Isolation and identification of bacterial enteropathogens in children
under five years of age attending Mbagathi District Hospital,
Nairobi, Kenya
Alice Akeyo Ndege
A thesis submitted in partial fulfillment for the degree of Master
of Science in Medical Microbiology in the Jomo Kenyatta
University of Agriculture and Technology
2012
DECLARATION
This thesis is my original work and has not been presented for a degree in any other
University.
Signature………………………………….
Date………………………..
Alice Akeyo Ndege
This thesis has been submitted for examination with our approval as University
supervisors:
Signature……………………………
Date………………………..
Dr. Samuel Kariuki
KEMRI, Kenya
Signature………………………………….
Date………………………..
Prof. Anne W. T. Muigai
JKUAT, Kenya
ii
DEDICATION
This work is dedicated my loving parents Eunice and Henry Ndege for the love, courage
and inspiration that I drew from them during the entire course of my study. This work is
also dedicated to my sister Priscah from whom I drew strength to move on.
iii
ACKNOWLEDGEMENT
I am grateful to the Almighty God for His grace, mercy, love and care that took me
through the whole project until this submission.
I wish to thank Dr. Samuel Kariuki, who was my supervisor, sponsor of the study and
whose support has been great throughout the study period.
I also wish to thank Prof. Anne Muigai for her acceptance, supervision and massive
assistance accorded in the development of this research project.
I extend my sincere gratitude to the Medical personnel at the Mbagathi District Hospital
and Centre for Microbiology Research at Kenya Medical Training Institute.
Special thanks to my parents, Eunice and Henry for the love, support, encouragement
and strength that I drew from them each moment of the way.
Last but not least, I am thankful to the parents and guardians of the study participants
who gave consent for samples to be collected for this study.
May God bless you all.
iv
TABLE OF CONTENTS
DECLARATION ............................................................................................................... ii
DEDICATION .................................................................................................................. iii
ACKNOWLEDGEMENT ............................................................................................... iv
LIST OF TABLES ............................................................................................................ x
LIST OF FIGURES ......................................................................................................... xi
LIST OF PLATES .......................................................................................................... xii
LIST OF APPENDICES ............................................................................................... xiii
LIST OF ABBREVIATIONS ....................................................................................... xiv
ABSTRACT ..................................................................................................................... xv
CHAPTER ONE ............................................................................................................... 1
1.0 INTRODUCTION ....................................................................................................... 1
1.1 Morbidity and mortality of children under the age of five years ................................... 1
1.2 Impacts of bacterial pathogen infections........................................................................ 1
1.2.1 Salmonella infections .................................................................................................. 1
1.2.2 Cholerae infections...................................................................................................... 2
1.2.3 Campylobacteriosis ..................................................................................................... 3
1.2.4 Clostridium infections ................................................................................................. 4
1.2.5 Aeromonas infections.................................................................................................. 5
1.2.6 Plesiomonas infections ................................................................................................ 5
v
1.2.7 Klebsiella infections .................................................................................................... 6
1.3 Transmission of pathogens ............................................................................................. 6
1.4 Enteric pathogens ........................................................................................................... 7
1.4.1 Effects of Salmonella species...................................................................................... 9
1.4.2 Effects of Shigella species ........................................................................................ 10
1.4.3 Effects of Campylobacter species ............................................................................. 11
1.5 Problem Statement ....................................................................................................... 12
1.6 Justification .................................................................................................................. 13
1.7 Research questions ....................................................................................................... 14
1.8 General objective ......................................................................................................... 14
1.8.1 Specific objectives .................................................................................................... 14
CHAPTER TWO ............................................................................................................ 15
2.0 LITERATURE REVIEW......................................................................................... 15
2.1 Diarrhoea ...................................................................................................................... 15
2.1.1 Epidemiology of diarrhea .......................................................................................... 15
2.1.2 Etiology of diarrhea................................................................................................... 16
2.1.3 Bacterial diarrhoea and its causes ............................................................................. 17
2.1.4 Management of diarrhea ........................................................................................... 17
2.2 Classical Bacterial Pathogen Detection Methods ........................................................ 17
2.3 Polymerase Chain Reaction ......................................................................................... 21
2.4 Types of Polymerase Chain Reaction .......................................................................... 22
vi
2.5 Advantages of Multiplex PCR ..................................................................................... 27
2.6 Limitations of multiplex PCR ...................................................................................... 28
2.7.0 Multiplex PCR in detection of bacterial pathogens .................................................. 29
2.7.1 Multiplex PCR in the detection of Salmonella species ............................................. 29
2.7.2 Multiplex PCR in the detection of Shigella species .................................................. 30
2. 7.3 Multiplex PCR in the detection of Vibrio species ................................................... 30
2. 7.4 Multiplex PCR in the detection of Clostridium species ........................................... 31
2.7.5 Multiplex PCR in detection of E. coli ...................................................................... 31
2.7.6 Multiplex PCR in the detection of Plesiomonas ....................................................... 32
CHAPTER THREE ........................................................................................................ 34
3.0 MATERIALS AND METHODS ............................................................................. 34
3.1 Study Site ..................................................................................................................... 34
3.2 Study design ................................................................................................................. 34
3.3 Study Population .......................................................................................................... 34
3.4 Inclusion Criteria .......................................................................................................... 35
3.5 Exclusion criteria ......................................................................................................... 35
3.6 Ethical considerations .................................................................................................. 35
3.7 Sample size estimation ................................................................................................. 35
3.8 Sample collection, transportation and storage ............................................................. 36
3.9 Phenotypic identification of the isolated organisms .................................................... 37
3.10 DNA isolation from stool samples ............................................................................. 37
vii
3.11 DNA Amplification .................................................................................................... 40
3.12 Gel electrophoresis ..................................................................................................... 41
3.13 Data analysis and storage ........................................................................................... 41
3.14 Limitations of the study ............................................................................................. 41
CHAPTER FOUR ........................................................................................................... 42
4.0 RESULTS .................................................................................................................. 42
4.1 Demographic characteristics ........................................................................................ 42
4.2 Bacterial pathogen detection using mPCR with regard to age of the patients ............. 43
Organisms ......................................................................................................................... 44
4.3 Bacterial pathogen detection with regard to sex of the patients................................... 44
4.4 Isolation of bacterial pathogens using culture method ................................................. 46
4.5 Detection of the enteric bacterial pathogens using Multiplex PCR ............................. 46
4.6 E. coli detection using mPCR using Seeplex Diarrhoea-B2 ACE ............................. 47
4.7 Multiple bacterial infections ........................................................................................ 48
4.8 Detection of bacterial multiple infections using Multiplex PCR ................................. 50
CHAPTER FIVE ............................................................................................................. 53
5.0 DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS ....................... 53
5.1 Detection of enteric bacterial pathogens ...................................................................... 53
5.2 Salmonella, Shigella and E. coli species detection ...................................................... 54
5.3 Campylobacter spp. detection ...................................................................................... 56
5.4 Vibrio spp. detection .................................................................................................... 57
viii
5.5 Clostridium spp. detection ........................................................................................... 58
5.6 Conclusions .................................................................................................................. 58
5.7 Recommendations ........................................................................................................ 59
3. REFERENCES .......................................................................................................... 60
APPENDICES ................................................................................................................. 73
ix
LIST OF TABLES
Table 1.1
Estimates of diarrhoea deaths among children under the age of five years
in low and middle income regions of the world, 2004 …..…………... 8
Table 4.1
Demographic characteristics …………………………………………...42
Table 4.2
Biochemical characteristics of bacterial pathogens isolated……….…...44
Table 4.3
Percentage number of organisms detected using Multiplex PCR ……..45
Table 4.4
Organisms isolated using culture method...……………………….…....46
Table 4.5
Organisms detected in association with the age group of the patients ....47
Table 4.6
Bacterial pathogen detection with regard to sex of the patients ……..….48
Table 4.7
Bacterial Co-infections from the 100 stool samples collected …...........49
x
LIST OF FIGURES
Figure 2.1
Worldwide distribution of deaths caused by diarrhoea in children
under the age of five years in 2000…………………................... 9
xi
LIST OF PLATES
Plate 1
mPCR results for enteric pathogens for samples 29-39 …………….…...51
Plate 2
Agarose gel electrophoresis of products from mPCR for samples 1-9 for
E. coli detection….….……………………………………….……...52
Plate 3
mPCR results for enteric pathogens for samples 69-76………….…..…...79
Plate 4
mPCR results for enteric pathogens in samples 50-59………….…………80
Plate 5
Multiplex PCR results for enteric pathogens in samples 60-68..………….81
Plate 6
mPCR results for enteric pathogens for samples 1-10…..………………....82
xii
LIST OF APPENDICES
APPENDIX 1:
Informed consent document .……………………………………..74
APPENDIX 2:
Multiplex PCR plates..…………………………………………….78
APPENDIX 3:
Approval letter from Scientific Steering Committee/KEMRI ..........83
APPENDIX 4:
Approval letter from Ethical Review Committee/KEMRI..………84
APPENDIX 5:
Composition of culture medium……………………………………85
xiii
LIST OF ABBREVIATIONS
CDC
Centres for Disease Control and Prevention
CHERG
Child Health Epidemiology Research Group
CMR
Centre for Microbiology Research
DNA
Deoxyribonucleic acid
EPEC
Enreropathogenic E. coli
ETEC
Enterotoxigenic E. coli
HHV
Human herpes viruses
HUS
Haemolytic uremic syndrome
KEMRI
Kenya Medical Research Institute
KNH
Kenyatta National Hospital
MDH
Mbagathi District Hosiptal
MIC
Minimum inhibitory concentration
mPCR
Multiplex Polymerase Chain Reaction
PCR
Poymerase Chain Reaction
UPEC
Uropathogenic E. coli
USA
United States of America
UTI
Urinary tract infections
WHO
World Health Organization
xiv
ABSTRACT
Bacterial pathogens continue to be the most common cause of enteric infections that
leads to diarrhoea especially in poor-resource settings and is rated by the World Health
Organization as the second contributory factor in the mortality and morbidity of
children. Diagnostic techniques for detection of these pathogens is mainly through
culture that takes long and may not often useful be for instituting immediate treatment.
Multiplex polymerase chain reaction (mPCR) is a technique which enables simultaneous
amplification of many targets of interest in one reaction by using more than one pair of
primers. This study was aimed at isolating and identifying enteropathogens in children
under five years of age attending Mbagathi District Hospital, Nairobi, Kenya
One
hundred stool samples from the children under the age of five years with diarrhoea were
collected in polypots and used for the study. The stool samples were enriched on
Selenite F and then subcultured on XLD and MacConkey agar. Biochemical tests were
carried out to further identify the bacterial species. For evaluation of the mPCR
technique, the Seeplex Diarrhoea-B1 ACE detection was used in a multiplex assay that
permitted simultaneous amplification of target DNA of Salmonella spp. (S.
and S.
enterica), Shigella
dysenteriae), Vibrio spp.
spp.
(S.
flexneri, S.
boydii, S.
(V. cholerae, V. parahamolyticus and V.
bongori
sonnei and S.
vulnificus),
Clostridium difficile toxin B, Campylobacter spp. (C. jejuni and C. coli) and Internal
Control (IC). Out of the 100 samples that were analysed in this study Salmonella spp,
Shigella spp and E. coli (VTEC) showed the highest prevalence of 73%. This was
xv
followed by Vibrio spp at 26%, then Campylobacter spp at 6%. The study found mPCR
to be relatively easy to perform, reproducible and rapid, therefore recommends it in
rapid disease diagnosis and timely decision making for treatment of infection.
xvi
CHAPTER ONE
1.0 INTRODUCTION
1.1 Morbidity and mortality of children under the age of five years
It is estimated that more than 10 million children below five years of age die each year
worldwide, with only six countries accounting for half of these deaths (Black et al.,
2003).
