondary end points were the
proportions of homeless persons free of body lice on days 14 and 45, respectively. Mutations
associated with permethrin resistance in the body lice were also identified.
RESULTS Significantly more homeless persons receiving permethrin-impregnated underwear
than homeless persons receiving the placebo were free of body lice on day 14 in the
intent-to-treat population (28% vs 9%; P = .04), with a between-group difference of 18.4
percentage points (95% CI, 1.4-35.4), and in the per-protocol population (34% vs 11%;
P = .03), with a between-group difference of 23.7 percentage points (95% CI, 3.6-43.7). This
difference was not sustained on day 45. At baseline, the prevalence of the permethrinresistant haplotype was 51% in the permethrin group and 44% in the placebo group. On day
45, the permethrin-resistant haplotype was significantly more frequent in the permethrin
group than in the placebo group (73% vs 45%, P < .001).
CONCLUSION AND RELEVANCE Permethrin–impregnated underwear is more efficient than
placebo at eliminating body louse infestations by day 14; however, this difference was not
sustained on day 45. The use of permethrin may have increased the resistance to permethrin
in body lice and thus must be avoided.
TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT01287663
JAMA Dermatol. doi:10.1001/jamadermatol.2013.6398
Published online December 4, 2013.
Author Affiliations: Unité de
Recherche sur les Maladies
Infectieuses et Tropicales
Émergentes, Aix-Marseille Université,
Marseille, France (Benkouiten, Drali,
Badiaga, Veracx, Raoult, Brouqui);
Institut Hospitalo-Universitaire
Méditerranée Infection, Marseille,
France (Benkouiten, Drali, Veracx,
Giorgi, Raoult, Brouqui); Service des
Entérobactéries et Hygiène de
l'Environnement, Institut Pasteur
d'Algérie, Alger, Algérie (Drali);
Institut National de la Santé et de la
Recherche Médicale, Aix-Marseille
Université, Marseille, France (Giorgi).
Corresponding Author: Philippe
Brouqui, MD, PhD, Aix Marseille
Université, URMITE, 27 bd Jean
Moulin, 13005 Marseille, France
(philippe.brouqui@univ-amu.fr).
E1
179
Copyright 2013 American Medical
Association. All rights reserved.
Downloaded From: http://archderm.jamanetwork.com/ by American Medical Association, June Robinson on 12/30/2013
Research Original Investigation
Permethrin–Impregnated Underwear
H
omelessness is a major social and public health problem worldwide. The prevalence of body lice in sheltered homeless persons varies from 7% to 22%.1 Body
lice are known vectors of Bartonella quintana, Rickettsia prowazekii, and Borrelia recurrentis, which cause trench fever, epidemic typhus, and relapsing fever, respectively. 2 Consequently, all measures that can be used to decrease the burden
of body lice infestation in homeless persons, and more generally in persons living in crowded and unhygienic environments, are warranted to avoid the spread and/or outbreak of
these diseases.
Pediculus humanus humanus, the human body louse, is a
host-specific hematophagous ectoparasite that lives in the
clothes. Body lice are extremely contagious and can be spread
through body contact, shared clothing, or shared bedding in
overcrowded conditions.3 The classic therapeutic measures for
body lice infestations are the frequent changing or washing of
the infected person’s clothes and blankets at 50°C and the frequent treatment of bedding with insecticides.4 However, in our
experience with the sheltered homeless persons in Marseille,
France, these measures have had little success.5 Oral ivermectin reduces the prevalence of body lice infestations and pruritus in homeless persons, but the effect is transient.6,7 These
findings suggest that the complete eradication of this ectoparasite in homeless persons remains a challenge.8
The pyrethroids are the major commercially available pediculicides. All World Health Organization–recommended insecticide-treated mosquito nets are pyrethroid based.9 The impregnation of clothing with a pyrethroid emulsion has been
reported to eradicate body lice after a single application to military uniforms, even after 20 washes,10 and may provide longlasting protection. The clinical safety and effectiveness of topical permethrin in humans have been reported previously.11,12
We conducted our randomized, double-blind, placebocontrolled study to determine whether the use of longlasting permethrin–treated underwear provides long-term protection against louse proliferation in sheltered homeless
persons. Our secondary aim was to assess the mutations associated with permethrin resistance in the body lice.
Methods
Study Design
Our study was a double-center, double-blind, randomized, placebo-controlled intervention trial. Homeless persons were
given underwear treated with permethrin or an identicalappearing placebo for 45 days. The protocol was approved by
our institutional review board (January 24, 2011; reference
2010-A01406-33), and the study was performed in accordance with the good clinical practices recommended by the
Declaration of Helsinki and its amendments. All participants
provided written informed consent. This study is registered
with clinicaltrials.gov (identifier NCT01287663).
Underwear Preparation
An 8% (8-g/L) permethrin formulation for impregnation, which
is commercially available under the label Barrage Insect (S.P.C.I.
E2
S.A., Paris, France), was prepared as a 1:20 emulsion in
water as recommended by the manufacturer. The impregnation was performed by an independent person in the Public
Hospitals of Marseille laundry. Sets of underwear (T-shirt,
underpants, and socks) were placed into the emulsion for 15
minutes, completely saturated, removed, and allowed to
dry. Once dry, the underwear was odorless. According to the
manufacturer’s instructions, the permethrin–impregnated
underwear is effective up to 6 months and even after 6
washes. Other sets of underwear were treated identically
but without the permethrin formulation. The permethrinimpregnated and placebo underwear were identical in
appearance but were labeled discreetly and then stored in 2
separate boxes until use.
Participants
To recruit a sufficient number of participants, 2 independent
study cohorts of homeless persons were performed. In study
A, homeless persons were recruited in February 2011 from 2
shelters (Madrague Ville and Forbin) in the city of Marseille.
In study B, different homeless persons were recruited in December 2011 from the same 2 shelters. Each facility provides
nighttime shelter for a mean of 300 homeless persons who stay
in the shelter overnight and leave it in the morning. Homeless persons with a self-reported diagnosis of pruritus and/or
with body lice were screened. Homeless persons were eligible for inclusion in the study if they were 18 years or older,
were able to provide consent, declared that they slept at least
3 nights per week in 1 of the 2 shelters, and had at least 1 live
body louse recovered on examination. The exclusion criteria
were the presence of cutaneous superinfection or intravenous drug use.
Randomization and Interventions
Homeless persons were randomly assigned to the intervention group with sealed, opaque envelopes in a 1:1 ratio with a
permuted block size of 10. Participants and investigators were
unaware of the treatment assignments throughout the study.
Visits were scheduled on days 14 and 45. Data on the presence or absence of live body lice, whether the clothes had been
changed between visits, and the occurrence of adverse events
were collected.
All participants received their protocol underwear on day
1 (baseline) and at each follow-up visit (on days 14 and 45) under the supervision of the investigators. The underwear could
also be changed between the follow-up visits in the shelters
at the request of the individual (with respect to the assigned
group). The used underwear was collected by the same entomologist for detailed visual inspection. This evaluator was
trained in the technique for detecting and counting live body
lice from the infested underwear. Dead and living lice were differentiated; lice were considered to be dead if they were not
moving. Homeless persons were excluded from further study
if they had any manifestations suggesting adverse effects, and
specific treatment was given as needed. The final visit on day
45 was regarded as the end of the study for every participant.
If persistent live body lice were found at this visit, homeless
persons were offered a single dose of oral ivermectin (12 mg).6
JAMA Dermatology Published online December 4, 2013
180
Copyright 2013 American Medical
Association. All rights reserved.
Downloaded From: http://archderm.jamanetwork.com/ by American Medical Association, June Robinson on 12/30/2013
jamadermatology.com
Original Investigation Research
Permethrin–Impregnated Underwear
Figure. Study Flow of Participants
125 Homeless persons assessed for eligibility
52 Excluded
52 Not infested with body lice
73 Randomized
40 Randomized to permethrin
33 Randomized to placebo
8 Lost to follow-up at day 14
5 Lost to follow-up at day 14
Included in analysis of primary end point
40 Included in intent-to-treat population
27 Included in per-protocol population
Included in analysis of primary end point
33 Included in intent-to-treat population
28 Included in per-protocol population
13 Lost to follow-up at day 45
9 Lost to follow-up at day 45
Included in analysis of secondary end point
40 Included in intent-to-treat population
27 Included in per-protocol population
Outcome Measures and Safety End Points
The primary efficacy end point was the proportion of homeless persons free of body lice (defined as absence of living body
lice in the underwear) 14 days after treatment. The secondary
efficacy end point was the same assessment 45 days after treatment. End points were assessed on the basis of the exhaustive examination of live body lice in all collected underwear.
The pruritus that normally accompanies body lice infestation may be exacerbated temporarily after dermal exposure to permethrin.13 Physical examinations were performed
at scheduled visits, and adverse events were recorded during
the 45-day study period. The prevalence and severity of pruritus, graded from 0 to 3 (0, none; 1, mild; 2, moderate; and 3,
severe), were assessed at each visit.
Another objective was to investigate the evolution of permethrin resistance in the body lice. The permethrin resistance of body lice was determined in a representative random sample of body lice collected from all body lice–infested
homeless persons and stratified by the level of infestation of
the homeless persons (see eTable 1 in the Supplement). A melting curve analysis genotyping method,14 based on a previously reported real-time polymerase chain reaction using hybridization probes, was used to detect the 3 mutations (M815I,
T917I, and L920F), identified in the voltage-sensitive sodium
channel α-subunit gene, responsible for knockdown resistance (kdr). According to the literature, these 3 mutations define the RRR haplotype, which confers permethrin resistance
in head lice.15,16
Statistical Analysis
We estimated that approximately 60 body lice–infested homeless persons (30 in each group) would need to be enrolled to
provide 90% power to detect a difference of 40 percentage
jamadermatology.com
Screening and inclusion process for
participants of the randomized
controlled trial and flow of
participants through each stage of
the study.
Included in analysis of secondary end point
33 Included in intent-to-treat population
24 Included in per-protocol population
points between the permethrin and placebo groups when calculating the proportion of homeless persons free of body lice
on day 14 with a 2-sided α = .05, assuming an anticipated effect between 20 and 40 percentage points in the placebo group.
In our experience, approximately 70% of pruritus symptoms
are due to body lice infestations, and presuming that the rate
of individuals lost to follow-up could be up to 30%, we predicted that we would need to screen 122 homeless persons.
Analyses were conducted in accordance with the intent-totreat and per-protocol principles. In the intent-to-treat analysis, only the homeless persons who were present at the scheduled follow-up visits were included. For the intent-to-treat
analysis, loss to follow-up was considered a treatment failure. The Pearson χ2 test and Fisher exact test, as appropriate,
were applied to analyze the primary and secondary end points
of efficacy, and 95% CIs for the difference between the success rates in the study groups were calculated. The t test for
independent groups and Mann-Whitney test, as appropriate,
were used to investigate the safety end point of mean pruritus score and the continuous variables. P ≤ .05 (2-tailed test)
was established as the level of significance for all tests. Statistical analyses were performed using SPSS statistical software, version 17.2 (SPSS Inc).
Results
Participants
The trial profile is summarized in the Figure. Of the 125
homeless persons screened for eligibility in February and
December 2011, 73 (58%) were eligible on the basis of the
presence of live body lice (40 in the permethrin group and
33 in the placebo group) and were consequently randomized
JAMA Dermatology Published online December 4, 2013
181
Copyright 2013 American Medical
Association. All rights reserved.
Downloaded From: http://archderm.jamanetwork.com/ by American Medical Association, June Robinson on 12/30/2013
E3
Research Original Investigation
Permethrin–Impregnated Underwear
Table 1. Demographic and Baseline Characteristics of the Study Groups
No. (%) of Study Participantsa
Characteristic
Permethrin
(n = 40)
Age, mean (SD), y
56.4 (14)
Men
Placebo
(n = 33)
57.62 (12)
37 (92)
33 (100)
P Value
.69
.24
Madrague Ville Shelter
36 (90)
31 (94)
.68
Marginal homelessb
20 (50)
20 (61)
.36
Homeless with >50 lice
18 (45)
19 (58)
.28
Duration of homelessness ≤24 mo
15 (38)
19 (58)
.08
Pruritus
40 (100)
33 (100)
a
Data are presented as number
(percentage) of study participants
unless otherwise indicated.
b
Classified by shelter staff.
Table 2. Effect of Treatment on Days 14 and 45 in Study A and Study B Combined
No./Total (%)
Outcome Measure
Permethrin
Placebo
Body lice–free homeless after 14 d
11/40 (28)
3/33 (9)
Body lice–free homeless after 45 d
11/40 (28)
9/33 (27)
Body lice–free homeless after 14 d
11/32 (34)
3/28 (11)
Body lice–free homeless after 45 d
11/27 (41)
9/24 (38)
Difference, % (95% CI)
P Value
Intent-to-treat population
18.4 (1.4 to 35.4)
0.2 (−20.3 to 20.8)
.04
.98
Per protocol
into the control and treatment groups (Figure). They were
predominantly male (96%), were mostly from the Madrague
Ville shelter (92%), and had a mean (SD) age of 56.9 (13.3)
years (age range, 20-79 years). Approximately 45% reported
being homeless for less than or equal to 24 months. Baseline
characteristics were similar between the treatment groups
(Table 1).
