Infection, Genetics and Evolution 17 (2013) 269276

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Infection, Genetics and Evolution

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Multilocus genotyping of Giardia duodenalis in Malaysia

Choy Seow Huey a, Mohammed A.K. Mahdy a,b,, Hesham M. Al-Mekhla a,b, Nabil A. Nasr a,
Yvonne A.L. Lim a, Rohela Mahmud a, Johari Surin a
a
b

Department of Parasitology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia
Department of Parasitology, Faculty of Medicine, Sanaa University, Sanaa, Yemen

a r t i c l e

i n f o

Article history:
Received 18 December 2012
Received in revised form 6 April 2013
Accepted 9 April 2013
Available online 25 April 2013
Keywords:
Multilocus genotyping
Giardia
tpi
bg
gdh
Malaysia

a b s t r a c t
Giardia duodenalis is considered the most common intestinal parasite in humans worldwide. In Malaysia,
many studies have been conducted on the epidemiology of giardiasis. However, there is a scarcity of
information on the genetic diversity and the dynamics of transmission of G. duodenalis. The present study
was conducted to identify G. duodenalis assemblages and sub-assemblages based on multilocus analysis
of the glutamate dehydrogenase (gdh), beta-giardin (bg) and triose phosphate isomerase (tpi) genes. Faecal specimens were collected from 484 Orang Asli children with a mean age of 7 years and examined
using light microscopy. Specimens positive for Giardia were subjected to PCR analysis of the three genes
and subsequent sequencing in both directions. Sequences were edited and analysed by phylogenetic
analysis. G. duodenalis was detected in 17% (84 of 484) of the examined specimens. Among them, 71 were
successfully sequenced using at least one locus. Genotyping results showed that 30 (42%) of the isolates
belonged to assemblage A, 32 (45%) belonged to assemblage B, while discordant genotype results were
observed in 9 specimens. Mixed infections were detected in 43 specimens using a tpi-based assemblage
specic protocol. At the sub-assemblages level, isolates belonged to assemblage A were AII. High nucleotide variation found in isolates of assemblage B made subtyping difcult to achieve. The nding of
assemblage B and the anthroponotic genotype AII implicates human-to-human transmission as the most
possible mode of transmission among Malaysian aborigines. The high polymorphism found in isolates of
assemblage B warrants a more dening tool to discriminate assemblage B at the sub-assemblage level.
2013 Elsevier B.V. All rights reserved.

1. Introduction
Giardia duodenalis (syn. G. intestinalis; Giardia lamblia) is a agellate enteric parasite that infects the intestinal tract of humans
and a wide variety of other mammals through ingestion of infective cysts (van der Giessen et al., 2006). It is a major cause of
non-viral/bacterial diarrhea affecting both developed and developing countries and more frequently encountered in areas with substandard environment, poor hygienic practices and inadequate
water treatment system (Daly et al., 2010; Robertson et al.,
2009). G. duodenalis infections are most often asymptomatic. For
symptomatic cases, the degree of symptoms and severity varies
among different individuals (Buret, 2008).
Knowing the genetic diversity of Giardia is an essential component in enhancing our understanding on the taxonomy, epidemiology and population genetics of this parasite. Therefore, molecular
characterization and in the recent years, multilocus genotyping

Corresponding author at: Department of Parasitology, Faculty of Medicine,

University of Malaya, 50603 Kuala Lumpur, Malaysia. Tel.: +60 3 7949 4746; fax:
+60 3 7949 4754.
E-mail address: alsharaby9@yahoo.com (M.A.K. Mahdy).
1567-1348/$ - see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.meegid.2013.04.013

(MLG) have become the prevailing tool used for genotyping and
subtyping of G. duodenalis. This has in turn facilitated in outbreak
surveillance, contamination source-tracking, and unveiling zoonotic potential, dynamic transmission, and relationship of genotypes and hosts (Feng and Xiao, 2011). Molecular analyses
revealed that G. duodenalis isolated from humans and animals is
a species complex with at least 8 distinct assemblages designated
as A to H, which demonstrate similarity in morphologic characteristics but are phenotypically and genotypically heterogeneous
(Plutzer et al., 2010). Of these, only assemblages A and B can cause
infection in humans with the exception of a small fraction of cases
where animal host specic assemblages CF were reported in human (Sprong et al., 2009). Besides, both assemblages A and B are
also capable of infecting animals. The subdivision into AIAIII has
provided greater insights on the dynamic of transmission of these
assemblages. Sub-assemblage AII has been regarded as anthroponotic whereas AI and AIII are predominant in livestock and wildlife,
respectively (Feng and Xiao, 2011; Nolan et al., 2010; Sprong et al.,
2009; Thompson et al., 2000).
In Malaysia, giardiasis is an endemic disease and is associated
with malnutrition among children in the rural areas resulting in
stunting, wasting and vitamin A deciency (Al-Mekhla et al.,