Pneumonia and diarrhoea are the predominant causes, with malnutrition as an
underlying cause in most cases (Lana et al., 2008). Although most mortality under five
years of age occurs in India, Nigeria and China, of the 20 countries with the highest
mortality rates for individuals under five years of age, 19 are in Africa (Lana et al.,
2008).
The World Health Organization (WHO) estimated diarrhoeal deaths among
children aged less than five years in Africa in 2004 to be 17.5% (Lana et al., 2008).
Diarrhoea causes substantial illness among rural sub-Saharan Africans (WHO, 2005).
Children are very susceptible to environmental health related diseases such as diarrhoea
and worm infestations due to their under developed immune systems.
Children bear
almost seventy per cent of the diarrhoeal disease burden in Kenya and this is largely
attributed to unsafe water and poor sanitation (WHO, 2005).
1.2 Impacts of bacterial pathogen infections
1.2.1 Salmonella infections
The frequency of life threatening infections caused by consumption of untreated water
has increased worldwide and is becoming an important cause of mortality in developing
countries (Al-Bari et al., 2006).
1
Salmonella species typically cause an intestinal infection that is accompanied by fever,
abdominal cramps, and diarrhoea with symptoms lasting over one week (Hohmann,
2001). The main exception is typhoid fever, a systemic infection with serious medical
implications (Bhan et al., 2005).
Typhoid is common in the developing world, and
typical symptoms include severe headaches and high fevers, but not diarrhoea.
Salmonellosis is a food and water-borne bacterial infection of man and animals.
Salmonella causes a wide range of human diseases such as enteric fever, gastroenteritis
and bacteremia.
It is estimated that food-borne Salmonella infections are responsible
for 1.3 million illnesses annually worldwide, resulting in 16,000 hospitalizations and
600 deaths (CDC, 2006).
Humans are the only carriers, and infected persons may be
asymptomatic.
Headache, fatigue and rose spots (visible in light skinned people) are
also possible.
These symptoms can be severe especially in young children,
immunocompromised and the elderly.
Symptoms last generally up to a week, and
may/can appear 12 to 72 hours after ingestion of the bacterium (CDC, 2006).
1.2.2 Cholerae infections
Cholera is an important enteric disease, which is endemic to different regions of the
world and has historically been the cause of severe pandemics. Vibrio, a diverse genus
of aquatic bacteria, currently includes 72 species, 12 of which occur in human clinical
samples. Of these 12, three species—Vibrio cholerae, Vibrio parahaemolyticus, and
Vibrio vulnificus—account for the majority of Vibrio infections in humans. Rapid and
2
accurate identification of Vibrio species has been problematic because phenotypic
characteristics are variable within species and biochemical identification requires 2 or
more days to complete (Cheryl et al., 2007). Cholera disease usually caused by Vibrio
cholerae O1 and O139 and is endemic in most of sub-Saharan Africa (Bhattacharya et
al., 2009).
The main symptom of cholera is the massive loss of body fluids through
watery diarrhoea, leading to loss of electrolytes and severe dehydration that may result
in death if untreated. Vibrio cholerae is a natural inhabitant of aquatic environment and
toxigenic strains are causative agents of potentially life-threatening diarrhoea.
Vibrio
cholerae O1 is responsible for the life threatening secretory diarrhoea, mostly associated
with epidemic outbreaks when sanitary conditions are not optimum ; the Asiatic cholera
outbreaks have been linked to consumption of unsafe food and water such as drinking
lake and river water, food sold by the roadside and feasting at funeral gathering (Acosta
et al., 2001).
1.2.3 Campylobacteriosis
In the United States, the incidence of Campylobacter infections was 12.7 cases per
100,000 populations in 2009; this was not a significant change when compared to the
previous three years (Mahmud and Burke, 2009).
Among all age groups, the highest
incidence occurred in children under the age of four years, whereas the highest rate of
hospitalization was in persons older than 50 years.
In developing and developed
countries, continuous increase in the number of C. jejuni has been seen, with incidence
rates as high as 73 cases per 100,000 population reported.
3
Campylobacter
gastroenteritis is especially common during the first five years of life.
The vast
majority of patients fully recover from C. jejuni infection within five days (range, 2-10
d), either spontaneously or after appropriate antimicrobial therapy.
Infection with C.
fetus is a concern in immunocompromised patients, pregnant women, and neonates
(Mahmud and Burke, 2009).
Almost all persons infected with Campylobacter recover without any specific treatment.
Patients should drink extra fluids as long as the diarrhea lasts. In more severe cases,
antibiotics such as azithromycin or erythromycin can shorten the duration of symptoms
if given early in the illness (CDC 2010).
1.2.4 Clostridium infections
Clostridium difficile is one of the common nosocomial and antibiotic resistant infections
in children. It also causes associated diarrhoea in adults; it thus represents a major public
health issue (Jose et al., 2011).
The infection was initially considered clinically
unimportant, buthas recently been seen to have an increased incidence in hospitalized
children (Jose et al., 2011). Both healthy children without significant risk factors and
children with complex medical problems have been reported to suffer significant
morbidity from C. difficile infections. C. difficile is a bacterial enteric pathogen that
causes a broad range of clinical disease from asymptomatic colonization or mild
diarrhoea to life-threatening pseudomembranous colitis. C. difficile produces disease by
toxin production in the colon via two toxins, toxin A and toxin B.
presentation is watery diarrhoea and cramps associated with antibiotic use.
4
The usual
1.2.5 Aeromonas infections
Bacteria of Aeromonas genus belong to the Vibrionaceae Family. They are Gramnegative short rods with rounded ends, aerobe-anaerobe facultative; they are motile by a
single polar flagellum, with the exception of A. salmonicida which is non-motile. Over
the years much work has been carried out in order to identify the virulence factors or the
pathogenetic mechanisms in man and animals; however, at present, only a single factor the 'S' layer of A. salmonicida, can be related to the virulence of this species (Kay et al.,
1981). Most of the other factors have been related to pathogenicity by induction, since
the same factors play an important role in the pathogenesis of infections caused by other
bacterial species (as E. coli). The most common syndrome is confined to the gastroenteric tract; sometimes it runs a course identical to the traveller's diarrhoea (Yamada et
al., 1997). The clinical findings are similar, although less dramatic, to the ones of
cholera: watery diarrhoea, fever and vomit; occasionally the disease can be more severe,
with mucus and/or blood in the faeces. In compromised patients, the gastroenteric form
can develop into a severe abdominal or septicemic infection (Saito and Schick, 1973).
1.2.6 Plesiomonas infections
Plesiomonas shigelloides is an oxidase-positive, fermentative, gram-negative rod.
Plesiomonas shigelloides (family Enterobacteriaceae) has been implicated in
gastroenteritis outbreaks in travelers to tropical regions and in persons who have
ingested contaminated food or water (Adams and Moss, 2008). For persons native to
5
tropical regions, however, case–control studies have found little or no association
between P. shigelloides infection and diarrhea (Bodhidatta et al., 2010).
1.2.7 Klebsiella infections
The most clinically important species of this genus is Klebsiella pneumoniae. This large,
non-motile bacterium produces large sticky colonies when plated on nutrient media.
Klebsiella's pathogenicity can be attributed to its production of a heat-stable enterotoxin.
K. pneumoniae infections are common in hospitals where they cause pneumonia
(characterized by emission of bloody sputum) and urinary tract infections in catheterized
patients. In fact, K. pneumoniae is second only to E. coli as a urinary tract pathogen
(Podschun and Ullmann, 1998).
1.3 Transmission of pathogens
The transmission of bacterial pathogens occurs from person to person through the
faecal-oral pathway, and also by contaminated food and water (Schroeder and Hilbi,
2008). The symptoms of Shigella infection range from mild watery diarrhoea to severe
bacillary dysentery with fever, abdominal pain, blood and mucus in stool samples
(Schroeder and Hilbi, 2008).
C. fetus have affinity for the genital tract (and by the tropism for fetal tissue). C fetus
and rarely C. jejuni are associated with perinatal infection (Mahmud and Burke, 2009).
Abortion or stillbirth and premature labor have been described.
Infants are often
premature and develop signs and symptoms suggestive of sepsis, including fever, cough,
6
respiratory distress, vomiting, diarrhea, cyanosis, convulsions, and jaundice (Mahmud
and Burke, 2009). Infection typically progresses to meningitis, which may be rapidly
fatal or may result in serious neurologic sequelae. The source of the organism in these
cases has been the mother (Mahmud and Burke, 2009).
1.4 Enteric pathogens
The WHO ranks diarrhoeal disease as the second most common cause of morbidity and
mortality in children in the developing world as seen in Table 1.1 (WHO, 2005). This
is mainly due to the underdeveloped immune system of the children to the infections.
The Child Health Epidemiology Research Group (CHERG), created by the WHO in
2001, has shown worldwide distribution of death caused by diarrhoea in children under
the age of five years as seen in Figure 1.1.
A wide array of microbes cause diarrhoea in
children (Haque et al., 2003; Brooks et al., 2003; Steiner et al., 2006).
7
Table 1.1: Estimates of diarrhoeal deaths among children under the age of five years in
low- and middle-income regions of the world, 2004.
WHO region
Mortality
Average
of Estimated
Uncertainty
stratuma
diarrhoea-
diarrhoea deaths ranges
proportional
(thousands)
(thousands)
mortality (%)
D
17.8
402
346–455
E
17.5
365
315–413
B
13.3
35
30–40
D
14.9
14
12–16
B
13.4
12
10–14
D
16.9
221
190–250
B
22.3
44
34–53
D
24.5
651
500–793
Western Pacific (WPR) B
13.8
105
90–118
World
18.7
1870
1558–2193
a
WHO subregions are defined on the basis of levels of child and adult mortality: A, very
low child and very low adult mortality; B, low child and low adult mortality; C, low
child and high adult mortality; D, high child and high adult mortality; E, high child and
very high adult mortality.
8
Figure 1.1: Worldwide distribution of deaths caused by diarrhoea in children less than
five years of age in 2000.
1.4.1 Effects of Salmonella species
In Europe, Spain experiences the highest out breaks of enteric pathogen infections and
the most important serotypes causing disease are Salmonella enterica serotypes
Enteritidis, Typhimurium, Hadar, and subsp.
9
I serotype 4, 5, 12: i: – (Usera et al.,
2001).
Salmonella enterica is one of the major bacterial agents that cause foodborne
infections in humans all over the world (Herikstad et al., 2002).
1.4.2 Effects of Shigella species
Although 99% of the cases of shigellosis occur in the developing world, industrialized
countries such as Norway, Denmark, Australia, Portugal and the United States of
America, among others, have reported outbreaks of foodborne shigellosis in the last
decade. In the United States, Shigella species is reported as the third highest cause of
foodborne disease since 1997, after Salmonella and Campylobacter species (Lee and
Puthucheary, 2002)
In Malaysia, Shigella species was reported to be the third commonest bacterial agent
responsible for childhood diarrhoea.
Members of the genus Shigella, namely S.
flexneri, S. dysenteriae, S. sonnei and S. boydii have caused and continue to be
responsible for mortality and/or morbidity in high risk populations such as children
under five years of age, senior citizens, toddlers in day-care centres, patients in custodial
institutions, homosexual men and, war- and famine-engulfed people.
A study in 2002
by Lee and Puthucheary on bacterial enteropathogens in childhood diarrhoea in a
Malaysian urban hospital showed that Shigella species was the third most common
bacteria isolated.
