Molecular Characterization of Echinococcus granulosus from Hydatid Cysts Isolated from Human and Animals in Golestan Province,North of Iran

Background

The aim of the present study was to determine the molecular characteristics of Echinococcus granulosus from paraffin-embedded tissues of hydatid cysts isolated from human and protoscoleces of hydatid cysts from sheep, cattle and camel isolates using PCR- RFLP of ITS1- rDNA analysis in Golestan Province, northern Iran.

Methods

E. granulosus isolates from human patients infected with hydatid cyst and protoscoleces from hydatid cysts of sheep, cattle and camel isolates were collected from different hospitals and the abattoir throughout the Golestan Province. In all, 60 E. granulosus genomic DNA were extracted and examined by PCR - ITS1 of rDNA and amplified using BD1 / 4S and EGF1 / EGR2 primers, followed by RFLP using Alu1, Msp1 and TaqI restriction enzymes.

Results

The PCR-ITS1 products obtained from sheep, cattle and human isolates were similar to sheep strain (1000 bp and 391 bp). Majority of the camel samples yielded 295 bp DNA bands. RFLP -ITS1 of E. granulosus with Taq1 in human, sheep and cattle isolates showed similar patterns in the number and size of DNA. RFLP methods in camel isolates showed a different genotype, using Taq1, whereas no DNA bands were observed using Alu1 in camel and human isolates. Therefore, two clearly distinguishable banding patterns of E. granulosus were obtained with the three enzymes, which separating human, sheep and cattle isolates from the camel origin.

Conclusion

The results indicate the possible of transmission of the G1 and G6 genotypes of E. granulosus between livestock animals and human in Golestan Province.     

Serological Evaluation of EgAgB16 kDa,a Recombinant Antigen from Echinococcus granulosus for Diagnosis of Human Hydatidosis

Background

Regarding that accurate diagnosis of human hydatidosis still needs more investigations, the present study was conducted to clone, express, and evaluate the gene encoding AgB subunits (EgAgB16 kDa) from Echinococcus granulosus (Iranian G1 strain) and its evaluation by ELISA test.

Methods

DNA was extracted from protoscoleces and was utilized by PCR for strain identification. Total RNA was prepared with RNeasy protect mini kit from E. granulosus (Iranian G1 strain) protoscoleces collected from naturally infected sheep with hydatid cyst. Recombinant AgB16 kDa was produced using pETDuet as vector and evaluated by ELISA method. A panel of sera including hydatid cyst-infected individuals (n=72), healthy individual (n=48), toxoplasmosis (n=4), strongyloidosis (n=4), kala-azar (n=5) and tuberculosis (n=5) were examined using this recombinant antigen.

Results

Recombinant protein was purified by affinity chromatography using His-Tag column. After purification, recombinant protein was confirmed by western blot analysis using His Tag monoclonal antibody or hydatid positive human serum. The sensitivity, specificity; positive and negative predictive values were calculated as 93.5%, 95.6%, 96% and 92.9%, in that order. The cut-off point was detected 0.3 for rAgB16.

Conclusion

While the produced recombinant AgB16 kDa showed promising results in diagnosing human hydatidosis, but more investigations should be implemented to reach an accurate gold standard.     

Performance of Antigen B Isolated from Different Hosts and Cyst Locations in Diagnosis of Cystic Echinococcosis

Background

The aim of this study was to assess the performance of Antigen B (AgB) isolated from different Echinococcus granulosus intermediate hosts and from different cyst locations for immunodiagnosis of human cystic echinococcosis (CE).

Methods

Hydatid cyst fluids were collected from lung and liver cysts of sheep, liver cysts of goats, lung cysts of camels and cattle, and liver cysts of human. AgB was purified from each of these hydatid cysts fluids. Serum samples obtained from 47 pathologically confirmed cases of CE along with 30 sera samples from non-CE patients and 40 sera from healthy individuals were tested by ELISA using AgB prepared from different hosts or cyst locations.

Results

The highest sensitivity (97.8%) for diagnosis of CE was seen with AgB prepared from human liver cysts. This maximal sensitivity was followed by AgB isolated from those of sheep liver and lung cysts. The least sensitivity was found with AgB prepared from bovine lung cysts. The highest specificities (97.1%) were observed with AgB isolated from human liver cysts followed by those of sheep and goat liver cysts while the lowest specificity was seen with AgB isolated from bovine lung cysts. In view of the specificities and sensitivities of the different AgB, the best validity was found for AgB prepared from human liver cysts while the least validity was found with AgB prepared from bovine lung cysts.

