Next generation amplicon sequencing improves detection of Blastocystis mixed subtype infections

Blastocystis is a highly prevalent enteric protist parasite of humans and animals. Transmission occurs via the fecal-oral route through ingestion of contaminated food or water. Genetic diversity studies have identified numerous subtypes (STs) within the genus Blastocystis based on polymorphism at the SSU rRNA gene. Although there is evidence of frequent mixed subtype infections, the extent of within-host subtype diversity remains largely unexplored. Accurate assessment of Blastocystis ST diversity is crucial to understand epidemiology and sources of Blastocystis transmission to humans. Here, we report the application of next generation sequencing (NGS) for detection and characterization of Blastocystis subtypes to investigate intra-host Blastocystis diversity. A total of 75 specimens obtained from cattle feces, previously identified as Blastocystis positive, were examined using next generation amplicon sequencing. A fragment of the SSU rRNA gene was amplified using Blastocystis-specific primers and resulting amplicons were used for NGS. Comparison of Sanger and NGS results suggest greater sensitivity using the NGS approach. Using Sanger sequencing, mixed infections were suspected in 18 specimens but only confirmed through cloning in three, while NGS identified 49 mixed infections (16 times more). In addition, NGS revealed greater diversity of subtypes with 14 detected compared to 11 by Sanger. Nine more infections with potentially zoonotic STs were detected by NGS than Sanger. Indeed, subtype 3, the most common subtype found in humans, was found in 37% (28) of specimens tested by NGS but in only four specimens using Sanger. Our findings indicate that mixed Blastocystis infections may be far more common than previously thought due to the limitations of current detection methods. This next generation amplicon sequencing strategy improves detection of mixed subtype infections and low abundance subtypes and represents a valuable resource for future Blastocystis studies to improve our understanding of its epidemiology.     

Genetic Diversity of Human Blastocystis Isolates in Khorramabad,Central Iran

Background

There are some genetic differences in Blastocystis that show the existence of species or genotypes. One of these genes that help in identifying Blastocystis is SSUrRNA. The aim of this study was assessment of genetic diversity of Blastocystis by PCR with seven pairs of STS primers.

Methods

This study was done on 511 stool samples collected from patients referred to the health care centers of Khorramabad, Central Iran, in 2012. Genomic DNA was extracted and in order to determine the Blastocystis subtype in contaminated samples, seven pairs of primers STS (subtype specific sequence-tagged site) were used.

Results

Out of 511 samples, 33 (6.5%) samples were infected with Blastocystis. Subtype (ST) of 30 samples was identified and three subtypes 2, 3 and 4 were determined. Mix infection was reported 10% which 3.33% of the infection was for the mixture of ST 3 and ST5 and 6.67% was for the mixture of ST 2 and ST 3.

Conclusion

The predominant subtype was ST3 that is the main human subtype. The dominance of ST2 and 5 are important in this study. This superiority has been reported in some of the studies in ST 2 which is different from the studies in other countries, because they have announced priorities of the ST1 and ST6 after ST3.     

Epidemiology and subtype distribution of Blastocystis in humans: A review

Blastocystis is a commonly encountered gastrointestinal protozoan in humans and animals with uncertain pathogenicity. Despite its potential public health impact, epidemiological data regarding the prevalence and molecular subtype (ST) distribution of Blastocystis have been rarely reported. Among Blastocystis STs, ST1–ST4 are common in humans, including healthy and immunodeficient populations. According to the Chi-squared (χ²) association based on the data compiled for this cross-sectional study, the presence of ST1 is associated with asymptomatic infection, whereas the presence of ST4 is associated with symptomatic infection. However, cross-sectional studies cannot clarify the potential pathogenicity of Blastocystis, unlike in vivo and in vitro studies. Poor hygiene, poor sanitation and zoonotic transmission are possible factors associated with high Blastocystis prevalence, although this protozoan may be part of the normal healthy human gastrointestinal microbiota. This review covers the prevalence, STs and distribution of Blastocystis infection in humans. Thus, future epidemiological and subtyping studies could reveal new STs in humans as well as possible associations of STs with disease, drug resistance and related mechanisms such as protease activity. These associations with proper ST identification may facilitate the control of potential threats to host health, including the direct pathogenic effects of Blastocystis or alterations of the gastrointestinal microbiome.     

