Search

Background: A lizard Leishmania has been isolated from a lizard (Agama agilis) in Iran. Its genome sequence has not been determined, so far. Methods: The study was done at Shahid Beheshti University of Medical Sciences, Tehran, Iran in 2017-2023. Leishmania promastigotes were cultured in RPMI1640 culture medium and collected at logarithmic phase by centrigugation. Parasite RNA was extracted by the Qiagene standard kit and its quantity and quality was determined and sequenced by NGS method with Illumina PE machine at BGI Company (China). Results: The number of 8316 mRNA, 83 tRNA, 63 rRNA, 83 ncRNA, 5 snRNA, 1039 snoRNA, 36 region, and 3 repeat regions, 8343 CDS, 9597 Exon and 9292 Genes were identified in promastigote of Iranian lizard Leishmania. Conclusion: Genomic elements of Iranian lizards Leishmania (with unique characteristics) were determined and identified by NGS system. [ABSTRACT FROM AUTHOR]

Mediterranean type of visceral leishmaniasis (VL) is a zoonotic parasitic infection. Some provinces of Iran are endemic for VL while other parts are considered as sporadic areas. This study aimed to assess a combination of recombinant K26 and rK39 antigens as well as crude antigen (CA), derived from an Iranian strain of L. infantum, compared to direct agglutination test (DAT) for the detection of VL in humans and domestic dogs as animal reservoir hosts of the disease. A combination of rK26 and rK39 antigens and also CA was evaluated using indirect ELISA on serum samples of 171 VL confirmed humans (n = 84) and domestic dogs (n = 87) as well as 176 healthy humans (n = 86) and domestic dogs (n = 90). Moreover, 36 serum samples of humans (n = 20) and canines (n = 16) with other potentially infectious diseases were collected and tested for finding cross- reactivity. The results of ELISA were compared to DAT, currently considered as gold standard for the serodiagnosis of VL. The sensitivity and specificity, positive predictive and negative predictive values were calculated compared to DAT. The positive sera had previously shown a positive DAT titer ≥ 1:800 for humans and ≥ 1:80 for dogs. Analysis was done by MedCalc and SPSS softwares. Using the combination of rK26 and rK39 in ELISA, a sensitivity of 95.2% and a specificity of 93.0% % were found in human sera at a 1:800 (cut-off) titer when DAT-confirmed cases were compared with healthy controls; a sensitivity of 98.9% and specificity of 96.7%% were found at a 1:80 (cut-off) titer compared with DAT. A good degree of agreement was found between the combined rK39 and rK26-ELISA with DAT in human (0.882) and dog serum samples (0.955) by kappa analysis (p < 0.05). The ELISA using the CA test showed 75% sensitivity in human and 93.1% in dog serum samples as well as 53.5% specificity in human and 83.3% in dog,s sera, respectively. The combination of rK26 and rK39 recombinant antigen prepared from Iranian strain of Leishmania infantum showed high accuracy for the serodiagnosis of VL in human and domestic dogs. Further extended field trial with a larger sample size is recommended. [ABSTRACT FROM AUTHOR]

In this study, fluorescence in situ hybridization (FISH) and PCR-amplified fragments of the 16SrDNA gene were used to determine prokaryotes diversity in Urmia Salt Lake. Prokaryote cell population in Urmia lake range from 3.1 ± 0.3 × 106, 2 ± 0.2 × 108, 4 ± 0.3 × 108, and 1.8 ± 0.2 × 108 cells ml−1 for water, soil, sediment, and salt samples by DAPI (4́, 6-diamidino-2-phenylindole) direct count, respectively. The proportion of bacteria and archaea in the samples determinable by FISH ranged between 36.1 and 55% and 48.5 and 55.5%, respectively. According to the DGGE method, some bands were selected and separated from the gel, then amplified and sequenced. The results of sequences were related to two phyla Proteobacteria (16.6%) and Bacteroidetes (83.3%), which belonged to four genera Salinibacter, Mangroviflexus, Pseudomonas, and Cesiribacter, and the archaeal sequences were related to Euryarchaeota phyla and three genera Halonotius, Haloquadratum, and Halorubrum. According to our results, it seems that prokaryotic populations in this hypersaline environment are more diverse than expected, and bacteria are so abundant and diverse and form the metabolically active part of the microbial population inhabiting this extreme environment. Molecular dependent and independent approaches revealed a different aspect of this environment microbiota. [ABSTRACT FROM AUTHOR]

Background: Visceral leishmaniasis (VL) is the most severe form of leishmaniasis in Iran with high mortality rates in the case of inaccurate diagnosis and treatment. This study aimed to prepare and evaluate a new rk39 recombinant antigen from an Iranian strain of Leishmania infantum for diagnosis of VL in humans and dogs. Methods: rK39-based enzyme-linked immunosorbent assay (ELISA) was compared with the direct agglutination test (DAT) for the detection of anti L. infantum antibodies. We screened 84 human sera and 87 dog sera from clinical cases in the endemic area of Meshkin-Shahr, Iran along with 176 sera from healthy controls (collected from 86 humans and 90 dogs) during 2013 -2016. Results: Using the rK39 ELISA, a sensitivity of 85.7% (95% CI, 95-99%) and a specificity of 86.0% (95% CI, 95%-99%) were detected in human sera at a 1:800 (cut-off) titer when DAT-confirmed cases were compared with healthy controls; a sensitivity of 96.6% (95% CI, 95%-99%) and specificity of 94.4% (95% CI, 95%-99%) were found at a 1:80 (cut-off) titer compared with DAT. Kappa analysis indicated agreement between the rK39 ELISA and DAT (0.718) when using human sera at a 1:800 (cut-off) titer as well as (0.910) at a 1:80 (cut-off) titer when using dog sera (P<0.05). Conclusion: New rk39 recombinant antigen from an Iranian strain of Leishmania infantum seems to be used for diagnosis of VL in humans and dogs. Further extended field studies are recommended. [ABSTRACT FROM AUTHOR]