Primary and Secondary Outcomes
In the intent-to-treat population, 11 of 40 homeless persons
(28%) were free of live body lice on day 14 (primary end point)
in the permethrin group compared with 3 of 33 (9%) in the placebo group (P = .04), with a between-group difference of 18.4
percentage points (95% CI, 1.4-35.4) (Table 2). This proportion was also significantly greater in the permethrin group than
in the placebo group in the per-protocol population (34% vs
11%; P = .03), with a between-group difference of 23.7 percentage points (95% CI, 3.6-43.7).
With respect to the secondary efficacy end point, in the
intent-to-treat population, 11 of 40 homeless persons (28%)
were free of live body lice on day 45 in the permethrin group
compared with 9 of 33 (27%) in the placebo group (28% vs 27%;
P = .98), with a between-group difference of 0.2 percentage
points (95% CI, –20.3 to 20.8). In addition, no significant difference was found between the 2 proportions in the perprotocol population (41% vs 38%; P = .81), with a betweengroup difference of 3.2 percentage points (95% CI, –23.6 to 30.0)
(Table 2).
Significant reductions from the baseline in the mean number of body lice were observed on day 14 and day 45 in the permethrin and placebo groups (see eTable 2 in the Supplement). However, no significant difference was found between
the 2 groups on day 14 (mean [SD], 176.1 in the permethrin
group vs 104 [202.1] in the placebo group; P = .18) or on day
E4
23.7 (3.6 to 43.7)
3.2 (−23.6 to 30.0)
.03
.81
45 (mean [SD], 148.4 [427.6] in the permethrin group vs 147.6
[272.3] in the placebo group; P = .68) (data not shown).
Adverse Events
No adverse events were reported in any treated homeless persons. The prevalence of pruritus was reduced in both groups,
with no significant differences in the proportion of homeless
persons free of pruritus between the permethrin group and the
placebo group on day 14 (8 of 32 [25%] vs 6 of 27 [22%]; P = .80),
with an odds ratio of 1.16 (95% CI, 0.34–3.91), or on day 45 (8
of 27 [30%] vs 8 of 24 [33%]; P = .77), with an odds ratio of 0.84
(95% CI, 0.25-2.75) in the per-protocol population. The mean
(SD) pruritus score at baseline was 2.53 (0.69) in the permethrin group and 2.24 (0.90) in the placebo group. No significant differences were found in the mean reduction in pruritus score from baseline between the permethrin group and the
placebo group on day 14 (–0.68 vs –0.28; 95% CI, –0.97 to 0.17;
P = .17) and on day 45 (–0.92 vs –0.45; 95% CI, –1.15 to 0.21;
P = .17).
Permethrin Resistance of Body Lice
Of the 34 035 live body lice that were collected, 371 were used
to assess permethrin resistance because at least 1 louse per infested homeless persons was selected (see eTable 1 in the
Supplement): 187 were collected in study A (91 on day 1 [44
from 18 homeless persons from the permethrin group and 47
from 17 homeless persons from the placebo group] and 96 on
day 45 [43 from 8 homeless persons from the permethrin group
and 53 from 7 homeless persons from the placebo group]) and
184 in study B (67 on day 1 [32 from 16 homeless persons from
the permethrin group and 35 from 14 homeless persons from
the placebo group] and 117 on day 45 [56 from 8 homeless persons from the permethrin group and 61 from 8 homeless persons from the placebo group]).
JAMA Dermatology Published online December 4, 2013
182
Copyright 2013 American Medical
Association. All rights reserved.
Downloaded From: http://archderm.jamanetwork.com/ by American Medical Association, June Robinson on 12/30/2013
jamadermatology.com
Original Investigation Research
Permethrin–Impregnated Underwear
Table 3. Evolution of the Permethrin-Resistant Haplotype in Body Lice During the Survey Period
Permethrin
Haplotype
Day 1
Day 45
Placebo
P Value
Day 1
Day 45
P Value
Study A
RRR, No./total (%)a
RRR, % (95% CI)a,b
17/44 (39)
41.9 (40.4-43.4)
31/43 (72)
67.4 (66.4-68.4)
.002
18/47 (38)
17/53 (32)
21.1 (20.8-21.4)
26.2 (25.3-27.0)
.51
Study B
RRR, No./total (%)a
RRR, % (95% CI)a,b
22/32 (69)
77.8 (76.1-79.6)
41/56 (73)
75.0 (74.1-76.0)
18/35 (51)
.65
34/61 (56)
56.9 (55.1-58.6)
59.9 (58.8-61.0)
.68
Studies A and B
RRR, No./total (%)a
RRR, % (95% CI)a,b
39/76 (51)
62.8 (61.9-63.7)
72/99 (73)
71.8 (71.3-72.3)
.004
36/82 (44)
51/114 (45)
34.3 (33.8-34.9)
a
Considered permethrin resistant.
b
Inference based on the subrandom sample of body lice stratified based on the initial infestation level of the individual.
44.8 (44.3-45.4)
.90
Table 4. Evolution of the Allele Frequency of the T917I and L920F Mutations in Body Lice During the Survey Period
No./Total (%)
Permethrin
Mutation
Day 1
Day 45
Placebo
P Value
Day 1
Day 45
P Value
Study A
T917I
18/44 (41)
31/44 (72)
.003
21/47 (45)
17/53 (32)
.19
L920F
43/44 (98)
43/43 (100)
.99
37/47 (79)
53/53 (100)
<.001
T917I
22/32 (69)
45/56 (80)
.21
18/35 (51)
34/61 (56)
.68
L920F
32/32 (100)
56/56 (100)
>.99
34/35 (97)
61/61 (100)
.77
T917I
40/76 (53)
76/99 (77)
.008
39/82 (48)
51/114 (45)
.69
L920F
75/76 (99)
99/99 (100)
.89
71/82 (87)
114/114 (100)
<.001
Study B
Studies A and B
At baseline, the prevalence of the permethrin-resistant
haplotype (Table 3) among the lice collected was 51% in the permethrin group and 44% in the placebo group. On day 45, the
permethrin-resistant haplotype was significantly more frequent in the permethrin group than in the placebo group (73%
vs 45%; P < .001).
At baseline, the prevalence of permethrin-resistant mutations (Table 4) was established as 99% for the L920F mutation and 53% for the T917I mutation in the permethrin group
and 87% for the L920F mutation and 48% for the T917I mutation in the placebo group. The prevalence of the T917I mutation increased significantly from baseline to day 45 in the permethrin group (from 53% to 77%; P = .008) but remained stable
in the placebo group (from 48% to 45%; P = .69). The prevalence of the L920F mutation increased significantly in the placebo group from baseline to day 45 (from 87% to 100%;
P < .001). The prevalence of the M815I mutation was established as 100% for all samples.
Discussion
In this randomized controlled trial, long-lasting permethrin–
treated underwear is more efficient in the elimination of body
louse infestations than placebo in the short term (ie, on day
14), but the difference with the placebo was not sustained by
jamadermatology.com
day 45 and is accompanied by increasing permethrin resistance in body lice collected from homeless persons.
Resistance to permethrin has been reported in the head louse
Pediculus humanus capitis in many parts of the world.17-20
This is the first trial, to our knowledge, that tests permethrin–
impregnated underwear on body lice in sheltered homeless
persons, combined with molecular detection of mutations associated with permethrin resistance in body lice. The kdr allele frequency in the body lice population at baseline was unexpectedly
high in study A (averaging 38%). The relative ease with which
this group of mutations was identified within the modern
body louse population may be related to prior exposure to
dichlorodiphenyltrichloroethane, which likely involved
kdr-like mechanisms in some cases, in a manner similar to the
rapid increase of permethrin resistance in the head louse
populations shortly after the introduction of synthetic pyrethroids in Europe and worldwide.17-20 Resistance of the body
louse to dichlorodiphenyltrichloroethane was reported at the
end of the 1940s and early 1950s.21-23 This finding could suggest that, although no resistance to permethrin was reported
in the body lice in Western Europe before this study, some lice
are currently resistant to pyrethroids through dormant crossresistance attributed to the kdr mechanism.
In our study, the increase of the prevalence of the T917I
mutation interestingly coincided with the loss of permethrin efficacy. This result is coherent with that obtained in a
JAMA Dermatology Published online December 4, 2013
183
Copyright 2013 American Medical
Association. All rights reserved.
Downloaded From: http://archderm.jamanetwork.com/ by American Medical Association, June Robinson on 12/30/2013
E5
Research Original Investigation
Permethrin–Impregnated Underwear
previous study16 that concluded that the T917I mutation
alone was responsible for most of the target site insensitivity reported in the resistant RRR haplotype. In addition, a
recent study 24 reported a prevalence of 5% of the T917I
mutation, against 70% for both the M815I and L920F mutations, in an Egyptian head louse population for which selection with pyrethroid-based pediculicides was expected to
be low. These findings suggested that head lice may have
acquired the M815I and L920F mutations first and then,
once these 2 mutations are present, rapidly acquired the
T917I mutation, high levels of nerve insensitivity, and resistance after they were again placed under pyrethroid selective pressure, leading to control failure.
Our trial had several limitations that merit consideration. First, we believe that head and body lice are transferred among people when they come in close personal contact in the shelter, and thus the resistance to permethrin is
also shared. Indeed, of the 11 homeless persons who were
free of live body lice on day 14 in the permethrin group, 2
were reinfected on day 45. Moreover, the frequency of the
L920F mutation increased significantly from baseline to day
45 in the placebo group in study A, suggesting that L920Fmutated lice had been transferred between the permethrin
and placebo groups.
ARTICLE INFORMATION
Accepted for Publication: June 27, 2013.
Published Online: December 4, 2013.
doi:10.1001/jamadermatol.2013.6398.
Author Contributions: Dr Brouqui had full access
to all of the data in the study and takes
responsibility for the integrity of the data and the
accuracy of the data analysis.
Study concept and design: Benkouiten, Badiaga,
Raoult, Brouqui.
Acquisition of data: Benkouiten, Drali, Badiaga,
Veracx.
Analysis and interpretation of data: Benkouiten,
Drali, Giorgi.
Drafting of the manuscript: Benkouiten, Drali,
Badiaga, Giorgi, Raoult.
Critical revision of the manuscript for important
intellectual content: Veracx, Raoult, Brouqui.
Statistical analysis: Benkouiten, Giorgi.
Obtained funding: Brouqui.
Administrative, technical, or material support:
Benkouiten, Badiaga, Raoult.
Study supervision: Benkouiten, Drali, Badiaga,
Brouqui.
Conflict of Interest Disclosures: None reported.
Funding/Support: This study was supported by
national grant PHRC 2010 from the French Health
Ministry to Dr Brouqui.
Role of the Sponsor: The French Health Ministry
had no role in the design and conduct of the study;
collection, management, analysis, and
interpretation of the data; and preparation, review,
or approval of the manuscript; decision to submit
the manuscript for publication.
Additional Contributions: We thank the homeless
individuals who were involved in this study; the
medical and pharmacy students, interns and
fellows, and researchers of the Unité de Recherche
sur les Maladies Infectieuses et Tropicales
E6
Second, recent studies25 suggest that head and body lice
can be mixed in persons infested with both head and body lice.
Whether these lice are conspecific remains controversial,25-30
and although head and body lice do not interbreed in the
wild,31 fertile hybrids that have intermediate morphologic
characteristics 32 have been reported under laboratory
conditions.33,34 Moreover, several observational studies35-38
have also suggested that head lice could become body lice when
raised under the laboratory conditions.
Third, it appears that only a change of underwear could reduce body lice infestation (equivalent efficiency of placebo at
day 45 compared with permethrin). This occurrence would not
be expected outside a controlled study. In fact, at the start of
this study, the mean number of body lice per homeless persons was high (approximately 450 body lice per subject), and
despite the availability of the protocol underwear in shelters,
most homeless persons changed their clothes only during scheduled follow-up visits and had not washed them between visits.
In conclusion, this trial clearly demonstrates that the use
of permethrin–impregnated underwear had the consequence
of increasing the percentage of permethrin-resistant body lice
in sheltered homeless persons. These findings lead us to recommend avoiding the use of permethrin to treat body lice infestations, although implementing new strategies is crucial.
Émergentes; and the infectious diseases specialists
for helpful discussions and active participation in
the study. We also thank the directors and the staff
of the 2 shelters for their assistance.
9. WHO recommended long-lasting insecticidal
mosquito nets. http://www.who.int/whopes/Long
_lasting_insecticidal_nets_Jul_2012.pdf. Accessed
November 19, 2012.
