Genetic Characterization of Giardia intestinalis Strains from Patients Having Sporadic Giardiasis by Using PCR Assay
Kareem H. Nahavandi,
In this study, 325 stool samples from sporadic cases giardiasis were
examined by conventional techniques for parasite diagnosis. A simple and
rapid procedure for the extraction of DNA from fecal samples was developed.
Triose phosphate isomerase (tim) based PCR assay was applied for
definitive identification and genetic characterization of Giardia
intestinalis strains collected from Tabriz Reference Laboratory
and pediatric Hospital in Tabriz. Among 34 DNA samples extracted, the
tim gene was amplified from 31 (91.1%). Of these, 13 (41.9%) samples
contained assemblage B, 17 (54.8%) contained assemblage A and one (3.2%)
contained a mixture of assemblage A and assemblage B. Of these, three
samples (8.8%) were negative. The results indicated that PCR technique
provides an applicable and feasible method for detection and identification
of Giardia cysts in stool samples. The results of furthermore, demonstrated
that Giardia intestinalis assemblage A and B exist in East
Azerbaijan province of Iran.
Giardia intestinalis [synonym of G. lamblia, G. duodenalis
(Haque et al., 2007; Di Matteo et al., 2008; Sokmen
et al., 2008; Ponce-Macotela et al., 2008)] is a common
cause of parasitic gastroenteritis and is a major health concern
worldwide (Wolfe, 1992). Giardiasis is believed to be responsible
for 2.5 million diarrhoea-associated deaths and nutritional deficiencies
in children in developing countries (WHO, 1998). This organism is endemic
throughout the world, with the highest prevalence occurring in the tropics
and subtropics (Jones, 1998). Worldwide incidence is believed to range
from 20 to 60% (Tripathi et al., 1999). The incidence rate is 2-7%
in industrialized nations (Upcroft and Upcroft, 2001). The prevalence
of giardia in Iran has been reported to 10.9% (Sayyari et al.,
2005). This infection is diversely dispersed throughout allover Iran,
such as East Azerbaijan province. The incidence rate in this province
is variable among 15.2% (Tabriz City) to 43.8% (country of Naghadeh) (Saebi,
2005). This protozoan produces robust cysts, which are voided in the feces
and transmitted directly through faecal/oral contact, or by ingesting
the contaminated water or food (Gelanew et al., 2007). Although
morphologically identical isolates of G. intestinalis have been
shown to be phenotypically and genotypically heterogeneous (Thompson et
al., 2000), the majority of G. intestinalis isolates from
humans and some species of domestic animals can be grouped into two distinct
genetic assemblages, A and B (Homan et al., 1992; Thompson et
al., 2000). The assemblage A isolates have been further grouped into
two distinct clusters AI and AII. Cluster A-I consists of a mixture of
closely related animal and human isolates that appear to have undergone
a recent global dispersion. Much of the focus regarding the potential
for zoonotic transmission of Giardia has centered on this group
and to a lesser extent, genotypes in Assemblage B. The assemblage B isolates
have been separated into cluster BIII and BIV. In contrast, Cluster AII
consists entirely of human isolates (Thompson et al., 2000). Isolates
of G. intestinalis have been shown to differ in their pathogenicity,
virulence and other biological characteristics (Read et al., 2002).
In the present study, we used tim-based PCR technique to characterize
and determine distribution of human Giardiasis assemblages A and B in
fecal samples collected from humans from the Tabriz city, Iran.
MATERIALS AND METHODS
Collection and purification of cysts: Three hundred and twenty
five faecal samples were collected from individuals in two localities
at the Tabriz (Center of East Azerbaijan province at the Northwest of
Iran), including: patients with acute gastroenteritis hospitalized in
the Paediatric Hospital and infected humans referred to the Tabriz Reference
Laboratory (TRL). These samples were collected from sporadic cases giardiasis
between May and October 2007. The most of faecal samples of the collected
from TRL were related to workers that they were coming from suburb to
center place of Tabriz. The children`s samples of collected from pediatric
hospital related to children belonging to households which they were settlement
of areas that characterized by a high number of children per household,
poor sanitation and personal hygiene and lack of health education. Stool
samples were collected in plastic cups; approximately 5 g were then transferred
to the Parasitology Laboratory of the Faculty of Medicine of the Tabriz
Medical University. Fecal samples were examined using light microscopic
examination by Lugol`s iodine-stained wet mounts and formalin-ethyl acetate
concentration technique for parasite diagnosis (Amar et al., 2002).
Thirty-four fecal samples (10.46%) containing G. intestinalis
cysts were obtained during this study. Of the 34 patients, 80% were adults
aged between 20 to 60 years and 73% were males. Cysts were partially purified
from faecal material by sucrose density gradient method (Barazesh et
al., 2006) and washing with sterile distilled water and then all samples
were stored at 4°C without preservatives for up to 7 weeks (Bertrand
et al., 2005).