Background: Visceral leishmaniasis (VL) or kala-azar is a parasitic disease caused by the species of Leishmania donovani complex. Mediterranean type of the disease is endemic in some parts of Iran and more than 95% of cases were reported in children up to 12 years of age. This study was performed to determine the seroprevalence of VL in the rural areas of the Dashti district from Bushehr province. Materials and Methods: In this cross-sectional study, a randomized cluster sampling method was used for the collection of blood samples from children up to 12 years old from rural areas of Dashti district. Before sampling; a questionnaire was filled out for each case. All the collected blood samples were examined after the serum separating by Direct Agglutination Test (DAT) for detection of anti-Leishmania infantum antibodies. The cutoff titers of ≥1: 3200 with specific clinical features were supposed to be considered as VL. Results: Altogether, 24 out of 1221 (1.96%) blood samples showed titers between 1:800 and 1:1600 which considered as suspicious cases. None of the suspicious cases had a history of kala-azar. None of 1221 collected blood samples showed anti Leishmania infantum (L. infantum) at titer ≥1:3200. Conclusion: This study confirms the circulation of L. infantum in Dashti district and highlights the sporadic pattern of VL in the studied areas which necessitates the surveillance system to be monitored by health authorities. [ABSTRACT FROM AUTHOR]

Background: Visceral leishmaniasis (VL), caused by Leishmania infantum (L. infantum), is a life-threatening vector-borne parasitic disease is distributed in some parts of the world. The disease is endemic in some parts of Iran. This study was aimed to determine the seroprevalence of VL among children and domestic dogs (as a reservoir of the parasite) in Dehloran, west of Iran. Materials and Methods: This cross-sectional study was carried out in Dehloran County. The blood samples of 872 children up to 12 years old and 52 dogs were collected from 10 villages of Dehloran using randomlyclustered sampling method. Sera were separated from all peripheral blood samples and tested by direct agglutination test (DAT). Anti-Leishmania infantum antibodies at titers of ≥1:800 and ≥1:80 were considered as Leishmania infantum infection in human and dog, respectively. Results: In general, among 872 human samples, 1.03% of samples had anti-Leishmania antibody with 1:1600 titers and 1.26% had 1:800 titers. In addition, from 52 dog samples, 21.15% of dogs had a titer of ≥1:320 and 25% had 1:80 and 1:160 titers. Conclusion: Our findings indicate that the seropositive dogs in the studied areas are considerable and L. infantum may be circulated between human and domestic dog in the studied area. Further study of isolation and molecular identification of Leishmania spp. is recommended. [ABSTRACT FROM AUTHOR]

Objectives: This study was aimed to evaluate the role of toxoplasmosis in etiology of abortion and stillbirth based on molecular and serological techniques.Material and Methods: A total of 110 pregnant women with abortion and stillbirth were enrolled as the case group, and 110 pregnant women with normal delivery were enrolled as the control group. Serological and molecular detections of Toxoplasma gondii were assessed by ELISA and PCR methods.Results: The seroprevalence of IgG was 25.5% in the case group (26.8% in abortion and 21.4% in stillbirth) and 26.4% in the control group. IgM seropositivity was detected in 2.7% of the case group (3.6% in abortion and 0% in stillbirth) and 0.9% of the control group (p = 0.37). Toxoplasma gondii DNA was detected in 6.4% of the case group (7.3% in abortion and 3.6% in stillbirth) and 1.8% of the control group by PCR (p = 0.17). The major risk factor of congenital toxoplasmosis was the history of eating undercooked meat (p = 0.06).Conclusion: Results of this study revealed that the rate of PCR positive in women with abortion and stillbirth was 3.7 times higher than that in normal delivery, but the difference was not statistically significant. These findings suggest that toxoplasmosis can be involved in etiology of abortion and stillbirth. [ABSTRACT FROM AUTHOR]

Background: Free-living amoebae such as Acanthamoeba species may act as carriers of Cryptosporidium and Toxoplasma oocysts, thus, may play an important role in the water-borne transmission of these parasites. In the present study, a loop mediated isothermal amplification (LAMP) method for detection of Toxoplasma and a PCR assay were developed for investigation of Acanthamoeba in environmental water samples. Methods: A total of 34 samples were collected from the surface water in Guilan Province. Water samples were filtrated with membrane filters and followed by DNA extraction. PCR and LAMP methods used for detection of the protozoan parasites Acanthamoeba and Toxoplasma respectively. Results: Totally 30 and 2 of 34 samples were positive for Acanthamoeba and Toxoplasma oocysts respectively. Two samples were positive for both investigated parasites. Conclusion: The investigated water supplies, are contaminated by Toxoplasma and Acanthamoeba (oo)cystes. Acanthamoeba may play an important role in water-borne transmission of Toxoplasma in the study area. For the first time in Iran, protocol of LAMP method was used effectively for the detection of Toxoplasma in surface water samples in Iran. [ABSTRACT FROM AUTHOR]