Correction: This article was corrected on
December 5, 2013, for an error in the first sentence
of the Results section of the Abstract.
10. Sholdt LL, Rogers EJ Jr, Gerberg EJ, Schreck CE.
Effectiveness of permethrin-treated military
uniform fabric against human body lice. Mil Med.
1989;154(2):90-93.
REFERENCES
1. Brouqui P, Stein A, Dupont HT, et al.
Ectoparasitism and vector-borne diseases in 930
homeless people from Marseilles. Medicine
(Baltimore). 2005;84(1):61-68.
2. Raoult D, Roux V. The body louse as a vector of
reemerging human diseases. Clin Infect Dis.
1999;29(4):888-911.
3. Badiaga S, Brouqui P. Human louse-transmitted
infectious diseases. Clin Microbiol Infect.
2012;18(4):332-337.
4. Izri A, Chosidow O. Efficacy of machine
laundering to eradicate head lice:
recommendations to decontaminate washable
clothes, linens, and fomites. Clin Infect Dis.
2006;42(2):e9-e10.
5. Badiaga S, Raoult D, Brouqui P. Preventing and
controlling emerging and reemerging transmissible
diseases in the homeless. Emerg Infect Dis.
2008;14(9):1353-1359.
6. Foucault C, Ranque S, Badiaga S, Rovery C,
Raoult D, Brouqui P. Oral ivermectin in the
treatment of body lice. J Infect Dis.
2006;193(3):474-476.
7. Badiaga S, Foucault C, Rogier C, et al. The effect
of a single dose of oral ivermectin on pruritus in the
homeless. J Antimicrob Chemother.
2008;62(2):404-409.
8. Chosidow O. Scabies and pediculosis. Lancet.
2000;355(9206):819-826.
11. Tomalik-Scharte D, Lazar A, Meins J, et al.
Dermal absorption of permethrin following topical
administration. Eur J Clin Pharmacol.
2005;61(5-6):399-404.
12. Young GD, Evans S. Safety and efficacy of DEET
and permethrin in the prevention of arthropod
attack. Mil Med. 1998;163(5):324-330.
13. Hollister LE. AMA Drug Evaluations Annual
1991. JAMA. 1991;266(3):424.
14. Drali R, Benkouiten S, Badiaga S, Bitam I, Rolain
JM, Brouqui P. Detection of a knockdown resistance
mutation associated with permethrin resistance in
the body louse Pediculus humanus corporis by use
of melting curve analysis genotyping. J Clin
Microbiol. 2012;50(7):2229-2233.
15. Lee SH, Gao JR, Yoon KS, et al. Sodium channel
mutations associated with knockdown resistance in
the human head louse, Pediculus capitis (De Geer).
Pestic Biochem Physiol. 2003;75:79-91.
16. SupYoon K, Symington SB, Hyeock Lee S,
Soderlund DM, Marshall Clark J. Three mutations
identified in the voltage-sensitive sodium channel
alpha-subunit gene of permethrin-resistant human
head lice reduce the permethrin sensitivity of house
fly Vssc1 sodium channels expressed in Xenopus
oocytes. Insect Biochem Mol Biol. 2008;38(3):
296-306.
17. Clark JM. Permethrin resistance due to
knockdown gene mutations is prevalent in human
JAMA Dermatology Published online December 4, 2013
184
Copyright 2013 American Medical
Association. All rights reserved.
Downloaded From: http://archderm.jamanetwork.com/ by American Medical Association, June Robinson on 12/30/2013
jamadermatology.com
Original Investigation Research
Permethrin–Impregnated Underwear
head louse populations. Open Dermatol J.
2010;4:63-68.
18. Durand R, Bouvresse S, Andriantsoanirina V,
Berdjane Z, Chosidow O, Izri A. High frequency of
mutations associated with head lice pyrethroid
resistance in schoolchildren from Bobigny, France.
J Med Entomol. 2011;48(1):73-75.
19. Gao JR, Yoon KS, Lee SH, et al. Increased
frequency of the T929I and L932F mutations
associated with knockdown resistance in
permethrin-resistant populations of the human
head louse, Pediculus capitis, from California,
Florida, and Texas. Pestic Biochem Physiol.
2003;77(3):115-124.
20. Yoon KS, Gao JR, Lee SH, Clark JM, Brown L,
Taplin D. Permethrin-resistant human head lice,
Pediculus capitis, and their treatment. Arch
Dermatol. 2003;139(8):994-1000.
21. Hurlbut HS, Altman RM, Nibley C Jr. DDT
resistance in Korean body lice. Science.
1952;115(2975):11-12.
22. Eddy GW, Cole MM, Couch MD, Selhime A.
Resistance of human body lice to insecticides.
Public Health Rep. 1955;70(10):1035-1038.
23. McLINTOCK J, Zeini A, Djanbakhsh B.
Development of insecticide resistance in body lice
in villages of North-Eastern Iran. Bull World Health
Organ. 1958;18(4):678-680.
jamadermatology.com
24. Hodgdon HE, Yoon KS, Previte DJ, et al.
Determination of knockdown resistance allele
frequencies in global human head louse
populations using the serial invasive signal
amplification reaction. Pest Manag Sci.
2010;66(9):1031-1040.
man [in Russian]. Med Parazitol (Mosk).
1995;(1):23-25.
31. Busvine JR. Evidence from double infestations
for the specific status of human head lice and body
lice (Anoplura). Syst Entomol. 1978;3:1-8.
25. Veracx A, Rivet R, McCoy KD, Brouqui P, Raoult
D. Evidence that head and body lice on homeless
persons have the same genotype. PLoS One.
2012;7(9):e45903.
26. Olds BP, Coates BS, Steele LD, et al.
Comparison of the transcriptional profiles of head
and body lice. Insect Mol Biol. 2012;21(2):257-268.
27. Li W, Ortiz G, Fournier PE, et al. Genotyping of
human lice suggests multiple emergencies of body
lice from local head louse populations. PLoS Negl
Trop Dis. 2010;4(3):e641.
28. Leo NP, Hughes JM, Yang X, Poudel SK,
Brogdon WG, Barker SC. The head and body lice of
humans are genetically distinct (Insecta:
Phthiraptera, Pediculidae): evidence from double
infestations. Heredity (Edinb). 2005;95(1):34-40.
29. Leo NP, Campbell NJ, Yang X, Mumcuoglu K,
Barker SC. Evidence from mitochondrial DNA that
head lice and body lice of humans (Phthiraptera:
Pediculidae) are conspecific. J Med Entomol.
2002;39(4):662-666.
32. Busvine JR. The head and body races of
Pediculus humanus L. Parasitology.
1948;39(1-2):1-16.
33. Bacot AW. A contribution to the bionomics of
Pediculus humanus (vestimenti) and Pediculus
capitis. Parasitology. 1917;9:228-258.
34. Mullen G, Durden LA. Medical and Veterinary
Entomology. San Diego, CA: Academic Press; 2009.
35. Howlett FM. Notes on head- and body-lice and
upon temperature reactions of lice and mosquitoes.
Parasitology. 1917;10:186-188.
36. Nuttall GHF. The biology of Pediculus humanus:
supplementary notes. Parasitology. 1919;11:201-221.
37. Alpatov VV, Nastukova OA. Transformation of
the head form of Pediculus humanus into the body
form under changed conditions of existence. Bull
Soc Nat Moscow. 1955;60:79-92.
38. Levene H, Dobzhansky T. Possible genetic
difference between the head louse and the body
louse. Am Nat. 1959;93:347-353.
30. Khudobin VV. The adaptive potentials of
human head and clothes lice when parasitizing on
JAMA Dermatology Published online December 4, 2013
185
Copyright 2013 American Medical
Association. All rights reserved.
Downloaded From: http://archderm.jamanetwork.com/ by American Medical Association, June Robinson on 12/30/2013
E7
Supplementary Online Content
Benkouiten S, Drali R, Badiaga S, et al. Effect of Permethrin-Impregnated Underwear on
Body Lice in Sheltered Homeless Persons: A Randomized Controlled Trial
eTable 1. Stratified random sampling of the levels of body lice infestations
eTable 2. Evolution of the mean number of body lice between days 1 and 14 and days 1 and 45 in
each subgroup
This supplementary material has been provided by the authors to give readers additional
information about their work.
186
eTable 1. Stratified random sampling of the levels of body lice infestations
Day 1
Day 45
Homeless, No.
33.33
Lice , %
21/404
12/41
Lice tested , No. /total
4
11
5
Homeless, No.
3.75
7.50
15
100
Lice , %
44/2360
56/1485
20/272
47/316
17/17
Lice tested , No./total
c
Lice , Min–Max
16
5
21/832
6
1.88
b
1–9
19
2.50
44/3554
3
c
10–49
12
1.25
37/5711
29/3099
b
50–99
13
0.63
0.94
a
100–499
8
2
500–999
23/7571
5
0.31
1000–1999
213/7549
31
73
158/18113
Total
a
Total number of body lice per homeless person
Percentage of body lice drawn per homeless person
Number of body lice tested
b
c
Lice, No. (%)
269.5 (431.0)
7545 (100)
278.3 (472.7)
8907 (100)
Day 1
104 (202.1)
2912 (38.6)
176.1 (500.9)
5460 (61.3)
Day 45
P Value
0.001
0.001
253.3 (440.8)
6078 (100)
308.8 (502.8)
8339 (100)
Day 1
147.6 (272.3)
3542 (58.3)
148.4 (427.6)
4008 (48.1)
Day 45
eTable 2. Evolution of the mean number of body lice between days 1 and 14 and days 1 and 45 in each subgroup
Permethrin
Lice, Mean (SD)
Lice, No. (%)
Lice, Mean (SD)
Total number of body lice per homeless person
Placebo
a
P Value
0.001
0.04
187
Conclusions et perspectives
Cette thèse s’inscrit dans la continuité des nombreux travaux sur le
pou humain réalisés à l’URMITE au sein des équipes dirigées par le
Professeur Didier Raoult, un pourvoyeur essentiel des connaissances
scientifiques dans ce domaine durant les vingt dernières années.
A travers cette thèse, nous avons pu répondre à certaines questions
concernant les poux humains restées en débat depuis longtemps.
Ainsi, nous avons mis en place un outil moléculaire qui permet de
différencier pour la première fois entre le pou de tête et le pou de
corps du clade mitochondrial le plus distribué à travers le monde
(Clade A). Un outil qui a démontré son utilité sur le terrain notamment
pour déterminer le génotype des poux infectés par des pathogènes.
Nous avons mis en évidence l’existence d’un nouveau clade
mitochondrial, le Clade D renfermant des poux de tête et des poux de
corps susceptibles de vectoriser B. quintana et Y. pestis. Nous avons
mis en place un système pour pouvoir retracer les migrations des
humains et des pathogènes à travers l'analyse de poux anciens
provenant de différentes périodes et de différentes localisations. Nous
avons démontré que P. mjobergi était à l’origine un pou humain qui a
été transféré aux primates du Nouveau Monde par les premiers
189
Hommes à avoir atteint le continent américain il y a des milliers
d’années. Cela fût possible grâce aux premières analyses génétiques
effectuées sur P. mjobergi. Nous avons mis en place un outil de
détection et de contrôle de la résistance moléculaire des poux à la
perméthrine. Outil qui fût particulièrement utile dans l'étude clinique
que nous avons menée pour déterminer si l'utilisation de sousvêtements imprégnés d'insecticide offrait une protection efficace à
long terme contre les poux de corps infestant les personnes sans-abri.
Durant cette thèse, j’ai eu la chance d’accéder à un matériel
biologique d’une rareté exceptionnelle, et comme on l’a constaté, dans
chacune des thématiques où ce matériel fut impliqué, les résultats
obtenus étaient surprenants. A l’ère du séquençage à haut débit, il
serait très intéressant de séquencer le maximum de poux provenant de
différents endroits à travers le monde pour effectuer une analyse
globale des génomes. Ce qui permettra d’en savoir un peu plus sur
l’odyssée de l’espèce humaine et des pathogènes vectorisés par les
poux. Car comme on le soupçonne, ces informations doivent être
enregistrées et bien gardées par le plus intimes de nos parasites.
190
Références
1. Johnson KP, Yoshizawa K, Smith VS (2004) Multiple origins of
parasitism
in
lice.
Proc
Biol
Sci
271:
1771-1776.
10.1098/rspb.2004.2798 [doi];HM75VQVC7RH4PK25 [pii].
2. Lyal CH (1985) Phylogeny and classification of the Psocodea, with
particular reference to the lice (Psocodea: Phthiraptera). Systematic
Entomology 10: 145-165.
3. Barker SC, Whiting M, Johnson KP, Murrell A (2003) Phylogeny
of the lice (Insecta, Phthiraptera) inferred from small subunit
rRNA. Zoologica Scripta 32: 407-414.