Genomic DNA extraction: Genomic DNA of G. intestinalis
isolates were extracted by freeze-thawing technique and then by method
of modified proteinase K, SDS and CTAB. This method was done before (Van
Soolingen et al., 1994), but we did set up the modified method
Suspend 300 μL of suspension of cysts obtained from the feces in
150 μm of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) by vortexing.
Then, 60 μL of 10% SDS and 10 μL of 20 mg mL-1 proteinase
K added and vortex and incubate (overnight) at 60°C. After than, add
100 μL of 5 M NaCl and vortex. Which followed by added 80 μL
of CTAB/NaCl solution (1:7) (was warmed in 65°C), vortex until the
liquid content becomes milky and incubate for 10 min at 65°C. In this
stage add 700 μL of Chloroform/Isoamyl Alcohol (24:1), vortex for
10 sec and centrifuge for 8 min at 11,000 g and precipitate the nucleic
acid by adding 0.6 volume (420 μL) of 2-propanol to the aqueous supernatant
and keep the mixture for 30 min at -20°C. Which followed by 15 min
centrifugation at 12,000 g at room temperature (RT). Then, wash the DNA
pellet by adding 1 mL of cold 70% ethanol and centrifuge for another 5
min at 12,000 g. Carefully remove the supernatant and permit the pellet
to dry at room temperature for approximately 15 min. Finally redissolve
the pellet in 30 μL of deionized water. The DNA is stored at -20°C.
Primers: Primers sets A-for/A-rev (A-PCR) and B-for/B-rev (B-PCR)
were used for amplification of the G. intestinalis Triose
phosphate isomerase (tim) gene, as previously (Bertrand et
al., 2005). The primers used for assemblage A amplification were:
A-for 5`-GGAGACCGACGAGCAAAGC-3` (positions 839 to 857 on the WB sequence,
GenBank no. L02120) and A-rev 5`-CTTGCCAAGCGCCTCAA-3` (positions 970 to
986 on the WB sequence). The primers used for assemblage B amplification
were: B-for 5`-AATAGCAGCACARA ACGTGTATCTG-3` (positions 126 to 150 on
the BAH-12 sequence) and B-rev 5`-CCCATGTCCAGCAG CATCT-3` (positions 188
to 206 on the BAH-12 sequence) (Cinnagen, Iran). A 148 bp fragment of
the assemblage A gene with primers A-for and A-rev (A-PCR) and a 81 bp
fragment of assemblage B gene with primers B-for and B-rev (B-PCR) were
PCR amplification for identification of G. intestinalis assemblages
A and B: PCR amplification of the Triose phosphate isoerase (tim)
gene was performed by using conventional thermocycler (model Eppendrof-Germany).
Two separated PCR amplification were done with primers A-for/A-rev and
B-for/B-rev as described above. Amplification reactions (20 μL) contained
2 μL of template DNA, 1xPCR reaction buffer corresponding to a final
concentration of 1.5 mM MgCl2 (Fermentas, Lithuania), 50 mM
KCl, 20 mM Tris-HCl (Cinnagen, Iran), each deoxynucleotide triphosphate
at a concentration of 100 μM (Fermentas, Lithuania), 0.5 μM
of each primer (F/R) and 2.5 U of Taq DNA polymerase (Cinnagen, Iran)
For A-PCR and B-PCR. Samples were subjected to an initial denaturation
of 94°C for 10 min, 50 cycles of 94°C for 35 sec, 63°C for
35 sec and 72°C for 45 sec and a final extension at 72°C for 7
min. For B-PCR, cycling parameters were 10 min at 95°C (initial heat
activation step), followed by 50 cycles of 35 sec at 94°C, 30 sec
at 65°C and 40 sec at 72°C, with a final extension of 7 min at
72°C. Both positive and negative controls (distilled water) were included
in A-PCR and B-PCR to validate results. For positive control, first the
genotype of Giardia intestinalis cysts from one fecal sample classified
in assemblage A with GDHiF/GDHiR (GDH-PCR) (Read et al., 2004)
was confirmed by sequencing analysis (Unpublished data). This analysis
showed 100% matches between the amplified product obtained with GDH-PCR
(432 bp) and the sequence of G. intestinalis assemblage A.
Gel electrophoresis: PCR products were separated by electrophoresis
in 2% agarose gel, stained with ethidium bromide (0.5 μg mL-1)
and visualized under a UV transilluminator. A 100 bp, plus DNA ladder
(Fermentas, Lithuania) was included as a size marker.