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2010, 2005). The prevalence of human Giardia infection varies between 0.2% and 29.2% (Lim et al., 2008; Noor Azian et al., 2007).
Most of the epidemiological studies detected Giardia on the basis
of microscopic examination without employing molecular approach. Data on genotypes of G. duodenalis up to the assemblage level remains scarce. In a previous genotyping study using SSU rRNA
locus, one specimen was identied as assemblage A in 42 specimens and the rest were assemblage B (Mahdy et al., 2009). In a
study on immunocompromised patients, assemblage A was identied in four of the microcopy-positive Giardia specimens using tpi
gene (Lim et al., 2011). Assemblage A was also isolated from environmental samples including recreational lake water and water
bodies in a zoo (Lim et al., 2009a,b). In addition, genotyping study
was conducted on animals and assemblages A and E were detected
among goats (Lim et al., 2013). However, subtyping of assemblages
A and B in the previous studies was not conducted which limit our
understanding on the transmission dynamics and the source of
infection of giardiasis in this country. Thus, the present study
was aimed to identify G. duodenalis assemblages and sub-assemblages based on multiloci genes which included gdh, bg and tpi
genes to attain better understanding of the genetic diversity and
transmission of giardiasis in Malaysia. The study also aimed at
determining the occurrence of mixed infections using primers targeting tpi gene specic for assemblages A and B.
2. Materials and methods
2.1. Source of samples
A total of 484 children (51% males, 49% females) with the mean
age of 7 years were involved in the present study. The sample collection was conducted from April to September 2011 in 13 Orang
Asli villages located in Lipis district, Pahang state, Peninsular
Malaysia. The study protocol was approved by The Medical Ethics
Committee of University of Malaya Medical Center, Kuala Lumpur,
Malaysia under the MEC Ref. No.: 788.74. Informed consents were
obtained from the participants parents or guardians prior to the
collection. Single sample was collected from each participant.
Upon receipt, the samples were transferred to the Department of
Parasitology, University of Malaya, preserved in 2.5% potassium
dichromate and stored in cold room (4 C) until further analysis.
2.2. Microscopy
The faecal samples were concentrated based on formal-ether
technique. Briey, a small amount of stool samples (pea size)
was emulsied with 7 ml of 10% formalin and sieved through a
double-layered gauze and collected in a beaker. The suspension
was transferred into 15 ml conical centrifuge tube and topped up
with three ml of diethyl ether. The centrifuge tube with the suspension was capped and mixed by shaking before centrifuging
for 5 min at 2500 rpm. The top three layers (ether, debris and formalin water) were removed and the sediment was examined by
light microscope under x100 and x400 magnication for the presence of Giardia cysts and other intestinal parasites after staining
with iodine.
2.3. Molecular analysis
DNA was extracted directly from samples positive for Giardia
cyst using PowerSoilDNA Isolation Kit (MoBio Laboratories Inc.,
Carlsbad, California) following the manufacturers instructions.
Elution step was accomplished by adding reduced volume of solution C6 (10 mM Tris) to obtain a nal volume of 50 ll and the DNA
was stored in freezer under 20 C.