4. Wappler T, Smith VS, Dalgleish RC (2004) Scratching an ancient
itch: an Eocene bird louse fossil. Proc Biol Sci 271 Suppl 5: S255S258. 10.1098/rsbl.2003.0158 [doi].
5. Grimaldi D, Engel MS (2006) Fossil Liposcelididae and the lice
ages (Insecta: Psocodea). Proc Biol Sci 273: 625-633.
6. Smith VS, Ford T, Johnson KP, Johnson PC, Yoshizawa K, Light
JE (2011) Multiple lineages of lice pass through the K-Pg
boundary.
Biol
Lett
7:
782-785.
rsbl.2011.0105
[pii];10.1098/rsbl.2011.0105 [doi].
7. Barker SC (1991) Evolution of host-parasite associations among
species of lice and rock-wallabies: coevolution? (J. F. A. Sprent
Prize lecture, August 1990). Int J Parasitol 21: 497-501. 00207519(91)90053-A [pii].
191
8. Hafner MS, Nadler SA (1988) Phylogenetic trees support the
coevolution of parasites and their hosts. Nature 332: 258-259.
10.1038/332258a0 [doi].
9. Brooks DR (1979) Testing the context and extent of host-parasite
coevolution. SysT Zool 28: 229-307.
10. Johnson KP, Adams RJ, Page RD, Clayton DH (2003) When do
parasites fail to speciate in response to host speciation? Syst Biol
52: 37-47. JF8959128JU409RB [pii].
11. Page RD, Charleston MA (1998) Trees within trees: phylogeny and
historical associations. Trends Ecol Evol 13: 356-359. S01695347(98)01438-4 [pii].
12. Ewing HE (1938) The Sucking Lice of American Monkeys. The
Journal of Parasitology 24: 13-33.
13. Reed DL, Light JE, Allen JM, Kirchman JJ (2007) Pair of lice lost
or parasites regained: the evolutionary history of anthropoid
primate lice. BMC Biol 5: 7. 1741-7007-5-7 [pii];10.1186/17417007-5-7 [doi].
14. De Geer C (1778) Mémoires pour servir à l'histoire des Insectes.
62-68.
15. Hopkins GHE (1952) The correct names of the body and head lice
of man. 91-92.
16. Sangare AK, Boutellis A, Drali R, Socolovschi C, Barker SC,
Diatta G, Rogier C, Olive MM, Doumbo OK, Raoult D (2014)
192
Detection of Bartonella quintana in African Body and Head Lice.
Am J Trop Med Hyg 91: 294-301.
17. Chosidow O, Chastang C, Brue C, Bouvet E, Izri M, Monteny N,
Bastuji-Garin S, Rousset JJ, Revuz J (1994) Controlled study of
malathion and d-phenothrin lotions for Pediculus humanus var
capitis-infested schoolchildren. Lancet 344: 1724-1727.
18. Nuttall GH (1919) The systematic position, synonymy and
iconography
of
Pediculus
humanus
and
Phthirus
pubis.
Parasitology 11: 329-346.
19. Hindle E (1917) Notes on the biology of Pediculus humanus L.
Parasitology 9: 259-265.
20. Ewing HE (1926) A revision of the American lice of the genus
Pediculus, together with a consideration of the significance of their
geographical and host distribution. Proc US Nat Mus 68: 1-30.
21. Nuttall GH (1919) The biology of Pediculus humanus L.
(Supplementary notes). Parasitology 11: 201-220.
22. Veracx A, Boutellis A, Merhej V, Diatta G, Raoult D (2012)
Evidence for an African cluster of human head and body lice with
variable colors and interbreeding of lice between continents. PLoS
One 7: e37804. 10.1371/journal.pone.0037804 [doi];PONE-D-1205586 [pii].
23. Reed DL, Smith VS, Hammond SL, Rogers AR, Clayton DH
(2004) Genetic analysis of lice supports direct contact between
193
modern
and
archaic
humans.
PLoS
Biol
2:
e340.
10.1371/journal.pbio.0020340 [doi].
24. Boutellis A, Abi-Rached L, Raoult D (2014) The origin and
distribution of human lice in the world. Infect Genet Evol 23: 209217.
25. Boutellis A, Drali R, Rivera MA, Mumcuoglu KY, Raoult D
(2013) Evidence of sympatry of clade a and clade B head lice in a
pre-Columbian Chilean mummy from Camarones. PLoS One 8:
e76818.
26. Raoult D, Reed DL, Dittmar K, Kirchman JJ, Rolain JM, Guillen
S, Light JE (2008) Molecular identification of lice from preColumbian mummies. J Infect Dis 197: 535-543. 10.1086/526520
[doi].
27. Johnston JS, Yoon KS, Strycharz JP, Pittendrigh BR, Clark JM
(2007) Body lice and head lice (Anoplura: Pediculidae) have the
smallest genomes of any hemimetabolous insect reported to date. J
Med Entomol 44: 1009-1012.
28. Kirkness EF, Haas BJ, Sun W, Braig HR, Perotti MA, Clark JM,
Lee SH, Robertson HM, Kennedy RC, Elhaik E, Gerlach D,
Kriventseva EV, Elsik CG, Graur D, Hill CA, Veenstra JA,
Walenz B, Tubio JM, Ribeiro JM, Rozas J, Johnston JS, Reese JT,
Popadic A, Tojo M, Raoult D, Reed DL, Tomoyasu Y, Kraus E,
Mittapalli O, Margam VM, Li HM, Meyer JM, Johnson RM,
Romero-Severson J, Vanzee JP, Alvarez-Ponce D, Vieira FG,
Aguade M, Guirao-Rico S, Anzola JM, Yoon KS, Strycharz JP,
194
Unger MF, Christley S, Lobo NF, Seufferheld MJ, Wang N, Dasch
GA, Struchiner CJ, Madey G, Hannick LI, Bidwell S, Joardar V,
Caler E, Shao R, Barker SC, Cameron S, Bruggner RV, Regier A,
Johnson J, Viswanathan L, Utterback TR, Sutton GG, Lawson D,
Waterhouse RM, Venter JC, Strausberg RL, Berenbaum MR,
Collins FH, Zdobnov EM, Pittendrigh BR (2010) Genome
sequences of the human body louse and its primary endosymbiont
provide insights into the permanent parasitic lifestyle. Proc Natl
Acad
Sci
U
S
A
107:
12168-12173.
1003379107
[pii];10.1073/pnas.1003379107 [doi].
29. Olds BP, Coates BS, Steele LD, Sun W, Agunbiade TA, Yoon KS,
Strycharz JP, Lee SH, Paige KN, Clark JM, Pittendrigh BR (2012)
Comparison of the transcriptional profiles of head and body lice.
Insect Mol Biol 21: 257-268. 10.1111/j.1365-2583.2012.01132.x
[doi].
30. Raoult D, Roux V (1999) The body louse as a vector of reemerging
human diseases. Clin Infect Dis 29: 888-911. 10.1086/520454
[doi].
31. Houhamdi L, Lepidi H, Drancourt M, Raoult D (2006)
Experimental model to evaluate the human body louse as a vector
of
plague.
J
Infect
Dis
194:
1589-1596.
JID36487
[pii];10.1086/508995 [doi].
32. Piarroux R, Abedi AA, Shako JC, Kebela B, Karhemere S, Diatta
G, Davoust B, Raoult D, Drancourt M (2013) Plague epidemics
195
and lice, Democratic Republic of the Congo. Emerg Infect Dis 19:
505-506.
33. Bonilla DL, Kabeya H, Henn J, Kramer VL, Kosoy MY (2009)
Bartonella quintana in body lice and head lice from homeless
persons, San Francisco, California, USA. Emerg Infect Dis 15:
912-915. 10.3201/eid1506.090054 [doi].
34. Boutellis A, Veracx A, Angelakis E, Diatta G, Mediannikov O,
Trape JF, Raoult D (2012) Bartonella quintana in head lice from
Senegal.
Vector
Borne
Zoonotic
Dis
12:
564-567.
10.1089/vbz.2011.0845 [doi].
35. Angelakis E, Diatta G, Abdissa A, Trape JF, Mediannikov O,
Richet H, Raoult D (2011) Altitude-dependent Bartonella quintana
genotype C in head lice, Ethiopia. Emerg Infect Dis 17: 23572359. 10.3201/eid1712.110453 [doi].
36. Sasaki T, Poudel SKS, Isawa H, Hayashi T, Seki S, Tomita T,
Sawabe K, Kobayashi M (2006) First Molecular Evidence of
Bartonella quintana in Pediculus humanus capitis (Phthiraptera:
Pediculidae), Collected from Nepalese Children. J Med Entomol
43: 110-112.
37. Boutellis A, Mediannikov O, Bilcha KD, Ali J, Campelo D, Barker
SC, Raoult D (2013) Borrelia recurrentis in head lice, Ethiopia.
Emerg Infect Dis 19: 796-798. 10.3201/eid1905.121480 [doi].
38. Mumcuoglu KY, Zias J (1988) Head lice, Pediculus humanus
capitis (Anoplura, Pediculidae) from hair combs excavated in
196
Israel and dated from the first century B.C. to the eighth century
A.D. J Med Entomol 25: 545-547.
39. Clark JM, Yoon KS, Lee SH, Pittendrigh BR (2013) Human lice:
Past, present and future control. Pesticide Biochemistry and
Physiology 106: 162-171.
40. Pariser DM, Meinking TL, Ryan WG (2013) Topical ivermectin
lotion
for
head
lice.
N
Engl
J
Med
368:
967.
10.1056/NEJMc1215548 [doi].
41. Yoon KS, Strycharz JP, Baek JH, Sun W, Kim JH, Kang JS,
Pittendrigh BR, Lee SH, Clark JM (2011) Brief exposures of
human body lice to sublethal amounts of ivermectin overtranscribes detoxification genes involved in tolerance. Insect Mol
Biol 20: 687-699. 10.1111/j.1365-2583.2011.01097.x [doi].
42. Drali R, Boutellis A, Raoult D, Rolain JM, Brouqui P (2013)
Distinguishing body lice from head lice by multiplex real-time
PCR analysis of the Phum_PHUM540560 gene. PLoS One 8:
e58088.
43. Veracx A, Raoult D (2012) Biology and genetics of human head
and
body
lice.
Trends
Parasitol
28:
563-571.
S1471-
4922(12)00163-8 [pii];10.1016/j.pt.2012.09.003 [doi].
44. Alpatov WW, Nastjukova OK (1955) Transformation of the head
form of Pediculus humanus L. into the body form under the
influence of changed living conditions. Bull Soc Nat Moscow 60:
79-92.
197
45. Bacot A (1917) A contribution to the bionomics of Pediculus
humanus (vestimenti) and Pediculus capitis. Parasitology 9: 228258.
46. Drali R, Sangare AK, Boutellis A, Angelakis E, Veracx A,
Socolovschi C, Brouqui P, Raoult D (2014) Bartonella quintana in
body lice from scalp hair of homeless persons, France. Emerg
Infect Dis 20: 907-908. 10.3201/eid2005.131242 [doi].
47. Kittler R, Kayser M, Stoneking M (2003) Molecular evolution of
Pediculus humanus and the origin of clothing. Curr Biol 13: 14141417.
48. Light JE, Toups MA, Reed DL (2008) What's in a name: the
taxonomic status of human head and body lice. Mol Phylogenet
Evol 47: 1203-1216.
49. Boutellis A, Abi-Rached L, Raoult D (2014) The origin and
distribution of human lice in the world. Infect Genet Evol 23: 209217. S1567-1348(14)00020-3 [pii];10.1016/j.meegid.2014.01.017
[doi].
50. Hafner MS, Sudman PD, Villablanca FX, Spradling TA, Demastes
JW, Nadler SA (1994) Disparate rates of molecular evolution in
cospeciating hosts and parasites. Science 265: 1087-1090.
51. Brouqui P, Stein A, Dupont HT, Gallian P, Badiaga S, Rolain JM,
Mege JL, La SB, Berbis P, Raoult D (2005) Ectoparasitism and
vector-borne diseases in 930 homeless people from Marseilles.
198
Medicine (Baltimore) 84: 61-68. 00005792-200501000-00006
[pii].
52. Badiaga S, Foucault C, Rogier C, Doudier B, Rovery C, Dupont
HT, Castro P, Raoult D, Brouqui P (2008) The effect of a single
dose of oral ivermectin on pruritus in the homeless. J Antimicrob
Chemother 62: 404-409. dkn161 [pii];10.1093/jac/dkn161 [doi].
53. Foucault C, Ranque S, Badiaga S, Rovery C, Raoult D, Brouqui P
(2006) Oral ivermectin in the treatment of body lice. J Infect Dis
193: 474-476. JID35187 [pii];10.1086/499279 [doi].
54. Drali R, Benkouiten S, Badiaga S, Bitam I, Rolain JM, Brouqui P
(2012) Detection of a knockdown resistance mutation associated
with permethrin resistance in the body louse Pediculus humanus
corporis by use of melting curve analysis genotyping. J Clin
Microbiol
50:
2229-2233.
