Mini-Exon Genotyping of Leishmania Species in Khuzestan Province, Southwest Iran

Background: Leishmaniasis is a protozoan disease cause by Leishmania genus. Anthroponotic and zoonotic cutaneous leishmaniasis are endemic in Iran. The aim of this study was to identify the causative agent of cutaneous leishmaniasis by mini-exon gene in five regions of Khuzestan Province, southwest of Iran. Methods: From 2007 to 2008 in this cross-sectional study, cutaneous samples were collected from patients referred to Health Centers and Hospitals of the Khuzestan Province for cutaneous leishmaniasis diagnosis and cultured in Novy-MacNeal-Nicolle (NNN) and RPMI 1640. The propagated promastigotes were harvested and Leishmania species of cutaneous leishmaniasis were identified by RFLP and DNA sequencing of the PCR generated fragments. Results: L. major and L. tropica were the causative agents of cutaneous leishmaniasis by predominantly of L. major species. The alignment of the mini-exon sequencing isolates with reported sequencing of L. major and L. tropica revealed 92-99 identity. Conclusion: Our study showed that mini-exon PCR-RFLP was useful method to identify the causative species of cutaneous leishmaniasis

Molecular characterization of the Iranian isolates of Giardia lamblia: Application of the glutamate dehydrogenase gene

Background: This study was conducted to determine of molecular epidemiology of the Giardia lamblia by PCR-RFLP method in Tehran, capital of Iran. Methods: Thirty eight stool samples were randomly selected from 125 patients diagnosed with giardiasis using microscopy in Tehran. DNA extraction of some samples were performed by phenol/chloroform/isoamyl alcohol method and to raise the sensitivity of the PCR assay, the genomic DNA of the others were extracted using glass beads and the QIAamp Stool Mini Kit in order to effectively remove the PCR inhibitors. A single step PCR-RFLP assay, targeting the glutamate dehydrogenase (gdh) locus, was used to differentiate within and between assemblages A and B that have been found in humans. Results: Of the 38 isolates, 33 samples (87) were found as G. lamblia (genotype AII), 3 (7.8) belonged to assemblage B, genotype BIII, the mixed of genotype AII and B were detected only in two samples (5.2). Conclusions: PCR-RFLP is a sensitive and powerful analytical tool that allows effective genotype discrimination within and between assemblages at targeting gdh gene, and makes it possible to identify the presence of mixed genotypes. Our data suggest that there is an anthroponotic origin of the infection route, assemblage A group II, in Tehran so it seems that the main reservoir of Giardia infection is humans in the area studies

Cloning of Leishmania major P4 gene

Objective: Leishmania major P4 gene is normally expressed during amastigote form of the parasite and can be good candidate for producing an effective vaccine. In this study we cloned this gene in suitable vector (pQE-30) for further vaccine preparation studies. Materials and Methods: Leishmania promastigotes were grown in N.N.N.medium and culture in RPMI 1640 cell culture medium. Total genomic DNA was extracted by centrifugation of promastigotes. The pellet was suspended in lysis buffer and followed by boiling method. PCR was carried out using P4 gene specific primers. PCR product was detected by agaros gel electrophoresis and cloned into Bluescript plasmid via T/A cloning method. Reaction was transformed into XL1- Blue competent cell and recombinant plasmid screened using agar plate contained X-gal and IPTG. The product was extracted, digested by restriction enzyme and electrophoresed on agarose gel. Results: Plasmid was extracted and cloned gene was released by restriction enzyme and subcloned into pQE-30 expression vector. Conclusion: This construct is ready for protein expression in-vitro

Frequency of Intestinal Parasites in Tehran

"nBackground: For a long time, intestinal parasite infections are among the major problems of public health in Iran. Our aim was epidemiological studies on the frequency of intestinal parasites in patients re­ferred to three hospitals in Tehran during 2007-2008."nMethods: During 2007-2008, by simple random selection, 1000 stool samples were collected from Mi­lad, Hazrat-e-Rasoul and Shahid Fahmideh hospitals in Tehran, Iran. We examined the samples using di­rect smear, formol-ethyl acetate concentration, Agar-plate culture and Ziehl-Neelsen staining tech­nique."nResults: The frequency of intestinal parasites were: Blastocystis hominis 12.8%, Giardia lamblia 2.5%, En­tamoeba coli 4.8%, Iodamoeba butschlli 0.9%, unknown 4 nuclei cysts 0.4%, Endolimax nana 3.2%, Chilomastix mesnili 0.4%, Strongyloides stercoralis 0.1%, Hymenolepis nana 0.2% and Taenia sagi­nata 0.2%. Coccidian parasites were not found. Results show that infection with intestinal parasites does not statistically significant according to sex and age."nConclusion: The intestinal parasites, especially helminthic infections have been decreased during re­cent years