RESULTS AND DISCUSSION
Studies were performed with DNA extracted from purified cysts by using
method of modified proteinase K, SDS and CTAB. A 148 bp fragment of the
assemblage A gene and a 81 bp fragment of assemblage B gene were amplified
along A-PCR and B-PCR, respectively (Fig. 1). Among the
one hundred fecal samples collected from children hospitalized in pediatric
hospital, no cyst was observed and all of the 34 positive samples were
related to patients referred to the Tabriz Reference Laboratory. Among
fecal samples of sporadic cases of giardiasis identified by conventional
techniques (n = 34), the tim gene was amplified from 31 samples
(91.1%) with A-PCR and B-PCR (two separated amplification step) developed
in our laboratory. Of these, 13 (41.9%) samples contained assemblage B,
17 (54.8%) contained assemblage A, one
||Differentiation of G. intestinalis assemblages
A and B with the tim-based on PCR technique with the primer
pairs A-for/A-rev and B-for/B-rev in Tabriz city. The figure shows
a 2% low-melting-point ethidium bromide-stained agarose gel of A-PCR
and B-PCR products (148 and 81 bp). Lanes 1, Giardia intestinalis
assemblage A group II (positive control); lanes 2, assemblage A; lane
3; assemblage B of G. intestinalis specimens from patients
with giardiasis; lane 4; Negative control and Lane M, 100 bp DNA ladder
(Cinnagen/Iran) (the 500 bp fragments are indicated)
||Results of PCR analysis of the G. intestinalis
tim gene amplified from DNA extracted from feces
|aTotal No. = 325, bNot amplified
(3.2%) contained a mixture of assemblage A and assemblage B and three
(8.8%) samples were negative (Table 1). The negative
results observed could be explained by the presence of parasites at a
very low level or degradation of parasite DNA during transport of the
samples to our laboratory (Bertrand et al., 2005). The tim gene
fragment from assemblages A and B could be amplified by using 0.5 and
0.05 pg of DNA per reaction mixture, respectively, equivalent to 50 and
5 copies of the tim gene, respectively, based on a genome size
of 1.2x107 bp (Adam, 2000). The mixture of these assemblages
has been reported previously in a few studies (Amar et al., 2002;
Guy et al., 2004; Lalle et al., 2005). Amar et al.
(2002) observed a mixture of assemblage A and assemblage B in 9% of 35
samples, The observation that the majority of sporadic giardiasis case
isolates were assemblage A genotype (54.8%) in contrast with the findings
of several studies conducted in France [61.5%, n = 26 (Bertrand et
al., 2005)], India [100%, n = 10 (Sulaiman et al., 2003)],
Peru [76%, n = 25 (Sulaiman et al., 2003)], United States [80%,
n = 15 (Weber et al., 1992)] and United Kingdom [64%, n = 35 (Amar
et al., 2002)]. However, an Italian study reported 80% assemblage
A in 30 stool samples examined by sequencing or PCR-RFLP analysis of the
β-giardin gene (Caccio et al., 2002). These differences in
the prevalence of assemblages A and B may be attributed to the geographical
locations of the populations studied. The predominance of assemblage B
in samples collected in sewage treatment facilities was shown in one study
(Guy et al., 2003). With respect to different pathogenicity (Read
et al., 2002; Aydin et al., 2004) and zoonotic aspects (Thompson
et al., 2000) of assemblages A and B, this study provides, for
the first time, information on the distribution of the genotypes of G.
intestinalis from humans with sporadic giardiasis in East Azerbaijan
province of Iran. Present findings agree with the findings of other investigators
which have identified two very distinct genotypes of G. intestinalis
each of which appear to be distributed throughout the world (Van Keulen
et al., 1995) according to Polish and Belgian (Homan et al.,
1992) and groups ½ and 3
(Nash, 1995), although, assemblage B isolates appear to be less widespread
and restricted to localized endemic foci (Meloni et al., 1995).
Higher frequency of assemblage A was shown in this study. From a public
health importance view, among the G. intestinalis strains, assemblage
A subgroups AI and assemblage B subgroups BIII and BIV have zoonotic importance.
Thus, further studies by application of appropriate genotyping tools in
endemic foci where predictive assessments can be made about transmission
patterns are needed to characterise the full genetic nature of parasites
from human and animal to identify the source of contamination and human-infective
potential of species. In summary, we developed a highly sensitive PCR
technique to detect and distinguish G. intestinalis assemblages
A and B. This study provides the first information about the distribution
of the two major assemblages of G. intestinalis in sporadic human
giardiasis in Iran. However, further studies with a larger series of fecal
or environmental samples could lead to better knowledge of the distribution
of these assemblages in humans as well as the role of domestic animals
and livestock as a potential source of infection for humans. The results
of this study might help public health care systems in management of Giardiasis.
We thank colleagues in Tabriz Reference Laboratory and Tabriz Paediatric
Hospital Laboratory for providing stool samples.
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