Design and expression of polytopic construct of cathepsin-L1, SAP-2 and FhTP16.5 proteins of Fasciola hepatica.

The enzyme-linked immunosorbent assay (ELISA) technique can play an important role in the early detection of fascioliasis. However, they have some diagnostic limitations, including cross-reaction with other helminths. It seems that the combination of recombinant parasite proteins as antigen can reduce these problems. Hence, the present study was aimed to design and confirm the antigenic recombinant multi-epitope (rMEP) construct of three protein epitopes (linear and conformational B-cell epitopes) of the parasite using immunoinformatic tools. For this purpose, the tertiary structures of Fasciola hepatica cathepsin-L1, saposin-like protein 2 and 16.5-kDa tegument-associated protein were predicted using the I-TASSER server. Validation of the modelled structures was performed by Ramachandran plots. The antigenic epitopes of the proteins were achieved by analysing the features of the IEDB server. The synthesized gene was cloned into the pET-22b (+) expression vector and transformed into the Escherichia coli BL21. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to verify and analyse the expression of the rMEP protein. Western blotting was utilized to confirm rMEP protein immunogenicity in two forms, one using an anti-His tag antibody and the other with human pooled sera samples (fascioliasis, non-fascioliasis and negative control sera). Our results demonstrated that the rMEP designed for the three proteins of F. hepatica was highly antigenic, and immune-detection techniques confirmed the antigen specificity. In conclusion, the presented antigenic multi-epitope may be very helpful to develop serodiagnostic kits such as indirect ELISA to evaluate the proper diagnosis of fascioliasis in humans and ruminants.

Vitamin D deficiency and functional response to CRT in heart failure patients.

BACKGROUND: Vitamin D deficiency has been associated with a poor outcome in patients with heart failure (HF). We examined the role of vitamin D in the response of HF patients to cardiac resynchronization therapy (CRT). METHODS: The study comprised 50 patients (30 men and 20 women) with HF undergoing CRT implantation who were prospectively enrolled. Response to CRT was defined as a combination of ≥15% reduction in left ventricular end-systolic volume (LVESV) and ≥10% improvement in the 6­Minute Walk Test within 6 months. Patients were grouped based on their levels of vitamin D prior to CRT implantation. Clinical and echocardiographic examinations were performed prior to and 6 months after the procedure. RESULTS: Of the patients, 11 (22%) failed to respond to CRT; two patients died within 6 months and an additional nine patients showed no improvement in the 6­Minute Walk Test and no reduction in their baseline LVESV. A comparison was made between 25 patients with sufficient levels of vitamin D and 25 patients with insufficient levels. Nine patients (36%) in the "insufficient" group and two patients (8%) in the "sufficient" group failed to respond to CRT implantation (p = 0.037). CONCLUSION: Adequate serum concentrations of vitamin D play a significant role in improving the functional status of patients with systolic HF following CRT implantation.

Coinhibition of overexpressed genes in acute myeloid leukemia subtype M2 by gold nanoparticles functionalized with five antisense oligonucleotides and one anti-CD33(+)/CD34(+) aptamer.

The aim of this study was to evaluate an engineered nanostructure to silence five important oncogenes, including BAG1, MDM2, Bcl-2, BIRC5 (survivin) and XIAP, in acute myeloid leukemia subtype 2 (AML-M2). The smart nanostructures were functionalized gold nanoparticles (FGNs) containing five antisense oligonucleotides (AOs) and one anti-CD33(+)/CD34(+) aptamer. First, the best AO for each gene was selected with the OligoWalk online software, and then different arrangements of AOs were evaluated with the RNAstructure software. Thereafter, naked gold nanoparticles (NGNs) were synthesized by the reaction of 1000 mm HAuCl4 with 10 µg ml(-1) ascorbic acid. Next, five AOs and one anti-CD33(+)/CD34(+) aptamer were attached to NGNs through serial reactions. Later, 5 ml of heparinized blood samples from five AML-M2 patients were prepared, cancerous cells were isolated and then incubated with three concentrations (75, 150 and 300 µg ml(-1)) each of FGNs, NGNs, gold nanoparticles functionalized with scrambled oligonucleotides (GNFSONs) and doxorubicin. Finally, cell death percentage and gene expressions were measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and real-time PCR, respectively. This study showed that FGNs and doxorubicin led to more cell death compared with NGNs and GNFSONs (P<0.05). Interestingly, all concentrations of FGNs led to a decrease in gene expression. As an important finding, although all concentrations of doxorubicin could also inhibit the expression of genes, FGNs had more effect (P<0.05). Moreover, both NGNs and GNFSONs could silence all genes only at a concentration of 300 µg ml(-1). For BCL2 and XIAP, a dose-dependent pattern was observed, but there was no similar pattern for others.