Conclusion

For any AgB-based tests, obtaining of the antigen from one of these sources will significantly increase the diagnostic sensitivity and specificity of the assay.     

Identification of Echinococcus granulosus Strains in Isolated Hydatid Cyst Specimens from Animals by PCR-RFLP Method in West Azerbaijan – Iran

Background

The aim of this study was DNA extraction from protoscolecses of Echinococcus granulosus and identification of these strains in West-Azerbaijan Province, north western Iran.

Methods

Thirty one livestock isolates from sheep and cattle were collected from abattoirs of the province. To investigate the genetic variation of the isolates, after DNA extraction by Glass beads-phenol chloroform method; PCR-RLFP analysis of rDNA-ITS1 was performed using three different restriction enzymes of Taq 1, Rsa 1 and Alu 1.

Result

Amplified PCR products for all isolates were 1000bp band which is expected band in sheep strains (G1-G3 complex). The results of RFLP analysis also were the same for all isolates. PCR-RFLP patterns restriction enzymes were identical as follows, Rsa1 bands under UV showed two bands approximately 655bp and 345bp. Alu1 bands were as follows: two approximately 800bp and 200bp and Taq1 did not cut any region and bands were approximately 1000 bp in all samples.

Conclusions

Based on PCR-RFLP patterns of ITS1 fragment produced with endonucleases enzyme digestion in animal isolates, it can be concluded that a single strain of E. granulosus (sheep strain or G1-G3 complex) is dominant genotype in this province.     

Molecular Cloning and Expression of EG95 Gene of Iranian Isolates of Echinococcus granulosus

Background

Echinococcosis or hydatidosis is a chronic, zoonotic worldwide infection that occurs by the larval stages of taeniid cestodes of the genus Echinococcus. Iran is known as endemic region for this infection in the world. Vaccination has been considered as a good prevention method for this disease. Recombinant vaccines containing EG95 protein, against E. granulosus, has shown a high degree of protection against E. granulosus infection. In this study EG95 gene was extracted from Iranian isolates of E. granulosus and then cloned and expressed in expression vector.

Methods

Protoscoleces were collected from sheep hydatid cysts. Then DNA and RNA were extracted from protoscoleces, and amplified by PCR and RT-PCR with specific primer. Afterward the purified RT-PCR products were successfully ligated into pTZ57R/T plasmid vector. The pcDNA3 plasmid was used as expression vector and Eg95 fragment sub cloned into this plasmid. The pcEG95 plasmid was digested by restriction enzymes to confirm cloning of this gene in pcDNA3 plasmid. In last step, the subcloned gene was expressed in CHO as eukaryotic cell.

Results

EG95 fragment successfully was subcloned in pcDNA3 and EG95 protein was expressed by eukaryotic cell. The recombinant EG95 protein was confirmed by SDS-PAGE and Western blot.

Conclusion

Recombinant plasmid of pcEG95 was constructed successfully and express of recombinant EG95 protein was confirmed.     

Genetic Variation of MSP-1 Gene in Plasmodium vivax Isolated from Patients in Hormozgan Province,Iran using SSCP-PCR

Background

The main goal of present study was to detect polymorphism in MSP-1 gene which is a major blood stage candidate for vaccine in Plasmodium vivax by Single Strand Conformational Polymorphism-Polymerase Chain Reaction (SSCP-PCR).

Methods

During 2008 to 2010 fifty samples were collected from Iranian patients with P. vivax in Hormozgan Province, southern Iran. All of the samples were detected by microscopical examination. Amplification of MSP-1 gene was done by PCR after DNA extraction. Single strand DNAs due to using in SSCP, was electrophoresed on polyacrylamid- Bisacrylamid gel then banding patterns were revealed by silver-staining method. Sequencing as a typing method was performed for some isolates.

Results

All of the 50 isolates were positive microscopically. Totally 12 (24%) isolates showed 440 bp and 38 (76%) showed 500 bp in PCR assay. SSCP analysis revealed four banding patterns. Pattern I (10/50), Pattern II (12/50), Pattern III (27/50), and Pattern IV (1/50). The results sequencing analysis of the MSP-1 gene in 19 isolates revealed diversity in nucleotides and amino acid in Iranian P. vivax isolates.