A partial sequence of gdh gene (432-bp) was amplied using

semi-nested PCR as described by Read et al. (2004). For primary
PCR, the forward primer GDHeF (50 -TCA ACG TYA AYC GYG GYT
TCC GT-30 ) and the reverse primer GDHiR (50 -GTT RTC CTT GCA
CAT CTC C-30 ) were used. In the secondary PCR, the forward primer
GDHiF (50 -CAG TAC AAC TCY GCT CTC GG-30 ) and the reverse primer
GDHiR were used. Primary and secondary PCR reactions were performed in a 50 ll PCR master mix comprising 0.5 lM of each primer
(Bioneer Q-Oligos, Korea), 2.5 U of HotStarTaq Plus DNA Polymerase (Qiagen, Hilden, Germany),1 PCR buffer (Qiagen, Hilden, Germany), 200 lM of dNTP (Fermentas, Ontario, Canada), 1.5 mM
MgCl2 (Qiagen, Hilden, Germany), 5% dimethyl sulfoxide (SigmaAldrich, USA) and 0.4 mg/ml BSA (New England Biolabs, Ipswich, USA).
2 ll of DNA template were used in both amplications that were run
in the MyCycler thermal cycler (Bio-Rad, Hercules, USA) under the
following conditions: Initial activation at 95 C for 5 min, 1 cycle at
94 C for 2 min, 56 C for 1 min and 72 C for 2 min, followed by 55
amplication cycles at 94 C for 30 s, 56 C for 20 s, 72 C for 45 s,
and a nal extension at 72 C for 7 min. For secondary PCR, the number of cycles was reduced to 33.
A partial sequence of tpi (530-bp) was amplied using nestedPCR protocol according to Sulaiman et al. (2003). Primary PCR was
run using forward primer AL3543 (50 -AAA TIA TGC CTG CTC GTC G30 ) and reverse primer AL3546 (50 -CAA ACC TTI TCC GCA AAC C-30 ).
For secondary PCR, forward primer AL3544 (50 -CCC TTC ATC GGI
GGT AAC TT-30 ) and reverse primer AL3545 (50 -GTG GCC ACC ACI
CCC GTG CC-30 ) were used. Primary and secondary PCRs were performed in a 50 ll PCR mix comprising 0.2 lM of each primer (Bioneer Q-Oligos, Korea), 1 U of HotStarTaq Plus DNA Polymerase
(Qiagen, Hilden, Germany), 1 PCR buffer (Qiagen, Hilden, Germany), 200 lM dNTP (Fermentas, Ontario, Canada), 1.5 mM MgCl2
(Qiagen, Hilden, Germany), and 0.2 mg/ml BSA (New England Biolabs, Ipswich, USA). 2 ll of DNA template were used and the prepared master mix was incubated in the MyCycler thermal cycler
(Bio-Rad, Hercules, USA) under the following conditions: Initial
hot start at 95 C for 5 min, 35 amplication cycles at 94 C for
45 s, 50 C for 45 s (58 C for secondary PCR), 72 C for 60 s and a
nal extension at 72 C for 10 min.
In addition, the rst PCR product of the reaction described by
Sulaiman et al. (2003) underwent further amplication using a
set of separate A (Geurden et al., 2007) and B (Geurden et al.,
2009) assemblage-specic primers. Presence of mixed infection
was detected by visualizing the occurrence of bands in the agarose
gel at 332 bp for assemblage A amplied using primers AssAF (50 CGC CGT ACA CCT GTC-30 ) and AssAR (50 -AGC AAT GAC AAC CTC
CTT CC-30 ) and at 400 bp for assemblage B amplied using primers
AssBF (50 -GTT GTT GTT GCT CCC TCC TTT -30 ) and AssBR (50 -CCG
GCT CAT AGG CAA TTA CA-30 ). The PCR reaction mix consisted of
0.2 lM (0.4 lM for assemblage B) of each primer (Bioneer Q-Oligos, Korea), 1.25 U of HotStarTaq Plus DNA Polymerase (Qiagen,
Hilden, Germany), 1 PCR buffer (Qiagen, Hilden, Germany),
200 lM dNTP (Fermentas, Ontario, Canada), 1.5 mM MgCl2 (Qiagen, Hilden, Germany) and 0.1 mg/ml BSA (New England Biolabs,
Ipswich, USA) to a nal volume of 25 ll. 1 ll of DNA template
was added for assemblage A and 2 ll was added for assemblage
B for the PCR amplications following the cycle parameter: Initial
hot start at 95 C for 5 min, initial denaturation at 94 C for 10 min,
and 35 amplication cycles at 94 C for 45 s, 64 C for 45 s (62 C
for secondary PCR) and 72 C for 45 s.
A partial sequence of bg gene (511-bp) was amplied using
PCR protocols described by Caccio et al. (2002) and Lalle et al.
(2005). The primers for primary PCR were G7 (50 -AAG CCC GAC
GAC CTC ACC CGC AGT GC-30 ) and G759 (50 -GAG GCC GCC CTG
GAT CTT CGA GAC GAC-30 ). For secondary PCR, BG511F (50 -GAA
CGA ACG AGA TCG AGG TCC G-30 ) and BG511R (50 -CTC GAC GAG
CT TCG TGT T-30 ) were used. The PCR master mix consisted of

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Table 1
The distribution of assemblages A and B based on the different loci and mixed
infection.
Genes

tpi
gdh
b-giardin
MLGsa
tpi-Mixedb

Assemblages n (%)
A

34 (58)
7 (18)
18 (64)
3 (27)
1 (2)

25
31
10
8
23

(42)
(82)
(36)
(73)
(34)

A+B

Total

43 (64)

59
38
28
11
67

a
Multilocus genotypes (isolates with the same assemblage identied using tpi
and gdh and b-giardin).
b
tpi-based PCR used assemblage-specic primers to identify the mixed infection.

0.4 lM of each primer (Bioneer Q-Oligos, Korea), 2.5 U HotStarTaq

Plus DNA Polymerase (Qiagen, Hilden, Germany), 1 PCR buffer
(Qiagen, Hilden, Germany) and 200 lM dNTP (Fermentas, Ontario,
Canada) in a total volume of 50 ll. 2 ll of DNA template were
added and the mixture was run in the MyCycler thermal cycler
(Bio-Rad, Hercules, USA) under the following conditions: Initial
hot start at 95 C for 5 min, initial denaturation at 95 C for
15 min, followed by 35 amplication cycles at 95 C for 30 s,
65 C for 30 s (55 C for secondary PCR), 72 C for 60 s, and a nal
extension at 72 C for 7 min.
In all the PCR reactions, a Giardia-positive DNA specimen
and distilled water were used as positive and negative control.
The PCR products were analyzed using 2% agarose gel (Vivantis)
electrophoresis stained with SYBR Safe DNA (Invitrogen, Auckland, New Zealand).