S.
flexneri and S.
dysenteriae type 1 infections are usually
characterized by frequent passage of small amounts of stool and mucus or blood.
At
times, watery stool followed by typical dysenteric stool maybe present with S.
10
dysenteriae type I infection.
S. sonnei and S.
boydii infections are less severe with
watery faeces but little mucus or blood (Lee and Puthucheary, 2002)
In Costa Rica, Shigella has been isolated from faecal, water and food samples
(Barrantes, et al., 2006; Barrantes et al., 2004).
Other studies confirm that Shigella
species is an endemic pathogen in Central American countries causing severe diarrhoeal
disease that requires hospitalization in young children.
Enteric infections remain an
important cause of morbidity in Chile. Incidence rates of diarrhoea in Chilean children
range from 1.3 to 4.5 per 100,000 inhabitants (Sotomayor, 2000).
Episodes of shigellosis globally have been estimated to be 164.7 million and of these,
163.2 million were in developing countries and the remaining in industrialized nations.
The mortality rate was approximately 0.7% (Kotloff et al., 1999).
1.4.3 Effects of Campylobacter species
Campylobacter infections are among the most common bacterial infections in humans.
They produce both diarrhoeal and systemic illnesses. In industrialized regions, enteric
campylobacter infections produce an inflammatory, sometimes bloody, diarrhoea or
dysentery syndrome.
Campylobacter jejuni is usually the most common cause of
community-acquired inflammatory enteritis. In developing regions, the diarrhoea may
be watery. Campylobacter bacteria, usually transmitted in contaminated food or water,
can infect the gastrointestinal tract and cause diarrhoea, fever, and cramps. Good handwashing and food safety habits will help prevent campylobacter infections (or
campylobacteriosis), which usually clear up on their own but sometimes are treated with
11
antibiotics.
Campylobacter infects over 2 million people each year, and it's a leading
cause of diarrhoea and food-borne illness.
Babies under 1 year old, teens, and young
adults are most commonly affected (Mahmud and Burke, 2009).
Symptoms of campylobacter infections usually appear one to seven days after ingestion
of the bacteria.
The main symptoms of campylobacteriosis are fever, abdominal
cramps, and mild to severe diarrhoea. Diarrhoea can lead to dehydration, which should
be closely monitored.
Signs of dehydration include: thirst, irritability, restlessness,
lethargy, sunken eyes, dry mouth and tongue, dry skin, fewer trips to the bathroom to
urinate, and (in infants) a dry diaper for several hours. In cases of campylobacteriosis,
the diarrhoea is initially watery, but later may contain blood and mucus.
Sometimes,
the abdominal pain appears to be a more significant symptom than the diarrhoea. When
this happens, the infection may be mistaken for appendicitis or a problem with the
pancreas (Mahmud and Burke, 2009).
1.5 Problem Statement
Diarrhoea is a preventable major cause of morbidity and mortality in children less than
five years and yet it is preventable when preventive measures are put in place (WHO,
2005). Members of the genus Shigella have caused and continue to be responsible for
mortality and/or morbidity in high risk populations including children less than five
years of age (Kotloff et al., 1999).
Salmonella species typically causes an intestinal
infection that is accompanied by fever, abdominal cramps, and diarrhoea with symptoms
lasting over one week (Hohmann, 2001).
There is need for the rapid identification of
12
these bacterial pathogens in respective stool samples. This will facilitate giving of the
right treatment and establishment of prevention measures to avert the complications
resulting from these infections.
1.6 Justification
Infections due to bacterial pathogens have a vast effect on systems of patients involved;
children being very susceptible to environmental health related diseases such as
diarrhoea and worm infestations due to their under developed immune systems (Garrett
et al., 2008).
The major factors contributing to diarrhoea morbidity and mortality in
developing countries are lack of access to safe clean water and sanitation (Garrett et al.,
2008).
Although mortality rates have declined in many countries in recent years,
largely as a result of environmental improvements (e.g. in access to effective sanitation
and safe drinking water) and advances in health care and treatment (e.g. oral rehydration
therapy), outbreaks of diarrhoeal diseases continue to affect many millions of children.
Traditional detection methods are based on cultures using selective media and
characterization of suspicious colonies by biochemical and serological tests.
methods are generally time-consuming.
These
Thus the development of a rapid multiplex
assay to identify the presence of bacterial pathogens within a specimen will have a
profound impact on the efficient management of disease, treatment and prevention,
clinical follow-up studies, and the development of new therapies of prophylactic or
therapeutic vaccines.
Hence the cases of morbidity and mortality will be reduced if
there is a rapid method of diagnosing a broad spectrum of bacterial pathogens causing
13
diarrhoea and when the results obtained are acted upon on time to assist in the treatment
of the patients. Therefore, a rapid method is necessary for identification of Salmonella
serotypes from clinical specimens.
1.7 Research questions
1. Can enteric bacterial pathogens be simultaneously detected using multiplex PCR directly
from stool samples?
2. What is the prevalence of enteric bacterial pathogens causing diarrhoeal cases in
children less than five years seeking health care at the Mbagathi District Hospital?
3. What are the demographic and clinical characteristics of children less than five years
with these enteric pathogens seeking health care at the Mbagathi District Hospital?
1.8 General objective
To isolate and identify enteropathogens in children under five years of age attending
Mbagathi District Hospital, Nairobi, Kenya
1.8.1 Specific objectives
1. To identify enteric bacterial pathogens associated with diarrhoea using mPCR in
children under the age of five years attending Mbagathi District Hospital, Nairobi.
2. To determine the prevalence of enteric bacterial pathogens associated with diarrhea in
children less than five years seeking health care at the Mbagathi District Hospital.
3. To evaluate the utility of mPCR in the detection of enteric bacterial pathogens associated
with diarrhoea in children under the age of five years attending Mbagathi district
Hospital.
14
CHAPTER TWO
2.0 LITERATURE REVIEW
2.1 Diarrhoea
Diarrhoea is the passage of 3 or more loose or liquid stools per day, or more frequently
than is normal for the individual. It is usually a symptom of gastrointestinal infection,
which can be caused by a variety of bacterial, viral and parasitic organisms. Infection is
spread through contaminated food or drinking-water, or from person to person as a result
of poor hygiene. Severe diarrhoea leads to fluid loss, and may be life-threatening,
particularly in young children and people who are malnourished or have impaired
immunity (WHO, 2012). There are three clinical types of diarrhoea: acute watery
diarrhoea – lasts several hours or days, and includes cholera; acute bloody diarrhoea –
also called dysentery; and persistent diarrhoea – lasts 14 days or longer (WHO, 2012).
2.1.1 Epidemiology of diarrhea
Every year there are about two billion cases of diarrhoeal disease worldwide. Diarrhoeal
disease is a leading cause of child mortality and morbidity in the world, and mostly
results from contaminated food and water sources. Worldwide, around 1 billion people
lack access to improved water and 2.5 billion have no access to basic sanitation.
Diarrhoea due to infection is widespread throughout developing countries (WHO, 2012).
In 2004, diarrhoeal disease was the third leading cause of death in low-income countries,
causing 6.9% of deaths overall. In children under five years old, diarrhoeal disease is the
second leading cause of death – second only to pneumonia (WHO, 2012). Out of the 1.5
15
million children killed by diarrhoeal disease in 2004, 80% were under two years old. In
developing countries, children under three years old experience on average three
episodes of diarrhoea every year. Each episode deprives the child of the nutrition
necessary for growth. As a result, diarrhoea is a major cause of malnutrition, and
malnourished children are more likely to fall ill from diarrhoea (WHO, 2012).
2.1.2 Etiology of diarrhea
Diarrhoea is a symptom of infections caused by a host of bacterial, viral and parasitic
organisms, most of which are spread by faeces-contaminated water. Infection is more
common when there is a shortage of clean water for drinking, cooking and cleaning.
Rotavirus and Escherichia coli are the two most common causes of diarrhoea in
developing countries. Children who die from diarrhoea often suffer from underlying
malnutrition, which makes them more vulnerable to diarrhoea. Each diarrhoeal episode,
in turn, makes their malnutrition even worse. Diarrhoea is a leading cause of
malnutrition in children under five years old. Water contaminated with human faeces,
for example, from sewage, septic tanks and latrines, is of particular concern. Animal
faeces also contain micro organisms that can cause diarrhoea. Diarrhoeal disease can
also spread from person-to-person, aggravated by poor personal hygiene. Food is
another major cause of diarrhoea when it is prepared or stored in unhygienic conditions.
Water can contaminate food during irrigation. Fish and seafood from polluted water may
also contribute to the disease.
16
2.1.3 Bacterial diarrhoea and its causes
The main etiology of the diarrhea is related to a wide range of bacteria (such as
Campylobacter jejuni, Escherichia coli, Salmonella species, Vibrio cholerae, Yersinia
enterocolitica,
and
Aeromonas
species),
enteroparasites
(Giardia
species,
Criptosporidium species, and Entamoeba histolytica), and viruses (adenovirus, Norwalk
virus, and rotavirus).
2.1.4 Management of diarrhea
In 2003 the Center for Disease Control (CDC), put forth new recommendations for the
management of acute pediatric diarrhea in both the outpatient and inpatient settings
including indication for referral. Oral rehydration therapy is the cornerstone of treatment
for small bowel infections that produce a large volume of watery stool output.
Antimicrobial therapy is indicated for some non viral diarrhoea because most is self
limiting and does not require therapy (CDC 2003).
2.2 Classical Bacterial Pathogen Detection Methods
Traditionally, the detection test of food-borne micro organisms is made by plating a food
homogenate on highly selective media, although in the case of some bacteria a preenrichment step is required (Hayes, 1985). However, positive identification cannot be
confirmed by growth on a selective medium alone because many enteric bacteria possess
similar biochemical characteristics. A second culture medium is often used for
confirmation, and as a result, the analysis time is increased. After several days of
17
incubation, the presence or absence of the microorganism or the number of colonies is
determined (Hayes, 1985).
Pathogenic bacteria are often present in very low numbers therefore several plates may
be needed to streak the entire sample (Hill, 1996).
Moreover, the simultaneous
detection and identification of several pathogens by use of one differential medium plate
is not possible.
2002).
As a result, culture methods are time-consuming and tedious (Osek,
This plating technique, based on the phenotype of the bacteria, is labour-
intensive and can take several weeks to obtain results (Hill et al., 1985).
Shigella is considered a fastidious pathogen for bacteriological isolation, which in the
context of indigenous micro flora and other substances makes detection less feasible.
Immunologic methods of detection are usually specific, but cross-reactions can occur
(Gryko et al., 2002).
Currently, international guidelines and regulations for the detection of Salmonella
species in foods are based on traditional culture methods, which take at least five days
for confirmation of results. Despite its widespread use, serotyping has deficiencies that
limit its utility, including that it often takes three or more days to generate a result and
approximately 5 to 8% of isolates are partially typed or untyped (Bopp et al., 2003).
This can be caused by several factors which include the blocking of exposure of the
surface O antigens due to capsular polysaccharides in mucoid strains and in “rough”
18
strains that produce partially formed O antigens that can cross-react with different O
antisera (Bopp et al., 2003).
These types of isolates can be partially typed by their H antigens (Bopp et al., 2003).
With H-antigen typing, both flagellar phases must be assayed; this entails testing one
flagellar phase at a time. Nonmotile isolates can be partially typed by their O antigens.
Further problems with serotyping include that prolonged subculturing can affect the
antigenic properties of the strain, highly trained laboratorians are required to type strains
accurately, antisera must be available, and the high costs of producing and validating
specific antisera to rare antigens are problematic. Delays caused by identification can
hinder the response to an outbreak of disease and/or its epidemiologic surveillance (Kim
et al., 1999).