Cryptosporidium genotypes and subtypes distribution in river water in Iran.

Little is known about the diversity and public health significance of Cryptosporidium species in river waters in Iran. In the present study, we determined the genotype and subtype distribution of Cryptosporidium spp. in river water samples in Iran. A total of 49 surface water samples were collected from rivers and surface water in Guilan and Tehran provinces during 2009-2010. Water samples were filtrated through a 1.2-µm pore size membrane filter or by Filta-Max filter followed by immunomagnetic separation or sucrose purification methods. Genotype and subtype of Cryptosporidium were identified by sequence analysis of the 18S rRNA and 60 kDa glycoprotein (gp60) genes, respectively. A total of 24 (48.97%) water samples were positive for Cryptosporidium species by the 18sRNA-based polymerase chain reaction (PCR)-sequencing technique. DNA sequencing revealed the presence of five species of Cryptosporidium (C. parvum, C. hominis, C. muris, C. andersoni, and C. canis) in the water samples of the study area and, to our knowledge, the first report of C. muris in Iran. The results of GP60 gene analysis showed that all C. parvum and C. hominis isolates belonged to the IId and Id subtype families, respectively. The investigated river water supplies were heavily contaminated by pathogenic species of Cryptosporidium from humans and livestock. There is potential risk of waterborne cryptosporidiosis in humans and animals.

Comparison of the RE and B1 gene for detection of Toxoplasma gondii infection in children with cancer.

Early, accurate and effective diagnosis of toxoplasmosis can make an important contribution to the prevention and control of disease, especially in people who are at risk. In this study, two commonly used genomic repeats of Toxoplasma gondii, RE (GenBank accession number AF146527) and B1, were compared to each other in nested-PCR assay. Five hundred and thirty-five blood samples from children with leukemia were tested for the presence of T. gondii antibodies using enzyme immunoassays. One hundred and ten DNA samples of these patients (50 IgM+, IgG+, 10 IgM-, IgG+, and 50 IgM-, IgG-) were analyzed by nested-PCR. The specificity of two nested PCR assays was determined using the DNA samples of other parasites and human chromosomal DNA. As a result, 82% (41/50) and 68% (34/50) of the IgM+, IgG+ samples were positive on duplicate RE and B1-nested PCR analyses, respectively. None of the 10 IgM-, IgG+ seropositive samples was detected positive after testing RE and B1-nested PCR assays in duplicate. One (2%) of the 50 seronegative samples was positive by duplicate RE-nested PCR but none of them were positive by duplicate B1-nested PCR. The detection limit of the RE-nested PCR assay was 640 fg of T. gondii DNA whereas this rate for B1-nested PCR was 5.12 pg of the DNA template. No cross-reactivity with the DNA of other parasites and human chromosomal DNA was found. The results indicate that an RE-based nested PCR assay is more sensitive than B1 genomic target, of those tested, for detection of T. gondii. It is noteworthy that in comparison with B1-nested PCR, RE-nested PCR could detect the T. gondii DNA in seronegative samples too.

Gene regulation of pteridine reductase 1 in leishmania promastigotes and amastigotes using a full-length antisense construct.