Background: Leishmaniasis is a group of diseases that are created by intracellular parasites of Leishmania. Cutaneous leishmaniasis is considered as one of the health problems in some provinces of Iran. Methods: In this study, a total of 178 Giemsa-stained slides from confirmed cases of cutaneous leishmaniasis were examined. The slides were prepared from the patients with cutaneous leishmaniasis that referred to health centers and infected during the epidemic of cutaneous leishmaniasis in Poldokhtar city, Lorestan Province, Iran in 2006.Genomic DNA from each slide was extracted. After DNA extraction, ITS-PCR was used. Results: Out of 178 slides, 129 (72.47%) samples had a band in the range of 485 bp and 49 (27.53%) samples 626 bp that matched L. tropica and L. major standard samples, respectively. Conclusion: This study showed that Leishmania DNA could be efficiently extracted and amplified even from old Giemsa-stained microscopic slides that were stored more than 6 yr. In this study was shown that both L. tropica and L. major species exist in Lorestan Province. [ABSTRACT FROM AUTHOR]

Ovine theileriosis is an important hemoprotozoal disease of sheep and goats in tropical and subtropical regions that leads to economic losses in these animals. A nested PCR-restriction fragment length polymorphism (RFLP) was carried out to identification Theileria species in sheep in some area in western half of Iran (Sari, Rasht, Urmia, Ilam, and Ahvaz). Two hundred and fifty blood samples were taken from sheep during tick activating season (summer of 2008). Microscopic examination revealed that 9.2% (23/250) sheep were infected by Theileria spp. piroplasms. Parasitemia ranged from 0.011% to 0.015%. In nested PCR assessment of DNA samples, 32.8% (82/250) sheep were positive. The negative samples were confirmed by amplifying of ovine beta-actin gene as an internal control. The differentiation of Theileria species was based on RFLP patterns using three restriction enzymes: HpaII, Rsa1, and Bsh 1285I. Out of 82 positive samples, 54.8% (45/82) and 40.2% (33/82) were positive for Theileria lestoquardi and Theileria ovis respectively. Mixed infection was detected in 4.8% (4/82) cases. Based on their PCR product digestion pattern with HpaII (1178, 900, 278, and 106 bp), it seemed to be mixture of Theileria annulata and T. lestoquardi. The presence of T. annulata was supported by sequence analysis. This is the first report of naturally infected sheep with T. annulata in Iran. Geographical distribution of Theileria species in sheep is shown according to the result of microscopy and nested PCR and RFLP data. [ABSTRACT FROM AUTHOR]

Aim: The aim of the present study was to determine the species and genotypes of Cryptosporidium spp. among children with diarrhea by PCR- RFLP using the TRAP-C2 gene. Background: Cryptosporidium is a globally distributed protozoan parasite and one of the most common causes of infection and diarrhea in humans. Patients and methods: Four hundred and sixty nine stool samples were collected from children less than 12 years with diarrhea who had been referred to Pediatrics Medical Centers in Gazvin provinces. The presence of Cryptosporidium oocysts was determined by Ziehl-Neelsen acid fast staining, then, genomic DNA was extracted from positive samples and nested PCR-RFLP was performed to amplify the TRAP-C2 gene. Results: The overall prevalence of Cryptosporidium infection in children was 2.5 %. Results of nested PCR amplification showed that of 12 positive children samples, 10 (83.3%) were belonged to C. parvum, followed by C. hominis in 1 (8.3%) and mixed infection in 1 isolate (8.3%). Conclusion: This study showed that Cryptosporidium parvum (the zoonotic genotypes) is more prevalent than other Cryptosporidium species in children from this area. This suggests that zoonotic transmission is the main mode of transmission of Cryptosporidium infection in Iran. [ABSTRACT FROM AUTHOR]

Background: Leishmania is an obligate interacellular protozoa and sand fly, as a vector, transmits infectious forms of the parasite to vertebrate host. In this way it is important to find candidate antigens which could tend to prevent the disease. Methods: The gene coding mannose 1 phosphate guanyl transferase enzyme was amplified from genomic DNA isolated from the Iranian strain of L. major (MRHO/IR/75/ER) as a template. The Polymerase Chain Reaction (PCR) product was ligated into the pTZ57R plasmid and the recombinant gene was digested using restriction enzymes, BamHI and EcoRI. The fragment was ligated into the pET32a plasmid, as an expression vector. The cloned pET32a-GDP mannose was confirmed using restriction enzyme digestion method and the DNA fragment was sequenced. Findings: Electrophoresis method confirmed the PCR product is related to the enzyme mannose 1 phosphate guanyl transferase. After the ligation of the product into the pTZ57R and pET32a, and the restriction enzyme digestion by BamHI and EcoRI, the correct frame of cloned gene in vectors was confirmed. Conclusion: There was 92 percent homology between the cloned gene coding enzyme mannose 1 phosphate guanyl transferase in this study and the ones present in gene bank. It is suggested that the gene encoding mannose 1 phosphate guanyl transferase enzyme is conserved among different genera of Leishmania. [ABSTRACT FROM AUTHOR]

Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Correction to: Current Microbiology https://doi.org/10.1007/s00284-021-02583-w The original version of this article unfortunately contain mistake in the co-authors affiliation. [Extracted from the article]

Summary: We investigated the prevalence of intestinal protozoan parasites in patients with gastrointestinal complaints in medical centers in Zahedan, Iran. A total of 1562 stool samples was examined from July 2004 to January 2006 using microscopy (direct smear, formalin-ether concentration), xenic culture and PCR techniques. Four hundred and twenty-seven (27.3%) of the patients were infected with one or more intestinal parasites. Giardia lamblia (10.1%), Entamoeba coli (10%), E. hartmanni (1.7%), Blastocystis hominis (2.2%), Chilomastix mesnili (1.7%), Trichomonas hominis (0.7%), E. histolytica/E. dispar (0.51%) and Iodamoeba butschlii (0.45%) were the most prevalent protozoa detected with microscopy. Of the eight microscopy-positive E. histolytica/E. dispar samples, six were identified as E. dispar by PCR/gel electrophoresis, whereas E. histolytica was not detected at all. Although Zahedan is an area with poor hygiene located in a tropical area near the border of Pakistan and Afghanistan, the prevalence of E. histolytica and E. dispar here compared with other parasites and infectious diseases is unexpectedly low. [Copyright &y& Elsevier]