Identification of diarrhoeagenic Salmonella, Shigella and E. coli strains requires that
these organisms be differentiated from non pathogenic members of the normal flora.
Serogrouping of O antigen is not sufficient to identify a strain as diarrhoeagenic,
because it does not correlate, in most cases, with the presence of virulence factors.
Conventional methods used to detect and classify cholera-causing vibrios isolated from
clinical and environmental samples require several days to complete and involve culture
in alkaline peptone water, thiosulfate citrate bile sucrose agar, slide agglutination with
specific antisera, and assay for production of cholera toxin (Sakazaki, 1992). However,
V. cholerae O1 or O139 can be detected rapidly by using probes or primers employing
19
the rfb region responsible for O-antigen biosynthesis (Albert et al., 1997). PCR and
multiplex PCR including two genes have also been used to detect the presence of
virulence-associated and regulatory genes (Fields et al., 1992).
From a diagnostic point of view, toxigenic- pathogenic and nontoxigenic-nonpathogenic
strains of V. cholerae can be differentiated by the presence of the cholera toxin and
toxin-coregulated pilus genes. However, it is not known whether non-cholera toxinproducing strains possess other virulence genes, such as zot, ace, a part of the cholera
toxin genetic element, and ompU. Although ompU has no role in the adhesion of these
methods, it can be relied on individually to identify all V. cholerae strains and detect
virulence genes in a one-step PCR. Recently, a PCR-based method targeted to the toxR
gene was developed for species-specific identification of Vibrio parahaemolyticus (Kim
et al., 1999).
The optimum method for the detection of campylobacters from stool samples in the
diagnostic laboratory remains selective culture. campylobacter species detection PCR
based methods
CCDA, charcoal cefoperazone desoxycholate agar, enzyme linked
immunosorbent assay ELISA.
Campylobacter were first isolated from humans in 1938 from the blood cultures of
patients suffering from diarrhoea. They could not be isolated from the faeces because of
the overgrowth of plates by commensal faecal flora and because of their own fastidious
nature. The breakthrough of their successful isolation from faeces came in 1972 when
20
Butzler used the technique of membrane filtration( Dekeyser et al., 1972). This was
followed in 1977 by the development of a selective medium by Skirrow, which enabled
the isolation of campylobacter with greater ease ( Skirrow, 1977). The selective medium
was designed to isolate the two species known at the time to cause gastroenteritis,
Camylobacter jejuni and C. coli.
Moreover, biochemical identification to the species level is limited and unreliable for
campylobacters; hence they are identified only to the genus level. The combination of
not being able to detect the unusual species by the current method, and not identifying to
the species level the common species that are isolated, has contributed to a limited
understanding of the epidemiology of campylobacter gastroenteritis
2.3 Polymerase Chain Reaction
Polymerase Chain Reaction (PCR) is used to amplify a specific DNA (target) sequence
lying between known positions (flanks) on a double-stranded (ds) DNA molecule. The
PCR can be used to amplify both double and single stranded DNA. PCR amplifies small
DNA targets 100-1000 base pairs (bp) long. It is technically difficult to amplify targets
>5000 bp long. A pair of single stranded oligonucleotide primers, which have DNA
sequences complementary to the flanking regions of the target sequence, must be
synthesized. The primers are complementary to either end of the target sequence but lie
on opposite strands. The primers are usually 20-30 nucleotides long and bind to
complementary flanking region at 3' end.
21
2.4 Types of Polymerase Chain Reaction
Allele-specific PCR is a variation of the PCR which is used as a diagnostic or cloning
technique, to identify or utilize single-nucleotide polymorphisms (SNPs) (single base
differences in DNA). It requires the sequence of the target DNA sequence, including
differences between the alleles. It is used as a diagnostic or cloning technique, to
identify or utilize single-nucleotide polymorphisms (SNPs). It uses primers whose 3′
ends encompass the SNP. An allele-specific oligonucleotide (ASO) will only anneal to
sequences that match it perfectly, a single mismatch being sufficient to prevent
hybridization under appropriate conditions. Hot start PCR is a variation of the PCR
which reduces non-specific amplification during the initial set up stages of the PCR. It
inhibits the polymerase’s activity at ambient temperature, either by the binding of an
antibody. Methylation-specific PCR. is a variation of the PCR which can rapidly assess
the methylation status of virtually any group of CpG sites within a CpG island,
independent of the use of methylation-sensitive restriction enzymes. It analysis uses
bisulfite-treated DNA but avoids the need to sequence the area of interest. Primer pairs
are designed to be “methylation-specific” by including sequences complementing only
unconverted 5-methylcytosines, or “unmethylation-specific”, complementing thymines
converted from unmethylated cytosines. Methylation is determined by the ability of the
specific primer to achieve amplification.
22
Reverse transcription PCR is a sensitive method for the detection of mRNA expression
levels. It involves two steps: RNA is first reverse transcribed into cDNA using a reverse
transcriptase and then the resulting cDNA is used as templates for subsequent PCR
amplification using primers specific for one or more genes. RT-PCR is widely used in
expression profiling, which detects the expression of a gene. Reverse Transcription PCR
is also used for insertion of eukaryotic genes into prokaryotes. Quantitative PCR is used
widely to detect and quantify specific DNA sequences in scientific fields that range from
fundamental biology to biotechnology and forensic sciences. It quantitatively measures
starting amounts of DNA, cDNA, or RNA. Multiplex PCR uses multiple primer sets
within a single PCR mixture to produce amplicons of varying sizes that are specific to
different DNA sequences. By targeting multiple genes at once, additional information
may be gained from a single test-run that otherwise would require several times the
reagents and more time to perform. Annealing temperatures for each of the primer sets
must be optimized to work correctly within a single reaction, and amplicon sizes. That
is, their base pair length should be different enough to form distinct bands when
visualized by gel electrophoresis.
Assembly PCR is a PCR variation that artificial synthesizes long DNA sequences by
performing PCR on a pool of long oligonucleotides (primers) with short overlapping
segments. Helicase-dependent amplification PCR is almost similar to the traditional
PCR but uses a constant temperature rather than cycling through denaturation and
23
annealing/extension cycles. DNA helicase is used to unwind DNA. Inverse PCR is a
variant of PCR, and is used when only one internal sequence of the target DNA is
known. It is therefore very useful in identifying flanking DNA sequences of genomic
inserts. Intersequence-specific PCR amplifies the region between simple sequence
repeats to produce a unique fingerprint of amplified fragment lengths. It is an efficient
tool in phylogenetic classification of prokaryotic genomes in general and diagnostic
genotyping of microbial pathogens in particular. Ligation-mediated PCR is a technique
that amplifies DNA fragments between an insertion sequence element and an MspI
restriction site.
Miniprimer PCR uses miniprimers which are about 9 to 10 nucleotide s for detecting
sequences beyond those detected by standard methods using longer primers and Taq
polymerase. Solid phase PCR is where DNA oligonucleotides complementary to a
soluble template are immobilized on a surface are extended in situ. Although primarily
used for pathogen detection, SP-PCR has the potential for much broader application,
including disease diagnostics, genotyping, and expression studies. Touchdown PCR is
another modification of conventional PCR that may result in a reduction of nonspecific
amplification. It involves the use of an annealing temperature that is higher than the
target optimum in early PCR cycles. The annealing temperature is decreased by one
degree centigrade every cycle or every second cycle until a specified or ‘touchdown’
annealing temperature is reached. The touchdown temperature is then used for the
24
remaining number of cycles. This allows for the enrichment of the correct product over
any non-specific product.
PCR has revolutionized the field of infectious disease diagnosis to overcome the
inherent disadvantage of cost and to improve the diagnostic capacity of the test.
Nucleic-acid based diagnostic techniques typically rely on unbiased PCR amplification
of the targets, which requires a set of general PCR amplification primers to hybridize
upstream and downstream of the variable region, followed by either real-time PCR,
hybridization assays or template sequencing for read-out interpretation. Multiplex PCR
has the potential to decrease cost, time and effort in pathogen diagnostics (Elnifro et al.,
2000). Engineering an efficient multiplex PCR requires laborious strategic planning in
primer design, nucleotide concentrations, optimal salt and buffer conditions, and use of
chemical adjuvants and is rarely capable of achieving multiplex degrees greater than a
20-plex.
The field of molecular diagnostics is experiencing a revolution in regards to
theranostics, the integration of diagnostic technologies with therapeutic applications
(Bissonnette and Bergeron, 2006).
Ligase-based technology has led to myriad promising techniques such as the padlock
probe and the proximity ligation assay which have both been investigated as potential
pathogen diagnostic methods (Gustafsdottir et al., 2006; Szemes et al., 2005). Presence
of inhibiting substances found in complex biological samples can interfere with cell lysis
25
or inactivate the DNA polymerase, and DNA extraction procedures are usually necessary
to remove them (Al-Soud and Radstrom, 2000).
The utility of multiplex PCR as a tool for pathogen detection in clinical and
environmental samples is well documented (Lee et al., 2003).
Over the last 20 years,
alternative strategies to replace or complement traditional serotyping methods have been
proposed. These include ribotyping (Esteban et al., 1993), ribosomal DNA intergenic
spacer amplification (Jensen and Hubner, 1996), random amplification of DNA
polymorphism (Shangkuan and Lin, 1998), IS200 analysis (Ezquerra et al., 1993, Uzzau
et al., 1999), real-time PCR (Hoorfar, et al., 2000), PCR-single-strand conformation
polymorphism analysis (Nair, et al., 2002), amplified fragment length polymorphism
(Torpdahl and Ahrens, 2004), sequence analysis (Mortimer, et al., 2004 ), multiplex
PCR (Alvarez et al., 2004.), and DNA micro arrays (Porwollik et al., 2004.).
Other
laboratories have taken protein-based approaches to type Salmonella enterica by
methods including protein arrays (Cai et al., 2005.) and mass spectrometry (Wilkes et
al., 2005).
Multiplex PCR is a variant of PCR which enables simultaneous amplification of many
targets of interest in one reaction by using more than one pair of primers. Typically, it
is used for genotyping applications where simultaneous analysis of multiple markers is
required, detection of pathogens or genetically modified organisms, or for microsatellite
analyses (Bissonnette and Bergeron, 2006). Multiplex assays can be tedious and timeconsuming to establish, requiring lengthy optimization procedures.
26
It
was first
described by Chamberlain and others in 1988 and since then, been applied in many areas
of DNA testing including, analyses of deletions, mutations, and polymorphisms, or
quantitative assays and reverse transcription.
2.5 Advantages of Multiplex PCR
Polymerase chain reaction (PCR) has revolutionalized the field of disease diagnosis as it
is more sensitive and specific. As the number of biomarkers for microbial agents and
disease markers (Ji et al., 2006) increases, simultaneous detection of multiple agents or
risk factors implicated in particular clinical syndromes or diseases that share similar
epidemiological features become highly desirable to avert severity of infections.
PCR
has the potential to decrease cost, time and effort in pathogen diagnostics (Elnifro et al.,
2000).
The quality of the template may be determined more effectively in multiplex than in
single locus PCR.
Degraded templates give weaker signals for long bands than for
short. A loss in amplification efficiency due to PCR inhibitors in the template samples
can be indicated by reduced amplification of an abundant control sequence in addition to
the amplification of rarer target sequences in an otherwise standardized reaction,
(Hofreiter et al., 2001).
The exponential amplification and internal standards of multiplex PCR can be used to
assess the amount of a particular template in a sample.