BACKGROUND: Pteridine metabolic pathway is unusual features of Leishmania, which is necessary for the growth of parasite. Leishmania has evolved a complex and versatile pteridine salvage network which has the ability of scavenging a wide area of the conjugated and unconjugated pteridines especially folate and biopterin. In this study, we focus on the inhibition of ptr1 gene expression. METHODS: L. major ptr1 gene was cloned into pcDNA3 and digested using KpnI and BamHI. The gene was subcloned so that antisense will transcribe and called pcDNA-rPTR. Leishmania major was cultured and late logarithmic-phase promastigotes were harvested. The promastigotes were divided into two groups. One group was transfected with 50 µg of pcDNA-rPTR, whereas the other group was transfected with pcDNA3. Transfected cells were cultured and plated onto semi-solid media. Mouse pritonean macrophages were transfected using pcDNA-rPTR-tansfected promastigotes. Western blotting was performed on mouse transfected pritonean macrophages and extracts from transfected promastigotes of L. major using a L. major ptr1 antibody raised in rabbits. RESULTS: The PTR1 protein was not expressed in pcDNA-rPTR- tansfected promastigotes and mouse macrophage transfected with pcDNA-rPTR- tansfected promastigotes. CONCLUSION: This approach might be used to study the pteridine salvage pathway in Leishmania or to assess the possibility of using gene expression inhibition in the treatment of leishmaniasis.

Double-Stranded RNA Viral Infection in Tehran Trichomonas vaginalis Isolates.

BACKGROUND: Trichomonas vaginalis is a pathogenic protozoon and may be contaminated with dsRNA virus called Trichomonas vaginalis virus (TVV). The viral infection is an important factor for its pathogenesis and sensitivity to metronidazole. The presence of TVV is associated with qualitative and quantitative expression of cysteine proteinases and surface immunogenic; P270. The purpose of this study was to determine TVV frequency in T. vaginalis clinical isolates in Tehran, Iran. METHODS: The 46 T. vaginalis isolates were collected from Tehran Province and cultured in TYI-S-33 culture medium. Viral RNA was extracted and RT-PCR was done. RESULTS: Of 46 T. vaginalis isolates, 8 isolates (17.39%) were infected with TVV-1. There was not any association between patient age and TVV- infected T. vaginalis. There were 17.39% viral infection in T. vaginalis isolates which was lower than that reported by other researchers. CONCLUSION: This is the first report on T. vaginalis isolates infection by TVV-1 in Iran.

Canine visceral leishmaniasis: a comparative study of real-time PCR, conventional PCR, and direct agglutination on sera for the detection of Leishmania infantum infection.

Canine visceral leishmaniasis (CVL) is endemic in northwestern Iran. This study aimed to compare real-time PCR, conventional PCR, and the direct agglutination test (DAT) for the diagnosis Leishmania infantum infection in 167 serum samples of domestic dog. Bone marrow was used for parasitological examination (smears and/or culture) in symptomatic visceral leishmaniasis, and serum was used for detection of L. infantum kinetoplast DNA (kDNA) by both conventional PCR and real-time PCR, while anti-L. infantum antibodies in sera were measured by DAT. The sera were collected from 37 symptomatic and 112 asymptomatic dogs during April to May 2011. Eighteen presumed negative samples were obtained from healthy dogs kept in non-endemic areas with no history of CVL and used as controls. All 18 samples were negative by DAT and Dipstick rK39. DAT confirmed previous exposure to L. infantum for all 149 serum samples collected from symptomatic and asymptomatic dogs in CVL endemic areas of Iran. Among the 37 symptomatic dogs, 20 (54%), 25 (67.6%), 36 (97.3%), and 37 (100%) showed L. infantum infection by parasitological methods, conventional PCR, real-time PCR, and DAT (≥ 1:80), respectively. Of 112 asymptomatic dogs, 79 (70.5%), 111 (99.1%), and 112 (100%) were shown to be positive by conventional PCR, and DAT (≥ 1:80), respectively. For ethical reasons, no asymptomatic or healthy control dogs were examined by parasitological methods. Three (16.7%) control dogs were positive by real-time PCR, but were negative by DAT, dipstick rK39, and conventional PCR methods. Parasitemia levels were measured by real-time PCR targeting kDNA using SYBR(®) green assay. This quantitative technique detected infection in 89.9% (150/167) of the domestic dogs that harbored L. infantum kDNA, ranging from 0.01 49 to 310.1 parasites/ml. The average was 16.60 parasites/ml. A good agreement (0.97) was found between real-time PCR and DAT at ≥ 1:80 titer, used as cut-off value by Kappa analysis. Thus, real-time PCR as a quantitative PCR assay on serum samples represents a valuable tool for initial diagnosis of CVL when whole blood is not available.

