Molecular epidemiology and multilocus genotyping of Giardia duodenalis in individuals attending major public hospitals in Shiraz, southwestern Iran: A public health concern

Giardia duodenalis is one of the most common causes of waterborne disease worldwide, and is often associated with outbreaks of diarrhea in areas with poor sanitation and hygiene. This study aimed to assess the prevalence and genetic diversity of G. duodenalis assemblages in individuals attending major public hospitals in Shiraz, southwestern Iran. From August 2022 to May 2023, a total of 614 stool samples from individuals were collected and initially examined for G. duodenalis cysts using parasitological techniques, sucrose flotation, and microscopy. Microscopy-positive samples were validated by SSU-PCR amplification of the parasite DNA. A multilocus genotyping (MLG) scheme, which focused on the triose phosphate isomerase (tpi) and the glutamate dehydrogenase (gdh) genes, was employed for genotyping purposes. G. duodenalis cysts were found in 7.5% (46/614) and 8.5% (52/614) of samples through microscopy and SSU-PCR, respectively. Successful amplification and sequencing results were obtained for 77.3% (17/22) and 45.5% (10/22) of the infected samples at the tpi and gdh loci, respectively. MLG data for the two loci were available for only five samples. Out of the 22 samples genotyped at any loci, 54.5% (12/22) were identified as assemblage A, while 45.5% (10/22) were identified as assemblage B. AII was the most predominant sub-assemblage identified [54.5% (12/22)], followed by BIII [27% (6/22)], discordant BIII/BIV [13.6% (3/22)], and BIV [4.5% (1/22)]. In the present study, no assemblages suited for non-human animal hosts (e.g., C—F) were detected. This suggests that the transmission of human giardiasis in Shiraz is primarily anthroponotic. Further molecular-based analyses are necessary to confirm and expand upon these findings. These analyses will also help determine the presence and public health importance of the parasite in environmental samples, such as drinking water.

Giardia duodenalis is one of the most common causes of waterborne disease worldwide, and is often associated with outbreaks of diarrhea in areas with poor sanitation and hygiene.This study aimed to assess the prevalence and genetic diversity of G. duodenalis assemblages in individuals attending major public hospitals in Shiraz, southwestern Iran.From August 2022 to May 2023, a total of 614 stool samples from individuals were collected and initially examined for G. duodenalis cysts using parasitological techniques, sucrose flotation, and microscopy.Microscopy-positive samples were validated by SSU-PCR amplification of the parasite DNA.A multilocus genotyping (MLG) scheme, which focused on the triose phosphate isomerase (tpi) and the glutamate dehydrogenase (gdh) genes, was employed for genotyping purposes.G. duodenalis cysts were found in 7.5% (46/614) and 8.5% (52/614) of samples through microscopy and SSU-PCR, respectively.Successful amplification and sequencing results were obtained for 77.3% (17/22) and 45.5% (10/22) of the infected samples at the tpi and gdh loci, respectively.MLG data for the two loci were available for only five samples.Out of the 22 samples genotyped at any loci, 54.5% (12/22) were identified as assemblage A, while 45.5% (10/22) were identified as assemblage B. AII was the most predominant sub-assemblage identified [54.5% (12/22)], followed by BIII [27% (6/22)], discordant BIII/BIV [13.6% (3/22)], and BIV [4.5% (1/22)].In the present study, no assemblages suited for non-human animal hosts (e.g., C-F) were detected.This suggests that the transmission of human giardiasis in Shiraz is primarily anthroponotic.Further molecular-based analyses are necessary to confirm and expand upon these findings.These analyses will also help determine the presence and public health importance of the parasite in environmental samples, such as drinking water.