Blastocystis subtypes detected in humans and animals from Colombia

Blastocystis is a common enteric protist colonizing probably more than 1 billion people along with a large variety of non-human hosts. This protist has been linked to symptoms and diseases such as abdominal pain, constipation, diarrhea, flatulence and irritable bowel syndrome (IBS). Remarkable genetic diversity has been observed, leading to the subdivision of the genus into multiple subtypes (ST), some of which are exclusively found in non-human hosts. The aim of this study was to determine the distribution of Blastocystis STs in different Colombian hosts. We obtained fecal samples positive for Blastocystis by microscopy from 277 humans, 52 birds, and 117 mammals (25 cattle, 40 opossums, 40 dogs, 10 rats and 2 howler monkeys). The samples were submitted to DNA extraction, PCR and sequencing using primers targeting the small subunit rRNA gene, and ST identification was performed according to DNA barcoding. We observed the occurrence of ST1 (34%) and ST2 (23%) and lower proportions of STs 3 (11.4%), 4 (0.8%), 6 (19.8%) and 8 (10.5%). Domesticated mammals shared the same STs as those usually seen in humans (ST1, ST2, ST3), while birds and marsupials had STs, which are usually rare in humans (ST6, ST8). Further studies implementing high-resolution molecular markers are necessary to understand the phylodynamics of Blastocystis transmission and the role of this stramenopile in health and disease in Colombian populations, and to expand on the phylogeographic differences observed so far with a view to exploring and understanding host–parasite co-evolution.     

Geographic distribution of human Blastocystis subtypes in South America

Blastocystis is a cosmopolitan enteric protist colonizing probably more than 1 billion people. This protozoan exhibits genetic diversity and is subdivided into subtypes (STs). The aim of this study was to determine the distribution of Blastocystis STs in symptomatic and asymptomatic human samples from different countries of South America. A total of 346 fecal samples were genotyped by SSU rDNA showing ST1 (28.3%), ST2 (22.2%), ST3 (36.7%), ST4 (2%), ST5 (2.3%), ST6 (2%), ST7 (2.3%), ST8 (0.6%), ST12 (0.9%) and a novel ST (2.7%). These findings update the epidemiology of Blastocystis in South America and expand our knowledge of the phylogeographic differences exhibited by this stramenopile.     

Blastocystis subtypes isolated from travelers and non-travelers from the north of Poland – A single center study

Blastocystis is a common, enteric protist of humans and animals with a worldwide distribution and unclear clinical relevance. Nine out of 17 genetically diverse subtypes occur in humans. We analysed the distribution of Blastocystis subtypes and the intensity of invasion in relation to the gastrointestinal tract disorders and travels to different continents. 122 Blastocystis stool cultures were subtyped via polymerase chain reaction (PCR) with seven pairs of subtype-specific, sequence-tagged-site (STS) primers. Five subtypes of Blastocystis were detected: ST3 (59%), ST2 (19.7%), ST1 (13.1%), ST6 (3.3%), ST7 (3.3%), and two mixed infections with ST1/ST3 (1.6%). ST1 was detected exclusively in travelers to hot climate zones and ST2 was found more frequently in people visiting other continents compared to those who never left Poland. We found no correlation between gastrointestinal tract disorders, Blastocystis STs, and parasite load. There was no age predisposition to the Blastocystis infection. We established the distribution of Blastocystis STs among Poles traveling to different continents and never leaving Poland. Our study sheds more light on the problem of importing Blastocystis infection. It shows that certain subtypes detected in Europe can be imported due to travel or migration. Collecting data on the travel history of the surveyed persons is necessary to clarify this matter.     

Evolutionary and phylogenetic analyses of the barcoding region suggest geographical relationships among Blastocystis sp., ST3 in humans

Blastocystis sp., has 21 distinct subtypes of which ST3 thought to be the most prevalent subtype. This study aims to analyze the global variations of ST3. In total, 496 sequences with more than 400 nucleotides from Asia, Europe, Africa, and America were included in this study. Results show that allele 34 was the most prevalent allele in all continents. The lowest and highest allele diversity were observed in Europe and Africa, respectively. The nucleotide diversity ranged from 0.0077 in Europe to 0.02 in Africa, and haplotype diversity ranged from 0.461 in America to 0.6 in Africa. The haplotype network and Bayesian structure showed at least two major clusters including Asia and Europe-Africa-America. Tajima's D values for all continents were negative and statistically significant, indicating an excess of rare nucleotide variants. Similarly, the Fu's FS test showed negative values for all regions, indicating an excess of rare haplotypes. Pairwise F_ST exhibited a high genetic differentiation between Asia and other continents. Mismatch analysis for all populations showed a unimodal distribution. Our findings indicate that there are two probable major clusters of Blastocystis sp. ST3, a cluster which is shared between Europe, Africa, and America, and a cluster which is restricted to Asia.     