Comparison of real-time PCR and conventional PCR with two DNA targets for detection of Leishmania (Leishmania) infantum infection in human and dog blood samples.

Zoonotic visceral leishmaniasis (VL) is endemic in northwestern Iran. Real-time PCR, conventional PCR, and the direct agglutination test (DAT) were used to diagnose Leishmania infantum infection in blood samples from 100 domestic dogs and 100 humans. Based on clinical evaluation, 82 humans and 72 dogs from the endemic area were categorized as having asymptomatic infection, DAT positive with no clinical signs of VL, or symptomatic infection, DAT positive with at least one sign of VL. Eighteen human samples containing no Leishmania antibodies (DAT(-)) and 28 dog DAT(-) sera from non-endemic areas with no history of VL constituted negative controls. All 46 DAT(-) samples were also negative by Dipstick rK39. Bone marrow material was used for parasitological examinations in symptomatic VL, and peripheral blood samples were used for detection of L. infantum infection using conventional PCR and real-time PCR in non-symptomatic subjects. Two DNA targets (ITS1 kDNA) were used for conventional PCR. L. infantum antibodies in sera were detected by DAT. Parasitemia was measured by real-time PCR targeting kDNA using Taqman Assay. All 72 (100%) symptomatic (38/38) and asymptomatic (34/34) dog DAT(+)samples, 45 of 48 (93.8%) symptomatic human DAT(+) samples, and 32 of 34 (94.1%) human asymptomatic cases were identified by real-time PCR. The mean (59.19 vs 12.38 parasite equivalents/mL of blood) and median (16.15 vs 1 parasite equivalents/mL of blood) ranges of parasitemia were higher in dogs than in humans (P<0.05). The highest agreement was obtained between real-time PCR and DAT (99% in dogs and 95% in humans). Sensitivity of 100% and 93.9%, specificity of 96.4% and 100%, positive predictive values of 98.6% and 100%, and negative predictive values of 100% and 78.3% were found by real-time PCR for dog and human samples, respectively.

Inhibition of Leishmania major PTR1 Gene Expression by Antisense in Escherichia coli.

BACKGROUND: Protozoa related to Trypanosome family including Leishmania, synthesize enzymes to escape from drug therapy. One of them is PTR1 that its enzymatic activity is similar to dihydrofolate reductase (DHFR). Dihydrofolate reductase - thymidylate synthase has a major role in DNA synthesis, if it is inhibited, the result would be the death of parasite. Since PTR1 activity is similar to DHFR, causes the decrease of inhibition effect of drug. The aim of this study was inhibition of Iranian L. major PTR1 expression with mRNA antisense in prokaryotic system as an approach to appear of the drugs therapeutic effects more. METHODS: PTR1 gene was ligated to pACYCDuet-1 and pcDNA3 plasmids as sense and antisense plasmids, respectively. Simultaneously transfer of sense and antisense plasmids was done in E. coli strain M15. SDS-PAGE and western blot analysis were carried out to analyze the expression. RESULTS: Sense and antisense plasmids were prepared and confirmed by restriction analysis and PCR then simultaneously transfer of them was done. SDS-PAGE and western blot analysis showed PTR1 gene was inhibited by mRNA antisense in bacterial cells. CONCLUSION: Expression of PTR1 gene in sense plasmid was inhibited successfully by antisense plasmid.

Expression and Purification of P43 Toxoplasma gondii Surface Antigen.

BACKGROUND: Toxoplasma gondii is an obligate intracellular protozoan parasite, capable of infecting all species of mammals including man. Congenital toxoplasmosis is more important during pregnancy for the first time. In this study we expressed and purified P43 Toxoplasma gondii tachyzoite and bradyzoite specific surface antigen. METHODS: The recombinant pGEMEX-1 contained Toxoplasma P43 coding sequence was transformed into E. coli and mass cultured in LB medium contained 100 µg/ml ampicillin at 37°C over night. The T7 promoter was induced by 1mM isopropyl-1-thio-ß-D-galactopyranoside (IPTG. Recombinant protein was purified by affinity chromatography and confirmed by gel diffusion dot blot and western blot,-using specific anti Toxoplasma antibodies. RESULTS: Recombinant plasmid was induced by IPTG and analyzed by SDS-PAGE. Recombinant protein was confirmed by Western-blot and dot blot using anti human Toxoplasma antibody. CONCLUSION: Recombinant Toxoplasma P43 was produced successfully.