JCM.00808-12
[pii];10.1128/JCM.00808-12 [doi].
55. Benkouiten S, Drali R, Badiaga S, Veracx A, Giorgi R, Raoult D,
Brouqui P (2014) Effect of permethrin-impregnated underwear on
body lice in sheltered homeless persons: a randomized controlled
trial.
JAMA
Dermatol
150:
[pii];10.1001/jamadermatol.2013.6398 [doi].
199
273-279.
1782130
Annexes
201
Article X:
Matrix-Assisted Laser Desorption Ionization–Time
of Flight Mass Spectrometry for Rapid
Identification of Tick Vectors
Journal of Clinical Microbiology, 51(2), 522-528.
203
Matrix-Assisted Laser Desorption Ionization
−Time of Flight Mass Spectrometry for
Rapid Identification of Tick Vectors
Amina Yssouf, Christophe Flaudrops, Rezak Drali, Tahar
Kernif, Cristina Socolovschi, Jean-Michel Berenger, Didier
Raoult and Philippe Parola
J. Clin. Microbiol. 2013, 51(2):522. DOI:
10.1128/JCM.02665-12.
Published Ahead of Print 5 December 2012.
These include:
REFERENCES
CONTENT ALERTS
This article cites 32 articles, 4 of which can be accessed free at:
http://jcm.asm.org/content/51/2/522#ref-list-1
Receive: RSS Feeds, eTOCs, free email alerts (when new
articles cite this article), more»
Information about commercial reprint orders: http://journals.asm.org/site/misc/reprints.xhtml
To subscribe to to another ASM Journal go to: http://journals.asm.org/site/subscriptions/
205
Downloaded from http://jcm.asm.org/ on October 24, 2014 by guest
Updated information and services can be found at:
http://jcm.asm.org/content/51/2/522
Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass
Spectrometry for Rapid Identification of Tick Vectors
Amina Yssouf,a Christophe Flaudrops,b Rezak Drali,a Tahar Kernif,a Cristina Socolovschi,a Jean-Michel Berenger,a Didier Raoult,a
Philippe Parolaa
Aix Marseille Université, Unité de Recherche en Maladies Infectieuses et Tropicales Emergentes, UM63, CNRS 7278, IRD 198, Inserm 1095, WHO Collaborative Center for
Rickettsioses and Other Arthropod Borne Bacterial Diseases, Faculté de Médecine, Marseille, Francea; Assistance Publique des Hôpitaux de Marseille, CHU Timone, Pôle
Infectieux, Marseille, Franceb
T
icks are obligate hematophagous parasites of the order Acari.
These arthropods can feed on every known class of vertebrate
and can bite people (1). Ticks are currently the second leading
vector of human infectious diseases and can carry bacterial (1),
viral (1a), and protozoan pathogens (2). However, only in 1982,
with the identification of Borrelia burgdorferi as the etiological
agent of Lyme disease, was the major effect of ticks on public
health recognized, leading to an increased awareness of tick-borne
diseases (3). Since then, more than 15 tick-borne rickettsioses
have emerged throughout the world (4).
The removal of a tick from the human body is a common
situation, and patients may visit a physician with an attached or
removed tick. Certain tick species are well-known vectors of human diseases, such that identifying the species, which will alert the
physician to the diseases that may have been transmitted, is clinically helpful if such information is obtained quickly (1). Indeed,
recent studies confirm that the use of doxycycline prophylaxis
following an Ixodes tick bite is useful for the prevention of Lyme
disease (4). Similar postexposure regimens could also prevent
tick-borne relapsing fever in areas of endemicity (5). However, for
prophylactic treatment to be effective, it must be delivered shortly
after potentially infectious ticks are removed from patients (6).
Ticks species can be morphologically identified using taxonomic keys for endemic species in several geographic regions (1).
However, morphological identification can be difficult because it
requires some entomological expertise, and it is difficult to identify a specimen that is damaged or at an immature stage of its life
cycle (1). Molecular methods, such as the sequencing of the mitochondrial 12S (7), 18S (8), and 16S ribosomal DNAs (rDNAs) (7),
mitochondrial cytochrome oxidase subunit 1 (COX1), and nu-
522
jcm.asm.org
clear internal transcribed spacer 2 (ITS2), have been developed to
identify arthropods, including ticks (9). However, there is currently no PCR assay that can distinguish tick species, and ideal
PCR primer pairs that can amplify the relevant gene fragments are
not available.
In addition to the technical and logistical drawbacks of PCR
assays, this approach is further limited by the availability of gene
sequences in GenBank. Protein profiling by matrix-assisted laser
desorption ionization–time of flight mass spectrometry (MALDITOF MS) is now increasingly common for the routine identification of microorganisms in clinical microbiology (10). This revolutionary, reliable, and cost-effective technique is simpler and
faster than conventional phenotypic and molecular methods for
the identification of human pathogens (10).
The MALDI-TOF MS approach was first applied to arthropods
for the differentiation of Drosophila species (11). It was found that
protein extracts obtained from whole specimens generated reproducible spectra (11). Species-specific protein profiles have also
been be used to differentiate three species of aphids (insects that
feed on plants) (12). In 2011, a blind test in which 111 wild specimens were compared to the database profiles of Culicoides species
Received 4 October 2012 Returned for modification 25 October 2012
Accepted 18 November 2012
Published ahead of print 5 December 2012
Address correspondence to Philippe Parola, philippe.parola@univ-amu.fr.
Copyright © 2013, American Society for Microbiology. All Rights Reserved.
doi:10.1128/JCM.02665-12
Journal of Clinical Microbiology
p. 522–528
206
February 2013 Volume 51 Number 2
Downloaded from http://jcm.asm.org/ on October 24, 2014 by guest
A method for rapid species identification of ticks may help clinicians predict the disease outcomes of patients with tick bites and
may inform the decision as to whether to administer postexposure prophylactic antibiotic treatment. We aimed to establish a
matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) spectrum database based on the
analysis of the legs of six tick vectors: Amblyomma variegatum, Rhipicephalus sanguineus, Hyalomma marginatum rufipes, Ixodes ricinus, Dermacentor marginatus, and Dermacentor reticulatus. A blind test was performed on a trial set of ticks to identify
specimens of each species. Subsequently, we used MALDI-TOF MS to identify ticks obtained from the wild or removed from patients. The latter tick samples were also identified by 12S ribosomal DNA (rDNA) sequencing and were tested for bacterial infections. Ticks obtained from the wild or removed from patients (R. sanguineus, I. ricinus, and D. marginatus) were accurately
identified using MALDI-TOF MS, with the exception of those ticks for which no spectra were available in the database. Furthermore, one damaged specimen was correctly identified as I. ricinus, a vector of Lyme disease, using MALDI-TOF MS only. Six of
the 14 ticks removed from patients were found to be infected by pathogens that included Rickettsia, Anaplasma, and Borrelia
spp. MALDI-TOF MS appears to be an effective tool for the rapid identification of tick vectors that requires no previous expertise
in tick identification. The benefits for clinicians include the more targeted surveillance of patients for symptoms of potentially
transmitted diseases and the ability to make more informed decisions as to whether to administer postexposure prophylactic
treatment.
MALDI-TOF MS Identification of Ticks
TABLE 1 Arthropods used to establish the reference database of MALDI-TOF spectra and arthropods used in the blind test
No. of specimens used to
create the database
No. of specimens used for the blind test
procedure
Sourcea
Total
Sex and/or stageb
Total (score range)
Sex and/or stageb
LC
LC
Vegetation
Vegetation
LC
LC
LC
LC
Vegetation
Animal
Vegetation
18
10
6 M, 5 F, 7 N
5 M, 5 F
10
10
12
7
5 M, 5 F
5 M, 6 F
6 M, 6 F
N
Rhipicephalus sulcatus
Haemaphysalis concinna
Senegal
France
France
France
France
Croatia
Senegal
France
France
Senegal
France
2 (2.248–2.357)
2 (1.995- 2.135)
3 (1.715–1.745)
2 (1.701–1.884)
2 (2.232–2.519)
3 (2.119- 2.281)
2 (1.992–2.352)
2 (1.706–1.831)
7 (1.304–1.878)
2 (1.009–1.166)
2 (0.838–1.062)
1 M, 1F
1 M, 1 F
M
F
1 M, 1 F
1 M, 2 F
1 M, 1 F
1 M, 1 F
3 M, 2 F, 2 N
1 M, 1 F
1 M, 1 F
Other arthropods
Ctenocephalides felis
Pediculus humanus corporis
Triatoma infestans
Cimex lectularius
Culex pipiens
Apis mellifera
Pyrrhocoris apterus
Blaptica dubia
Tenibrio molitor
England
USA
Bolivia
France
France
France
France
India
France
LC
LC
LC
LC
LC
Field
Field
Field
Field
17
12
12
12
7
11 F, 6 M
7 M, 6 F
6 M, 6 F
6 M, 6 F
F
2 (2.103–2.123)
2 (1.788–1.797)
2 (1.733–1.779)
2 (2.145–2.429)
2 (1.655–1.749)
2 (0.732–0.778)
2 (0.759–0.998)
2 (0.707–1.066)
2 (0.755–0.958)
1 M, 1 F
1 M, 1 F
1 M, 1 F
1 M, 1 F
F
1 M, 1 F
1 M, 1 F
1 M, 1 F
1 M, 1 F
Arthropod for database
Ticks
Amblyomma variegatum
Rhipicephalus sanguineus
Dermacentor marginatus
Dermacentor reticulatus
Hyalomma marginatum rufipes
Ixodes ricinus
a
b
LC, laboratory colonies.
M, male; F, female; N, nymph stage.
showed that MALDI-TOF MS can differentiate species of Culicoides biting midges collected in the field (13). More recently, a
MALDI-TOF MS study of seven ticks reported that whole ticks or
body parts, excluding the legs, generate spectra that are sufficient
for species identification (14).
The objective of the present study was to investigate the use of
MALDI-TOF MS for the rapid differentiation of tick species using
only their legs. Our goals were to establish a reference database, to
evaluate the MALDI-TOF MS-based identification system in a
blind test, and to evaluate this new identification tool using ticks
removed from patients.
MATERIALS AND METHODS
Arthropods. To establish a reference database, we used laboratory-reared
hard ticks, fleas, lice, triatomines, mosquitoes, and bedbugs (Table 1). All
specimens were fresh and nonengorged and maintained at room temperature for less than 1 week postmortem. The tick specimens included Amblyomma variegatum, Hyalomma marginatum rufipes, Rhipicephalus sanguineus, Ixodes ricinus, Dermacentor marginatus, and Dermacentor
reticulatus. For the blind test, other specimens of the same species and
specimens of other arthropod families were used as controls. Starved ticks
collected in the field or removed from animals were characterized by
MALDI-TOF MS (Table 1). A total of 14 ticks removed from 14 patients
in France from June to August 2012 were morphologically characterized
using standard taxonomic keys as D. marginatus, R. sanguineus, I. ricinus,
Rhipicephalus sp., and Ixodes sp. (Table 2). One extensively damaged hard
tick removed from a patient was also tested. After the morphological
identification procedure, four legs were removed for MALDI-TOF MS
(see below) and sequencing assays. DNA was extracted from tick body
halves, and a 360-bp fragment of the mitochondrial 12S rDNA sequence
was amplified by PCR and sequenced (14a). The sequences were analyzed
using ChromasPro, version 1.34 (Technelysium Pty, Ltd., Tewantin,
February 2013 Volume 51 Number 2
Queensland, Australia), and were compared with sequences from GenBank. The unused tick body parts were stored at �80°C for subsequent
analyses.
MALDI-TOF procedure. (i) Preparation of samples. Each specimen
was placed in a 1.5-ml microcentrifuge tube and immobilized or anesthetized at �20°C for 30 min (11). Subsequently, the specimens were rinsed
once with distilled water. After the specimens were dried with paper, four
legs were removed with scalpels. One of two different homogenization
solutions was used depending on the specimen’s size (11). The tick legs
were manually homogenized in 60 �l of 70% formic acid and 60 �l of 50%
acetonitrile in 1.5-ml microcentrifuge tubes using pellet pestles (Fischer
Scientific). The legs of mosquitoes, fleas, and lice were also homogenized
in 20 �l of formic acid and 20 �l of acetonitrile. Triatomine legs were
homogenized in 100 �l of 70% formic acid and 100 �l of 50% acetonitrile.
All homogenates were centrifuged at 10,000 rpm for 20 s, and 1 �l of each
supernatant was spotted onto a steel target plate (Bruker Daltonics) in
quadruplicate (13). One microliter of a CHCA matrix suspension composed of saturated �-cyano-4-hydroxycinnamic acid (Sigma), 50% acetonitrile, 10% trifluoroacetic acid, and high-performance liquid chromatography (HPLC)-grade water was directly spotted onto each sample on
the target plate to allow cocrystallization (15). The target plate was dried
for several minutes at room temperature before insertion into the
MALDI-TOF MS instrument.