Inter- and intra-subtype variation of Blastocystis subtypes isolated from diarrheic and non-diarrheic patients in Iran

Blastocystis is a common intestinal parasitic protist infecting birds and mammals. Blastocystis comprises at least 17 subtypes (ST), of which ST1–ST9 have been detected in humans. Significant correlation between certain subtypes and pathogenicity remains to be established. Nevertheless, some studies suggest a potential linkage between subtypes (inter- and intra-subtype variation) and clinical manifestations. The aim of this study was to identify intra-subtype genetic variation of subtypes of Blastocystis in stools samples submitted by diarrheic and non-diarrheic patients. A 550-bp fragment of the nuclear small subunit ribosomal rRNA gene was amplified from 58 culture-positive samples isolated from diarrheic and non-diarrheic Iranian patients. PCR products were sequenced and sequences subjected to phylogenetic analysis. Intra-and inter-subtype variation was calculated. Based on comparison with reference sequences in GenBank, ST1, ST2 and ST3 were found in 18 (31.03%), 21 (36.22%), and 19 (32.75%) of the samples, respectively. Diarrheic stools were observed in eight (44.44%), 10 (47.61%), and nine (47.36%) patients with ST1, ST2, and ST3, respectively. No statistically significant correlation was found between subtypes and diarrhea (P = 1.000). Multiple sequence alignment exhibited a within-subtype similarity of 98.76%, 97.17%, and 99.78% in ST1, ST2, and ST3, respectively. Highest similarity was seen among ST3 isolates, while lowest similarity was seen among ST2 isolates. Phylogenetic analysis did not suggest any correlation between diarrhea and intra-subtype variation. Inter- and intra-subtype variation in SSU rRNA gene appears not to reflect differences in the clinical outcome of Blastocystis carriage.     

Epidemiological and Diagnostic Features of Blastocystis Infection in Symptomatic Patients in Izmir Province,Turkey

Background

The aims of this study were to identify Blastocystis subtypes (STs) in a cohort of Turkish patients with various gastrointestinal symptoms using a novel Real Time PCR method developed recently for Blastocystis detection and assess the relationship between Blastocystis STs and patient symptoms.

Methods

Totally, 617 stool samples of patients with gastrointestinal symptoms were examined with microscopy and inoculated in Jones medium. Blastocystis-positive samples were further assessed to identify coinfections with other possible pathogens, including bacteria and viruses. Diagnostic efficacies of microscopy, culture and Real-Time PCR were compared. PCR products were sequenced to identify the subtypes of Blastocystis isolates.

Results

Totally 94 (15.24%) samples were positive for Blastocystis after all methods. Among these, 83 of 94 (88.3%) samples were identified with all methods, while 11 were positive only with Real Time PCR. Diarrhea and abdominal pain were the leading symptoms in the patients. The only pathogenic agent identified in 76 of 94 (80.9%) patients was Blastocystis. Subtype 3 was the leading Blastocystis subtype (44.6%), while subtypes 6 and 7 were firstly isolated from symptomatic patients in our region.

Conclusion

Comparison of three diagnostic methods indicated Real Time PCR as the most sensitive and specific method. Blastocystis was the only pathogenic agent among symptomatic patients, with subtype 3 being predominant. Patients with subtypes 6 and 7 need further assessments concerning the zoonotic potential of Blastocystis.     

Molecular Detection and Identification of Zoonotic Microsporidia Spore in Fecal Samples of Some Animals with Close-Contact to Human