• Certain cytokine gene polymorphisms associate with cutaneous leishmaniasis (CL) susceptibility. • Significant differences exist between CL patients and endemic controls for TGF-β1, IFN-γ, and IL-10 variants. • The TGF-β1 (rs1800469) SNP links to over 5-fold greater CL risk in southeastern Iran. • IFN-γ (rs2430561) AA genotype demonstrates a 4-fold increased frequency in CL cases. • A nearly 2-fold heightened risk occurs with the IL-10 (rs1800871) CC genotype among patients. Cutaneous leishmaniasis (CL) is a complex, multifactorial disease that results from environmental factors such as parasite polymorphism, phlebotomine vectors, and host genetic factors. Some studies have identified specific genetic factors that may be associated with cutaneous leishmaniasis. The objective of this research was to resolve the association of 8 cytokine polymorphisms, including TNF-α −308 A/G (rs 1800629), TNF-α −238 A/G (rs 361525), TGF-β1 −509 T/C (rs 1800469), TGF-β1+ 915 G/C (rs 1800471), IFN-γ −874 T/A (rs 2430561), IFN-γ −179 G/A (rs 2069709), IL-10 −819 C/T (rs 1800871), and IL-10 −592 A/C (rs 1800872) with susceptibility to CL. A total of 152 patients with designated CL and 100 healthy controls were selected from those referred to Sistan and Baluchestan hospitals. CL was diagnosed by microscopic examination of Giemsa-stained samples and culture. Leishmania species were identified using ITS2 gene PCR amplification with universal primers. Genetic polymorphism was determined by the ARMS PCR method on extracted genomic DNA of individuals. Eight SNPs cytokines were genotyped. Most of the Genotypic and allelic frequency comparisons between patients with CL and healthy subjects showed no difference, except 3. Individual SNP analysis showed highest association of TGF-β1 −509 (rs1800469) –CC genotype (P = 0.03, OR = 7.05, 95 % CI = 3.3–15) with 5.7-fold increase, IFN-γ −874 (rs 2430561) -AA genotype (P = 0.04, OR = 4.72, 95 % CI = 1.6–14) with 4.2-fold increase, and IL10 −819 (rs1800871) –CC genotype (P = 0.05, OR = 3.63, 95 % CI = 2.5–5.3) with 1.9-fold increase, with CL. Odds ratios (ORs) and 95 % confidence intervals (CIs) were evaluated to assess the association power. Our results conclude that rs1800469 (TGF-β1), rs2430561 (INF-γ), and rs1800872 (IL10) polymorphisms are associated with CL in southeastern Iranian people. [ABSTRACT FROM AUTHOR]

Trichomonas vaginalis is a human urogenital pathogen that causes trichomoniasis, the most common nonviral, parasitic sexually transmitted infection in the world. At present, little is known regarding the degree of strain variability of T. vaginalis . A classification method for T. vaginalis strains would be a useful tool in the study of the epidemiology, drug resistance, pathogenesis and transmission of T. vaginalis . Eight different types of actin genes have been identified by PCR-RFLP in T. vaginalis ; the purpose of this study is to determine the genotypes of this parasite in Karaj city, Iran. Forty-five clinical T. vaginalis isolates from vaginal secretions and urine sediment were collected from Karaj city from 2012 through 2014. DNA was extracted and the actin gene was amplified by nested-PCR; all samples were positive. To determine the genetic differences, sequencing on seven samples was conducted. Then, all PCR products were digested with HindII, MseI, and RsaI restriction enzymes. Of 45 isolates, 23 samples (51.1%) were of actin genotype G, 11 samples (24.4%) of genotype E, six samples (13.3%) of genotype H, three samples (6.6%) of genotype I, and two samples (4.4%) were mixed genotypes of G and E. Genetic diversity of T. vaginalis isolates is notable. The actin genotype G may be the dominant genotype in Karaj city, Iran. [ABSTRACT FROM AUTHOR]

Visceral leishmaniasis (VL) is a protozoan disease caused by Leishmania infantum in the Mediterranean region including Iran. In 95% of cases, the disease can be fatal if not rapidly diagnosed and left untreated. We aimed to identify immunoreactive proteins of L. infantum (Iranian strain), and to design and evaluate a recombinant multi-epitope antigen for serodiagnosis of human VL. To detect the immunoreactive proteins of L. infantum promastigotes, 2DE immunoblotting technique was performed using different pooled sera of VL patients. The candidate immunoreactive proteins were identified using MALDI-TOF/TOF mass spectrophotometry. Among 125 immunoreactive spots detected in 2-DE gels, glucose-regulated protein 78 (GRP78), ubiquitin-conjugating enzyme E2, calreticulin, mitochondrial heat shock 70-related protein 1 (mtHSP70), heat shock protein 70-related protein, i/6 autoantigen-like protein, ATPase beta subunit, and proteasome alpha subunit 5 were identified. The potent epitopes from candidate immunodominant proteins including GRP78, mtHSP70 and ubiquitin-conjugating enzyme E2 were then selected to design a recombinant antigenic protein (GRP-UBI-HSP). The recombinant antigen was evaluated by ELISA and compared to direct agglutination test for detection of anti L. infantum human antibodies. We screened 34 sera of VL patients from endemic areas and 107 sera of individuals without L. infantum infection from non-endemic area of VL. The recombinant protein-based ELISA provided a sensitivity of 70.6% and a specificity of 84.1%. These results showed that GRP78, ubiquitin-conjugating enzyme E2, and mtHSP70 proteins are potential immunodominant targets of the host immune system in response to the parasite and they can be considered as potential candidate markers for diagnosis purposes. Image 1 • The immunoproteomics approach provides a tool for the discovery of antigenic proteins. • The antigenic proteins are suggested as biomarkers for serodiagnosis of human VL. • The multiepitope antigen can potentially improve the diagnosis efficiency. [ABSTRACT FROM AUTHOR]

