Multilocus genotyping of Giardia duodenalis isolates from preschool
Egyptian children
Hanan Mahmoud Abou-Seri1, Christian Klotz2, Toni Aebischer2, Khalifa El Sayed Khalifa Mohamed1
1Faculty of Medicine, Ain Shams University, 11591, Cairo, Egypt.
2Mycotic and parasitic agents and mycobacteria, Department of Infectious Diseases, Robert Koch-Institute, 13353 Berlin, Germany.
Introduction
 Giardia duodenalis, an enteric protozoan, is common among children
especially in developing countries. Chronic infection is associated with
malnutrition, wasting and stunting.
 In Egypt a prevalence rate of 27.3% among children complaining of chronic
diarrhea was recorded.
 Genetic studies have revealed that at least 8 assemblages (A-H) of G.
duodenalis exist, where assemblages A and B are found primarily in human
beings and occasionally in animals.
 Studies on G. duodenalis genetic diversity in preschoolers are limited in
Egypt.
Genetic variants and relatedness using distance matrix analysis of
assemblage A & B subtypes at the in silico concatenated gene level
Objective
The aim of the present study was to determine the genetic diversity of G.
duodenalis isolates among preschool Egyptian children using multilocus
genotyping of triose phosphate isomerase (tpi), β- giardin (bg) and glutamate
dehydrogenase (gdh) genes of the parasite according to previously described
protocols (1, 2, 3).
Methods
Clinical Labs in
Greater Cairo
Multilocus
Nested
PCR
Alignment with
reference strains
+ ve Stool
Samples for
Giardia by Light
Microscope
DNA extraction
Sequencing of
amplification
products
Sequences
editing using
Geneious
software
63 (83%) isolates
were succ. amplified
and sequenced at
least at 1 locus
Assemblage
typing &
subtyping
Distance Matrix
analysis
Phylogenetic
Confirmation
by
Real- time PCR
tree analysis
Results
(A)
Frequency of G. duodenalis assemblages among preschoolers (B)
Distribution of assemblages as determined by multilocus sequence
typing (MLST) based on the different loci (tpi, bg and gdh)
 (MLG): have similar assemblage at the three
loci.
Assemblage of Giardia isolates using one, two or three marker
Marker
Tpi
Bg
Gdh
Tpi-bg
Bg-gdh
Tpi-gdh
Tpi-bg-gdh
Assemblage
A
B
A
B
A
B
A
B
A
№
1
1
1
2
1
-
B
A
B
A
B
2
1
2
15
35
Numbers at the top represent nucleotide substitutions from the start of the
gene, which differentiate between subtypes.
44 samples with complete information at all three gene loci were in silico
concatenated in the order of the tpi-bg-gdh (1358 bp) sequences and
subsequently aligned using MUSCLE integrated within Geneious 9.0.5 Software
program.
15 were identified as assemblage A, of there only 27% were unique, 29 were
identified assemblage B and all (100%) were unique.
Phylogenetic relationships of the in silico concatenated
genes of G. duodenalis. The alignment was generated with
ClustalX and analyzed using neighbor-joining algorithm with
maximum likelihood composite method performed in
(MEGA 6). Phylogenetic analysis formed a monophyletic
group for each of assemblage B and A. A large genetic
variability was revealed in assemblage B (but not
assemblage A) isolates of the parasite, which is consistent
with the occurrence of intra-assemblage recombination in
G. duodenalis.
Isolate No.70 was identified as assemblage B at tpi and gdh genes and as assemblage A at bg gene.
 Isolate No. 82 was found to be possibly mixed at tpi gene, as assemblage A at bg gene and as
assemblage B at gdh gene.

Acknowledgment
The study was supported by German Egyptian Scientific Projects (GESPs) and a scientific
conjoint cooperation between the German Academic Exchange Service (DAAD) and the
Science & Technology Development Fund (STDF, project no. 5387), who assigned
resources for the study. Special thanks must go to Petra Gosten-Heinrich at Robert KochInstitute for the excellent technical assistance during the sequence typing procedure.
References
1. Sulaiman IM, Fayer R, Bern C, Gilman RH, Trout JM, Schantz PM, Das P, Lal AA, Xiao L (2003) Triosephosphate isomerase gene
characterization and potential zoonotic transmission of Giardia duodenalis. Emerg Infect Dis 9(11):1444–14524.
2. Lalle M, Pozio E, Capelli G, Bruschi F, Crotti D, Cacciò SM(2005)Genetic heterogeneity at the beta-giardin locus among human and
animal isolates of Giardia duodenalis and identification of potentially zoonotic subgenotypes. Int J Parasitol 35(2):207–213.
3. Read CM, Monis PT, Thompson RC (2004) Discrimination of all genotypes of Giardia duodenalis at the glutamate dehydrogenase
locus using PCR-RFLP. Infect Genet Evol 4(2):125–130.