2.4. DNA sequencing and sequences analysis

Successfully amplied PCR products with the nested-PCR for
each locus (except for tpi assemblage-specic PCR) were sequenced
in both directions using Applied Biosystems 3730 xl DNA Analyzer
(Applied Biosystems, USA). The chromatograms and sequences generated from this study were viewed and assembled using BioEdit Sequence Alignment Editor Programme (http://www.mbio.ncsu.edu).
Preliminary similarity comparison of the consensus sequence with
the sequences in GenBank database was made using Basic Local
Alignment Search Tool (BLAST) (http://blast.ncbi.nlm.nih.gov). The
isolate sequences were genotyped into assemblage and sub-assemblage using multiple alignments implemented by ClustalW (Thompson et al., 1994) with previously dened reference sequences
retrieved from GenBank database. Phylogenetic analysis was performed in MEGA 5 (www.megasoftware.net) using neighbour-joining (NJ) algorithms with evolutionary distances calculated by
Kimura-2 parameter method (Kimura, 1980) and 1000 bootstrap value. Sequences from this study were deposited in the GenBank under
accession numbers KC313907- KC313948.

3. Results
3.1. Identication of G. duodenalis assemblages and mixed
assemblages
From a total of 484 participants, 84 (17.0%) were found infected with Giardia using microscopy. The 84 microscopy-posi-

Table 2
The distribution of assemblages A and B based on the different loci and mixed infection.
Isolate

tpi

gdh

bg

tpi-Mixed

Isolate

tpi

gdh

bg

tpi-Mixed

TB11.6
TG2.5
TG13.2
TB13.3
SB14.1
ST15.1
KK16.4
KK16.5
TB7.3
SE10.1
SE17.1
TB15.3
TG9.2
PD1.1
PD11.2
SE29.3
KK11.3
KM4.1
TB3.3
TB13.2
SE25.2
KK1.6
CK1.2
CK10.2
SB1.1
SE25.4
ST10.3
TB12.3
KK7.5
SE29.4
KM15.1
TB2.3
TB4.1
TB5.1
CK20.2
SE7.1
SE12.1
SE24.2
KN7.4

A (A2)
A (A2)
A(A2)
A (A2)
A (A2)
A (A2)
A (A2)
A (A2)
A (A2)
A (A2)
A (A2)
A (A2)
A (A2)
A (A2)
A (A2)
A (A2)
A (A2)
A (A2)
A (A2)
A (A2)
A (A2)
A (A2)
A (A2)
A (A2)
A (A2)
A

B
B
B
B
B
B
B
B
B

A
A
A
A
A
A

A
A
A

A
A
A
A
A
A
A
A
A
A

A+B
B
B
A+B

A+B

A+B
A+B
B
A
A+B
A+B
A+B
A+B

A+B

A+B
A+B
A+B
A+B
B
A+B
A+B
A+B
B

B
B
A+B
A+B
A+B
A+B
B
B
A+B

SM1.2
KM13.2
TB9.2
CK7.1
SE13.1
TB15.1
SE9.3
KN6.4
TB9.1
UM3.1
SE8.4
SE27.6
SE27.7
KK2.3
KK2.5
KM7.3
KM8.1
KM15.2
ST13.2
ST20.3
KM8.4
CK1.4
SB4.1
ST8.1
KN7.2
SM6.2
KM3.2
KM12.3
TB11.2
SB15.1
PD20.1
SE18.4
KK6.1
CK20.1
ST1.3
ST3.2
ST21.1
TB2.5
SE13.2

B
B
B
B
B
B
B
B
B
B
B
B
B
B
B

A
A
A
A
A
A
A
B
A

B
B
B
B
B
B

B
B
B
B
B
B
B

B
B
B
B
B
B
B
B
B

B
A (A2)

A (A3)

A+B
A+B
A+B
A+B
B
A+B
A+B
A+B
A+B
A+B
A+B
B
B
A+B
A+B

B
B
B

A+B

B
B
A+B
B
A+B
A+B
A+B
A+B
A+B
A+B
A+B
B
B
B
B
B

(A2)
(A2)
(A2)
(A2)
(A2)
(A2)

A (A2)

B
B
B
B
B
B
B
B
B

(A2)
(A3)
(A3)

(A2)
(A2)
(A2)
(A3)
(A2)
(A2)
(A2)
(A3)
(A2)
(A2)

A (A2)
A (A3)
A (A2)
B
B
B
B
B
B
B
B

(A2)
(A2)
(A2)
(A2)
(A2)
(A2)
(A2)