Introduction
Giardia duodenalis (syn.Giardia lamblia, Giardia intestinalis) is a parasite that infects the small intestine of mammals, including humans.G. duodenalis is a globally distributed parasitic protozoan with a prevalence of 0.4-7.5% in developed countries and 8-30% in developing countries (Hashemi-Hafshejani et al., 2022;Heyworth, 2016).G. duodenalis infections are caused by ingesting cysts in contaminated food or water.Asymptomatic G. duodenalis infections are common in humans and usually clear within weeks without treatment (Rafiei et al., 2020).Asymptomatic infections can lead to malabsorption syndrome, characterized by poor growth in children in developing countries (Mahdavi et al., 2022;Pouryousef et al., 2023).Symptomatic disease causes gastrointestinal symptoms such as diarrhea, discomfort, flatulence, nausea, and bloating (Mahdavi et al., 2021).The G. duodenalis complex is composed of eight genotypes, referred to as assemblages A-H, that are morphologically identical but have diverse molecular lineages.G. duodenalis assemblages A and B infect humans and various mammals (Asghari et al., 2022b;Asghari et al., 2022a).While, assemblages C -H are typically found in dogs and other canids (C, D), hoofed livestock (E), cats (F), rodents (G), and marine mammals (H) (Asghari et al., 2023;Zajaczkowski et al., 2021).Nevertheless, recent evidence suggests that livestock-only circulating assemblages (i.e., E) may also infect humans, suggesting some assemblages have less strict host-specificity, potentially enabling transmission from non-human mammals to humans (Dixon, 2021;Fu et al., 2022).An allozyme examination specified four sub-assemblages within assemblages A and B (AI-AIV and BI-BIV), of which AI, AII, BIII, and BIV have been particularly recognized in humans.Following nucleotide sequence and phylogenetic analysis have confirmed sub-assemblages AI-AIII within assemblages A, with AI being isolated mostly from animals, whereas AII is primarily determined in humans.Additionally, AIII has mainly been reported in wild mammals (e.g., deer), with only two human cases reported recently (Hashemi-Hafshejani et al., 2022;Seabolt et al., 2022).Moreover, multilocus sequence typing (MLST) has distinguished 9-12 subtypes/genotypes at each of the individual loci within the three main sub-assemblages A. However, the analysis of genetic loci sequences has not found distinct sub-assemblages within assemblage B, likely due to high sequence diversity unsupported by bootstrap analysis.G. duodenalis assemblages A and B infect various mammalian hosts, including humans.Hence, these infections are of zoonotic significance (Costache et al., 2020;Klotz et al., 2022;Song et al., 2021).
Genetic markers such as small subunit ribosomal RNA (SSU-rRNA), glutamate dehydrogenase (gdh), triosephosphate isomerase (tpi), and b-giardin (bg) genes are frequently utilized to identify G. duodenalis in various hosts (Asghari et al., 2024), enabling the identification of genetic diversity and population dynamics among G. duodenalis assemblages (Köster et al., 2021).The SSU-rRNA gene is a highly conserved and multicopied locus, which makes it a potential substitute for identifying and distinguishing G. duodenalis assemblages.However, it is less useful in determining genetic diversity within assemblages due to its conserved nature and short A. Asghari et al. amplified fragments in most PCR assays.On the other hand, the single-copy tpi, bg, and gdh loci are more sensitive in detecting genetic variation and categorizing G. duodenalis populations at the sub-assemblage and genotype levels.However, these loci are not considered potential candidates for identifying G. duodenalis in clinical settings.Although there is agreement on the ability of these loci to genetically classify different G. duodenalis assemblages, conflicting results have been reported regarding the efficacy of a single locus in distinguishing G. duodenalis populations into assemblages and sub-assemblages.Thus, in order to enhance accuracy, a numeric multilocus genotyping (MLG) procedure was introduced, which involves the simultaneous analysis of at least two genes (tpi, bg, and gdh) (Dan et al., 2019;Hashemi-Hafshejani et al., 2022;Heng et al., 2022;Köster et al., 2021;Oh et al., 2021;Rafiei et al., 2020).
Hence, due to the importance of giardiasis in humans, especially in immunocompromised patients and children under 5 years of age, and the lack of extensive multilocus genotyping study of Giardia among the people of Shiraz city, the present study aims to determine the prevalence, distribution of assemblages, and investigate the genetic diversity between human isolates of G. duodenalis among people referring to public hospitals in Shiraz city.

Ethics approval
The study protocol was reviewed and approved by the Ethics Committee of Shiraz University of Medical Sciences (Approval No. IR.SUMS.REC.1400.460),Shiraz, Fars Province, Iran.

Study area
This is a descriptive cross-sectional study that investigated multilocus genotyping, assemblage distribution, and phylogeny of G. duodenalis infection in individuals attending major public hospitals in Shiraz at 29.5926 N latitude and 52.5836 E longitude between August 2022 and May 2023 (Fig. 1).Shiraz is a metropolis in Iran and the capital of Fars Province in the southwest of the country.According to the census 2021, the population of Shiraz metropolis was 1,955,500 people.Shiraz is the fourth largest and most populous city in Iran and the most populous city in the south of the country.This city is located in the central part of Fars Province, at an altitude of 1486 m above sea level and in the mountainous region of Zagros with a mild climate.