Conclusion

Our study confirms that the SSCP-PCR is a rapid method for detecting polymorphism in MSP-1 gene in P. vivax. The presence of different haplotypes in MSP-1 gene shows that several P. vivax strains exist in malaria endemic areas of Iran.     

Seroprevalence of Human Hydatidosis Using ELISA Method in Qom Province,Central Iran

Background

The objective of this study was to determine the prevalence of cystic echinococcosis (CE) in Qom Province, central Iran using ELISA test.

Methods

Overall, 1564 serum samples (800 males and 764 females) were collected from selected subjects by randomized cluster sampling in 2011-2012. Sera were analyzed by ELISA test using AgB. Before sampling, a questionnaire was filled out for each case. Data were analyzed using Chi-square test and multivariate logistic regression for risk factors analysis.

Results

Seropositivity was 1.6% (25 cases). Males (2.2%) showed significantly more positivity than females (0.9%) (P= 0.03). There was no significant association between CE seropositivity and age group, occupation, and region. Age group of 30-60 years encompassed the highest rate of positivity. The seropositivity of CE was 2.1% and 1.2% for urban and rural cases respectively. Binary logistic regression showed that males were 2.5 times at higher risk for infection than females.

Conclusion

Although seroprevalence of CE is relatively low in Qom Province, yet due to the importance of the disease, all preventive measures should be taken into consideration.     

Sequence Diversity in tRNA Gene Locus A-L among Iranian Isolates of Entamoeba dispar

Background

A number of methods for detecting diversity in Entamoeba have been described over the years. In the present study the genetic polymorphism of noncoding locus A-L was analyzed using PCR and sequencing in order to clarify the genotypic differences among E. dispar isolates.

Methods

A total of 28 E. dispar from patients with gastrointestinal symptoms were determined and the genomic DNA was extracted directly from stool. For genotype analysis; Locus A-L was amplified by PCR and PCR products were sequenced. The sequences obtained were edited manually and aligned using Gene Runner software.

Results

With sequencing of PCR products a reliable genetic diversity in size, number and position of the repeat units were observed among the Iranian E. dispar isolates in locus A-L gene. Sequences showed variation in length from 448bp to 507bp and seven distinct types were identified.

Conclusion

The genetic diversity of loci like A-L shows them to be suitable for epidemiological studies such as the characterization of the routes of transmission of these parasites in Iran.     

Morphometric Analysis of Larval Rostellar Hooks in Taenia multiceps of Sheep in Iran and Its Association with Mitochondrial Gene Variability

Background

The purposes of the present study were morphometric characterization of rostellar hooks of Taenia multiceps and to investigate the association of hook length variation and the variability within two mitochondrial genes of sheep isolates of the parasite.

Methods

Up to 4500 sheep brains were examined for the presence of C. cerebralis. Biometric characters based on the larval rostellar hook size were measured for each individual isolate. Representative mitochondrial CO1 and 12S rRNA gene sequences for each of the isolates were obtained from NCBI GenBank. Morphometric and genetic data were analyzed using cluster analysis, Interclass Correlation Coefficient (ICC) and random effects model.

Results

One hundred and fourteen sheep (2.5%) were found infected with the coenuri. The minimum and maximum number of scoleces per cyst was 40 and 550 respectively. Each scolex contained 22–27 hooks arranged in two rows of large and small hooks. The average total length of the large and small hooks was 158.9 and 112.1 μm, respectively. Using ICC, statistically significant clusters of different hook sizes were identified within the isolates. The length of the large and small hooks was significantly associated with the variability in mitochondrial 12S rRNA gene.

Conclusion

Taenia multiceps, is a relatively important zoonotic infection in Iranian sheep with the prevalence rate of 2.5%. Hook length analysis revealed statistically significant difference among individual isolates. Associations between the rostellar hook length and variability in the mitochondrial 12S rRNA was documented.     

Molecular Identification and Differentiation of Fasciola Isolates Using PCR- RFLP Method Based on Internal Transcribed Spacer (ITS1, 5.8S rDNA,ITS2)

Background

In this study, we used both ITS1 and ITS2 for molecular identification of Fasciola species.

Methods

The region between 18S and 28S of ribosomal DNA was used in PCR-RFLP method for molecular identification of Fasciola species. Ninety trematodes of Fasciola were collected during abattoir inspection from livers of naturally infected sheep and cattle from Khorasan, East Azerbaijan, and Fars provinces in Iran. After DNA extraction, PCR was performed to amplify region ITS1, 5.8S rDNA, ITS2. To select a suitable restriction enzyme, we sequenced and analyzed the PCR products of F. hepatica and F. gigantica samples from sheep and cattle. Tsp509I fast digest restriction enzyme was selected for RFLP method that caused the separation specifically of Fasciola species.