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tive specimens were analyzed at three loci (tpi, gdh, and bg) and
a set of assemblage-specic PCRs was used to detect mixed
infection. PCR amplicons from 78 specimens using at least one
of the molecular markers were amplied and subsequently sequences were generated for 71 specimens. The alignment analysis of the sequences obtained from tpi, gdh, and bg genes with
reference sequences from the GenBank database identied
assemblages A and B in 30 (42%) and 32 (45%) specimens,
respectively. Discordant genotype results were noted in 9 (13%)
specimens. At tpi gene, PCR amplicons were produced from
70% of the examined specimens (59/84). Among them, assemblages A and B were found in 34 (58%) and 25 (42%) specimens,
respectively. At gdh gene, 38 (45%) specimens were positive with
assemblage B being more frequently observed compared to
assemblage A (31 vs 7). PCR analysis of bg gene had the lowest
detection rate [33% (28/84)]. The distribution of assemblage A
and B for bg gene was 18 (64%) and 10 (36%), respectively. MLGs
were successful in 11 specimens, which were identied as
Assemblages A and B, in 3 and 8 specimens, respectively (Table 1). As mentioned above assemblage discordant results were
observed in 9 specimens. One isolates (ST8.1) was identied as
assemblage A at tpi and bg genes and as assemblage B at gdh
gene. The specimen PD20.1 was identied as assemblage B at
tpi and gdh genes and as assemblage A at bg gene. Seven specimens (KN7.2, SM6.2, KM3.2, KM12.3, TB11.2, SB15.1 and SE18.4)
were found to harbour assemblage A at tpi gene and assemblage
B at gdh gene. The tpi-mixed protocol showed that 6 of the 9
specimens harboured mixed assemblages A and B (Tables 2).
The tpi-based PCR protocol for mixed infection using assemblage-specic primers was conducted on the 84 Giardia-positive
specimens. Of these, 67 specimens showed amplicons for G.
duodenalis assemblages; 43 specimens had mixed infections
with assemblages A and B, 23 specimens had single infection
with assemblage B and one case was identied as assemblage
A (Table 1 and 2). The specicity of this protocol was conrmed
by sequencing selected cases representing assemblages A and B.
When the chromatogram was scrutinized at the positions where
nucleotide polymorphism could differentiate assemblage A and
B, double peaks were observed in 14 of the mixed isolates.

3.2. Subtyping of G. duodenalis Assemblage A

Based on analysis targeting tpi gene, 34 of the assemblage A isolates (Table 2) were classied as subtype A2 based on the phylogenetic analysis (Fig. 1) and the substitution pattern (Table 3). The
neighbor-Joining tree placed the four representative sequences
(TB11.2, KK16.4, TB13.2 and SM6.2) in one cluster with AII sequence references with high bootstrap support.
At gdh gene, all sequences of the assemblage A showed complete homologous to the reference sequence of subtype A2
(Accession No. L40510) except for isolate ST10.3 where substitution at two positions were observed (AG at position 341 and C
T at position 485) (Table 4, Fig. 2). Subtype A2 based on tpi or
gdh belongs to sub-assemblage AII (Caccio et al., 2008; Feng
and Xiao, 2011).
At bg gene, 18 isolates were identied as assemblage A. Twelve
were distinguished as A2 from A1 at position 606 (CT) and from
A3 at positions 460 (CT) and 468 (TC). Three were typed as A3
and the remaining 3 isolates have the subtype similar to A3 but different by one position at 460 (CT/Y). Phylogenetic analysis demonstrated that six isolates were typed as A3 (100% bootstrap
value) while the remaining 12 isolates were grouped as A2
(Fig. 3). It should also be noted that subtypes A2 and A3 belong
to sub-assemblage AII (Caccio et al., 2008; Feng and Xiao, 2011).

Fig. 1. Phylogram of G. duodenalis genotypes constructed by neighbour-joining

analysis, based on the nucleotide sequences of tpi retrieved from this study
compared with reference sequences of known assemblages from Genbank. Bootstrap values obtained from 1000 replicates are indicated on branches in percentage.