Sample collection and G. duodenalis identification
According to the arrangements made with the officials and experts of the parasitology department of Shahid Beheshti, Saadi, Namazi, and Hafez hospitals in Shiraz, 614 stool samples were randomly collected from people who were referred to laboratories of these hospitals.Direct microscopy using formalin-ether concentration was used for screening all stool samples (Rafiei et al., 2020).After microscopy examination, the sucrose-flotation method was conducted to isolate the cysts of G. duodenalis from positive samples.In brief, almost 5 g of stool was blended with 50 ml of distilled water, sifted by means of a four-layer gauze, and centrifuged at 1000 ×g for 5 min.Following the supernatant deletion, the residue was then mixed with 30 ml of distilled water and added to a 15 ml cold sucrose solution (1 M).After centrifuging at 800 ×g for 5 min, the top two layers with G. duodenalis cysts were moved to a new falcon tube and the volume was carried to 50 ml with distilled water.After three centrifugations at 1000 ×g for five min, a portion of the pellet was examined under a light microscope at 400 × magnification.The remaining sediment was stored at − 20 • C for future molecular analysis.

DNA extraction
To facilitate cyst rupture and parasite DNA extraction, fecal pellets were subjected to five freeze-thaw cycles comprising 5 min in liquid nitrogen and 5 min in a boiling water bath.Genomic DNA was straightforwardly extricated employing a QIAamp DNA Stool Mini Kit (QIAgen, Hilden, Germany), according to the manufacturer's instruction.DNA was eluted in 100 mL of distilled water and prepared DNA was located at − 20 • C prior to utilize for polymerase chain reaction (PCR).

Molecular identification of G. duodenalis
All fecal samples were re-checked using molecular analyses after microscopic examination.The parasite's presence was initially confirmed using a nested PCR protocol to amplify a ~ 130-bp fragment of the SSU-rRNA gene (Gillhuber et al., 2013).Thus, primary and secondary reactions were conducted utilizing the outer primer pair RH11 and RH4, and the inner primer set GiarF and GiarR, respectively.The reaction mixture contained 2 μl of template DNA for both the primary and secondary PCR reactions.Cycling conditions were the same for the first and second steps of PCR, with primary denaturation at 95 • C for 3 min, followed by 35 cycles of amplification (denaturation at 95 • C for 30 s, annealing at 55 • C for 30 s), with a final extension of 7 min at 72 • C.
Positive G. duodenalis specimens at SSU-PCR were afterward analyzed utilizing a multilocus genotyping (MLG) method according to the amplification of partial sequences of the gdh and the tpi genes.To amplify a ~ 432-bp fragment of the gdh gene, a semi-nested PCR was accomplished (Read et al., 2004).The protocol contained the outer primer set GDHeF and GDHiR, and the inner primer set GDHiF and GDHiR.Both PCR reaction mixtures included 5 μl of template DNA.Both the primary and secondary PCRs were performed as follows: an initial denaturation step of 95 • C for 3 min, followed by 35 cycles of 95 • C for 30 s, 55 • C for 30 s, and 72 • C for 60 s, with A. Asghari et al. a final extension of 72 • C for 7 min.Regarding the tpi gene (Sulaiman et al., 2003), a ~ 530-bp fragment was amplified employing the outer primer pair AL3543 and AL3546, and the inner primer pair AL3544 and AL3545.The primary PCR conditions included an initial denaturation phase at 94 • C for 5 min followed by 35 cycles of 94 • C for 45 s, 50 • C for 45 s, and 72 • C for 60 s, with a final extension of 72 • C for 10 min.The conditions for the secondary PCR were identical to the primary PCR.
All aforementioned PCR reactions were performed in a FlexCycler2 PCR Thermal Cycler (Analytik Jena, Jena, Germany).PCR mixtures (25 μL) comprised 12.5 μL of 2× Master Mix RED already consisting Taq DNA polymerase (Ampliqon-Biomol, Hamburg, Germany), 200 nM of each forward and reverse primer, and nuclease-free water.Negative (no DNA template) and positive (known G. duodenalis PCR-positive sample) controls were routinely contained in all PCR steps.Secondary PCR products were investigated by electrophoresis on 1.5% agarose gels (Sinacolon, Tehran, Iran) and visualized after ethidium bromide staining.A. Asghari et al.