To quantitate templates
accurately by multiplex PCR, the amount of reference template, the number of reaction
27
cycles, and the minimum inhibition of the theoretical doubling of product for each cycle
must be accounted (Hofreiter et al., 2001). In the simplest method of quantitation, the
gene multiplexes for major deletions detect carriers or duplications in probands when the
band intensity of abnormal amplicons is compared with that of normal, homozygous
fragments in the multiplex.
2.6 Limitations of multiplex PCR
Potential problems in PCR include false negatives due to reaction failure or false
positives due to contamination.
False negatives are often revealed in multiplex
amplification because each amplicon provides an internal control for the other amplified
fragments, (Hofreiter et al., 2001).
For example, multiple exons may be amplified in
assays that survey for gene deletion. Unless the entire region scanned by the multiplex
PCR is deleted, amplification of some fragment(s) indicates that the reaction has not
failed.
Furthermore, because major deletions are usually contiguous, results that
suggest non-contiguous deletions based on the absence of bands usually reflect
artifactual failure of some fragments to amplify.
In multiplex assays where closely related templates such as pathogen strains are
distinguished by amplifying differing sequence, primers for a sequence common to all
templates provide a positive control for amplification. The main challenge of designing a
multiple PCR assay is the possibility for primer dimers and non specific products. So, it
is necessary to design primers with close annealing temperatures, to begin the program
28
with a hot start, and to use reference strains to determine reaction specificity (Hofreiter
et al., 2001).
2.7.0 Multiplex PCR in detection of bacterial pathogens
2.7.1 Multiplex PCR in the detection of Salmonella species
More recently, attention has focused on molecular based methods due to their
sensitivity, specificity and reduced assay time.
Conventional PCR based assays for
Salmonella detection in foods have been widely reported (Whyte et al., 2002; Myint et
al., 2006; Moreira et al., 2008).
In a study done in Ireland, real time multiplex PCR using four primer sets and four
Taqman probes was developed for the detection of multiple Salmonella serotypes in
chicken samples (Regan et al., 2008). The study aimed at developing a rapid detection
method for multiple Salmonella serotypes based on real-time multiplex PCR and to
compare it to the traditional culture method (Regan et al., 2008).
Poultry-associated
serotypes that were detected in the assay include Enteritidis, Gallinarum, Typhimurium,
Kentucky and Dublin (Regan et al., 2008).
A multiplex PCR method was developed to differentiate between the most common
clinical serotypes of Salmonella enterica subsp.
enterica encountered in Washington
State and the United States in general (Seonghan et al., 2006).
The results from a
blind test screening 111 clinical isolates revealed that 97% were correctly identified
using the multiplex PCR assay.
The assay showed that it can be easily performed on
29
multiple samples with final results in less than 5 hours and, in conjunction with pulsedfield gel electrophoresis, forms a very robust test method for the molecular subtyping of
Salmonella enterica subsp. enterica (Seonghan et al., 2006).
2.7.2 Multiplex PCR in the detection of Shigella species
In a study done in Kaeng-Khoi District, Saraburi Province, Thailand using Real Time
PCR to detect Shigellosis, the ipaH real time PCR assay was found to be a highly
sensitive method for Shigella species detection when compared to conventional culture
methods (Samosornsuk and Chaicumpa, 2007). The sensitivity of the assay reached to
100% in patients with dysentery.
The most likely explanation for the observed
differences in detection rates by real time PCR are variations in bacterial load. Shigella
species is more difficult to detect in patients who shed only a few organisms compared
to individuals who shed a large bacterial load and thus a large amount of Shigella DNA
(Samosornsuk and Chaicumpa, 2007).
2. 7.3 Multiplex PCR in the detection of Vibrio species
In a study to determine the prevalence of virulence genes (ctxA, tcpA, zot and ace)
among Vibrio cholerae isolated from outbreaks during 2005 in Iran by multiplex PCR
method, the PCR analysis of the strains revealed that 100, 100, 82.1 and 82.1% carried
the ctxA, zot, tcpA and ace genes, respectively (Sadeghifard, and Maleki 2005).
30
2. 7.4 Multiplex PCR in the detection of Clostridium species
Infants, particularly those younger than two years of age, frequently are colonized
without symptoms. Most studies have failed to demonstrate any relationship between C.
difficile and common forms of diarrheal illness in this age group (Tang et al., 2005). The
reported rates of detection of toxigenic C. difficile and C. difficile toxins have been
similar in infants with and without diarrhea. The causes of this apparent resistance to
illness are unknown. Among preschool-age children with diarrhea, one group found
those with diarrhea had fecal C. difficile or C. difficile toxin more often than those
without (Mitchell et al., 1996). However, most studies in this age group, both in
ambulatory settings and in hospital, have found no association between diarrhea and the
presence of C. difficile toxins (Vernacchio et al., 2006).
2.7.5 Multiplex PCR in detection of E. coli
Escherichia coli O157:H7 can cause serious gastrointestinal illness. These bacteria are
commonly found in untreated and ineffectively treated sewage and water and pose a
threat to public health. It is the leading cause of morbidity and mortality among children
in developing countries (Hill, 1996).
The ubiquitous and virulent nature of these
bacterial pathogens creates a need for specific, sensitive, and rapid detection techniques.
Molecular techniques such as PCR have been invaluable tools for the detection of
pathogens (Wang et al., 2002).
31
A number of E. coli O157:H7 genes have been targeted for diagnostic amplification by
PCR, including those encoding the Shiga toxins (stx1 and stx2), eaeA, hlyA, fliC, and
several genes from the E. coli O157 O-antigen synthesis operon (Wang et al., 2002).
Real-time PCR allows for quantification of the target, and when combined with a rapid
cycling platform, results can be generated in 30 minutes from the start of thermal
cycling. Because of the advantages of real-time and rapid-cycle real-time PCR, many
assays that perform better than the standard culture-based assays have been developed to
detect pathogenic organisms (Uhl et al., 2002).
A real-time multiplex PCR assay was developed for the detection of diarrhoeagenic E.
coli and it proved to be specific and rapid in detection of virulence genes from Shiga
toxin-producing E. coli (STEC) in stool samples (Vidal et al., 2004). In a study carried
out in Chilean children with diarrhoea in detection of diarrhoeagenic E. coli, the results
of this study indicated that it is possible to perform simultaneous amplification of
virulence
genes
from
different
enterohemorrhagic,
enterotoxigenic,
and
enteropathogenic E. coli strains (Vidal et al., 2004).
2.7.6 Multiplex PCR in the detection of Plesiomonas
A rapid and efficient procedure for quantitative detection of Plesiomonas shigelloides in
pure culture was developed, which is the first report for quantitative detection of P.
shigelloides by the polymerase chain reaction (PCR). A minimum of 4 CFU per PCR
reaction could be detected. There was a linear relationship between the relative
32
fluorescent intensities of the DNA bands and the log of the CFU from 4 to 1.2 × 10 3
CFU per PCR reaction. We compared the effects of two different nucleic acid dyes,
GelStar™ stain and ethidium bromide (EB), and evaluated the effects of two different
staining methods with each dye. Adding the dye to the agarose solution before gel
formation proved to yield superior results compared to staining the DNA bands after
electrophoresis. The GelStar™ stain was found superior to EB with minimum detection
levels of 4 CFU and 12 CFU per PCR reaction, respectivel (Weimin et al., 2006)
33
CHAPTER THREE
3.0 MATERIALS AND METHODS
3.1 Study Site
The study was carried out at the Paediatric Clinic in Mbagathi District Hospital, Nairobi.
The samples were processed at the Kenya Medical Research Institute, (KEMRI) Centre
for Microbiology Research (CMR), Nairobi. Mbagathi District Hospital is a government
hospital under the Ministry of Health, Kenya. It is located in Dagoretti Division in
Nairobi county which is the capital city of Kenya. It lies at an altitude of 1,670 meters
above sea level, latitude 36 degrees, 50 degrees east and longitude 1 degree, 17 degrees
south about 140 km south of the equator.
3.2 Study design
This was a laboratory based cross-sectional study.
3.3 Study Population
The study population included children under the age of five years attending the
Mbagathi District Hospital, Nairobi and were presenting with diarrheic stool.
These
were children who were seeking care at the hospital at the paediatrics clinic. They were
approached by the clinician attending to them and referred to the investigator for
explanation on the project and consent was sought from their parents or guardians.
They were given an extra plastic pot to put stool samples for those whose parents gave
consent to be part of the study.
34
3.4 Inclusion Criteria
All children presenting with diarrhoea who were less than five years of age and for
whom consent was obtained were included in the study.
3.5 Exclusion criteria
Children not presenting with diarrhoea were not included in the study
Children who were more than five years old were not be included in the study
Children whose parents did not give consent were not included in the study
3.6 Ethical considerations
Clearance to carry out the study was obtained from Kenya Medical Research Institute
(KEMRI), Scientific Steering Committee and Ethical Review Committee of KEMRI
(SSC/ERC Number-1602) (Appendix 3 and 4).
All procedures were carried out in
accordance to the CMR/ KEMRI Biosafety guidelines and waste disposal.
Informed
consent was also obtained from the parents or guardians of the children that participated
in the study.
The consent forms also captured the demographic characteristics of the
patients
3.7 Sample size estimation
According to a study done to determine the prevalence of enteropathogens in stools of
rural Maasai children under five years of age in the Maasai region of the Kenyan Rift
Valley, it was established that the prevalence is 13% (Mcguigan et al., 1996).
Therefore the following formula was used to determine sample size (Cochran, 1963);
n= Z2 X p q/ d2
35
Where n = Desired sample size (if target population is greater than 10,000)
Z = Standard normal deviation at the required confidence interval
(1.96)
p = proportion in the target population estimated to have measured character (0.13)
q=1–p
d = level of statistical significance at 95% confidence level = 0.05
Therefore sample size calculation is;
= (1.96) 2 X 0.13 X (1-0.13)/.052
=3.8416 x 0.13 x 0.87/0.0025
=3.8416 x 0.13 x 348
= 173.7939, rounded off 174 samples was used
3.8 Sample collection, transportation and storage
Fresh 100 stool samples were collected in clean poly pots from children under five years
of age with diarrhoea after consent had been obtained from the parents or guardian. 300
parents/ guardians were approached for this study. Only 178 parents/guardians
consented to the study and stool specimens were obtained from their children. The
acceptance rate was 59%. This was then transported whole to the laboratory at the
Centre for Microbiology Research where it was stored at -4o C prior to processing for
two hours.
36
3.9 Phenotypic identification of the isolated organisms
Samples were examined macroscopically to check for consistency, colour of the stool;
whether or not it was blood stained or had mucus.
The samples were enriched in
peptone water on Selenite F to enhance their growth for 18-24 hours at 37oC, then
subcultured on MacConkey agar to differentiate the lactose fermenters from non- lactose
fermenters.
The colonies obtained were bio-typed using Motility Indole Orythine
(MIO) media, Simmon’s Citrate Agar and Triple Sugar Iron (TSI). Colonies obtained
were purified on the Mueller Hinton agar.
MIO (Motility-Indole-Ornithine) is a
semisolid medium used for the differentiation of the Enterobacteriaceae group by
motility, ornithine decarboxylase activity and indole production. Xylose lysine
deoxycholate agar (XLD agar) is a selective growth medium used in the isolation of
Salmonella and Shigella species from clinical samples and from food (Composition of
culture media is attached at the appendix).
3.10 DNA isolation from stool samples
Deoxyribonucleic acid (DNA) was isolated from stool samples according to
manufacturer’s (Qiagen) manual (QIAamp DNA stool handbook for DNA purification
from stool samples, Germany).