(ii) MALDI-TOF MS parameters. Protein mass profiles were acquired
using a Microflex MALDI-TOF mass spectrometer (Bruker Daltonics)
with Flex Control software (Bruker Daltonics). We performed measurements in the linear positive-ion mode (15) between 2 and 20 kDa, and
each spectrum corresponded to ions obtained from 240 laser shots performed in six regions of the same spot. The obtained spectra were processed using Flex Analysis, version 3.3, and MALDI Biotyper, version 3.0,
software.
(iii) Spectral analysis and reference database creation. To study the
reproducibility of the species spectra, the average spectral profiles ob-
207
jcm.asm.org 523
Downloaded from http://jcm.asm.org/ on October 24, 2014 by guest
Geographical
origin
Yssouf et al.
TABLE 2 Identification of ticks removed from patients using morphological, MALDI-TOF MS, and molecular methods and the results of the
detection of Rickettsia, Borrelia, Anaplasma, and Bartonella DNA
Molecular identification (% similarity with the indicated GenBank sequence) of:a
Morphological
identification
MALDI-TOF MS identification
(score)
Ticke
D. marginatus
D. marginatus
D. marginatus
R. sanguineus
I. ricinus
Damaged
I. ricinus
Rhipicephalus sp.
Ixodes sp.
Rhipicephalus sp.
I. ricinus
I. ricinus
I. ricinus
D. marginatus (1.83)
D. marginatus (1.798)
D. marginatus (1.799)
R. sanguineus (1.957)
I. ricinus (1,737)
I. ricinus (1,376)
I. ricinus (1.321)
Not identifiedb
I. ricinus (1.308)
R. sanguineus (2.117)
I. ricinus (1.728)
I. ricinus (1.823)
I. ricinus (1.641)
D. marginatus (99.3% AM410570.1)
D. marginatus (99.4% AM410570.1)
D. marginatus (99.4% AM410570.1)
R. sanguineus (99.1% JX206975.1)
I. ricinus (99.5% JN248424.1)
Not obtainedc
I. ricinus (99.3% JN248424.1)
R. bursa (100% AM410572.1)
I. ricinus (99.2% JM248424.1)
R. sanguineus (99.2% AY559843.1)
I. ricinus (99.7% JN248424.1)
I. ricinus (99.2% JN248424.1)
I. ricinus (99.06% AY945481.1)
I. ricinus
D. marginatus
D. marginatus
D. marginatus
I. ricinus (1.668)
D. marginatus (1.83)
D. marginatus (1.798)
D. marginatus (1.799)
I. ricinus (100% JN248424.1)
D. marginatus (99.3% AM410570.1)
D. marginatus (99.4% AM410570.1)
D. marginatus (99.4% AM410570.1)
Bacteria
R. slovacad, R. raoultiid
R. massiliaed
B. garinii (99.7% JN828689.1)
R. conoriid
A. phagocytophilumd
R. slovacad, R. raoultiid
R. massiliaed
a
DNA extracted from laboratory colonies of uninfected ticks was used as a negative control. DNA extracted from Rickettsia montanensis, Bartonella elizabethae, Borrelia crocidurae,
and Anaplasma phagocytophilum was used as a positive control.
No R. bursa spectrum was available in our MALDI-TOF MS database.
c
After two attempts.
d
qPCR.
e
By 12S RNA sequencing.
b
tained from the four spots for each specimen of the same species were
analyzed and compared using the ClinProTools, version 2.2, program
(Bruker Daltonics). Using MALDI Biotyper, version 3.0, a database containing the studied arthropod groups was assembled from at least five
specimens of each sex for all of the studied arthropod species, excluding
mosquitoes, for which the majority of tested specimens were females. The
reproducible spectra were used in the database.
(iv) Study validation using a blind test and identification of ticks
from the field and patients. An intercomparison of the reference spectra
of each species was performed to interrogate the database (14, 16). The
“start identification” function in the MALDI Biotyper allows the identification of each specimen to be tested, and the results were presented as log
score values. A blind test was then performed with specimens from our
laboratory colonies for which there were corresponding reference spectra
in our database. For each species, two new specimens were used. We also
tested two specimens of arthropods not included in our database, including bees, firebugs, roaches, and beetles. Identification scores were obtained for each spectrum of the tested sample. All ticks collected in the
field and removed from patients were tested against the database. When
each tick was tested, particular attention was paid to determine if the
spectral profile matched profiles for the reference samples of the same sex
as the specimen. The Mantel Haenszel test (Epi Info, version 7, program)
was used to evaluate the quality of the sex identification.
(v) Cluster analysis. We performed hierarchical clustering of the mass
spectra of all tested species that were present in or absent from the database using the mean spectrum projection (MSP) dendrogram function of
MALDI Biotyper, version 3.0. The objective was to determine whether the
ticks could be clustered using this approach.
Molecular detection of bacteria in ticks removed from patients. All
DNA samples of ticks removed from patients were screened for Rickettsia
spp. by quantitative real-time PCR (qPCR) analysis of a fragment of the
gltA gene (17). To screen for Bartonella spp., an internally transcribed
spacer was targeted, whereas a fragment of the 16S rRNA gene was targeted for Borrelia spp. (18). Rickettsia massiliae-specific qPCR and Rickettsia conorii-specific qPCR were performed on positive Rhipicephalus
524
jcm.asm.org
sanguineus DNA samples (19). Rickettsia slovaca- and Rickettsia raoultiispecific qPCRs were performed on positive Dermacentor DNA samples
(17). Borrelia-positive samples were confirmed by Borrelia-specific qPCR
amplification of the internal transcribed spacer (ITS) (19), and identification was performed by sequencing a 750-bp gene fragment of the flaB
gene (20).
RESULTS
MALDI-TOF MS spectrum analysis and database assembly. A
total of 120 arthropod specimens, including 67 ticks representing
six species, were subjected to MALDI-TOF MS analysis. The protein spectral profiles of all tested arthropod species were very similar between specimens of the same species within the mass range
of 2 to 20 kDa, and the signal intensities were very strong. The
alignment of the spectra of several specimens of the same species
showed that the major identified protein peaks were present in
each specimen of the same species (Fig. 1). The analysis in ClinProTools of the tick protein profiles yielded spectra comprising
between 60 and 136 peaks in the mass range of 2 to 20 kDa, with an
average of 106 peaks per spectrum. The spectra corresponding to
the legs of other arthropods (Culex pipiens, Triatoma infestans,
Ctenocephalides felis, Pediculus humanus, and Cimex lectularius)
comprised 47 to 64, 94 to 120, 75 to 93, 46 to 76, and 87 to 132
peaks, respectively, in the mass range of 2 to 20 kDa.
The spectra for each species of ticks were shown to be reproducible (Fig. 2) for all tested groups after spectral analysis and
alignment. This reproducibility was observed for both the male
and female specimens. Subsequently, a database was created and
loaded with all of the spectra for these species.
Study validation using a blind test. Querying spectra in the
database yielded satisfactory results, with scores between 2.224
and 2.764. The blind test of the reference database, in which two
208
Journal of Clinical Microbiology
Downloaded from http://jcm.asm.org/ on October 24, 2014 by guest
Incompletely described bacterium (97.6% AY776167.1),
B. miyamotoi (100% FJ874925.1)
MALDI-TOF MS Identification of Ticks
specimens of each of the six tick species maintained in the laboratory as well as the remaining arthropod groups were assayed, successfully identified all species groups. The majority (69%) of specimens were identified to the species level with scores of �2. All
specimens tested had spectra that matched the reference spectra
with scores between 1.749 and 2.519. The spectra of bees, firebugs,
roaches, and beetles, for which references were not included in the
database, scored lower, between 0.778 and 1.066. Regarding the
sex of the specimen, it was not possible to definitively discriminate
male and female ticks based on their MS spectra. However, the
FIG 2 Comparison of the A. variegatum spectral profiles to assess reproducibility and to identify an A. variegatum specimen. (A) Alignment of the spectra of
several specimens of A. variegatum (gel view) showing that the major identified protein peaks were present in each specimen of this species. Arb u, arbitrary units.
(B) Average peak list for the A. variegatum spectral profiles. (C) Results obtained for the blind testing of an A. variegatum specimen against the database using
MALDI Biotyper, version 3.0, software (correct identification with a score of 2.357).
February 2013 Volume 51 Number 2
209
jcm.asm.org 525
Downloaded from http://jcm.asm.org/ on October 24, 2014 by guest
FIG 1 Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectral profiles for the legs of different tick species. (A) Hyalomma (Hy)
marginatum rufipes male and female. (B) Rhipicephalus (Rh) sanguineus male and female in the range of 2 to 20 kDa. Intens, signal intensity; au, arbitrary units.
Yssouf et al.
ticks’ spectral profiles more frequently matched those of the correct sex than the incorrect sex, with 16/22 (72%) matches for
males and 19/29 (65%) matches for females (P � 0.007).
MALDI-TOF MS using ticks from the field and patients. The
field-collected ticks for which the species were represented in the
database (I. ricinus and R. sanguineus) were identified correctly by
MALDI-TOF MS (scores of �1.8). R. sulcatus and Haemaphysalis
concinna ticks from the field (absent from our MALDI-TOF MS
database) did not match any known spectra. Eleven morphologically identified adult ticks (I. ricinus, R. sanguineus, and D. marginatus) removed from patients were correctly identified by
MALDI-TOF MS (identification scores between 1.321 and 2.117).
An Ixodes sp. nymph removed from a patient was identified as I.
ricinus using MALDI-TOF MS. Additionally, the tick that was
extensively damaged was identified as I. ricinus based on its four
spectra, with a score of 1.376. However, one specimen (Rhipicephalus sp.) removed from a patient could not be identified by
MALDI-TOF MS, as the spectrum did not match that of any specimens in the database.
For the phylogenetic analysis, we performed hierarchical clustering of the mass spectra of different species of tested ticks that
were present in or absent from our reference database. In the
generated dendrogram, specimens of the same tick species, including R. sanguineus, I. ricinus, and D. marginatus from the laboratory, field, and patients, clustered together with the exception
of one specimen (Fig. 3).
Molecular identification of ticks and the detection of bacteria. 12S gene sequencing was performed on 13 of 14 specimens
removed from patients. The results corroborated those obtained
by morphological analysis and MALDI-TOF MS for most samples
(Table 1). R. sulcatus and H. concinna collected from the field were
526
jcm.asm.org
not identified by MALDI-TOF MS (no reference spectra in our
database). After two attempts, we were unable to obtain a highquality 12S RNA gene sequence for the damaged tick that was
removed from a patient. This specimen was identified as I. ricinus
by MALDI-TOF MS. Finally, the tick that was morphologically
identified as Rhipicephalus sp. and was not identified by MALDITOF MS was identified by sequencing as Rhipicephalus bursa
(100% agreement with AM410572 in GenBank), for which no
spectrum was available in the MALDI-TOF database. Six of the 14
ticks removed from patients were found to be infected by bacteria,
including Rickettsia massiliae, R. conorii, R. slovaca, R. raoultii,
Borrelia garinii, Borrelia miyamotoi, and Anaplasma phagocytophilum (Table 1). DNA sequences closely related to those of an incompletely described bacterium (21) were also detected in one
specimen of I. ricinus.
DISCUSSION
Although other factors should be considered, the identification of
ticks that have bitten or been removed from patients is the first
step in assessing the risk of infection (1). In this study, all ticks
removed from patients were potential vectors of pathogens that
depend on the involved tick species for propagation. In Europe, D.
marginatus is one of the primary vectors of R. slovaca and R. raoultii, two spotted fever group rickettsiae responsible for tick-borne
lymphadenopathy (TIBOLA), also called Dermacentor-borne
necrosis erythema and lymphadenopathy (DEBONEL) or SENLAT,
which is defined as scalp eschar and neck lymphadenopathy after
a tick bite (21a). I. ricinus is a major vector of several bacterial
agents, including the causative agent of Lyme borreliosis, Borrelia
garinii (3), B. miyamotoi, which causes relapsing fever (21b), A.
210
Journal of Clinical Microbiology
Downloaded from http://jcm.asm.org/ on October 24, 2014 by guest
FIG 3 Dendrogram obtained by cluster analysis of spectra obtained from laboratory-reared ticks, field-collected ticks, and ticks removed from patients. The ticks
were analyzed using MALDI Biotyper software. M, male; F, female; N, nymph; L, ticks from laboratory colonies; F, ticks collected in the field; P, ticks collected
from patients.
MALDI-TOF MS Identification of Ticks
February 2013 Volume 51 Number 2
results of the MALDI-OF MS analysis of arthropod specimens. To
expand our database, we will test a large number of tick species.
Some species are available in collections. However, it seems that
long-term storage in 70% ethanol reduces the reproducibility of
the MALDI-TOF MS spectra (13, 25). It will also be useful to
evaluate the use of MALDI-TOF MS for the identification of frozen specimens.