After half a century, Trichinella infection has been reported in humans following ingestion of wild boar meat hunted in Northern Iran. We have performed a cross-sectional study to evaluate the prevalence of Trichinella spp. infections in wild boar and prevalence of anti- Trichinella antibodies among at-risk individuals for the first time in Iran. Muscle and sera samples were collected from 79 wild boars and 364 at-risk individuals. Trichinella infection has been investigated by artificial digestion and molecular identification (in wild boar muscles) and by serology (in humans). Trichinella larvae were isolated from three wild boars (3.7%; 95% CI, 2.9–4.3). The isolated larvae were identified as T. britovi using CO1 and 5S rRNA gene primers amplification. The percent identity regarding to 5S rRNA gene (98.7–100%) and divergence (0–1.9%) further confirmed the isolates as T. britovi . Of the 364 participants, anti- Trichinella IgG were detected in 8 (2.2%; CI 95%, 1.9–2.4). Risk factors associated with Trichinella infection seropositivity in humans, were hunter being (OR, 13.5; 95% CI, 3.1–59.4; P = 0.003) and high consumption (more than 7 time in a year) of wild boar meat (OR, 17.5; 95% CI, 3.2–93.6; P < 0.001). In conclusion, results of this study emphasized that consumption of wild boar meat could be important source of human trichinellosis as a completely neglected infection disease in Iran. We suggest that the additional studies should be performed in different parts of Iran to further clarify the prevalence of trichinellosis in wild animals to guide the development of appropriate public health interventions. [ABSTRACT FROM AUTHOR]

Background: Hepatitis infection represents one of the important causes of morbidity and mortality in developing countries, however there is not any effective vaccine against hepatitis C which is one of the significant problems in vaccine project. Objectives: The aim of the present study is to evaluate the role of HCV core protein in inducing IFN-Gamma secretion and TCL activities as a vaccine in Balb/C mice. Materials and Methods: Our previous cloned plasmid (HCV Core gene into pETDuet-1) applied for protein expression in bacteria. The expressed and purified recombinant protein together with Freund's adjuvant was injected to 15 Balb/c mice. The total IgG and IgG2a of immunized mice sera were evaluated after a week. Two weeks after booster injection, we studied the proliferation and IFNγ secretion of spleens, inguinal and popliteal lymph nodes lymphocytes by ELISA and ELISPOT. Results: The FSFC (Frequency of spot forming cells) of secreting cells of immunized mice with HCV/Core protein and sera IgG2a were considerably higher than the control groups. Conclusions: The core protein together with proper adjuvant can be a candidate vaccine against of HCV infection. [ABSTRACT FROM AUTHOR]

Abstract: Clinically infected dogs have been identified as the main reservoir hosts of visceral leishmaniasis (VL) caused by Leishmania infantum in the Mediterranean region. The objective of this study was to determine the potential of asymptomatic infected dogs compared with symptomatic ones as a source of L. infantum infection to golden hamster. For this purpose, anti-Leishmania antibodies were detected with direct agglutination test (DAT) in 13 symptomatic (7 seropositive=≥1:320) and 53 asymptomatic (9 seropositive=≥1:320 and 44 seronegative= <1:320) ownership dogs. DNA of Leishmania sp. was extracted from skin and peripheral blood tissues of each dog and tested by PCR. Sixty-six Syrian golden hamsters (Mesocricetus auratus) were used for the determination of infectivity and pathogenicity of L. infantum, isolated from the dogs. We used the internal transcribed spacer 2 (ITS 2) rDNA sequence analysis. The results showed that 22 and 11 out of 66 inoculated golden hamsters were positive by PCR and parasitological examinations, respectively. From 22 PCR positive hamsters, 17 were related to asymptomatic dogs and 5 were from symptomatic ones. There was no significant difference between symptomatic and asymptomatic dogs in producing Leishmania infection in the susceptible animal model (P =0.66). Smears and cultures of 5 dogs from 13 symptomatic dogs (38.5%) and 6 dogs from 53 asymptomatic ones (11.3%) were found to be positive at parasitological examination. All the L. infantum isolates from symptomatic and asymptomatic dogs were similar in sequencing. In conclusion, asymptomatic infected dogs as well as symptomatic ones can harbor L. infantum in their blood and skins which are virulent and infectious for inoculated golden hamster. [Copyright &y& Elsevier]

Abstract: The aim of this study was to determine the prevalence, variability with host age, and the genotypes of species of Cryptosporidium in cattle from 15 dairy farms in Qazvin province, Iran. Fecal samples, collected from 272 cattle during May 2006 to December 2007, were characterized microscopically. Oocysts from 51 positive samples were analyzed using PCR assay of 18S SSU rRNA, restriction fragment length polymorphism (RFLP) and sequencing. We identified 72.6% of the positive samples as Cryptosporidium parvum, 17.7% as Cryptosporidium andersoni, 7.8% as Cryptosporidium bovis and 1.9% as a novel genotype of C. parvum possessing a single mutation on MboII restriction. An infection rate of 19.5% of C. parvum among 174 pre-weaned calves was significantly higher than the 3.1% among 98 post-weaned calves (P <0.0006). This is the first report of C. bovis and the new subgenotype of C. parvum in Iranian cattle. [Copyright &y& Elsevier]