Background: Microsporidia species are obligatory intracellular agents that can infect all major animal groups including mammals, birds, fishes and insects. Whereas worldwide human infection reports are increasing, the cognition of sources of infection particularly zoonotic transmission could be helpful. We aimed to detect zoonotic microsporidia spore in fecal samples from some animals with close – contact to human. Methods: Overall, 142 fecal samples were collected from animals with closed-contact to human, during 2012-2013. Trichrome – blue staining were performed and DNA was then extracted from samples, identified positive, microscopically. Nested PCR was also carried out with primers targeting SSU rRNA gene and PCR products were sequenced. Results: From 142 stool samples, microsporidia spores have been observed microscopically in 15 (10.56%) samples. En. cuniculi was found in the faces of 3 (15%) small white mice and 1 (10%) laboratory rabbits(totally 2.81%). Moreover, E. bieneusi was detected in 3 (10%) samples of sheep, 2 (5.12%) cattle, 1 (10%) rabbit, 3 (11.53%) cats and 2 (11.76%) ownership dogs (totally 7.74%). Phylogenetic analysis showed interesting data. This is the first study in Iran, which identified E. bieneusi and En. Cuniculi in fecal samples of laboratory animals with close – contact to human as well as domesticated animal and analyzed them in phylogenetic tree. Conclusion: E. bieneusi is the most prevalent microsporidia species in animals. Our results can also alert us about potentially zoonotic transmission of microsporidiosis.Key Words: Laboratory animals, Enterocytozoon bieneusi, Encephalitozoon cuniculi, Zoonotic transmission     

Identification and Genetic Variation of Fasciola Species from Tabriz,North- Western Iran

Background

Fascioliasis is considered as the most important helminthic infection of cattle and sheep. Traditional approaches using morphological and biologic characters cannot cause a certainty in the accurate and precise identification and intra-specific differences of Fasciola spp. In this study, we identified Fasciola species using ITS-1 marker and described genetic variation of each species of the parasite in isolates from Tabriz slaughterhouse in West Azerbaijan Province, north- western Iran.

Methods

Overall, 100 samples (50 from sheep and 50 from cattle) morphologically detected as Fasciola worms were studied for identification of Fasciola species by PCR-RFLP method and intra-species variation of the parasite using RAPD-PCR technique.

Results

A region of approximately 460bp in all samples was successfully amplified. There were no identifiable variations among the size of PCR products. Two and three fragments in samples correspond to F. hepatica and F. gigantica was seen, respectively, through PCR-RFLP method. No difference was seen in digestion pattern according to host (sheep or cattle). Different types of each species of the parasite was observed using RAPD-PCR technique.

Conclusion

We could have an estimate of frequency of F. hepatica and F. gigantic and different genotypes of the parasite in isolates from one locality in north- western of Iran. By extension of such studies in future to other animal hosts (buffalo and goat) and including more regions to sampling, the reliability of the results and their application for control programs in zoonotic diseases will be increased.     

Molecular typing of Giardia duodenalis in cattle,sheep and goats in an arid area of central Iran

Giardia duodenalis is one of the most common intestinal parasites in humans as well as livestock and wildlife. It is of both public and veterinary health importance in developing nations. A molecular survey of Giardia duodenalis assemblages in ruminants from Yazd Province, Iran was conducted on 484 animal faecal samples collected per rectum from slaughtered ruminants including 192 cattle, 192 sheep and 100 goats from June to November 2017. Species-specific and assemblage-specific PCRs for assemblages A, B and E at the triose phosphate isomerase (tpi) gene were performed, and samples positive for Giardia were confirmed by sequencing. In total, 25 (5.16%) of examined faecal samples including eight cattle (4.2%), twelve sheep (6.2%) and five goats (5%) were infected with G. duodenalis. Assemblage-specific PCR detected G. duodenalis assemblage E in seven faecal samples (six in sheep and one in a goat). Assemblages A and B were not detected. This study provides the first insight into Giardia infection in slaughtered livestock in Iran. Although the prevalence of infection with Giardia in this hot-arid area of Iran was low, educating people about direct contact with livestock such as farmers and abattoirs workers about this zoonotic infection is important.     

Prevalence and subtype distribution of Blastocystis in healthy individuals in Sharjah,United Arab Emirates

Blastocystis is estimated to be one of the most common parasites of the intestinal tract of humans, comprising multiple subtypes (ST). Meanwhile, the distribution of Blastocystis ST in many communities and countries remains unknown. In the present work, we aimed to identify the prevalence of Blastocystis and the ST distribution in human stool samples collected from healthy expatriates from different geographical regions and residing in Sharjah, United Arabian Emirates (UAE). A total of 133 samples were screened and subtyped using partial small subunit ribosomal RNA gene sequencing. Fifty-nine (44.4%) samples were identified as positive. Among these, 39 were successfully sequenced and subtyped. The ST distribution was as follows: ST3, 58.9% (23/39); ST1, 28.2% (11/39); and ST2, 7.6% (3/39). No correlation between geographic origin and infection (χ² = 11.006; P = 0.528) nor gender and infection (χ² = 1.264; P = 0.261) was observed. The data were compared with those available for other Middle Eastern and North African neighboring countries. This study is the first to provide data concerning the prevalence of Blastocystis and the frequency of various STs in the UAE, confirming the absence of ST4 and the commonness of ST1, ST2, and ST3 in this geographical region.     