One hundred and eighty to two hundred and twenty milligrams of stool was weighed in a
2 ml microcentrifuge tube and placed on ice. For liquid sample, 200 μl of the sample
was pippeted into the microcentrifuge tube. The end of the pipette tip was cut to make
37
pipetting easier (Qiagen). Of the Buffer ASL, 1.4ml was added to each stool sample
then vortexed continuously for 1 min or until the stool sample was thoroughly
homogenized.
The suspension was heated for 5 min at 70°C. (this heating step
increases total DNA yield 3- to 5-fold and helps to lyse bacteria and other parasites
(Qiagen).
The suspension was then vortexed for 15 sec and sample centrifuged at
20,000 x g ( approximately 14,000rpm) for 1 min to pellet stool particles. Of the
supernatant , 1.2ml was pippeted into a new 2 ml microcentrifuge tube and the pellet
discarded.
One InhibitEX Tablet was then added to each sample and vortexed
immediately and continuously for 1 minute or until the tablet was completely suspended.
Suspension was then incubated for 1 minute at room temperature to allow inhibitors to
adsorb to the InhibitEX matrix. The sample was then centrifuged at 20,000 x g (
approximately 14,000rpm) for 3 minutes to pellet inhibitors bound to InhibitEX matrix.
All the supernatant was pipette into a new 1.5 ml microcentrifuge tube and the pellet
discarded. The sample was centrifuged at 20,000 x g ( approximately 14,000rpm) for 3
min. Fifteen μl proteinase K was pipetted into a new 1.5 ml microcentrifuge tube. Two
hundred μl of the resultant supernatant was pippeted into the 1.5 ml microcentrifuge tube
containing proteinase K. Two hundred μl of Buffer AL was added and vortexed for 15
sec.
The tube was then incubated at 70°C for 10 minutes then centrifuged briefly to
remove drops from the inside of the tube lid . Two hundred μl of ethanol (96–100%)
was added to the lysate, and mixed by vortexing, then centrifuged briefly to remove
drops from the inside of the tube lid. The lid of a new QIAamp spin column placed in a
38
2 ml collection tube was labeled and the complete lysate from step the previous step was
carefully transferred to the QIAamp spin column without moistening the rim. The cap
was closed and centrifuged at 20,000 x g ( approximately 14,000rpm) for 1 minute.
The QIAamp spin column was placed in a new 2 ml collection tube, and the tube
containing the filtrate discarded. The QIAamp spin column was carefully opened and
500 μl Buffer AW1 added to it.
The cap was closed and centrifuged at 20,000 x g (
approximately 14,000rpm) for 1 minute.
The QIAamp spin column was placed in a
new 2 ml collection tube, and the collection tube containing the filtrate discarded. The
QIAamp spin column was carefully opened and 500 μl of Buffer AW2 added. The cap
was closed and centrifuged at 20,000 x g ( approximately 14,000rpm) for 3 minute. The
collection tube containing the filtrate was discarded. The QIAamp spin column was
placed in a new 2 ml collection tube and the old collection tube with the filtrate
discarded.
minute.
It was then centrifuged at 20,000 x g ( approximately 14,000rpm) for 1
This step helped to eliminate the chance of possible Buffer AW2 carryover.
The QIAamp spin column was transferred into a new, labeled 1.5 ml microcentrifuge
tube.
The QIAamp spin column was carefully opened and 200 μl of Buffer AE
pippetted directly onto the QIAamp membrane. The cap was closed and incubated for 1
minute at room temperature, then centrifuged at 20,000 x g ( approximately 14,000rpm)
for 1 minute to elute DNA.
39
3.11 DNA Amplification
DNA isolated was amplified by use of Multiplex PCR. Two kits were used in the
amplification of the DNA, these were Seeplex Diarrhoea-B1 ACE and Seeplex
Diarrhoea-B2 ACE. The Seeplex Diarrhoea-B1 ACE detection was used in a multiplex
assay that permitted simultaneous amplification of target DNA of Salmonella species
(S. bongori and S. enterica), Shigella species (S. flexneri, S. boydii, S. sonnei and
S. dysenteriae), Vibrio species (V. cholerae, V.parahamolyticus and V. vulnificus),
Clostridium difficile toxin B, Campylobacter species (C.
jejuni and C. coli) and
Internal Control (IC). The Seeplex Diarrhoea-B2 ACE Detection is a multiplex assay
that permitted simultaneous amplification of target DNA of Clostridium perfringens
toxin, Yersinia enterocolitica, Aeromonas species (A. salmonicida, A. sobria, A.
bivalvium, A. hydrophila), E.coli O157:H7, Verocytotoxin-producing Escherichia coli
(VTEC) and Internal Control (IC).
For each sample a reaction mix was prepared by adding 4 μl of 5X DB PM1 / PM2, 3
μl of 8-Mop Solution, 10 μl of 2X Multiplex Master Mix.
volume of PCR master mix.
centrifuged for 2 minutes.
This gave a 17 μl total
The master mix was then mixed by quick vortexing and
17 μl of the reaction mix was dispensed into 0.2 ml PCR
tubes and, 3 μl of each sample’s nucleic acid was then added, giving a total volume of
reaction at 20 μl. For the negative control PCR, 3 μl of the DV/DB ACE NC was used
instead of sample’s nucleic acid.
Only internal control band is expected in negative
control. For the positive control PCR, 3 μl of the DV/DB ACE PC was used instead of
40
sample’s nucleic acid.
thermal cycler.
The tubes were then sealed and placed in a preheated (940)
The cycling conditions were an initial denaturation at 94o for 15
minutes, template denaturation at 94o for 50 seconds, annealing at 60o for1.5 min, and an
extension at 72o for 1.5 minutes for a total of 40 cycles with a final extension at 72o for 1
minute.
3.12 Gel electrophoresis
Seven micro litre samples of reaction mixtures were analyzed by gel electrophoresis in
0.8% agarose, dissolved in 1× TAE (40 mM Tris-Acetate, 1 mM EDTA at pH 8.3) for
60 minutes at 130V.
After staining the sample with ethidium bromide, the amplicons
were photographed by Gel Documentation Analyzer.
3.13 Data analysis and storage
The data entry took place both at the MDH and the KEMRI, CMR. The data were
entered into Microsoft Excel (2007) worksheets. This data was cleaned and then entered
into the Statistical Package for Social Sciences (SPSS®) version 17.0.
Data obtained
was presented as tables of frequencies and bar graphs. All data was stored in a secure
computer with a backup located in the CMR (KEMRI) server.
3.14 Limitations of the study
There is yet to be developed a multiplex kit that can detect all diarrhoea cases including
E. coli and viral pathogens which account for 30% of diarrheal cases in childhood.
PCR detects both dead and live pathogens hence data needs to be carefully interpreted
and in reference to clinical examination.
41
CHAPTER FOUR
4.0 RESULTS
4.1 Demographic characteristics
In this study a total of 100 samples was collected. Out of this 54 were from male patients
while 46 were from female patients. The rest of the demographic characteristics are as
shown below (Table 4.1).
Table 4.1 Demographic characteristics
Age group of the patients
Number of patients
0-6 months
9
7-12 months
7
13-18months
12
19-24 Months
9
25-30 Months
11
31-36 Months
10
37-42 Months
11
43-48 Months
10
49-54 Months
15
55- 60Months
6
Total
100
42
4.2 Bacterial pathogen detection using mPCR with regard to age of the patients
Salmonella infection was most prevalent at 21% (15/73) in patients aged 25-30 months
followed by 18% (13/73) in patients aged 37-42 months with the lowest prevalence (0%)
being in 0-6 age group (Table 4.2). Infection caused by Shigella species was found to be
highest at 21% (15/73) among patients aged 49-54 months, this was followed by 15%
(11/73) age 43-48 months and there was no Shigella infection detected at age 0-6
months (Figure 4.1). The highest prevalence of Campylobacter spp was at 34% (9/26) in
age group 0-6 months followed by 15% (4/26) at age groups 7-12months and 13-18
months (Table 4.2). The least prevalence was at 0% in age groups 49-54 months and
55-60 months (Table 4.2).The highest prevalence of Vibrio spp. detected was at 67%
(4/6) in 7-12 months of age followed by 17% (1/6) in patients aged 19-24months and 3136months (Table 4.2). There was no detection of Vibrio spp in the rest of the age
groups.
Vibrio spp was the only organism detected in patient aged 0-6 months.
Highest VTEC infection was detected in the 13-18 age group at 16% (12/73), followed
by 37-42 age group at 15% (11/73) (Table 4.2).
No VTEC was detected in 0-6 age
group. Highest E. coli O157; H7 was detected in 31-36 age group at 18% (6/33),
followed by 13-18 and 43-48 age group at 15% (5/33).
No E. coli O157; H7 was
detected in 0-6 age group. No Clostridium spp, Yersinia enterocolitica and Aeromonas
spp. (A. salmonicida, A. sobria, A. bivalvium, A. hydrophila) was detected in all the
100 samples processed in the study (Table 4.2).
43
Table 4.2 Bacterial pathogen detection using mPCR with regard to age of the patients
Salmonella
Shigella
Vibrio
Campy Clostridi VTEC
E.
lobact um
O157
coli
Organisms
er
Age group
0-6 months
0
0
9
0
0
0
0
7-12 months
5
1
4
4
0
7
2
13-18months
7
5
4
0
0
12
5
19-24 Months
4
9
3
1
0
5
3
25-30 Months
15
8
2
0
0
9
3
31-36 Months
10
10
1
1
0
10
6
37-42 Months
13
9
2
0
0
11
4
43-48 Months
9
11
1
0
0
8
5
49-54 Months
7
15
0
0
0
5
3
55- 60Months
3
5
0
0
0
6
2
Total
73
73
26
6
0
73
33
4.3 Bacterial pathogen detection with regard to sex of the patients
Out of the 100 stool specimens, 54 were from male patients while 46 were from female
patients (Table 4.3). Salmonella infection had overall prevalence at 73%, infection in
male children was at 45% (33/73), while in females it was at 55% (40/73) (Table 4.3).
Shigella infection had overall prevalence at 73%, infection in male children was at 48%
(35/73), while in females it was at 52% (38/73) (Table 4.3).
Campylobacter spp.
infection had overall prevalence at 26%, infection in male children was at 62% (16/26),
44
while in females it was at 38% (10/26) (Table 4.3).Vibrio spp. Had overall prevalence at
6%, infection in male children was at 67% (4/6) while in females it was at 33% (2/6).
VTEC infection was more prevalent in the male patients at 53% (39/73) compared with
the female patients which was at 47% (34/73). E. coli O157;H7 infection was more
prevalent in male patients at 64% (21/33) compared with the female patients which was
at 36% (12/33). No Clostridium spp. was detected in both male and female children
(Table 4.3).
Table 4.3: Bacterial pathogen detection with regard to sex of the patients
Sex
Males n (%)
Females n (%)
Salmonella
33 (45%)
40 (55%)
Shigella
35 (48%)
38 (52%)
Campylobacter
16 (62%)
10 (38%)
Vibrio
4 (67%)
2 (33%)
VTEC
39 (53%)
34 (47%)
E. coli O157;H7
21(64%)
12 (36%)
Clostridium
0 (0%)
0 (0%)
Organism Detected
45
4.4 Isolation of bacterial pathogens using culture method
Microbiological tests confirmed that majority (73%) of the patients who presented with
diarrhoeic stools had E. coli infections while 5% and 3% had Salmonella and Shigella
respectively (Table 4.4).