In Iran, both Plasmodium vivax and P. falciparum malaria have been detected, but P. vivax is the predominant species. Point mutations in dihydrofolate reductase (dhfr) gene in both Plasmodia are the major mechanisms of pyrimethamine resistance. From April 2007 to June 2009, a total of 134 blood samples in two endemic areas of southern Iran were collected from patients infected with P. vivax and P. falciparum. The isolates were analyzed for P. vivax dihydrofolate reductase (pvdhfr) and P. falciparum dihydrofolate reductase (pfdhfr) point mutations using various PCR-based methods. The majority of the isolates (72.9%) had wild type amino acids at five codons of pvdhfr. Amongst mutant isolates, the most common pvdhfr alleles were double mutant in 58 and 117 amino acids (58R-117N). Triple mutation in 57, 58, and 117 amino acids (57L/58R/117N) was identified for the first time in the pvdhfr gene of Iranian P. vivax isolates. All the P. falciparumsamples analyzed (n = 16) possessed a double mutant pfdhfrallele (59R/108N) and retained a wild-type mutation at position 51. This may be attributed to the fact that the falciparum malaria patients were treated using sulfadoxine-pyrimethamine (SP) in Iran. The presence of mutant haplotypes in P. vivax is worrying, but has not yet reached an alarming threshold regarding drugs such as SP. The results of this study reinforce the importance of performing a molecular surveillance by means of a continuous chemoresistance assessment.

Objective: To identify Leishmania using PCR., Methods: This study was conducted from April 2009 to March 2011 in order to identify Leishmania species in a new endemic area of CL in Lorestan, Iran. Samples were taken from 62 patients that referred to the health centers in different cities of Lorestan province, the presence of Leishmania was confirmed using direct smear and then grown in NNN media and mass cultured in RPMI 1 640 medium supplemented with 10% heat-inactivated fetal bovine serum. DNA was extracted from cultured promastigotes and used in ITS-PCR., Results: 45(72.6%) samples out of 62 showed a band in the range of 485 bp and 17 (27.4%) with a band in the range of 626 bp which were similar to standard strains of Leishmania tropica(L. tropica) and Leishmania major(L. major), respectively. 50 (65.80%) of samples were collected from people with no history of travel in at least a year prior to the onset which shows that indigenous source of infection., Conclusions: Since the vector and reservoir of the two species are different, so precise and extensive control and prevention methods should be designed and carried out., (Copyright © 2013 Hainan Medical College. Published by Elsevier B.V. All rights reserved.)

Background: Cryptosporidium and Giardia in water supplies is acknowledged as a public health problem. In the present study, we applied immunofluorescence assay (IFA), PCR and loop-mediated isothermal amplification (LAMP) for the detection of the two protozoa., Methods: Over a period of 12 months, surface water samples were collected from two rivers in the north of Iran, and filtrated by 142 mm membrane filters. At each sampling point 10 L water were used for IFT and the10 L were analysed using molecular methods., Results: In 15/40 samples, (oo)cysts were detected by one of the IFA, PCR or LAMP methods. Five samples that were Cryptosporidium-negative by IFA were positive by LAMP. A total of 10 out of 13 samples that were Giardia-positive by IFA were also positive by PCR. IFA revealed high levels of Giardia, with 1-1800 cysts and 1-16 Cryptosporidium oocysts detected per 10 L., Conclusion: The study reveals that the investigated water supplies were contaminated by Cryptosporidium and Giardia. The LAMP assay has advantages for detection and screening of these protozoa at relatively low concentration in water samples. The three assays applied are complimentary but no single one will give the true prevalence of these parasites in surface water samples. However, each method has its own advantages and disadvantages dependent of the aim and the study design; a combination of detection methods should be applied to discover whether water is, or is not, contaminated with (oo)cysts. This is the first report on the occurrence of (oo)cysts in Iranian surface waters to compare the results of parasite detection obtained with the different methods.