Emergence of Pseudomonas aeruginosa cross-infection in children with cystic fibrosis attending an Iranian referral pediatric center.

BACKGROUND AND OBJECTIVES: This study was carried out with the objective of determining the genomic variability of P. aeruginosa strains isolated from patients suffering from cystic fibrosis or from environmental cultures collected from different locations in the unit they admitted. MATERIALS AND METHODS: A total of 57 clinical and environmental P. aeruginosa isolates were genotyped by enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR), and antimicrobial susceptibility testing was performed using the Clinical and Laboratory Standards Institute method. RESULTS: One predominant ERIC profile (type A) was identified in 46 strains (81% of all typed isolates) which was responsible for thirty-nine of 44 clinical isolates (89%) and 7 of 13 environmental isolates (54%). All clinical isolates were susceptible to piperacillin-tazobactam, ceftazidime and cefepime followed by ticarcillin, aztreonam, amikacin and tobramycin (96.5%). CONCLUSIONS: In our country CF patients are not segregated from other patients, and transmission of bacteria between these patients and other patients might occur in the wards via personal contact or contaminated environment. Future evaluation for policy of patient segregation is necessary and the elimination of contaminated sources and control of environmental spread and recurrent contamination risk is needed.

Genetic variability of Blastocystis sp. isolated from symptomatic and asymptomatic individuals in Iran.

Blastocystis is an unusual enteric protozoan parasite of humans and many animals whose pathogenic potential is still controversial. To increase the understanding of the molecular epidemiology of this emerging parasite and due to its potential impact on public health, its subtypes (STs) in Iranian symptomatic and asymptomatic individuals were determined. A total of 100 Blastocystis isolates by microscopy and culture methods were obtained. DNA was extracted from the positive culture isolates, and the Blastocystis subtypes were identified using seven subtype-specific sequenced-tagged site (STS) primers. Four subtypes, ST3 as dominant (53 %), followed by ST1 (48 %), ST5 (33 %), and ST2 (7 %) were identified. In this study, ST1 in gastrointestinal patients compared to asymptomatic individuals was significantly dominant (p = 0.001). From 33 (33 %) mixed subtype infections, ST1, 3 (14 %) was significantly related to GI symptoms (p = 0.045), and eight mixed infections with three different STs, which are under reported, were also identified.

Evaluation of Recombinant SAG1 Protein for Detection of Toxoplasma gondii Specific Immunoglobulin M by ELISA Test.

BACKGROUND: Toxoplasmosis is a serious disease in immunocompromised patients and pregnant women. Differentiation of acute and chronic infection is a major challenge in serodiagnosis of the disease. Since the aim of this study was to assess the diagnostic utility of recombinant SAG1 (rec-SAG1) for the detection of Toxoplasma-specific IgM antibodies in human sera, by an enzyme-linked immunosorbent assay (ELISA). METHODS: The purified recombinant protein SAG1 was applied in house ELISA test and the ability of it in binding to specific immunoglobulin M in 30 serum samples of acute infected patients was evaluated. The results obtained by assays with the recombinant SAG1 and standard commercial assays were compared. RESULTS: The sensitivity and specificity of in house ELISA compared to a standard commercial ELISA (com-ELISA) were 80% and 90%, respectively. CONCLUSION: It was concluded that the rec-SAG1 could be an alternative marker for detection of anti Toxoplasma-specific IgM and diagnosis of acute infection.

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.

Isolation and detection of Leishmania species among naturally infected Rhombomis opimus, a reservoir host of zoonotic cutaneous leishmaniasis in Turkemen Sahara, North East of Iran.