Prevalence and Genetic Characterization of Cryptosporidium Spp. In Diarrheic Children from Gonbad Kavoos City,Iran

Background: Cryptosporidium is an intestinal protozean parasite causing waterborne and foodborne outbreaks of diarrheal diseases. The present study was performed in order to find prevalence and subtypes of Cryptosporidium among children with diarrhea in Gonbad Kavoos City, Northern Iran. Methods: Diarrheic samples were collected from 547 children. The initial parasitological diagnosis was made based on detection of oocysts using the modified Ziehl-Neelsen acid-fast staining method. The positive microscopically samples were selected for sequence analysis of partial 60 kDa glycoprotein (gp60) gene. Results: Out of 547 collected samples, 27 (4.94%) were positive for Cryptosporidium oocysts. Fifteen from 27 positive samples successfully amplified in PCR. Sequences analysis of gp60 gene in 15 Cryptosporidium isolates revealed that all of them (100%) were C. parvum. The results showed three subtypes of IIa subtype family (7 cases) including IIaA16G2R1, IIaA17G1R1, IIaA22G3R1 and one subtype of IId subtype family (8 cases). The most common allele was IId A17G1d (53.3%). Conclusion: The predominance of zoonotic subtype families of C. parvum species (IIa, IId) in the present study is in concordance with previous studies in Iran and emphasizes the significance of zoonotic transmission of cryptosporidiosis in the country.Key Words: Cryptosporidium, Subtypes, Gp60 gene, Children, Iran     

Seroprevalence of Toxoplasma gondii in Sheep,Cattle and Horses in Urmia North-West of Iran

Background

Toxoplasma gondii is a zoonotic protozoan parasite found worldwide and responsible for major economic losses in most classes of livestock. This study was aimed to determine the prevalence of T. gondii infection in sheep, cattle and horses in Urmia, north-west of Iran, using MAT.

Methods

Blood samples of 276 livestock and 26 horses were collected from July 2009 till April 2010. The data were analyzed by the Chi-square, Fisher''s Exact and Cochran''s and Mantel-Haenszel Tests. The level of significance was set at P<0.05.

Results

Thirty-three (21.1%) sheep, 2 (1.6%) cattle and 3 (11.5%) horses were seropositive to T. gondii. Analysis showed that sheep were 15 times more likely to be seropositive comparing to cattle also 2 times more likely to be seropositive than horses.

Conclusion

This study showed seroprevalence of equine T. gondii infection with a considerable rate in sheep in Urmia, northwest of Iran. More comprehensive studies on livestock toxoplasmosis are required for further analysis of the parasite reservoir for human infection.     

DNA barcoding of Iranian radicine freshwater snails begins to untangle the taxonomy and phylogeography of intermediate hosts of schistosomiasis and fasciolosis from the Middle East and across Central Asia

In the Middle East radicine snails are of considerable medical and veterinary importance acting as vectors of trematodes. In Iran, such snails are responsible for the transmission of the zoonotic trematodes Schistosoma turkestanicum and Fasciola gigantica. Historically, Radix gedrosiana has been incriminated as an important intermediate host for both trematodes, however, controversy remains over the snail’s true taxonomic status. This species has been determined using morphological characters that has resulted in erroneous identification of species, affecting understanding of population biology, and ultimately affecting vector incrimination. In this current study DNA barcoding using cox1 and phylogenetic analyses revealed that snails identified as R. gedrosiana from Iran split into two separate species, Radix euphratica and Ampullaceana sp. The cox1 also provided useful insights into the evolutionary history of R. euphratica populations. Phylogeographic analyses indicated that R. euphratica had an Iraqi/Iranian origin approximately 3.3 MYA and exists as a large stable population across the Middle East and Central Asia, and a lack of genetic differentiation between geographical isolates. Such molecular barcoding techniques are crucial for the identification of radicine snails of Iran being invaluable for the monitoring of zoonotic flukes, understanding the distribution of infection and the accurate incrimination of snail vectors.     