The diagnosis of visceral leishmaniasis (VL) in humans and animal reservoir hosts is difficult, particularly in rural areas where the disease is endemic and laboratory facilities are limited. This study aimed to develop a latex agglutination test (LAT) for the rapid detection of anti-Leishmania antibodies against the A2 antigen derived from the amastigote form as well as those against crude antigens derived from the promastigote form of an Iranian strain of Leishmania (Leishmania) infantum. The A2 antigen (42-100 kDa) was prepared from the amastigote form of L. infantum, purified with electroelution and compared with the crude antigen from the promastigote form of L. infantum. Both antigens showed appropriate intensity reactions, were selected using dot blotting of positive and negative pooled sera and used to sensitize 0.9-μm latex beads. The tests were carried out on sera from 43 symptomatic, human patients with VL confirmed by parasitological examination and direct agglutination test (DAT), 30 healthy controls and 32 patients with other infections but without VL. Canine sera were collected from 63 domestic dogs with VL confirmed using parasitological examinations and DAT and 31 healthy dogs from areas non-endemic for VL. Compared with the controls, human sera from DAT-confirmed patients yielded a sensitivity of 88.4% (95% CI, 82.1-94.5%) and specificity of 93.5% (95% CI, 87.0-99.7%) on A2-LAT (amastigote) when 1:3200 was used as the cut-off titre. A good degree of agreement was found between A2-LAT and DAT (0.914). LAT required 3-5 min to complete, versus the 12-18 h needed for DAT. Compared with the controls, A2-LAT of canine sera from DAT-confirmed cases yielded a sensitivity of 95.2% (95% CI, 95.0-95.4%) and specificity of 100% (95% CI 100%) when 1:320 was used as the cut-off titre. A good degree of agreement was found between A2-LAT and DAT (0.968). Similarly, the sensitivity and specificity of Pro.-LAT (promastigote) was calculated to be 88.4% and 91.9%, respectively for human sera and 96.8% and 90.3%, respectively for canine sera. No statistically significant differences were observed between A2-LAT and Pro.-LAT for the detection of human and canine L. infantum infections. In conclusion, A2-LAT and Pro.-LAT could be used in parallel to screen for L. infantum infections in humans and dogs in areas endemic for VL in Iran., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Prokaryotic diversity in Aran-Bidgol salt lake, a thalasohaline lake in Iran, was studied by fluorescence in situ hybridization (FISH), cultivation techniques, denaturing gradient gel electrophoresis (DGGE) of PCR-amplified fragments of 16S rRNA genes and 16S rRNA gene clone library analysis. Viable counts obtained (2.5-4 × 10(6) cells mL(-1)) were similar to total cell abundance in the lake determined by DAPI direct count (3-4×10(7) cells mL(-1)). The proportion of Bacteria to Archaea in the community detectable by FISH was unexpectedly high and ranged between 1:3 and 1:2. We analyzed 101 archaeal isolates and found that most belonged to the genera Halorubrum (55%) and Haloarcula (18%). Eleven bacterial isolates obtained in pure culture were affiliated with the genera Salinibacter (18.7%), Salicola (18.7%) and Rhodovibrio (35.3%). Analysis of inserts of 100 clones from the eight 16S rRNA clone libraries constructed revealed 37 OTUs. The majority (63%) of these sequences were not related to any previously identified taxa. Within this sampling effort we most frequently retrieved phylotypes related to Halorhabdus (16% of archaeal sequences obtained) and Salinibacter (36% of bacterial sequences obtained). Other prokaryotic groups that were abundant included representatives of Haloquadratum, the anaerobic genera Halanaerobium and Halocella, purple sulfur bacteria of the genus Halorhodospira and Cyanobacteria.

Objective: Comprehensive sequencing of the coding exons of the calcium-sensing receptor gene (CASR) was performed in a group of Iranian recurrent calcium kidney stone-formers and the results were compared with a control group., Material and Methods: Serum and urine parameters were evaluated in 99 males aged between 30 and 55 years old with idiopathic recurrent calcium urolithiasis and in 107 men as a control group. Products of polymerase chain reaction were sequenced using forward primer until a mutation was found in that exon. Then, other cases were analysed by single-strand conformation polymorphism., Results: Four polymorphisms were detected in CASR exons, all in the coding region of exon 7. These polymorphisms and their minor allele frequency were P748P (100%), A986S (1%), R990G (3%) and E1011Q (98%). There was a significantly higher count of 986S (p = 0.006), 990G (p = 0.006) and E1011 (p = 0.02) alleles in patients. The odds ratio (95% confidence interval) was 2.55 (1.31-4.96) in those at risk of stone disease for the 986S allele and 8.06 (1.80-35.9) for the 990G allele. Men with the RR genotype at R990G showed a significantly higher serum ionized calcium than the RG or GG group (p = 0.03). A significantly lower serum total calcium was found in subjects with the QQ than the EQ genotype with respect to the 1011 locus (p = 0.005). Furthermore, the 1011Q allele was marginally associated with hypercalciuria (p = 0.05)., Conclusion: The 986S, 990G and 1011Q alleles were associated with a recurrent calcium kidney stone-forming state. 986S and 1011Q alleles, but not 986S, were associated with hypercalcaemia.

Background: Currently, there are no effective vaccines against leishmaniasis, and treatment using pentavalent antimonial drugs is occasionally effective and often toxic for patients. The PTR1 enzyme, which causes antifolate drug resistance in Leishmania parasites encoded by gene pteridine reductase 1 (ptr1). Since Leishmania lacks pteridine and folate metabolism, it cannot synthesize the pteridine moiety from guanine triphosphate. Therefore, it must produce pteridine using PTR1, an essential part of the salvage pathway that reduces oxidized pteridines. Thus, PTR1 is a good drug-target candidate for anti-Leishmania chemotherapy. The aim of this study was the cloning, expression, and enzymatic assay of the ptr1 gene from Iranian lizard Leishmania as a model for further studies on Leishmania., Methods: Promastigote DNA was extracted from the Iranian lizard Leishmania, and the ptr1 gene was amplified using specific primers. The PCR product was cloned, transformed into Escherichia coli strain JM109, and expressed. The recombinant protein (PTR1 enzyme) was then purified and assayed., Results: ptr1 gene was successfully amplified and cloned into expression vector. Recombinant protein (PTR1 enzyme) was purified using affinity chromatography and confirmed by Western-blot and dot blot using anti-Leishmania major PTR1 antibody and anti-T7 tag monoclonal antibody, respectively. The enzymatic assay was confirmed as PTR1 witch performed using 6-biopterin as a substrate and nicotinamide adenine dinucleotide phosphate as a coenzyme., Conclusion: Iranian lizard Leishmania ptr1 was expressed and enzymatic assay was performed successfully.