Sequencing and phylogenetic analyses
Received gdh and tpi amplicons of the right size were instantly dispatched for bidirectional sequencing employing the related internal primer pairs at Microsynth AG (Balgach, Switzerland).Raw sequences were visually checked utilizing the free software Chromas version 2.1 as quality control and to identify the existence of single nucleotide polymorphisms (SNPs) including double peaks.Acquired consensus sequences were evaluated with those formerly deposited at the National Centre for Biotechnology Information (NCBI) using the BLAST tool (http://www.ncbi.nlm.nih.gov/blast).Designation of assemblages and sub-assemblages was conducted by sequence alignment using ClustalW in MEGA X (www.megasoftware.net).A phylogenetic investigation was conducted on the sequences acquired in the present study at the gdh and tpi loci and earlier published sequences of human origin recovered from GenBank utilizing the Neighbor-Joining (NJ) procedure.Evolutionary connections were computed by the Kimura-2-parameter model in MEGA X.The trustworthiness of these trees was estimated by using the bootstrap method with 1000 pseudoreplicates.Regarding haplotype analyses, genetic diversity was measured based on haplotype diversity (Hd) and nucleotide diversity (π).Values for the numbers of polymorphic sites, parsimony informative sites, haplotype frequencies, and the average number of nucleotide differences among sequences were estimated.These genetic diversity values were computed using the software DnaSP v5.0 (Xu et al., 2015).The Median-joining haplotype network of G. duodenalis sequences was built by PopART v1.7 software (Leigh and Bryant, 2015).Representative sequences at the gdh and tpi loci generated in the present survey were deposited in GenBank under accession numbers OR488681-OR488690 and OR488693-OR488709.

Microscopy and PCR-based prevalence of G. duodenalis
Based on formalin-ether concentration and saline/iodine wet mount examination of 614 fecal samples, 46 specimens (7.5%) were found to be infected with G. duodenalis by microscopy.In return, molecular analysis with SSU-rRNA gene revealed more sensitivity and detected 52 cases (8.5%).

Multilocus genotyping, sequencing, and assemblage distribution of G. duodenalis
Out of 52 molecular positive samples, 22 positive samples were randomly selected and evaluated for the amplification of ~530 and ~ 432 bp fragments of tpi and gdh genes by Nested-PCR.Among the 22 examined samples, PCR amplification and successful sequencing by tpi and gdh genes were 77.3% and 45.5%, respectively.The phylogenetic analysis using Neighbor-joining method and Kimura 2-parameter model demonstrated two assemblages (A and B) and three sub-assemblages (AII, BIII, and BIV) among infected people (Figs. 2 and 3).The assemblage/sub-assemblages of all 22 examined samples was characterized by at least one gene locus.Accordingly, 54.5% (12/22 samples) of the positive cases belonged to sub-assemblage AII, 27% (6/22 samples) to sub-assemblage BIII, 13.6% (3/22 samples) were identified as sub-assemblage BIII/BIV and 4.5% (1/22 samples) were detected as sub-assemblage BIV (Table 1).No A + B mixed infections, nor host-specific assemblages of canine, feline, or livestock (C -F) origin were detected.
Out of the 10 gdh sequences identified, seven were assigned to the sub-assemblage AII and three were discordant BIII/BIV results.Of the five AII sequences, one (OR488681) showed 100% identity with a previously described reference sequence (GenBank accession number L40510), and the remaining four varied from 1 to 4 SNPs with L40510.The three BIII/BIV sequences had 3 and 4 SNPs (Table 2).Out of the 17 tpi sequences identified, eight were assigned to the sub-assemblage AII, six to the BIII, one to the BIV, and three were discordant BIII/BIV results.Of the eight AII sequences, one (OR488698) showed 100% identity with a previously described reference sequence (GenBank accession number U57897), and the remaining sixteen varied from 2 to 5 SNPs with U57897.The six BIII, three BIII/BIV, and one BIV sequences had 1 and 6 SNPs (Table 2).