Results

The fragment approximately 1000bp in all of the Fasciola samples was amplified and then digested with the Tsp509I restriction endonuclease. Seventy F. hepatica and 20 F. gigantica were identified of total 90 Fasciola isolates.

Conclusion

The new PCR-RFLP assay using Tsp509I restriction enzyme provides a simple, practical, fast, low cost, and reliable method for identification and differentiation of Fasciola isolates.     

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.     

Sequence Analysis of the Second Internal Transcribed Spacer (ITS2) Region of rDNA for Species Identification of Trichostrongylus Nematodes Isolated From Domestic Livestock in Iran

Background

Infectivity of herbivores with Trichostrongylus nematodes is widespread in many countries, having a major economic impact on breeding, survivability, and productivity of domestic livestock. This study was carried out on Trichostrongylus species isolated from domestic livestock in order to develop an easy-to-perform method for species identification.

Methods

Trichostrongylus isolates were collected from sheep, goat, cattle, and buffaloes in Khuzestan Province, southwest Iran. Primary species identification was carried out based on morphological characterization of male worms. PCR amplification of ITS2-rDNA region was performed on genomic DNA and the products were sequenced. Phylogenetic analysis of the nucleotide sequence data was conducted employing Bayesian Inference approach. Consequently, a restriction fragment length polymorphism (RFLP) profile was designed to differentiate Trichostrongylus species.

Results

A consensus sequence of 238 nucleotides was deposited in the GenBank for Iranian isolates of Trichostrongylus species including T. colubriformis, T. capricola, T. probolurus and T. vitrinus. The designated RFLP using restriction enzyme TasI could readily differentiate among species having different ITS2 sequence. The molecular analysis was in concordance with morphological findings.

Conclusion

Phylogenetic analysis indicated a close relationship among the sequences obtained in this study and reference sequence of relevant species. ITS2-RFLP with TasI is recommended for molecular differentiation of common Trichostrongylus species.     

Sequence Analysis of Different Domains of Plasmodium vivax Apical Membrane Antigen (PvAMA-1 gene) Locus in Iran

Background

Plasmodium vivax is responsible for approximately 80 million malaria cases in the world. Apical membrane antigen1 (AMA-1) is a type I integral membrane protein present in all Plasmodium species. AMA-1 interferes in critical steps of invasion of human hepatocytes by sporozoites and red blood cells by merozoites and is one of the most immunodominant antigens for eliciting a protective immune response in human. It is considered as a promising antigen for inclusion in a vaccine against P. vivax. Since more knowledge is needed to lighten the scope of such antigen we compared genetic variation in P. vivax AMA-1from an Iranian isolate with those reported from some of the other malarious countries so far.

Methods

P. vivax genomic DNA was extracted from the whole blood of an Iranian patient with patent P. vivax infection. The nucleotide sequence for 446 amino acid (AA) residues (42–488 of PvAMA-1) was amplified by PCR and cloned in pUC19 vector for sequencing.

Results

Sequence analysis of the antigen showed a high degree of identity (99%) with strong homology to the PvAMA-1 gene of P. vivax S3 and SKO814 isolates from India and Korea (Asian isolates) respectively, and 96% similarity with P. vivax Sal-1 AMA-1 gene from El Salvador.

Conclusions

We cloned and characterized three domains of PvAMA-1 gene from an Iranian patient. Predicted protein sequence of this gene showed some discrepancies in corresponding protein in comparing with similar genes reported from other malarious countries.     

Protoscolecidal Effect of Berberis vulgaris Root Extract and Its Main Compound,Berberine in Cystic Echinococcosis

Background

Cystic echinococcosis (CE), a zoonotic parasitic infection caused by the metacestode (larvae) stage of dog tapeworm Echinococcus granulosus and recognized as a major economic and public health concern in the world. This study aimed to investigate the in vitro scolicidal effect of methanolic extract of Berberis vulgaris L. roots and its main compound, berberine against protoscoleces of hydatid cysts.

Methods

For this purpose, protoscoleces were aseptically aspirated from sheep livers having hydatid cysts. Various concentrations of the methanolic extract (0.25-2 mg/ml) and berberine (0.062- 0.5 mg/ml) were used for 5 to 30 min. Viability of protoscoleces was confirmed by eosin exclusive test.