Prevalence and Molecular Identification of Cryptosporidium Spp. in Pre-Weaned Dairy Calves in Mashhad Area,Khorasan Razavi Province,Iran

Background

Cryptosporidium parvum is a zoonotic pathogen transmissible from a variety of animals to humans and is a considerable public health concern. Dairy cattle have been identified in numerous reports as a major source of environmental contamination with this pathogen. The aim of study was to detect and isolate the Cryptosporidium spp. from fecal samples of naturally infected pre-wean calves in the Mashhad area

Methods

Overall, 300 fecal specimens from 1 to 30 days pre-weaned calves were collected from 10 farms in the Mashhad area the capital center of the Khorasan Razavi Province, Iran and microscopically examined for Cryptosporidium spp. All infected samples were also analyzed using nested –PCR. A polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis of the small-subunit (SSU) rRNA gene was also used to detect and identify Cryptosporidium spp. in PCR- positive samples.

Results

Eighty five (28.3%) of the specimens were positive for Cryptosporidium spp. The prevalence of Cryptosporidium spp. in 8-14 days old and diarrheic calves were significantly higher than other groups. Restriction digestion of the PCR products by SspI, VspI restriction enzymes and sequence analysis revealed the presence of C. parvum bovine genotype in all isolates.

Conclusions

Our results suggest that pre-weaned calves are likely to be an important reservoir of zoonotic C. parvum.     

First detection of tularaemia in domestic and wild mammals in Iran

During a study on the ecology of small-mammal-borne infections in Iran, over 4 600 wild mammals were collected at 47 localities. Attempts were made to isolate Francisella tularensis from the spleens of 3 548 of these animals. All were found to be negative. In addition, sera from 200 sheep and cattle and from 39 wild mammals were tested: 8 sheep, 3 oxen, and 1 hedgehog showed evidence of recent infection. This is the first report of tularaemia in Iran. The relationship of these findings to the potential distribution of natural foci in Iran and adjacent countries indicates that the infection in Asia may be more widespread than was previously thought.     

Studies on heterologous immunity in schistosomiasis. 6. Observations on cross-immunity to Ornithobilharzia turkestanicum, Schistosoma bovis, S. mansoni, and S. haematobium in mice, sheep, and cattle in Iran

Experiments were carried out in mice, cattle, and sheep to investigate the possibility that heterologous immune reactions may occur between the schistosomes prevalent in man and domestic animals in Iran. Immunization with Ornithobilharzia turkestanicum from cattle produced a considerable degree of immunity in mice against challenge with Schistosoma bovis, S. haematobium, and S. mansoni. The results of immunizing cattle with O. turkestanicum, S. bovis, and S. haematobium were even more striking; there was a reduction of 30-40% in the number of adult worms and a proportionally greater reduction in the tissue egg counts. Sheep developed a less marked immunity. Supplementary experiments on homologous immunity showed that mice developed a considerable degree of immunity against S. bovis. The results of the heterologous immunity experiments with S. haematobium and S. bovis are of particular interest as both parasites often occur in the same area and are often transmitted by the same snail host, man and cattle being exposed to the cercariae of both species simultaneously. The reciprocal immunity produced by these infections may be mutually beneficial in limiting the severity of schistosomiasis in man and domestic animals in the endemic areas.     

Molecular Epidemiology of Cryptosporidiosis in Iranian Children,Tehran, Iran

Background

Cryptosporidium is a worldwide protozoan parasite and one of the most common causes of infection and diarrhea in humans and cattle. The aim of the present study was determination of subtypes of Cryptosporidium among children with diarrhea in Tehran by sequence analysis of the highly polymorphic 60-kDa glycoprotein (GP60) gene.

Methods

Fecal samples were collected from 794 diarrheic children. Initial identification of Cryptosporidium was carried out on stool samples by Ziehl-Neelsen acid-fast staining method. DNA was extracted from positive microscopically samples and Cryptosporidium genotypes and subtypes were determined, accordingly.

Results

Out of 794 collected samples, 19 (2.40%) were positive for Cryptosporidium oocysts. Sequences analysis of GP60 gene showed that 17 (89.47%) of the positive isolates were Cryptosporidium parvum and 2 (10.52%) were C. hominis. All subtypes of C. parvum isolates belonged to allele families IIa (6/17) and IId (11/17). The most common allele in all 17 isolates belonged to IId A20G1a (41.18%). A22G1 (IF) subtype was detected in two C. hominis isolates of the children.

Conclusion

The predominancy of C. parvum species (specially, IId A20G1a subtype) in current study underlines the importance of zoonotic Cryptosporidium transmission in Iran.