3.3. Subtyping of G. duodenalis assemblage B

At tpi gene, 16 sequences representing 23 isolates which were
identied as assemblage B, were multiple aligned with 5 references
sequence representing BIV, BIV-like, BIII and BIII-like and analysed
for BIV/BIII specic substitutions according to Wielinga and
Thompson (2007). The 16 representative sequences had heterogeneous nucleotides at positions dening the sub-assemblages, making proposing specic sub-assemblages for assemblage B isolates
not possible (Table 3). Phylogenetic analysis conrmed the monophyletic group of assemblage B (bootstrap = 99%). The NeighborJoining formed tree sub-clusters of assemblage B. However, subclustering was not supported by bootstrap values (Fig. 1).
At gdh gene, 15 sequences representing 30 isolates had high
nucleotide variations, limiting the classication of assemblage B
to sub-assemblages (Table 4). Similar to tpi gene, phylogenetic

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C.S. Huey et al. / Infection, Genetics and Evolution 17 (2013) 269276

Table 3
Multiple alignments of tpi sequences from this study with reference sequences obtained from GenBank, representing sub-assemblages of assemblages A and B.
Isolates

GenBank accession no. Nucleotide position from the start of the gene

Assemblage A
AI
AII (A2) JH
AII (A2) TB11.6a

L02120
U57897
KC313923

129
T
C
C

399
C
T
T

Assemblage B
BIV
Ad 19
BIV
GS/M
BIV-Like 7115
BIII
2434
BIII-Like 2436
CK7.1, SE8.4, TB5.1
SE24.2, KN7.4,TB4.1
TB2.3
TB15.1
KM13.2
CK20.2
TB9.2
SE9.3
SE13.1
UM3.1
KN6.4
SE12.1, 27.6, TB9.1, SE7.1
KK2.3
SE27.7
SM1.2
KM15.1

AF069560
L02116
AY368167
AY368165
AY368153
KC313907
KC313908
KC313909
KC313910
KC313911
KC313912
KC313913
KC313914
KC313915
KC313916
KC313917
KC313918
KC313919
KC313920
KC313921
KC313922

39
A
.
.
G
G
.
.
G
G
G
G
G
.
.
.
.
.
G
.
G
.

45
T
.
.
.
C
.
.
C
C
C
.
C
.
.
C
.
.
C
.
.
.

91
T
.
.
C
.
.
.
C
C
.
C
.
.
.
.
.
C
C
C
C
.

100
C
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
t

109
G
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.

121
G
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
a

162
G
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.

165
T
.
.
C
.
.
.
.
.
.
.
.
.
.
.
.
.
.
C
C
C

168
T
.
.
C
.
.
.
.
.
.
C
C
C
C
C
C
C
C
C
C
C

210
A
.
G
G
.
.
G
G
G
G
G
G
G
.
G
G
G
G
G
.
G

216
C
.
.
.
T
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.

247
A
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
g
.

271
C
.
.
.
.
.
.
.
t
t
.
.
.
.
.
.
.
.
.
.
.

280
A
.
.
.
.
.
.
.
.
g
.
.
.
.
.
.
.
.
.
.
.

297
A
.
.
.
.
.
.
.
.
.
G
.
.
.
.
.
.
.
.
.
.

354
G
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
a

402
A
.
.
.
.
.
.
.
.
.
g
.
.
.
.
g
.
.
.
.
.

429
A
G
.
G
.
G
G
.
G
G
G
G
G
G
G
G
G
G
G
G
G

438
T
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.

483
A
.
.
A
G
.
.
.
.
.
G
.
.
G
.
.
.
.
.
.
.

Numbers in bold represent nucleotide substitutions from the start of the gene, which differentiate between subtypes according to Weilinga and Thompson (2007). Dots (.)
indicate nucleotides identity. Nucleotides in small letter indicate substitution not in the subtypes-dening positions.
a
Representative sequence of isolates identied as A2 (Listed in Table 2). Accession numbers in bold are reference sequences from the GenBank.

Table 4
Multiple alignments of gdh sequences from this study with reference sequences obtained from GenBank, representing sub-assemblages of assemblages A and B.
Isolates
Assemblage A
AI
(Prtland1)
AII
(A2)
AII
(A2) TB11.6,TG2.5,TG13.2, TB13.3, SB14.1, T15.1
ST10.3
Assemblage B
BIII (BAH-12)
BIV (Ad-7)
SM1.2
CK1.4
KM7.3,TB11.2,KM8.1, SM6.2
KM15.2,ST8.1,KM15.1, ST13.2,KM8.4,ST20.3,
TB2.3
SE13.1
KM3.2
TB15.1, SB15.1
SE12.1, TB4.1
TB5.1, SE7.1
KM13.2,CK7.1,TB9.2, SE24.2,SE18.4
PD20.1, KN7.4
CK20.2
KM12.3

GenBank accession
no.

Nucleotide position from the start of the gene

M84604
L40510
KC313924
KC313925

237
C
C
?
?

246
C
C
?
?

341
A
.
.
G

485
C
.
.
T

603
T
C
C
C

621
C
T
T
T

AF069059
L40508
KC313926
KC313927
KC313928
KC313929

309
C
T
T
T
T
T

357
T
.
.
.
.
.

360
G
.
.
.
.
.

429
T
C
.
.
C
C

447
T
C
C
.
C
C

519
C
.
T
T
T
.

540
C
T
.
.
.
.

561
C
T
.
.
.
.

570
C
.
.
.
.
.

576
G
.
.
.
.
.

582
G
.
.
.
.
.

597
C
.
.
.
.
.

612
G
A
.
.
.
.

KC313930
KC313931
KC313932
KC313933
KC313934
KC313935
KC313936
KC313937
KC313938

T
T
T
T
T
T
.
.
.