Entamoeba moshkovskii and Entamoeba dispar are impossible to differentiate microscopically from the pathogenic species Entamoeba histolytica. There are limited data on the prevalence of these commensal parasites in Iran. We utilized a single-round PCR assay to determine the prevalence of E. moshkovskii, E. dispar, and E. histolytica in stool samples from Iranian patients infected with gastrointestinal disorders. After culturing of microscopy-positive isolates and extraction of DNA, PCR was carried out to differentiate the Entamoeba isolates. Out of 3,825 stool samples examined by microscopy, 58 specimens (1.52%) were infected with E. histolytica, E. dispar, or E. moshkovskii. By PCR, 2 E. histolytica (3.45%), 53 E. dispar (91.37%), 2 E. moshkovskii (3.45%), and one mixed E. dispar/E. moshkovskii infection (1.73%) were detected. In view of the reporting of E. moshkovskii in this study in Iran and the difficulty in discriminating this ameba from two similar Entamoeba spp. by microscopy, we recommend the single-round PCR assay as an alternative tool in routine diagnosis and in epidemiological studies of amebiasis.

This study has identified and characterized the structure of locus D-A, a noncoding short tandem repeat (STR) region, also known as locus 1-2, in Iranian Entamoeba dispar isolates. This polymorphic locus has been shown to be potentially useful in investigating the molecular epidemiology of Entamoeba histolytica and E. dispar. The genetic polymorphisms in locus D-A in 28 isolates of E. dispar from three different geographic regions of Iran were distinguished using PCR and sequencing, and the results were compared with the E. dispar gene sequences available in GenBank. In all microscopy-positive E. histolytica/E. dispar samples, PCR with species-specific primers was used to amplify a 477-531 bp product, identifying the samples that had E. dispar. Analysis of the sequences revealed a remarkable degree of genetic diversity with regard to size, number and organization of the repeat units among the E. dispar isolates. The sequenced products showed 12 novel E. dispar genotypes, which have been submitted to the GenBank/EMBL/DDBJ database under accession numbers AB354125-AB354136.

Background: Patients with diabetes mellitus type II suffer from hyperglycemia because they are not able to use the insulin that they produce, often due to inadequate function of insulin receptors. There are some evidences that this deficiency is inherited in a dominant autosomal manner and leads to the malfunction of the pancreatic beta cells resulting in insulin excretion disorders. In this study, we sought to identify mutations in the insulin receptor (INSR) gene, which can cause insulin resistance in type II diabetic patients., Methods: DNA was extracted from peripheral blood cells of the patients (n = 128) diagnosed with type II diabetes. All 22 exons of the INSR gene of the patients were analyzed for mutations running PCR, conformation-sensitive gel electrophoresis and DNA sequencing, consecutively., Results: Approximately 26% of the patients had genetic mutations; however, most of them were not reported. These mutations include exon 2 (His171Asn, Ile172Ser, Cys196Ser and Ser210Arg), exon 3 (Gly227Asp and Gly232Ser), exon 8 (Thr543Ser), exon 9 (a heterozygote was observed with no change in phenylalanine at position 669), exon 13 (two heterozygotes: Arg890Pro with Asn865 remaining unchanged), exon 14 (Ala906Gly and Pro918Trp with Arg902 unchanged), exon 17 (Val1086Glu) and exon 19 (His1157Gln with Thr1172 unchanged)., Conclusion: The lack of similar mutation records in literature and genetic data banks may suggest a geographic pattern for these INSR gene variants in our population.

The aims of this study are to identify Leishmania species and compare and validate internal transcribed spacers (ITS) polymerase chain reaction (PCR) assay against parasitological methods for diagnosing cutaneous leishmaniasis (CL). We used the ITS-PCR, parasite culture, and microscopic evaluation of stained smears on 155 specimens from suspected cases of (CL) patients who referred to Mashhad Health Centers (northeast Iran). The PCR indicated the sensitivity (98.8%), correctly diagnosing 86 of the 87 confirmed positive specimens. Microscopy and parasite culture alone showed 79.3% sensitivity (69/87 positive) and 86.2% sensitivity (75/87 positive), respectively, while microscopy and culture in combination improved sensitivity totally to 100% (87/87). The results also revealed that Leishmania tropica species is dominant (96.5%) in the studied regions. This study suggests that both the parasitological techniques reliably were used for the diagnosis of CL, and the ITS-PCR assay without using RFLP analysis is useful for identifying Leishmania species in the area.

Introduction: MSX1 gene has a critical role in craniofacial development, the aim of this case-control study is to test the hypothesis that MSX1 mutation contributes to congenital tooth agenesis in Iranians., Materials and Methods: The study group consisted of 20 affected individuals with tooth agenesis of lower second premolars or upper lateral incisors with mean age of 24.6. The control group consisted of 20 unaffected individuals. DNA was extracted from all 40 individuals; the polymerase chain reaction (PCR) for MSX1 was carried out with Phenol: Chloroform: Isoamylalchol (PCI) extraction method. Ban II restriction digest and agarose gel electrophoresis of the 20 affected individuals verified the presence of mutation in all 20 affected individuals. The unaffected controls did not show any mutation. Statistical analysis performed by the chi-squared method., Results: Ban II did not digest PCR product (DNA) in the control group (195 bp band on electrophoresis gel) but digested the affected allele (106 bp and 89 bp bands). There is a statistically significant correlation between tooth agenesis and MSX1 mutation (P < 0.001)., Conclusion: The results indicated that MSX1 gene mutation contributes to tooth agenesis in Iranian individuals. As the timing of tooth calcification can vary, radiographic finding of congenital tooth agenesis can be confirmed by this molecular method during different dental ages to achieve certainty.