Predominance of Leishmania major and rare occurrence of Leishmania tropica with haplotype variability at the center of Iran

ABSTRACT Background Leishmania major is a causative agent of zoonotic cutaneous leishmaniasis in the center of Iran, Abarkouh district. Molecular characterization and precise incrimination of Leishmania species was carried out to perform controlling measurements and to design treatment programs for zoonotic cutaneous leishmaniasis. Methods All smears isolated from ulcers of suspected patients were examined under a light microscope and graded for amastigotes frequency. Extraction of DNA, PCR, RFLP and sequencing of ITS-rDNA genotype were done to increase the efficacy of Leishmania parasites identification at their species-specific level and to detect any Leishmania infections within. Results Humans were found to be infected with L. major with high infection frequency and also Leishmania tropica was identified with low occurrence for the first time as non-native species using molecular analyses. The rates of infections was considerable with microscopic observation (n= 65, 73%) out of 89 smears prepared from suspected patients. Molecular analyses showed that the density of L. major was significantly higher (n= 48, 53.93%) than L. tropica (n= 4, 4.49%) (Mann-Whitney U test: p< 0.05) and two samples (2.25%) remained ambiguous after several sequencing. L. major did not have diversity with two common haplotypes but L. tropica were found to exhibit high diversity with three novel haplotypes. Conclusion L. major was considered the causative agent of leishmaniasis in the region, but the identification of a non-native L. tropica revealed the importance of further isolation of Leishmania parasites following molecular analyses and confirmation, and also revealed the importance of further isolation of Leishmania parasites from patients of the field areas who do not have easily access to health care centers for specialized treatment strategies.

An overview of a diagnostic and epidemiologic reappraisal of cutaneous leishmaniasis in Iran

Cutaneous leishmaniasis (CL) is a widespread tropical infection which has a high incidence rate in Iran. Leishmania tropica, the causative agent of anthroponotic cutaneous leishmaniasis (ACL), and Leishmania major, which causes zoonotic cutaneous leishmaniasis (ZCL), are endemic in various parts of Iran with a high incidence rate. The aim of this study was to evaluate the reappraisal of the diagnosis and epidemiology of CL in Iran, by different clinical, parasitological and molecular assays among patients suspected of CL referred to the Department of Parasitology, at the Pasteur Institute of Iran during 2006-2009. Two hundred samples from patients with ulcerative skin lesions were collected, clinical analyses were applied, data questionnaire was completed and samples were examined for CL by using both direct microscopic and culture methods. Moreover, PCR assay was applied for detection of Leishmania species in CL isolates resulting from parasitological assay. Clinical observation revealed that the majority (58 percent) of lesions was single; double lesions were observed in 22 percent of patients, and only 20 percent of CL had multiple lesions. Out of 200 patients, Leishman body was observed in 77 samples (38.5 percent) by direct smear and 40 percent by cultivation assay. Most patients (21.3 percent) had a travel history to the Isfahan province, one of the most important endemic areas of CL located in center of Iran. PCR assay by kDNA indicated 32 and 18 out of 50 isolates respectively had similar patterns with standard L. major and L. tropica. In conclusion, clinical manifestations and an appropriate diagnostic assay with a parallel molecular characterization of CL may lead to a screening evaluation of disease, prognosis, treatment and control strategies.

[Evaluation of mulecular epidemiology of cutaneous leishmaniasis in Sabzevar]

Cutaneous leishmaniasis is a parasitic infection With an important health problem in many parts of Iran such as Sabzevar, in Khorasan Razavi province. Epidemiological and clinical findings aren't sufficient for identification of parasites. Because the host sources are different an accurate identification and diagnosis is necessary before treatment. DNA of every parasite such as every organism is specific. This facilitates extensive use of DNA for diagnostic and identification of parasite species. Molecular methods such as PCR seem to be very useful for this reason. We decided to identify different species of leishmania parasites causing Cutaneous leishmaniasis by PCR in Sabzevar A Total of 86 patients, whom diseases were confirmed by direct smear, were recruited and samples were isolated and cultured in NNN medium, followed by sub-cultured in RPMI-1640. Then DNA was extracted using four DNA extraction methods. Extracted kinetoplastic DNA was amplified by PCR method using two specific primers. Electrophoresis patterns from each isolate were compared with reference strains of L.major, L.tropica and the markerThe related bands to amplified products were detected on agarose gel in all samples expected of DNA extracted by boiling method. The results of kDNA gene templets in Electrophoresis gel indicated the leishmania parasite species, causing Cutaneous leishmaniasis, in Sabzevar as 32 samples L.tropica and 54 samples L.major. L.tropica and L.major both are Etiologic agents ofCutaneous leishmaniasis in Sabzevar and PCR technique is a suitable tool for the leishmania species characterization in epidemiological studies. The phenol-chloroform based methods are as valuable as DNeasy mini kit [QIAGEN] but more cost effective than kit

Protein profiling on meglumine antimoniate [Glucantime [R]] sensitive and resistant L. tropica isolates by 2-dimentional gel electrophoresis: a preliminary study

Glucantime[R] is the first- line drug for the treatment of all forms of leishmaniasis. Unfortunately, the prevalence of parasites becoming resistant to Glucantime[R] is increasing in several parts of the world including Iran. As protein is the most important target for drugs in response to a variety of signals including drugs so, it seems expression protein patterns in sensitive and resistant Leishmania parasites could greatly help us about the mechanisms of responses to antileishmanial drugs. In this study, we used 2-dimentional gel electrophoresis [2-DE] method to determine protein expression profiles between drug [Glucantime[R]] sensitive and resistant Leishmania tropica isolated from Iranian an throponotic cutaneous leishmaniasis [ACL] patients. We used from the two confirmed genetically of Glucantime[R] sensitive [Mash-4] and resistant [Mash-927] field strains of L. tropica, isolated from ACL patients in north eastern Iran. The two Leishmania isolates were cultured, promastigotes were harvested followed by protein extraction using TCA/Aceton to study protein profiling, 2-DE was done and gels stained with silver nitrate. At least 2236 distinct protein spots were detected. Twelve spots out of them, showed significant changes in expression in resistant compared to sensitive isolates. Of these, 11 protein spots were up- and one was down-regulated. This preliminary study has showed that a number of proteins differentially expressed in drug [Glucan-time [R]] resistance L. tropica and probably the role of these proteins are increasing the parasite resistance against the drug and delay in cell death