Results

In the present study, all of the various concentrations of the B. vulgaris methanolic extract (0.25, 0.5, 1 and 2 mg/ml) and berberine (0.062, 0.125, 0.25 and 0.5 mg/ml) revealed significant (P<0.05) scolicidal effects against protoscoleces of E. granulosus in a dose-dependent manner. Both berberine and methanolic extract exhibited 100% inhibition against protoscoleces of E. granulosus at the concentration of 2.0 and 0.5 mg/ml after 10 min incubation, respectively.

Conclusion

According to the results, both B. vulgaris methanolic extract and berberine alone demonstrated high scolicidal activities against protoscoleces of hydatid cysts in low concentration and short exposure time on in vitro model. However, in vivo efficacy of B. vulgaris and berberine also requires to be evaluated using an animal model with hydatid infection.     

Genetic Variation and Selection of Domain I of the Plasmodium vivax Apical Membrane Antigen-1(AMA-1) Gene in Clinical Isolates from Iran

Background

Apical Membrane antigen 1 (AMA-1) is positioned on the surface of merozoite and it may play a role in attack to red blood cells. The main aim of present study was to determine the genetic variation, as well as, to detect of selection at domain I of AMA-1 gene Plasmodium vivax isolates in Iran.

Methods

Blood samples were collected from 58 patients positive for P. vivax, mono infection and the domain I of AMA-1 gene was amplified by nested PCR and then sequenced.

Results

A total 33 different haplotypes were identified among 58 Iranian sequences. The 23 new haplotypes were determined in this study that was not reported previously in other regions of the world. There were totally observed 36 point mutations at the nucleotide level in the analyzed sequences. Sequences analyses indicated 25 amino acid changes at 20 positions in which 5 sites demonstrated thrimorphic polymorphism and the others were dimorphic in the domain I of the Iranian PvAMA-1 isolates.

Conclusion

Our findings indicated relatively high level of allelic diversity at the domain I of PvAMA-1 among P.vivax isolates of Iran. Since, PvAMA-1 is considering as vaccine candidate antigen, these data provide valuable information for the development of a PvAMA-1 based malaria vaccine.     

Evaluation of Benzimidazole Resistance in Haemonchus contortus Using Comparative PCR-RFLP Methods

Background

In order to deworm the ruminants especially of sheep in Iran, consumption of benzimidazoles has more than 2 decades history and today farmers are using imidazothiazoles, macrocyclic lactones and mostly benzimidazole compounds (BZs) to treat infected farm animals. It has been demonstrated that the most common molecular mechanism leading to BZsresistance in Haemonchus contortus is a single mutation at amino acid 200 (phenylalanine to tyrosine) of the isotype 1 of beta tubulin gene. According to the report of such mutations in Iranian Teladorsagia circumcincta isolates with Restriction Site Created PCR-RFLP, we decided to evaluate the frequency of such mutations in H. contortus in three different geographical areas of Iran.

Methods

A total of 102 collected adult male H. contortus were evaluated with PCR-RFLP (using PSP1406I as restriction enzyme). By means of a second step to compare function of different methods and to increase sensitivity of detection mechanism, a third of samples were examined by another PCR-RFLP method (using TaaI as restriction enzyme) and finally beta tubulin gene of two samples was sequenced.

Results

All of samples were detected as BZss homozygote. Finally, beta tubulin gene sequencing of two samples showed no point mutation at codon 200.

Conclusion

It seems that BZresistance of H. contortus in Iran is not a serious problem as anticipated before.     

Hematologic and Clinical Aspects of Experimental Ovine Anaplasmosis Caused by Anaplasma ovis in Iran

Background

Anaplasma ovis infections can cause clinical symptoms in acute phase and lead to huge economic losses in flocks. The aim of the present study was to investigate the hematological and parasitological changes in experimental anaplasmosis in sheep with Iranian strain of A. ovis.

Method

Five male sheep without any blood parasite infection were selected. One hundred ml heparinized blood was collected from splenectomised sheep that showed 6% A. ovis parasitemia. Inoculums of 20 ml blood were administered intravenously to each test animal. Hematological, parasitological and clinical changes of experimental anaplasmosis were studied in 0-38 days post infection.