In Iran, three species of Leishmania have been incriminated as the causative agents of human leishmaniasis, Leishmania (L.) major, Leishmania tropica, and Leishmania infantum.Rhombomis opimus have been incriminated as a principal reservoirs of the parasitic protozoan Leishmania major, the causative agent of rural zoonotic cutaneous leishmaniasis (ZCL) in Iran. Rodents captured and examined to find Leishmania species using conventional methods including direct impression smear and microscopic observation inoculation samples to Balb/c and culture in NNN medium. Also molecular method was employed to detect Leishmania in rodents by amplifying a region of the ribosomal RNA amplicon of Leishmania (ITS1-5.8S rRNA-ITS2) using Nested PCR. Leshmania species were specified by DNA sequences. 36 (38.3%) of R. opimus were Leishmania positive using at least one conventional methods. Many more ITS-rDNA fragments were amplified from R. opimus but only 65 out of 74 PCR products contained enough DNA for direct sequencing or readable sequences. The PCR assays detected in Iranian R. opimus not only Leishmania major in 59 (79.7%) rodents but also Leishmania turanica in 6 (8.1%) rodents, another parasite of the great gerbil. These parasites were found in Turkemen Sahara, North East of Iran, in a focus of rural (ZCL). L. major and L. turanica in R. opimus firmly identified from Turkemen Sahara. Nine rodents with Leishmania infections unidentified which some were unreadable sequences, these could be mixed infections of L. major, L. turanica, Leishmania gerbillisensu lato and Leishmania close to L. gerbilli or a related species reported in sandflies previously from this location. The haplotypes of L. major and L. turanica were found to be identical to that of isolates of L. major and L. turanica from Iran and in GenBank elsewhere. R. opimus is probably the key reservoir in this ZCL focus because of its abundance and its infection rates with both L. major and L. turanica.

Simultaneous and rapid differential diagnosis of Mycoplasma genitalium and Ureaplasma urealyticum based on a polymerase chain reaction-restriction fragment length polymorphism.

OBJECTIVES: The aim of this investigation was to simultaneously detect and differentiate Mycoplasma genitalium and Ureaplasma urealyticum in female patients suffering from genital complications by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP). MATERIALS AND METHODS: Genital swabs were taken from 210 patients. They were transported to the laboratory in phosphate-buffered saline. For PCR, samples were analysed with genus-specific MyUu-R and MyUu-F primers. This primer set, which was originally designed in our laboratory, amplified a 465 bp fragment (M. genitalium) and a 559 bp fragment (U. urealyticum). Samples containing a band of the expected sizes for the Mycoplasma strains were subjected to digestion with a restriction endonuclease enzyme of TaqI and Cac8I. RESULTS: Of the 210 samples, a total of 100 (47.6%) samples were found to be positive for Mycoplasmas (seven M. genitalium isolates, 3.3%; and 89 U. urealyticum isolates, 42.4%), and coinfections with both species were detected in four samples (1.9%). The PCR-RFLP results showed that M. genitalium and U. urealyticum are different by enzyme patterns. CONCLUSION: PCR-RFLP offers a rapid and easily applicable protocol to simultaneous detection and differentiation of M. genitalium and U. urealyticum from clinical samples when specific primers and restriction enzymes are used.

Detection of drug resistance gene in cutaneous leishmaniasis by PCR in some endemic areas of iran.

BACKGROUND: Cutaneous leishmaniasis is still a health problem in many rural and urban regions of Iran and drug resistance has emerged as a major impediment in the treatment of leishmaniasis. This study aims to determine the drug resistance gene in cutaneous leishmaniasis by PCR in some endemic areas of Iran. METHODS: Ninety seven samples were collected from ulcers of leishmaniasis patients from some endemic areas of Iran. The Giemsa stained samples were examined microscopically and cultured in NNN and RPMI 1640 mediums for parasite detection. After DNA extraction, PCR was done by a pair of specific primers. For detection of mutation in DNA, first PCR products were electrophoresed on CSGE gel. The suspected samples were compared by sequencing and RFLP results were demonstrated. Comparison of DNA derived from a wild type cell and mutant cell was undertaken by CSGE and sequencing methods. RESULTS: Among 90 isolates (92.8%) examined for detection of mutation in gene with CSGE and RFLP, 10 (11.1%) revealed a disorder in sequencing selection for unresponsive to drug. CONCLUSION: Drug resistance in cutaneous leishmaniasis to sodium stiboglocanat is probably due to a mutation in a genome. A field study is needed to determine the distribution of drug resistance and other gene mutations involved in unresponsiveness to drugs in leishmaniasis endemic areas of Iran.