Table 4.4: Organisms isolated using culture method
Organism isolated
Percentage number of organisms isolated (%)
E. coli
73
Salmonella
5
Shigella
3
4.5 Detection of the enteric bacterial pathogens using Multiplex PCR
Multiplex PCR was used to analyse the samples. Two different kits were used for this
reason. The Seeplex Diarrhoea-B1 ACE permitted simultaneous amplification of target
DNA of Salmonella species (S. bongori and S. enterica), Shigella species (S. flexneri,
S. boydii, S. sonnei and S.
dysenteriae), Vibrio species
(V. cholerae, V.
paraheamolyticus and V. ulnificus), Clostridium difficile toxin B, Campylobacter
species (C. jejuni and C. coli) and Internal Control (IC). Out of the 100 samples that
were analysed in this study Salmonella and Shigella showed the highest prevalence of
73%. This was followed by Vibrio at 26%, then Campylobacter species at 6%.
Clostridium species was detected using the Multiplex PCR (Table 4.5).
46
No
Table 4.5: Percentage number of organisms detected using Multiplex PCR using
Seeplex Diarrhoea-B1 ACE
Organism’s Name
Number
of
organisms
detected (%)
Salmonella
73 (73%)
Shigella
73 (73%)
Vibrio
26 (26%)
Campylobacter
6 (6%)
Clostridium
0(%)
4.6 E. coli detection using mPCR using Seeplex Diarrhoea-B2 ACE
E. coli being an important bacterial pathogen was detected using the Seeplex DiarrheaB2 ACE that permitted the simultaneous amplification of target DNA of Clostridium
perfringens toxin, Yersinia enterocolitica, Aeromonas species (A. salmonicida, A.
sobria, A.
bivalvium, A.
hydrophila), E.coli O157:H7, Verocytotoxin-producing
Escherichia coli (VTEC). Out of the 100 samples analyzed using mPCR, 33 (33%)
were positive for E.coli O157:H7 and 73 (73%) were positive for Verocytotoxinproducing Escherichia coli (VTEC).
No Clostridium perfringens toxin, Yersinia
enterocolitica and Aeromonas species (A. salmonicida, A. sobria, A. bivalvium, A.
hydrophila) was detected (Table 4.6).
47
Table 4.6: Percentage number of E. coli organisms detected using Multiplex PCR using
Seeplex Diarrhoea-B2 ACE
Organism detected
Percentage detected
VTEC
73
E. coli 0157; H7
33
Clostridium perfringens toxin
0
Yersinia enterocolitica
0
Aeromonas species
0
4.7 Multiple bacterial infections
Out of the 100 stool samples analysed 52% had Salmonella and Shigella co infection.
Of this 28/52 were females while 24/52 were males (Table 4.7). Salmonella and Vibrio
co infection was 18% with 11/18 males and 7/18 were females.
Salmonella and
Campylobacter co infection was 4% with males and females equally co infected at 2/4
(Table 4.7).
Shigella and Vibrio co infection was 17% with more females 10/17 co
infected compared to 7/17 males (Table 4.7). Shigella and Campylobacter co infection
was 4%, of this 3/4 were females while 1/4 were males. Campylobacter and vibrio coinfection was 3% with 1/3 females while 2/3 males co infected (Table 4.7). Out of the
100 stool samples analysed Salmonella, Shigella and Vibrio co- infections was at 14%,
of this eight were females while six were males. Salmonella, Shigella, Campylobacter
and Vibrio infections were at 2% both stool samples were from female patients (Table
4.7).
48
Table 4.7: Bacterial Co- infections from the stool samples collected from children less
than five years of age attending peadriatic clinic at MDH during the months of
September to December 2009
Percentage bacterial co infection (Odds ratio)
Salmonel
Shigella
VTEC
la
Salmonella
Shigella
Campylo
Clostri
O157
bacter
dium
52%
51%
22%
18%
4%
0%
(0.71)
(0.66)
(0.63)
(0.78)
(0.72)
(0.00)
48%
27%
17%
4%
0%
(0.15)
(2.05)
(0.61)
(0.72)
(0.00)
24%
17%
3%
0%
(0.98)
(0.61)
(0.34)
(0.00)
19%
2%
0%
(11.63)
(1.02)
(0.00)
3%
0%
(3.09)
(0.00)
VTEC
E.
E. coli Vibrio
coli
O157
Vibrio
Campyloba
0%
cter
(0.00)
Clostridium
49
4.8 Detection of bacterial multiple infections using Multiplex PCR
The plate below show Multiplex PCR amplification for detection of enteric bacterial
pathogens. The targeted pathogens were Salmonella, Shigella, Campylobacter, Vibrio
and Clostridium.
Salmonella and Shigella both at 73% were the most prevalent
organisms detected using the multiplex PCR, followed by Vibrio at 26% and
Campylobacter
at 6%.
No Clostridium organisms were detected in all samples
analysed.
In plate 4.1Shigella species was detected in all the 11 samples, Salmonella was detected
in 10 samples, Vibrio species in five of the samples, Campylobacter in 2 of the 11
samples (Plate 4.1). No Clostridium species was detected among these samples notably
samples 31, 33, 36, 37 and 39 had multiple infections of Salmonella, Shigella and
Vibrio. Sample 36 had the highest number of multiple isolates of Salmonella, Shigella,
Vibrio and Campylobacter, while sample 32 had only Shigella species
50
P
29
30
31
32
33
34
35
36
37
38
39
M
Plate 4.1: mPCR results for enteric pathogens for samples 29-39.
Key: M molecular size marker (1000bp, 900bp, 800bp, 700bp, 600bp, 500bp,
400bp,400bp, 300bp, 200bp, 100bp); 29-39 Sample results, P; Positive Control, NNegative Control, F; Campylobacter species 227bp, E; Shigella species 330bp,
D;Salmonella species 395bp, C; Clostridium difficile toxin B 476bp, B; Vibrio species
651bp, A; Internal control 1000bp
The plate below show Multiplex PCR amplification for detection of enteric bacterial
pathogens.
The targeted pathogens were Clostridium perfringens toxin, Yersinia
enterocolitica, Aeromonas spp.
(A.
salmonicida, A.
51
sobria, A.
bivalvium, A.
hydrophila), E.coli O157:H7, Verocytotoxin-producing Escherichia coli (VTEC). Out of
the 100 samples analyzed using mPCR, 33 (33%) were positive for E.coli O157:H7 and
73 (73%) were positive for Verocytotoxin-producing Escherichia coli (VTEC). No
Clostridium perfringens toxin, Yersinia enterocolitica and Aeromonas species
(A.
salmonicida, A. sobria, A. bivalvium, A. hydrophila) was detected
Plate 4.2: Agarose gel electrophoresis of products from mPCR for samples 1-9 for E.
coli detection
P; Positive control and N; negative control.
A; Internal Control (IC) (1000bp), B;
Clostridium perfringens toxin (700bp), C; Yersinia enterocolitica (580bp), D; E.coli
O157:H7 (370bp), E; Verocytotoxin-producing Escherichia coli (VTEC) (291bp), F:
Aeromonas species (217bp) (A. salmonicida, A. sobria, A. bivalvium, A. hydrophila)
52
CHAPTER FIVE
5.0 DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS
5.1 Detection of enteric bacterial pathogens
PCR has revolutionized the field of infectious disease diagnosis.
To overcome the
inherent disadvantage of cost and to improve the diagnostic capacity of the test, mPCR,
a variant of the test in which more than one target sequence is amplified using more than
one pair of primers, has been developed. Since its first description (Chamberlain et al.,
1988) PCR has been applied in many areas of DNA testing, including analyses of
deletions, mutations, and polymorphisms, or quantitative assays and reverse
transcription. mPCR detecting viral, bacterial, and/or other infectious agents in one
reaction tube have been described (Elnifro et al., 2009) . Early studies highlighted the
obstacles that can jeopardize the production of sensitive and specific multiplex assays,
but more recent studies have provided systematic protocols and technical improvements
for simple test design (Elnifro et al., 2009).
Several reports referring to rapid
identification of bacterial species by mPCR coupled to micro arrays detection
demonstrated the usefulness of this approach and the growing interest in implementing it
in routine diagnostics (Mikhailovich et al., 2008).
Multiplex assays can be tedious and time-consuming to establish, requiring lengthy
optimization procedures.
Multiplex assays used to identify the presence of a wide
variety of microbes within a specimen have a profound impact on the efficient
53
management of disease treatments and prevention, clinical follow-up studies, and the
development of new therapies of prophylactic or therapeutic vaccines (Akhras et al.,
2007).
In this study mPCR assay for detection and amplification of enteric bacterial pathogens,
Salmonella and Shigella showed the highest prevalence of 73%. The 27% of children
that had diarrhoea could have been due to other etiologies like drugs or even viral
infections. This was followed by Vibrio at 26%, then Campylobacter spp at 6%.
No
Clostridium spp was detected. The Multiplex PCR was standardized and compared to
the reference culture method.
5.2 Salmonella, Shigella and E. coli species detection
The results of this study are similar to those on Multiplex PCR for rapid detection of
different Salmonella enterica serovars that showed that all reference and clinical isolates
of S.
enterica were accurately identified with no cross reaction with other
enterobacterial strains tested (Karami et al., 2007). Another study that had similar
results was a study done by Seonghan et al in 2006 to compare PCR with conventional
serotyping, 111 clinical isolates were and that 97% were correctly identified using the
multiplex PCR assay. A study by Juan et al., (2004) was also in agreement with the
findings of this study, where a multiplex PCR was applied to clinical stool specimens,
the prevalent serotypes Enteritidis and Typhimurium were detected with a sensitivity of
93%, specificity of 100%, and efficiency of 98% the overall agreement of the multiplex
PCR with conventional culture-based techniques was 95% for Salmonella typing using
54
Cohen's kappa index. A similar study by Trafny et al.,(2006) where a novel multiplex
PCR assay was found to be specific for detection of S. enterica serovar Enteritidis in
human faeces.
It is also noteworthy that the results of this study were similar to another study (Lee and
Fairchild, 2006) where multiplex PCR was found to be a powerful diagnostic tool for
detection of micro organisms in clinical samples and the research laboratory hence was
used as an alternative to the conventional culture method in detecting Shigella and
enteroinvasive Escherichia coli (EIEC) virulence genes ipaH and ial in lettuce because
the diagnostic sensitivity and specificity was found to be 100% accurate while the
culture method detection limit was 106 CFU/ml, diagnostic sensitivity was 53% and
diagnostic specificity was 100%.
A study by Aranda et al, (2004), where a multiplex PCR was used to differentiate typical
and atypical enteropathogenic Escherichia coli (EPEC), enteroaggregative E. coli
(EAEC), enterotoxigenic (ETEC), enteroinvasive E. coli (EIEC) and Shiga toxinproducing E. coli (STEC) strains in Egyptian children found multiplex PCR to be
specific and sensitive for rapid detection of target isolates in stools. The findings by
Aranda et al, (2004) are in agreement with those that were found in this study where out
of the 100 target isolates in stools samples analyzed using mPCR, 33 (33%) were
positive for E.coli O157:H7 and 73 (73%) were positive for Verocytotoxin-producing
Escherichia coli (VTEC).
55
5.3 Campylobacter spp. detection
In this study Campylobacter spp was detected in 6 (6%) samples. Even though in this
study there were very few (one hundred) isolates, Campylobacter infection was found to
decrease with increase in age of the patients.
These findings were found to coincide
with other studies in developing countries, where symptomatic campylobacter infection
mainly affects children younger than five years and declines with age (Mahmud and
Burke, 2009). The reasons as to why Campylobacter infection was found to decrease
with increase in age is likely to be due to the development of protective immunity
secondary to a high level of exposure to the organism early in life. This could also be
the probably reason for the trend seen in the study. Campylobacter jejuni is usually the
most common cause of community-acquired inflammatory enteritis in developing
countries (Mahmud and Burke, 2009).