Result

Parasitemia was detected 3 days post infection and reached its maximum level on the day 12 of experiment in test animals. Then the parasitemia was declined, but the organism could be found persistently until the last day of study. The red cell counts, packed cell volume and hemoglobin concentration were decreased and mean corpuscular volume was increased significantly during the infection period. Reticulocytosis and basophilic stippling were also detected. No significant changes were observed in total and differential leukocyte count and animal body temperature.

Conclusion

Experimental A. ovis infection in sheep resulted in marked normocytic normochromic anemia at the beginning of the infection which became macrocytic normochromic by the development of the disease. There were negative correlations between parasitemia and RBC, PCV and Hb values, therefore hematological assessment can be considered as a practical diagnostic tool in ovine anaplasmosis.     

A Serological Survey of Cystic Echinococcosis in Equids in East of Turkey

Background

Cystic echinococcosis (CE), caused by hydatid cysts, is a widespread and hazardous disease in humans and animals worldwide. The disease is very common in Turkey, causing serious economic losses and public health problem. In this study, the seroprevalence of equine CE was determined by enzyme-linked immunosorbent assay (ELISA).

Methods

Partially purified cyst fluid antigen from sheep hydatid cyst fluid was used as antigen in ELISA. A total of 250 equids consisting of 206 donkeys and 44 horses from various regions of Erzurum province of Eastern Turkey.

Results

Anti- Echinococcus granulosus antibodies were detected in 78 (31.2%) of 250 equids. The prevalence rate was 20.4% for horses and 33.5% for donkeys. There was no statistically difference between sex and ages groups for both horses and donkeys (P>0.05).

Conclusion

Equine CE is quite endemic in Eastern Turkey. The high prevalence of CE suggests that equids in the transmission cycles is possible as a source of infection for definitive hosts.     

Study on ITS1 Gene of Iranian Trichomonas vaginalis by Molecular Methods

Background

Trichomoniasis is a worldwide protozoan parasitic disease and metronidazole is a choice drug for its treatment. Because of disease importance in public health and its controversial ideas about the prevalence of drug resistance, this study was carried out.

Methods

Fifty-two suspected vaginal samples were collected from 2006 to 2007 in Gynecology Maryam Hospital, Tehran, Iran. All isolates were examined by microscopic, culture and PCR techniques. The PCR products were analyzed by RFLP and CSGE methods and two suspected samples were sequenced.

Results

Trichomonas vaginalis was identified from all 52 samples. Of 52 isolates, 45 samples were successfully cultured and amplified by PCR except one. Seven were positive only by PCR. Finally, ITS1 fragment was successfully amplified in 51 of 52. CSGE analysis and PCR products digestion by MspI followed by sequencing showed nucleotide mutation at position 209 (C209T) of the ITS1 fragment in two (3.9%) of them.

Conclusion

The results showed mutation in ITS1 fragment of T. vaginalis in two (3.9%) of Iranian isolates which may be related to metronidazole resistance.     

Genotyping of Echinococcus granulosus Isolates from Human Clinical Samples Based on Sequencing of Mitochondrial Genes in Iran,Tehran

Background

The present study was aimed to investigate molecular diversity of Echinococcus granulosus isolates collected from human clinical samples using two mitochondrial genes cox1 and nad1 in Iran.

Methods

Forty seven human hydatid cysts were collected through surgery from two hospitals in Tehran during 2010-2012. To determine the fertility of protoscoleces, the cyst fluids were subjected to morphological microscopic examinations. Protoscoleces were removed from each cyst and their total genomic DNAs were extracted. PCR was performed to amplify fragments of 450 and 400 base pair (bp) for cox1 and nad1 genes, respectively. Genotype diversity and sequence variation of the strains were studied by bioinformatics software and in comparison with those mtDNA sequences already deposited in GenBank.

Results

Sixteen, (53.3%), 13 (43.3%), and 1 (3.3%) samples were related to lung, liver, and spleen, respectively. The remained 17 unfertile samples were excluded from the study. From the 29 isolates, 86.7% (n=26) and 10% (n=3) were related to G1, and G3 genotypes, respectively. The sole isolate with G6 genotype was obtained from lung sample. Analysis of concatenated sequences of cox1+nad1 indicated the presence of 11 haplotypes among our strains that were related to genotypes G1 (n=9), G3 (n=1) and G6 (n=1).

Conclusion

In consistent to other reports from Iran, genotypes G1, G3, and G6 were observed in our human isolates. The rate of G3 genotype was however higher than other studies implying that human can be considered as a new appropriate host for G3 genotype. Further studies with more sample size from different geographic areas of Iran are needed for E. granulosus mapping.