In conclusion, we showed in this study that MALDI-TOF MS is
an efficient approach for the rapid identification of tick vectors.
This method was used for the first time to identify ticks removed
from patients. The results were obtained rapidly relative to the
time required for molecular methods, and the completion of this
assay does not require any specific entomological expertise. One
damaged tick removed from a patient was successfully identified
using MALDI-TOF MS as I. ricinus, a vector of Lyme disease. The
knowledge of the tick species can be used to inform the clinician’s
decision as to whether to prescribe prophylactic doxycycline treatment. At the very least, the identity of the tick species can tell
physicians which specific clinical signs they should look for in
their patients. The rapid identification of ticks, and most likely of
other arthropod vectors, is now possible in any laboratory with a
MALDI-TOF MS system. In our unit, results are now available for
clinicians in less than 1 h, with no requirement for entomological
expertise. Our database contains reference spectra for ticks removed from humans in our area. This database can be shared and
used directly by any clinical microbiology laboratory equipped
with a MALDI Biotyper system. Because MALDI-TOF studies
with bacteria and yeast have demonstrated significant variation in
the protein profile based on geographical region (27), it will be
interesting to test ticks of the same species from a variety of geographical regions to determine if this technique can be used as a
regional or global tool. We will continue to add new reference
spectra to our database to test the ability of this MS method to
discriminate closely related species and to define a definitive cutoff
score for species identification. Finally, it will be informative to
determine whether MALDI-TOF MS can be used to identify not
only tick vectors but also the microorganisms with which the ticks
are infected.
ACKNOWLEDGMENTS
We thank Arnaud Canet for providing ticks collected in the field, Nicolas
Armstrong for technical assistance, and Guillaume Lacour for providing
mosquito specimens.
REFERENCES
211
1. Parola P, Raoult D. 2001. Ticks and tickborne bacterial diseases in humans: an emerging infectious threat. Clin. Infect. Dis. 32:897–928.
1a.Hubalek Z, Rudolf I. 2012. Tick-borne viruses in Europe. Parasitol. Res.
111:9 –36.
2. Gray J, Zintl A, Hildebrandt A, Hunfeld KP, Weiss L. 2010. Zoonotic
babesiosis: overview of the disease and novel aspects of pathogen identity.
Ticks Tick-Borne Dis. 1:3–10.
3. Stanek G, Wormser GP, Gray J, Strle F. 2012. Lyme borreliosis. Lancet
379:461– 473.
4. Parola P, Paddock CD, Raoult D. 2005. Tick-borne rickettsioses around
the world: emerging diseases challenging old concepts. Clin. Microbiol.
Rev. 18:719 –756.
5. Balicer RD, Mimouni D, Bar-Zeev Y, Levine H, Davidovitch N, Ankol
OH, Zarka SS 2010. Post exposure prophylaxis of tick-borne relapsing
fever. Eur. J. Clin. Microbiol. Infect. Dis. 29:253–258.
6. Piesman J, Hojgaard A. 2012. Protective value of prophylactic antibiotic
treatment of tick bite for Lyme disease prevention: an animal model. Ticks
Tick-Borne Dis. 3:193–196.
jcm.asm.org 527
Downloaded from http://jcm.asm.org/ on October 24, 2014 by guest
phagocytophilum, the causative agent of human granulocytic anaplasmosis (22), and Rickettsia helvetica, an emerging pathogen (4).
Rhipicephalus sanguineus, the brown dog tick, is a primary vector of Rickettsia conorii, the causative agent of life-threatening
Mediterranean spotted fever (23), Rickettsia rickettsii is the vector
of Rocky Mountain spotted fever in southern regions of the
United States (23a), and R. massiliae is an emerging pathogen
(23b). Rhipicephalus bursa is a known vector of several cattle parasites as well as a putative vector of Crimean-Congo hemorrhagic
fever in certain regions of the world, such as Turkey (24). The
potential for these ticks to transmit disease agents was illustrated
in this study by the detection of several pathogens in the ticks that
had bitten patients. This finding highlights the clinical need for the
species identification of ticks.
Our results suggest that the MALDI-TOF MS spectra of protein extracts from tick legs are a suitable tool for identifying ticks.
The results obtained using this method corroborated those from
morphological and molecular identification methods.
Overall, 63% of the laboratory tick specimens were identified
by MALDI-TOF MS with scores of �2, which are considered to be
reliable scores for the identification of bacterial species (15).
Ticks collected in the field or removed from patients were reliably identified with lower score values, but each specimen’s spectrum matched a reference spectrum. Only one tick was not identified by MALDI-TOF MS, R. bursa (identification confirmed by
12S sequencing). This tick was not identified because the corresponding spectrum was not present in our database. The species
not represented in our database, which were tested as controls
(fleas, mosquitoes, bugs, bees, and beetles), had low scores, less
than 1.1, and none of their spectra matched the reference spectra
in the database.
The establishment of a cutoff score for accurate identification
would be ideal. When ticks removed from patients were tested,
most (76%) were identified with scores of �1.7, and all were identified with scores of �1.3. However, it is not known if a score of 1.3
can be used as a definitive cutoff in other regions. The ticks used to
construct the database are representative of the tick species in
France and correspond to the species that have been removed
from French patients, including returned travelers, and sent to our
reference centers in the past 15 years (P. Parola, unpublished observations). In addition, the vectors included in this study are not
closely related, and it must be confirmed that this MALDI-TOF
MS method can differentiate closely related species, e.g., Ixodes
species. However, when D. marginatus specimens removed from
patients were tested against the database, which contained reference spectra for this species and for the closely related species D.
reticulatus, the identification of the specimens was unequivocal.
Using the MSP dendrogram function of MALDI Biotyper, version 3.0, all but one tick (R. bursa) clustered according to their
genera, species, and strain. A similar discrepancy was observed in
a preliminary study using the entire body of the tick (14). Therefore, although MALDI-TOF MS has opened new doors for the
phylogenetic study of arthropods and bacteria, additional data are
still needed, and detailed studies should be conducted.
Contrary to a recent report (14), we found that the use of tick
legs appears to be an effective means to identify tick vectors if a
reference database is available. The remainder of the body can be
reserved for other purposes, such as the detection of pathogens, as
performed in this study. However, various storage conditions (10,
25) and chemical extraction methods (26) appear to influence the
Yssouf et al.
528
jcm.asm.org
19. Socolovschi C, Reynaud P, Kernif T, Raoult D, Parola P. 2012. Rickettsiae of spotted fever group, Borrelia valaisiana, and Coxiella burnetii in
ticks on passerine birds and mammals from the Camargue in the south of
France. Ticks Tick-Borne Dis. 3:355–360.
20. Assous MV, Wilamowski A, Bercovier H, Marva E. 2006. Molecular
characterization of tickborne relapsing fever Borrelia, Israel. Emerg. Infect. Dis. 12:1740 –1743.
21. Skarphedinsson S, Jensen PM, Kristiansen K. 2005. Survey of tickborne
infections in Denmark. Emerg. Infect. Dis. 11:1055–1061.
21a.Parola P, Rovery C, Rolain JM, Brouqui P, Davoust B, Raoult D. 2009.
Rickettsia slovaca and R. raoultii in tick-borne rickettsioses. Emerg. Infect.
Dis. 15(7):1105–1108.
21b.Platonov AE, Karan LS, Kolyasnikova NM, Makhneva NA, Toporkova
MG, Maleev VV, Fish D, Krause PJ. 2011. Humans infected with relapsing fever spirochete Borrelia miyamotoi, Russia. Emerg. Infect. Dis. 17:
1816 –1823.
22. Dumler JS. 2012. The biological basis of severe outcomes in Anaplasma phagocytophilum infection. FEMS Immunol. Med. Microbiol.
64:13–20.
23. Rovery C, Raoult D. 2008. Mediterranean spotted fever. Infect. Dis. Clin.
North Am. 22:515–530.
23a.Demma LJ, Traeger MS, Nicholson WL, Paddock CD, Blau DM, Eremeeva ME, Dasch GA, Levin ML, Singleton J, Jr, Zaki SR, Cheek JE,
Swerdlow DL, McQuiston JH. 2005. Rocky Mountain spotted fever from
an unexpected tick vector in Arizona. N. Engl. J. Med. 353:587–594.
23b.Parola P, Socolovschi C, Jeanjean L, Bitam I, Fournier PE, Sotto A,
Labauge P, Raoult D. 2008. Warmer weather linked to tick attack and
emergence of severe rickettsioses. PLoS Negl. Trop. Dis. 2:e338. doi:10
.1371/journal.pntd.0000338.
24. Estrada-Pena A, Bouattour A, Camicas A, Walker AR. 2004. Ticks of
domestic animals in the Mediterranean region: a guide to identification of
species. University of Zaragoza, Zaragoza, Spain.
25. Kaufmann C, Ziegler D, Schaffner F, Carpenter S, Pfluger V, Mathis A.
2011. Evaluation of matrix-assisted laser desorption/ionization time of
flight mass spectrometry for characterization of Culicoides nubeculosus
biting midges. Med. Vet. Entomol. 25:32–38.
26. Carbonnelle E, Mesquita C, Bille E, Day N, Dauphin B, Beretti JL,
Ferroni A, Gutmann L, Nassif X. 2011. MALDI-TOF mass spectrometry
tools for bacterial identification in clinical microbiology laboratory. Clin.
Biochem. 44:104 –109.
27. Cassagne C, Ranque S, Normand AC, Fourquet P, Thiebault S, Planard
C, Hendrickx M, Piarroux R. 2011. Mould routine identification in the
clinical laboratory by matrix-assisted laser desorption ionization time-offlight mass spectrometry. PLoS One 6(12):e28425. doi:10.1371/journal
.pone.0028425.
212
Journal of Clinical Microbiology
Downloaded from http://jcm.asm.org/ on October 24, 2014 by guest
7. Norris DE, Klompen JS, Keirans JE, Black WC. 1996. Population genetics of Ixodes scapularis (Acari: Ixodidae) based on mitochondrial 16S
and 12S genes. J. Med. Entomol. 33:78 – 89.
8. Mangold AJ, Bargues MD, Mas-Coma S. 1998. 18S rRNA gene sequences
and phylogenetic relationships of European hard-tick species (Acari: Ixodidae). Parasitol. Res. 84:31–37.
9. Song S, Shao R, Atwell R, Barker S, Vankan D. 2011. Phylogenetic and
phylogeographic relationships in Ixodes holocyclus and Ixodes cornuatus
(Acari: Ixodidae) inferred from COX1 and ITS2 sequences. Int. J. Parasitol. 41:871– 880.
10. Seng P, Rolain JM, Fournier PE, La Scola B, Drancourt M, Raoult D.
2010. MALDI-TOF-mass spectrometry applications in clinical microbiology. Future Microbiol. 5:1733–1754.
11. Feltens R, Gorner R, Kalkhof S, Groger-Arndt H, and von Bergen M.
2010. Discrimination of different species from the genus Drosophila by
intact protein profiling using matrix-assisted laser desorption ionization
mass spectrometry. BMC Evol. Biol. 10:95. doi:10.1186/1471-2148-10-95.
12. Perera MR, Vanstone VA, Jones MG. 2005. A novel approach to identify
plant parasitic nematodes using matrix-assisted laser desorption/
ionization time-of-flight mass spectrometry. Rapid Commun. Mass Spectrom. 19:1454 –1460.
13. Kaufmann C, Schaffner F, Ziegler D, Pfluger V, Mathis A. 2012.
Identification of field-caught Culicoides biting midges using matrixassisted laser desorption/ionization time of flight mass spectrometry. Parasitology 139:248 –258.
14. Karger A, Kampen H, Bettin B, Dautel H, Ziller M, Hoffmann B, Suss
J, Klaus C. 2012. Species determination and characterization of developmental stages of ticks by whole-animal matrix-assisted laser desorption/
ionization mass spectrometry. Ticks Tick-Borne Dis. 3:78 – 89.
14a.Beati L, Keirans JE. 2001. Analysis of the systematic relationships among
ticks of the genera Rhipicephalus and Boophilus (Acari: Ixodidae) based on
mitochondrial 12S ribosomal DNA gene sequences and morphological
characters. J. Parasitol. 87:32– 48.
15. Fournier PE, Couderc C, Buffet S, Flaudrops C, Raoult D. 2009. Rapid
and cost-effective identification of Bartonella species using mass spectrometry. J. Med. Microbiol. 58:1154 –1159.
16. Sauer S, Freiwald A, Maier T, Kube M, Reinhardt R, Kostrzewa M,
Geider K. 2008. Classification and identification of bacteria by mass spectrometry and computational analysis. PLoS One 3:e2843. doi:10.1371
/journal.pone.0002843.
17. Bechah Y, Socolovschi C, Raoult D. 2011. Identification of rickettsial
infections by using cutaneous swab specimens and PCR. Emerg. Infect.