Genotyping of Toxoplasma Gondii Isolates from Soil Samples in Tehran, Iran

Background: The protozoan parasite Toxoplasma gondii can infect any warm blooded nucleated cells. One of the ways for human infection is ingestion of oocysts directly from soil or via infected fruits or vegetables. To survey the potential role of T. gondii oocyst in soil samples, the present study was conducted in Tehran City, Iran.Methods: A total of 150 soil samples were collected around rubbish dumps, children's play ground, parks and public places. Oocysts recovery was performed by sodium nitrate flotation method on soil samples. For molecular detection, PCR reaction targeting B1 gene was performed and then, the posi­tive results were confirmed using repetitive 529 bp DNA fragment in other PCR reaction. Finally, the positive samples were genotyped at the SAG2 locus.Results: Toxoplasma DNA was found in 13 soil samples. After genotyping and RFLP analysis in SAG2 locus, nine positive samples were revealed type III, one positive sample was type I whereas three samples revealed mixed infection (type, I & III).Conclusion: The predominant genotype in Tehran soil samples is type III

Bioconjugated fluorescent silica nanoparticles for the rapid detection of Entamoeba histolytica

Rapid detection of Entamoeba histolytica based on fluorescent silica nanoparticle (FSNP) indirect immunofluorescence microscopy was evaluated. Silica nanoparticles were synthesized using Stöber's method, with their surface activated to covalently bind to, and immobilize, protein A. For biolabeling, FSNP was added to conjugated E. histolytica trophozoites with monoclonal anti- E. histolytica IgG1 for microscopic observation of fluorescence. Fluorescent silica nanoparticle sensitivity was determined with axenically cultured E. histolytica serially diluted to seven concentrations. Specificity was evaluated using other intestinal protozoa. Fluorescent silica nanoparticles detected E. histolytica at the lowest tested concentration with no cross-reaction with Entamoeba dispar, Entamoeba moshkovskii, Blastocystis sp., or Giardia lamblia. Visualization of E. histolytica trophozoites with anti- E. histolytica antibody labeled with fluorescein isothiocyanate (FITC) was compared with that using anti- E. histolytica antibody bioconjugated FSNP. Although FITC and FSNP produced similar results, the amount of specific antibody required for FITC to induce fluorescence of similar intensity was fivefold that for FSNP. Fluorescent silica nanoparticles delivered a rapid, simple, cost-effective, and highly sensitive and specific method of detecting E. histolytica. Further study is needed before introducing FSNP for laboratory diagnosis of amoebiasis. © 2015 Elsevier B.V

Genotyping of Echinococcus granulosus from domestic animals and humans from Ardabil Province, northwest Iran

Cystic echinococcosis is endemic in Iran, particularly in Ardabil Province, where it causes health and economic problems. The genetic pattern of Echinococcus granulosus has been determined in most parts of Iran, except in this area. In the present investigation, 55 larval isolates were collected from humans (11), sheep (19), goats (4) and cattle (21). For analysis of the genetic characteristics of E. granulosus isolates, DNA sequencing of mitochondrial cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunit 1 (nad1) genes was applied. Fifty isolates were successfully analysed, with 92% (46) and 8% (4) identified as G1 and G3 genotypes, respectively. The sequence analyses of the isolates displayed nine characteristic profiles in cox1 sequences and eight characteristic profiles in nad1 sequences. Based on these results, the sheep strain (G1 genotype) was the most prevalent in humans, sheep, goats and cattle. The buffalo strain (G3 genotype) was not only demonstrated in sheep (1 isolate) and cattle (1 isolate), but also for the first time in two human isolates. These findings will provide information for local control of echinococcosis. © Cambridge University Press 2012

SYBR green-based detection of Leishmania infantum DNA using peripheral blood samples

Parasitological methods for the diagnosis of visceral leishmaniasis (VL) require invasive sampling procedures. The aim of this study was to detect Leishmania infantum (L. infantum) DNA by real time-PCR method in peripheral blood of symptomatic VL patient and compared its performance with nested PCR, an established molecular method with very high diagnostic indices. 47 parasitologically confirmed VL patients diagnosed by direct agglutination test (DAT > 3200), bone marrow aspiration and presented characteristic clinical features (fever, hepatosplenomegaly, and anemia) and 40 controls (non-endemic healthy control-30, Malaria-2, Toxoplasma gondii-2, Mycobacterium tuberculosis-2, HBV-1, HCV-1, HSV-1 and CMV-1) were enrolled in this study. SYBR-green based real time-PCR and nested PCR was performed to amplify the Kinetoplast DNA minicircle gene using the DNA extracted from Buffy coat. From among 47 patients, 45 (95.7 ) were positive by both nested-PCR and real time-PCR. These results indicate that real time-PCR was not only as sensitive as a nested-PCR assay for detection of Leishmania kDNA in clinical sample, but also more rapid. The advantage of real time-PCR based methods over nested-PCR is simple to perform, more faster in which nested-PCR requires post-PCR processing and reducing contamination risk. © 2014, Indian Society for Parasitology