C
C
C
C
C
C
.
C
.

.
.
.
.
.
.
.
.
.

C
C
C
C
C
C
.
.

C
C
C
C
C
C
C
C
C

T
T
.
.
.
.
.
.
.

.
.
.
T
.
T
T
.
.

.
.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.
.

.
.
.
.
A
A
.
.
.

Numbers in bold represent nucleotide substitutions from the start of the gene, which differentiate between subtypes according to Weilinga and Thompson (2007). Dots (.)
indicate nucleotides identity. Question mark (?) indicates missing nucleotides. Accession numbers in bold are reference sequences from the GenBank.

analysis formed a monophyletic group for assemblage B (Fig. 2).

Similar results were observed in bg gene (Table 5, Fig. 3).
4. Discussion
To the best of our knowledge, the present study was the rst
study of giardiasis using multi-locus genotyping (MLG) in Malaysia. In this study, the prevalence of giardiasis in the Orang Asli
communities was 17.0%. This is comparable with a recent study

conducted by Anuar et al. (2012) in Orang Asli communities in

Selangor state, which showed an overall prevalence of giardiasis
at 20.0% (n = 500).
Amplications of the three PCR assays (tpi, gdh, and bg) were
performed differently at different loci. Of the 84 Giardia microscopically-positive specimens, only 11 isolates were able to be amplied at three loci. The tpi locus achieved the highest percentage
of amplicons produced (70%), followed by gdh (45%) and bg
(33%). Similar occurrences were also reported in previous studies

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C.S. Huey et al. / Infection, Genetics and Evolution 17 (2013) 269276

Fig. 2. Phylogram of G. duodenalis genotypes constructed by neighbour-joining

analysis, based on the nucleotide sequences of gdh retrieved from this study
compared with reference sequences of known assemblages from Genbank. Bootstrap values obtained from 1000 replicates are indicated on branches in percentage.

(David et al., 2011; Lalle et al., 2009). Discordant genotyping results at the three loci (tpi, gdh, and bg) have been observed in 9 isolates (13%) in this study, which is in agreement with previous
reports (Caccio et al., 2008; Sprong et al., 2009; Thompson et al.,
2009; Yang et al., 2010a). An analysis of sequences from four genetic loci (tpi, gdh, bg and rDNA) has been conducted on 61 human
isolates and 29 animal isolates of G. duodenalis (Caccio et al., 2008).
In this study, incongruent assignment of ve human isolates and
one macaque isolates to G. duodenalis assemblages was reported
(Caccio et al., 2008). Yang et al. (2010) genotyped 124 human isolates of G. duodenalis at gdh and rDNA genes, and reported incongruent genotyping results in ve isolates, where three isolates
were classied as assemblage A at rDNA gene and assemblage B
at gdh gene, and two isolates were classied as assemblage B at
rDNA gene and assemblage A at gdh gene. Inconsistency in genotyping results of G. duodenalis has also been reported among dogs
and cats isolates, where 7 isolates (3 dogs and 4 cats) were classied as assemblage D at rDNA gene, and as assemblage B (3 isolates), assemblage C (3 isolates) and assemblage E (1 isolate) at
gdh gene. One dog isolate was classied as assemblage C at rDNA
and assemblage B at gdh gene (Read et al., 2004). Thompson
et al. (2009) genotyped G. duodenalis isolated from 70 cayotes at
rDNA and gdh, and reported four incongruent genotyping results.

Fig. 3. Phylogram of G. duodenalis genotypes constructed by neighbour-joining

analysis, based on the nucleotide sequences of bg retrieved from this study
compared with reference sequences of known assemblages from Genbank. Bootstrap values obtained from 1000 replicates are indicated on branches in percentage.

The implication of this inconsistency has provoked questions

about the strength and validity of genotyping relying on single locus and hence, the true picture of Giardia diversity has a high
chance of being masked by the data generated from analysis using
single marker (Feng and Xiao, 2011). Although the underlying
mechanisms remain uncertain, Caccio and Ryan (2008) have reviewed a number of factors that could contribute to the incongruent assignment of assemblages by different makers. These factors
include meiotic recombination, which suggests the potential of
sexual reproduction in Giardia, introgression that involves back
crossing of hybrids with parental species and retention of ancestral
polymorphism that could lead to presence of identical alleles in
genetically distinct groups. Another contributing factor which
would be discussed further in this context is the occurrence of
mixed infections. With the use of the assemblage-specic assays,
high prevalence (64%) of mixed infections was detected and further
proven the biasness of using a standard PCR assay alone as the
most plentiful assemblage would be amplied in preference (Wielinga and Thompson, 2007). Thus, the feature of mixed infections
could be applied in the present study to explain the discordant
genotype results.
The isolates with assemblage A were all identied as A2, which
belongs to sub-assemblage AII. This is similar with other studies

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C.S. Huey et al. / Infection, Genetics and Evolution 17 (2013) 269276

Table 5
Multiple alignments of bg sequences from this study with reference sequences obtained from GenBank, representing sub-assemblages of A and assemblages B.
Isolates
Assemblage A
AI-A1
AII-A2
AII-A3
A2
A3
A3
A3

Genbank accession no.