Dis. 17:83– 86.
18. Parola P, Diatta G, Socolovschi C, Mediannikov O, Tall A, Bassene H,
Trape JF, Raoult D. 2011. Tick-borne relapsing fever borreliosis, rural
Senegal. Emerg. Infect. Dis. 17:883– 885.
Mini review:
Typhus in World War I
Microbiology today 41 (2), 68 – 71.
213
Ty p h u s
in World War I
Fig. 1. Coloured electron micrograph of a bacterium
of the genus Rickettsia. CNRI/Science Photo Library
Rezak Drali, Philippe Brouqui & Didier Raoult
Epidemic typhus has always accompanied disasters striking
humanity. Famine, cold and wars are its best allies. Typhus, also
known as historical typhus, classic typhus, sylvatic typhus, red
louse disease, louse-borne typhus, and jail fever has caused
mortality and morbidity through the centuries, and on the Eastern
Front during World War I it led to the death of thousands.
68 Microbiology Today May 14 | www.sgm.ac.uk
215
T
he original description of typhus
is thought to have been made in
1546 by Fracastoro, a Florentine
physician, in his treatise of infectious
diseases: De contagione et contagiosis
morbis. His observations during the
Italian outbreaks in 1505 and 1528
allowed him to separate typhus from
the other pestilential diseases. It also
recognised the transmission of typhus
(a)
(b)
from human-to-human. The term
‘exanthematic typhus’ was introduced in
1760 by the French physician, Boissier
de Sauvages. Thanks to PCR testing
of dental pulp from ancient remnants
of bodies from graves, we now have
evidence that typhus and trench fever
were involved in the decimation of the
besiegers of Douai, 1710–1712, during
the War of the Spanish Succession,
and afflicted the soldiers of Napoleon’s
Grand Army in Vilnius in 1812 after their
retirement from Russia (Table 1).
In 1909, epidemic typhus was
found to be transmitted by Pediculus
humanus humanus, the body louse, by
Charles Nicolle, and he received a Nobel
Fig. 2. R. prowazekii-infected (a) or uninfected (b) dead P. humanus humanus. The louse infected with
R. prowazekii became red and developed rectal bleeding before dying. Reproduced from Houhamdi, L. & others
(2002), J Infect Dis 186, 1639–1646; license no. 3337041496430; Oxford University Press
Prize in 1928 for his findings. Nicolle
was able to transmit the typhus from
humans to chimpanzees and then to
macaques through blood transmission
and, finally, from macaque to macaque
via a body louse.
Between 1903 and 1908, Ricketts
identified Rickettsia rickettsii (Fig. 1), the
agent of spotted fever that is closely
related to the agent of typhus. In 1910,
he contracted typhus and died in Mexico
while conducting his experiments.
In 1914, von Prowazek in turn died
Table 1. Potential typhus outbreaks through the history of mankind
Period
Outbreak
Probability
15th century
Conquest of Granada
Likely
Other possible disease
16th century
Mexico
Likely
16th century
Hungarian disease
Likely
1710–1712
War of Spanish Succession
Proven
Trench fever
1812
Napoleonic Wars
Proven
Trench fever
1914–1918
World War I (Russia, Europe)
Proven
Trench fever
1917–1925
Bolshevik Revolution (Russia)
Proven
Other louse-borne
1940–1945
World War II (Europe, North Africa)
Proven
Trench fever
1997
Burundi Civil War (Central Africa)
Proven
Trench fever
Smallpox
(France, Europe)
(Vilnius, Eastern Europe)
diseases
from typhus after confirming Ricketts’
observations. In 1916, Rocha Lima
described the bacterium and named
it Rickettsia prowazekii in honour of
Ricketts and Prowazek.
Body lice infected by R. prowazekii
become red and die shortly thereafter
(Fig. 2). Humans are the principal
reservoir of typhus during outbreaks.
However, a zoonotic reservoir of R.
prowazekii exists. In addition to the
detection of antibodies against R.
prowazekii in a wide range of domestic
and wild animals, R. prowazekii was
isolated from the blood of Egyptian
donkeys and from the spleens, fleas and
lice of the flying squirrel in Florida, USA.
R. prowazekii was also isolated from
Hyalomma spp. ticks recovered from
livestock in Ethiopia and Amblyomma
spp. ticks in Mexico.
A typhus outbreak requires the
occurrence of both body louse outbreak
and a case of bacteraemic typhus
(Brill–Zinsser disease or epidemic
typhus) (Fig. 3). These two conditions
are often combined in wartime, where
stress, lack of hygiene and non-changing
of clothes during the winter months are
common.
Microbiology Today May 14 | www.sgm.ac.uk 69
216
“
“
organisation is disrupted
lesions on the skin. R. prowazekii enters
the skin through these lesions or by
the contamination of conjunctivae or
mucous membranes with louse faeces
containing rickettsiae. Infection through
the aerosols of faeces-infected dust has
also been reported and is the major risk
among physicians.
Body louse outbreak
Clinical manifestations of epidemic
typhus
The body louse is a blood-sucking
ectoparasite, specific to humans, that
lives and multiplies in clothing. During
its life cycle of approximately 35 days,
the female louse lays an average of 200
eggs, which can increase the number
of lice from a few to thousands on
the same individual. The body louse
ingests an average of five meals a day,
generating extremely dry dejections. It
injects various substances when biting
that cause itching, compelling the host
to scratch vigorously thereby generating
Epidemic typhus is a life-threatening,
acute exanthematic feverish disease
that is primarily characterised by the
abrupt onset of fever with painful
myalgia, a severe headache, malaise
and a rash. Non-specific symptoms
sometimes include a cough, abdominal
pain, nausea and diarrhoea. The rash
that is characteristic of epidemic typhus
classically begins a few days after the
onset of symptoms, appearing as a red
macular or maculopapular eruption on
the trunk that later spreads centrifugally
Epidemic typhus is an
unpredictable disease that can
suddenly re-emerge when social
(1)
(2)
to the extremities (Fig. 4). The rash,
which may be hard to see in darkerskinned individuals, except in the axilla,
is classically described as sparing the
palms and soles. Gangrene and necrosis
of toes and fingers that necessitates
amputation has been observed.
Neurologic symptoms include confusion
and drowsiness. Coma, seizures and
focal neurologic signs may develop in a
minority of patients. The mortality rate
varies from 0.7 to 60% for untreated
cases, depending on the age of the
patient, with a case fatality ratio lower
than 5% in patients less than 13 years
old. In self-resolving cases, R. prowazekii
can persist for life in humans, and under
stressful conditions recrudescence may
occur as a milder form of Brill–Zinsser
disease. R. prowazekii bacteraemia
occurs in Brill–Zinsser disease so it can
initiate an outbreak of epidemic typhus
when body lice are present on the
infected individuals.
(3)
Body lice infection
R. prowazekii-infected
body lice infestation
Wars
and cold
Crowding, lack of hygiene
and no clothing change
Body lice outbreak
Brill–Zinsser
Disease
Typhus outbreak
Fig. 3. Outbreak of epidemic typhus. When social organisation is disrupted, (1) body louse outbreak occurs
among defenceless populations and (2) the presence of a case of Brill–Zinsser can initiate (3) an outbreak of
epidemic typhus. Credit
70 Microbiology Today May 14 | www.sgm.ac.uk
217
Fig. 4. Diffuse petechial rash of epidemic typhus.
Credit
relapsing fever and caused by Borrelia
recurrentis.
Conclusion
Fig. 5. Soldiers’ kit bags being placed into gas chambers to be deloused during World War I. Otis Historical
Archives, National Museum of Health and Medicine/Science Photo Library
Typhus in the First World War
The declaration of war by Austria
against Serbia in 1914 following the
assassination of Archduke Ferdinand
quickly expanded into an uncontrollable
global conflict in World War I.
On the Eastern Front, intense
shelling of Serbian cities destroyed
the existing infrastructure and drove
the population to the streets, and at
least 20,000 Austrians were taken
prisoner by the Serbs. There was a
lack of physicians and other medical
professionals because they had been
seconded to the army, which led to the
rapid collapse of the health status of
defenceless populations. Malnutrition,
overcrowding and a lack of hygiene
paved the way for typhus. In November
1914, typhus made its first appearance
among refugees and prisoners, and it
then spread rapidly among the troops.
One year after the outbreak of hostilities,
typhus killed 150,000 people, of whom
50,000 were prisoners in Serbia. A third
of the country’s doctors suffered the
same fate. The mortality rate reached
an epidemic peak of approximately 60 to
70%. This dramatic situation dissuaded
the Germano-Austrian commandment
from invading Serbia in an attempt to
prevent the spread of typhus within their
borders. Drastic measures were taken,
such as the quarantine of people with
the first clinical signs of the disease,
but attempts were also made to apply
standards of hygiene among the troops
to prevent body lice infestations (Fig. 5).
On the Western Front, although
body lice were also endemic among
the troops, there was no outbreak of
typhus. The situation lacked the R.
prowazekii bacteraemia to trigger a
typhus epidemic, as had happened on
the Eastern Front. Another disease,
described for the first time and also
vectored by the body louse, was raging
in the trenches among the troops. It
is caused by the bacterium Bartonella
quintana and was named trench fever.
On the Russian front, throughout the
last two years of the conflict and during
the Bolshevik revolution, approximately
2.5 million deaths were recorded.
Typhus was latent in Russia long before
the beginning of World War I. The
mortality rate rose from 0.13 per 1,000
in peacetime to 2.33 per 1,000 in 1915.
Soldiers and refugees imported typhus
and propagated it across the country.
It was during the hard winter of 1917–
1918 that the biggest outbreak of typhus
in modern history began in a Russia that
was already devastated by famine and
war. The great epidemic started in the
big cities and eventually reached the
distant lands of the Urals, Siberia and
Central Asia.
After World War I, between 1919
and 1923, there were five million deaths
in Russia and Eastern Europe because
of a third disease vectored by body lice,
Epidemic typhus is an unpredictable
disease that can suddenly re-emerge
when social organisation is disrupted, as
was observed in 1997 among Burundi’s
Civil War refugees in central Africa.
Wars are optimal conditions for body
louse proliferation and their associated
diseases. Thus, the control of lice with
the combination of oral Ivermectin,
clean clothes and insecticides will help
to avoid disasters caused by typhus,
trench fever and relapsing fever during
humanitarian catastrophes.
Rezak Drali, Philippe Brouqui &
Didier Raoult
Aix Marseille Université, URMITE, UM63,
CNRS 7278, IRD 198, Inserm 1095, 13005
Marseille, France and Institut HospitaloUniversitaire Méditerranée Infection,
13005, Marseille, France; Corresponding
author Tel. +33 491 32 43 75
didier.raoult@gmail.com
Further reading
Bavaro, M. F. & others (2005). History of US
military contributions to the study of Rickettsial
diseases. Mil Med 170 (Suppl. 1), 49–60.
Bechah, Y. & others (2008). Epidemic typhus.
Lancet Infect Dis 8, 417–426.
Raoult, D., Woodward, T. & Dumler, J. S.
(2004). The history of epidemic typhus. Infect
Dis Clin N Am 18, 127–140.
Tschanz, D. W. (2008). Typhus Fever on the
Eastern Front in World War I. Insects, Disease
and History Website, Entomology Group of
Montana State University, microb.io/1kLupfa
(accessed 18 January 2008).
Zinsser, H. (1935). Rats, lice and history: A
chronicle of pestilence and plagues. New York:
Black Dog and Leventhal Publishers.
Microbiology Today May 14 | www.sgm.ac.uk 71
218
Remerciements
Je commencerai par remercier le Professeur Didier RAOULT pour avoir
accepté de m’accueillir dans son laboratoire. Je suis honoré et
extrêmement fier d’avoir travaillé sous votre direction. Permettez-moi de
vous exprimer mon profond respect.
Je remercie le Professeur Philippe BROUQUI, mon directeur de thèse et
avant cela mon responsable de Master. Je vous exprime ma gratitude pour
m’avoir dirigé, conseillé et aidé à mener à terme cette thèse.
Merci au Professeur Jean-Marc ROLAIN. J’ai beaucoup apprécié
d’avoir travaillé avec vous. Vous avez toujours répondu présent à mes
sollicitations.
Je remercie tous mes co-auteurs que je ne pourrais citer individuellement
car et ils sont nombreux.
Merci au Docteur Idir BITAM. Vos encouragements et vos conseils
m’ont toujours poussé à aller de l’avant. J’espère que l’avenir nous
réserve une longue et fructueuse collaboration.
Je remercie mes rapporteurs d’avoir accepté d’évaluer ce travail.
Je remercie toute l’équipe de l’URMITE et particulièrement tous les
membres avec qui j’ai été amené à travailler.
Enfin, je remercie vivement l’Institut Pasteur d’Algérie, l’institution à
laquelle j’appartiens qui m’a permis d’entreprendre et mener à terme cette
thèse.
219