Haplotype analyses
Regarding tpi-based sequences of assemblage A, 11 sites were variable (polymorphic) and one site was parsimony informative, resulting in the identification of six haplotypes from 38 G. duodenalis sequences isolated from Iran and various countries.Accordingly, average number of the nucleotide differences (k), haplotype diversity (Hd), and nucleotide diversity (π) were 0.629, 0.249, and 0.00136 respectively.Of the six obtained haplotypes, five were from Iran which were not shared with any of the investigated regions.Whereas, only one haplotype was shared among Iran and other countries.Of note, in the present study, haplotype 1 (H1) occurred in the most sequences of G. duodenalis with the most prevalence from Iran (Fig. 4).
Concerning tpi-based sequences of assemblage B, 20 sites were variable (polymorphic) and five site was parsimony informative, leading to the detection of 11 haplotypes from 63 G. duodenalis sequences isolated from Iran and various countries.The average number of nucleotide differences (k), haplotype diversity (Hd), and nucleotide diversity (π) were 0.629, 0.552, and 0.0043 respectively.From 11 identified haplotypes, nine were not shared among Iran and other countries.In terms of sub-assemblage BIII, haplotype 3 (H3) occurred in the most sequences of G. duodenalis (n = 39) and shared among Iran and other countries.Moreover, from 10 haplotypes of Genotype B which found in Iran, nine haplotypes were special to Iran.From two haplotypes of BIV (n = 14) only one haplotype (H5, n = 1) found in Iran, while the haplotype H4 with the most frequently (n = 13) shared between other countries.No BIV haplotype was shared between Iran and other countries (Fig. 5).

Discussion
Giardiasis is a major cause of diarrhea, particularly among children under the age of five in developing nations (Kiani et al., 2020).In Iran, the prevalence of giardiasis in humans has been documented to range from 5% to 23% in different regions of the country (Abasian et al., 2013).Among domestic animals, goats have a prevalence rate of 5-16%, while sheep have rates of 6-20%, and cattle have a rate of 4% (Asghari et al., 2022a;Rafiei et al., 2020).Nevertheless, there is a dearth of molecular epidemiological studies utilizing the MLG system in Iran.The available molecular data on giardiasis in Iran are limited to a few studies that utilized PCR-RFLP and nested-PCR techniques to examine specific loci such as gdh, tpi, and/or bg (Pestehchian et al., 2012;Tappeh et al., 2014).Previous investigations of human giardiasis in Iran, specifically in Shiraz, primarily relied on microscopy, with molecular methods being used ` 2 Genetic diversity, nucleotide variations, and accession numbers of G. duodenalis sequences isolated from the present study and reference samples registered in the GenBank based on tpi and gdh genes.A. Asghari et al.only for single-locus analyses (Bahrami et al., 2017;Kasaei et al., 2018;Mahmoudi et al., 2020;Rayani et al., 2014).Molecular findings in the country indicate that assemblage A and sub-assemblage AII are the primary genetic variants of G. duodenalis found in the Iranian population (Hashemi-Hafshejani et al., 2022;Mirrezaie et al., 2019).To our knowledge, this is the first investigation employing MLG schemes based on tpi and gdh loci in G. duodenalis-infected individuals in Shiraz, the capital of Fars Province, southwestern Iran.
In this study, G. duodenalis cysts were detected in 7.5% (46/614) and 8.5% (52/614) of samples using microscopy and SSU-PCR, respectively.This prevalence rate is considered moderate when compared to global reports on human giardiasis (Dixon, 2021).To further investigate, MLG and sequencing were conducted on 22 out of the 52 positive G. duodenalis isolates obtained from individuals referred to Saadi, Shahid Beheshti, Hafez, and Motahari hospitals in Shiraz.This analysis aimed to identify the infective assemblages/ sub-assemblages and assess the genetic diversity among the relevant sequences.Our findings revealed that individuals infected with G. duodenalis in Shiraz were infected with assemblages A and B, consistent with global reports of human giardiasis (Feng and Xiao, 2011).While a previous study (Ryan and Cacciò, 2013) indicated that assemblage B is more common than A worldwide, in line with our results, several studies in Iran (Babaei et al., 2008;Hooshyar et al., 2017;Rayani et al., 2014), Turkey (Tamer et al., 2015), Iraq (Turki et al., 2015), Syria (Skhal et al., 2017), Saudi Arabia (Al-Mohammed, 2011), Egypt (Helmy et al., 2009), Thailand (Traub et al., 2009), Italy (Cacciò et al., 2002), Czech Republic (Lecová et al., 2018), and Ethiopia (Gelanew et al., 2007) have reported a predominance of assemblage A. The discrepancies may result from the geographical distribution, the populations studied as well as the different genetic and molecular tools employed (Hashemi-Hafshejani et al., 2022;Rafiei et al., 2020), as the impact of loci is evident in the assemblage B results in tpi [52.9% (9/17)] compared to gdh [30% (3/10)] genes in the present study.Moreover, the amplification rates of the genetic loci mentioned were not uniform.Most primers could detect about 60% of the tpi genes and 40-60% of the gdh genes (Feng and Xiao, 2011).This could explain the differing amplification rates of tpi (77.3%) and gdh (45.5%) reported in the present study, which are consistent with previous findings (Feng and Xiao, 2011;Huey et al., 2013).In this study, three sub-assemblages, namely AII, BIII, and BIV, were identified among the individuals investigated in Shiraz.These findings shed light on the distribution and prevalence of different sub-assemblages of G. duodenalis in the human population.Notably, the AII sub-assemblage is closely associated with humans, while various sub-assemblages of B are primarily linked to zoonotic infections (Rafiei et al., 2020;Sprong et al., 2009).
In the current study, out of 22 positive samples tested with MLG schemes, 17 samples (77.5%) identified G. duodenalis by the tpi gene, 10 samples (45.5%) by the gdh gene, and 5 samples (22.7%) by both tpi and gdh genes.Of note, on a per-gene basis, some samples were not amplified, did not have optimal sequencing, or were not assigned to a specific assemblage/sub-assemblage; as mentioned in a previous study, this phenomenon may be due to the various factors including insufficient amount or suboptimal quality of parasite DNA, or inefficient removal of PCR inhibitors during the DNA extraction, purification process, and/or mixed infections (Cacciò and Ryan, 2008;Feng and Xiao, 2011;Huey et al., 2013).Based on the haplotyping findings, Iran exhibited high haplotype diversity for assemblage A, with six haplotypes identified among 17 examined sequences.Conversely, only one common haplotype was found among 21 sequences from various countries, highlighting the distinctiveness of Iran in terms of haplotype distribution for this gene.Furthermore, a shared haplotype was observed between Iran and other countries, indicating potential genetic connections.Additionally, Iran showed a significant presence of assemblage B haplotypes, highlighting the importance of Giardia parasites in the area and suggesting Iran as a potential origin for these assemblages, particularly BIII.Of note, the haplotyping aspect of the present study might not align entirely with statistical approaches, given the noticeable disparity in the number of Iranian sequences in comparison to other nations.Augmenting the sequences from other countries may lead to a potentially increased count of haplotypes.
Overall, our findings indicate that humans are likely a possible source of infection and person-to-person transmission probably takes place in Shiraz.However, the main limitation of this hypothesis is the limited data on non-human giardiasis in Iran.To address this issue, it is necessary to conduct extensive molecular analyses to identify the specific type of Giardia infection in humans, companion animals, and livestock that live together or exist in the same area.Additionally, the analysis should include environmental G. duodenalis isolates.