KK16.4a
KK7.5, TG2.5
SE10.1
PD20.1, PD1.1, TG13.2

X85958
AY072723
AY072724
KC313945
KC313946
KC313947
KC313948

450
C
.
.
.
.
.
.

460
C
.
T
.
T
Y
.

468
T
.
C
.
C
C
C

606
C
T
T
T
T
T
T

729
A
G
A
?
?
?
?

SB4.1, SE7.1
TB2.3
SE9.3
TB4.1
SE24.2
TB5.1, CK20.2, SE12.1

AY072727
KC313939
KC313940
KC313941
KC313942
KC313943
KC313944

210
C
T
T
.
Y
.
.

228
A
.

354
C
T
.
.
T
T
T

378
C
.
.
.
.
.
.

438
C
.
.
.
.
.
.

Portland 1
KC8

Assemblage B
B

.
.
.
.

540
G
A
A
A
A
.
A

564
T
.
.
.
.
.
.

Numbers in bold represent nucleotide substitutions from the start of the gene, which differentiate between subtypes according to Wielinga and Thompson (2007). Dots
indicate nucleotides identity. Question mark (?) indicates missing nucleotides. The dash () indicates deletion.
a
Representative sequence of isolates identied as A2. Accession numbers in bold are reference sequences from the GenBank.

where AII was the predominant sub-assemblage in human (Bonhomme et al., 2011; Hussein et al., 2009; Lebbad et al., 2011). Subassemblage AI which was absent from this study was reported to
have preference to cause infection in livestock or companion animals as opposed to AII which was found mainly in human cases
than animals (Sprong et al., 2009). The isolation of anthroponotic
assemblages and sub-assemblages (B and AII) in this study implicates human as a potential source of the infection. In accordance
with our ndings, previous studies conducted in Orang Asli communities in Malaysia found that the presence of other family members infected with G. duodenalis was either the only or the main
risk factor for giardiasis (Norhayati et al., 1998; Anuar et al.,
2012). However, the postulation of human-to-human transmission
is limited by the fact that animals were not included for analysis in
this study.
On the other hand, sub-assemblages were not able to assign to
isolates belonged to assemblage B due to high degree of nucleotides variation which was not uncommon among other molecular
studies (Bonhomme et al., 2011; Caccio et al., 2008; Lalle et al.,
2009; Lebbad et al., 2011; Levecke et al., 2009). This phenomenon
led us back to the focus of mixed infection, which is known to take
place at both the inter- and at the intra-assemblage levels (Caccio
and Ryan, 2008). Mixed infection can happen when a host ingests
Giardia cysts of different genetic proles or subsequent infection of
an infected host by genetically different Giardia cysts. This is especially common in areas where giardiasis is endemic (Caccio and
Ryan, 2008; Lebbad et al., 2011; Sprong et al., 2009). Therefore,
mixed subtype infections (intra-assemblage level) can stand as a
factor that causes the high heterogeneity. Besides, high polymorphism in assemblage B can also be attributed to intra-assemblage
recombination and allelic sequence heterozygosity (ASH). In order
to resolve the question whether the mixed genotype is due to
mixed infections or ASH, Ankarklev et al. (2012) had conducted
an investigation on single Giardia trophozoite and cyst. The result
based on molecular analysis positively exhibited the occurrence
of ASH at single cell level. However, high numbers of mixed subgenotypes infections (demonstrated by variable sequence patterns) were also observed in different cysts isolated from the same
sample indicating that mixed infection is also playing a role for the
high heterogeneity.
In conclusion, MLG was conducted to analyze the genetic diversity of Giardia infections in Orang Asli communities in Malaysia.
While assemblages A and B have almost equal frequency of infections, more than half of the isolates were mixed infections. The

presence of assemblage B and sub-assemblage AII in the samples

suggest that the mode of transmission of giardiasis in Malaysia
may be human-to-human. Nevertheless, the role of animals in
the dynamic of transmission needs to be further investigated
involving multilocus genotyping of parasites from animals and
humans.
Acknowledgments
We are grateful to the Orang Asli children and their parents for
their voluntary involvements and would like to acknowledge the
nancial support from UMRG grants RG302-11HTM and RG439/
12HTM, and the student grant (PV063- 2012A).
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