The results of this study where 6 (6%) of Campylobacter spp was detected in stool
samples by multiplex PCR assay and no detection by culture method are contradicting a
study by Persson and Katharina in 2005 where a multiplex-PCR method, developed for
the detection of Campylobacter coli and Campylobacter jejuni, the sensitivity was 100
cells per ml stool, indicating that culturing of campylobacters on modified charcoal
cefoperazone deoxycholate agar (mCCDA) plates was superior to direct DNA extraction
at least when fresh stool samples were analysed by PCR. The most likely reason for this
56
discrepancy is that the human stool sample was spiked and grown in mCCDA plates
before PCR was done.
In this study multiplex PCR assay rapidly detected 6 (6%) of Campylobacter spp in
stool samples. These findings a similar to a study by Yamazaki-Matsune et al., (2007),
where a multiplex PCR assay was developed for the identification of the six common
Campylobacter taxa associated with human gastroenteritis and/or septicaemia multiplex
PCR assay was a rapid, simple and practical tool for identification of the six
Campylobacter taxa and offered an effective alternative to conventional biochemicalbased assays. The results of this study are similar to Wang et al., (2002), where a
multiplex PCR assay to simultaneously detect Campylobacter was found to be rapid,
easy to perform, had a high sensitivity and specificity.
5.4 Vibrio spp. detection
Twenty six percent (26%) of the samples analysed by multiplex PCR were positive for
pathogenic Vibrio spp. Even though this study had very few (one hundred) isolates to
conclude as to whether the prevalence of 26% is high or low, the study attribute the
positive pathogenic Vibrio spp to the poor hygiene standards found among the habitat
surrounding Mbagathi hospital as the majority of them are the once visiting this health
facility.
A similar study by Huq et al., (2005) also acknowledges that cholera
57
epidemics to be related to environmental factors where clean drinking water is not
available to local populations.
5.5 Clostridium spp. detection
In this study no Clostridium difficile (C.
difficile) was detected by the mPCR.
likely reason as to why it was not detected is that C.
The
difficile is the mainly acquired
through nosocomial and antibiotic-associated diarrhoea in adults (Jose et al., 2011), no
child could have acquired C. difficile infection as all of them were out patients children
attending pediatric clinic and were not hospitalized.
A study by Jose et al., (2011)
indicates that children with C. difficile infection had longer hospitalizations compared
with those who didn't have the infection.
5.6 Conclusions
1. Salmonella, Shigella, Vibrio, Campylobacter and VTEC associated with
diarrhoea were identified using mPCR in children under the age of five years
attending Mbagathi District Hospital, Nairobi.
2. The Salmonella, Shigella and VTEC showed the highest prevalence of 73%.
This was followed by Vibrio at 26%, then Campylobacter species at 6%.
No
Clostridium species was detected using the Multiplex PCR
3. Multiplex PCR was found to be to be relatively easy to perform, reproducible
and rapid.
58
5.7 Recommendations
1. Patients less than five years of agepresenting with diarrhoea and attending
Mbagathi District Hospital be screened for enterobacterial pathogens
2. Community health education on personal hygiene, treatment of human waste and
access to treated water should be enhanced so as to reduce the incidence of
diarrhoea among pre- school children.
3. Multiplex PCR be used as an epidemiological and diagnostic purpose in
management of patients with enteric pathogens as it was found to be relatively
easy to perform and reproducible.
59
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72
APPENDICES
Appendix 1: Informed consent explanation and consent form
You are being invited to take part in this research study because your child has presented
with diarrhoea. Diarrhoea is a serious disease in young children. The study is aimed at
identifying major germs that could cause diarrhoea in children. The germs targeted are
Escherichia coli, Non-typhoidal Salmonella and Shigella which you can get from
contaminated food or water or from contact with people or animals that are infected with
them and they cycle between humans and sometimes between humans and animals.
If you can, please read all of the following information.
If you cannot read, please
listen to the person explaining this information to you very carefully.
Ask the
investigator, to explain to you any words, terms, or sections that are not clear to you.
You should also ask any questions that you have about this research study.
decide to take part in this research study, you will be asked to sign this form.
should keep your copy for your records.
If you
You
It has information, including important names
and telephone numbers, which you can use in the future either to ask questions, for
clarifications.
Project title:
Simultaneous detection of enteric bacterial pathogens in diarrhoeal stool specimens from
children less than five years attending Mbagathi District Hospital.
73
1. Purpose of the study
This study aims to simultaneously detect enteric bacterial pathogens in diarrhoea stool
specimens from children less than five years attending Mbagathi District Hospital.
2. Procedure’s to be followed
This is a non-invasive procedure where there is no pain.
In this study, watery stool
samples will be obtained from your child. Once investigation is over the results will be
communicated to you through the attending clinician for a period not exceeding one
month.
3. Risks
There are no risks involved
4. Benefits
The study that is being undertaken is expected to yield results on possible bacterial
pathogens which may be infecting the children leading to diarrhoea.
Once these
organisms are isolated and identified, it will assist attending clinician to manage problem
efficiently and proper medication will be given to the patient for speedy recovery.
In
case we detect the presence of infections your doctor will be informed for appropriate
treatment and management as is routine in this hospital.
5. Confidentiality of the records
The results that are related to this study will be maintained in confidence.
The
specimen will be given a number and thus the name will not be identified by any person.
74
No identity of any specific patient in this study will be disclosed in any public reports or
publications.
The results will only be submitted to the clinician and revealed to the
patient.
6. Obtaining additional information
Patient will be encouraged to ask any questions to clarify any issues at any time during
the participation in this study. Copy of this agreement will be given to the patient for
his/her own information. Contacts are; Alice Akeyo Ndege Cell; 0728716967; e-mail
alicinn@yahoo.com
7. Name of Subject …………………………
8. Date of Birth…………………Age……Yrs
Sex……………….
9. Address…………………………………………..Telephone…………………
10.E-Mail…………………………………………..Fax………………………..
11. Signatures
I have read the above information and have had the opportunity to ask questions and all
of my questions have been answered satisfactorily. I consent to participate in the study
as has been explained and as I have understood it.
I have been given a copy of this
consent form for my own records and future reference.
Signature……………………………………Date………………………….
75
Address of witness……………………………………..
I, the undersigned, have fully explained the relevant details of this study to the person
named above. By virtue of my training on how to conduct research in this field,
I’m qualified to perform this role.
Signature…………………………Name of PI …………………………..
Date…………………………….
Signature…………………………. Name of Witness…………………
76
Appendix 2; mPCR plates
In Plate 1 Shigella species was detected in six of the samples, Salmonella was detected
in eight samples, Vibrio species in seven of the samples, Campylobacter in one of the
10 samples.
No Clostridium species was detected among these samples. Notably in
this plate samples 41, 43, 44, 45 and 48 had multiple infections of Salmonella, Shigella
and Vibrio. Sample 41 had the highest multiple infections of Salmonella, Shigella,
Vibrio and Campylobacter. No organism was detected in sample 40 and 46 (Plate 4.2).
P
40
41
42
43
44
45
46
47
48
49
Plate 2: mPCR results for enteric pathogens for samples 40-49.
M molecular size marker (1000bp, 900bp, 800bp, 700bp, 600bp, 500bp, 400bp,400bp,
300bp, 200bp, 100bp); 40-49 sample results, P; Positive Control, N-Negative Control,
F; Campylobacter species 227bp, E; Shigella species 330bp, D;Salmonella species
77
M
395bp, C; Clostridium difficile toxin B 476bp, B;Vibrio species 651bp, A; Internal
control 1000bp
In plate 3 Shigella species and Salmonella species was detected all eight the samples,
Vibrio species in two of the samples, Campylobacter in 2 of the 11 samples.
No
Clostridium species was detected among these samples. Notably in this plate samples
70 and 74 had multiple infections of Salmonella, Shigella and Vibrio (Plate 3).
P
N
69
70
71
72
73
74
75
76
Plate 3: mPCR results for enteric pathogens for samples 69-76.
M molecular size marker (1000bp, 900bp, 800bp, 700bp, 600bp, 500bp, 400bp,400bp,
300bp, 200bp, 100bp); 69-76 sample results, P; Positive Control, N-Negative Control,
78
M
F; Campylobacter species 227bp, E; Shigella species 330bp, D;Salmonella species
395bp, C; Clostridium difficile toxin B 476bp, B;Vibrio species 651bp, A; Internal
control 1000bp
In plate 4, notably Shigella species, Salmonella species and Vibrio species isolates were
detected samples 51, 54 and 56 (Plate 4).
P
N
50
51
52
53
54
55
56
57
58
59
Plate 4: mPCR results for enteric pathogens in samples 50-59
M molecular size marker (1000bp, 900bp, 800bp, 700bp, 600bp, 500bp, 400bp,400bp,
300bp, 200bp, 100bp); 69-76 sample results, P; Positive Control, N-Negative Control,
F; Campylobacter species 227bp, E; Shigella species 330bp, D;Salmonella species
395bp, C; Clostridium difficile toxin B 476bp, B;Vibrio species 651bp, A; Internal
control 1000bp
79
M
In plate 5, only Salmonella species and Shigella species were detected in all the samples
P
N
60
61
62
63
64
65
66
67
68
Plate 5: Multiplex PCR results for enteric pathogens in samples 60-68.
M molecular size marker (1000bp, 900bp, 800bp, 700bp, 600bp, 500bp, 400bp,400bp,
300bp, 200bp, 100bp); 60-68 sample results, P; Positive Control, N-Negative Control,
F; Campylobacter species 227bp, E; Shigella species 330bp, D;Salmonella species
395bp, C; Clostridium difficile toxin B 476bp, B;Vibrio species 651bp, A; Internal
control 1000bp
80
M
Plate 6: mPCR results for enteric pathogens 1-10.
81
Appendix 3: Approval letter from Scientific Steering Committee/KEMRI
82
Appendix 4: Approval letter from Ethical Review Committee/KEMRI
83
Appendix 5; Composition of culture mediumComposition of simmon citrate
Agar-15 g/L
Ammonium dihydrogen phosphate - 0.2 g/L
Bromothymol blue - 0.08 g/L
Disodium ammonium phosphate - 0.8 g/L
Magnesium sulfate heptahydrate - 0.2 g/L
Composition of XLD agar
Yeast extract
3g/l
L-Lysine
5g/l
Xylose
3.75g/l
Lactose
7.5g/l
Sucrose
7.5g/l
Sodium deoxycholate
1g/l
Sodium chloride
5g/l
Sodium thiosulfate
6.8g/l
Ferric ammonium citrate 0.8g/l
Phenol red
0.08g/l
Agar
12.5g/l
84
Composition of MacConckey Agar
Peptone20g/l
Lactose 10g/l
Bile salts 5g/l
NaCl 0.05g/l
Neutral red 12.0g/l
Agar no. 2
Composition of TSI
Beef extract 3g/l
Yeast extract 3g/l
Balanced peptone 20g/l
NaCl 5g/l
Lactose 10g/l
Sucrose 10g/l
Dextrose 1g/l
Ferric citrate 0.3g/l
Na thiosulphate 0.3g/l
Phenol red indicator 0.025g/l
Composition of MIO (MOTILITY-INDOLE-ORNITHINE) MEDIUM
Gelatin Peptone 10 g/l
85
Dextrose 1 g/l
Casein Peptone 10 g/l
Bromocresol Purple 0.02 g/l
L-Ornithine 5 g/l
Bacteriological Agar 2 g/l
Yeast Extract 3 g/l
Final pH 6.5 ± 0.2 at 25ºC
86