Conclusion
This is the first MLG study to genetically identify human isolates of G. duodenalis in Shiraz, the capital of Fars Province, southwestern Iran.Our MLG analysis findings showed a higher occurrence of assemblage A compared to assemblage B in the human community residing in Shiraz.The absence of sub-assemblage AI in the studied individuals supports the hypothesis that most human giardiasis in Shiraz is acquired through anthroponotic sources.Overall, further advanced molecular-based epidemiological investigations are necessary to validate the extent of the aforementioned findings.

Fig. 1 .
Fig. 1.Map of Iran, Fars Province and Shiraz city showing sampling locality of the humans in the present study.

Fig. 2 .
Fig. 2. The phylogenetic tree was constructed from the sequence of positive cases of human G. duodenalis in the current study and sequences in GenBank based on the tpi gene using the Neighbor-Joining model.

Fig. 3 .
Fig. 3.The phylogenetic tree derived from the positive cases of human G. duodenalis in this study and sequences from GenBank based on the gdh gene using the Neighbor-Joining model.

Fig. 4 .
Fig. 4. Median-joining haplotype network of G. duodenalis assemblage A sequences based on triose-phosphate isomerase (tpi) gene.Circle size is relative to haplotype frequency (n); the black circle represents extinct or unsampled haplotypes.Hatch marks on the line represent mutational steps between haplotypes.Haplotype colors represent the geographic locations of haplotypes as indicated in the right corner of the figure (green=Iran, red=Iran, and other countries).

Fig. 5 .
Fig. 5. Median-joining haplotype network of G. duodenalis assemblage A sequences based on triose-phosphate isomerase (tpi) gene.Circle size is relative to haplotype frequency (n).Hatch marks on the line represent mutational steps between haplotypes.Haplotype colors represent geographic locations of haplotypes as indicated in the figure.

Table 1
Distribution of G. duodenalis assemblages/sub-assemblages among 22 positive human samples based on tpi and gdh genes.