Molecular Epidemiological Survey of Cutaneous Leishmaniasis in Two Highly Endemic Metropolises of Iran, Application of FTA Cards for DNA Extraction From Giemsa-Stained Slides

Abstract

Background:PCR has been used for confirmation of leishmaniasis in epidemiological studies, but complexity of DNA extraction and PCR approach has confined its routine use in developing countries.Objectives:In this study, recent epidemiological situation of cutaneous leishmaniasis (CL) in two hyper-endemic metropolises of Shiraz and Isfahan in Iran was studied using DNA extraction by commercial FTA cards and kinetoplastid DNA (kDNA)-PCR amplification for detection/identification of Leishmania directly from stained skin scraping imprints.Patients and Methods:Fifty four and 30 samples were collected from clinically diagnosed CL patients referred to clinical laboratories of leishmaniasis control centers in Isfahan and Shiraz cities, respectively. The samples were examined by direct microscopy and then scrapings of the stained smears were applied to FTA cards and used directly as DNA source in a nested-PCR to amplify kDNA to detect and identify Leishmania species.Results:Fifty four of 84 (64.2%) slides obtained from patients had positive results microscopically, while 79/84 (94%) of slides had positive results by FTA card-nested-PCR. PCR and microscopy showed a sensitivity of 96.4% and 64.2% and specificity of 100% and 100%, respectively. Interestingly, Leishmania major as causative agent of zoonotic CL was identified in 100% and 90.7% of CL cases from Isfahan and Shiraz cities, respectively, but L. tropica was detected from only 9.3% of cases from Shiraz city. All cases from central regions of Shiraz were L. tropica and no CL case was found in Isfahan central areas.Conclusions:Filter paper-based DNA extraction can facilitate routine use of PCR for diagnosis of CL in research and diagnostic laboratories in Iran and countries with similar conditions. Epidemiologic changes including dominancy of L. major in suburbs of Shiraz and Isfahan metropolises where anthroponotic CL caused by L. tropica had been established, showed necessity of precise studies on CL epidemiology in old urban and newly added districts in the suburbs.

Diagnostic performance of a qPCR for Leishmania on stained cytological specimens and on filter paper impressions obtained from cutaneous lesions suggestive of canine leishmaniosis

“…Study II showed an overall sensitivity of 84.6% for the Leishmania qPCR performed on cytological specimens from dogs with a diagnosis of CanL, whereas Leishmania amastigotes were visualized by microscopic examination from only 61.5% of clinical lesions. Similar findings were reported in a study of cutaneous leishmaniosis in humans, in which qPCR and microscopy showed sensitivities of 96.4% and 64.2%, respectively, both with 100% specificity . Other human studies have reported qPCR sensitivities ranging from 83.2 to 98% .…”

“…Similar findings were reported in a study of cutaneous leishmaniosis in humans, in which qPCR and microscopy showed sensitivities of 96.4% and 64.2%, respectively, both with 100% specificity. 44 Other human studies have reported qPCR sensitivities ranging from 83.2 to 98%. 42,45 A previous study of lymph node and bone marrow smears collected from subclinically infected dogs also showed microscopy to be inferior to qPCR for evaluation of glass slide preparations.…”

Population structures of Leishmania infantum and Leishmania tropica the causative agents of kala-azar in Southwest Iran

“…The first case of L. tropica VL came from SW Iran (Alborzi et al 2006), and other cases have been reported countrywide with varying frequencies, e.g., ranging from 1.4% in 1993/4 to 14% in our study (Alborzi et al 2006;Mohebali 2013;Hosseininasab et al 2014). Of our four cases, two were obtained from Shiraz and Kerman City, which are well established endemic foci of L. tropica-related ACL (Ghatee et al 2014;Izadi et al 2016), while the other two were from areas where L. tropica has not been reported previously. These limited data hint at trend of increasing L. tropica-related VL and emergence in the new regions in Iran that may be related to population movements or global warming that promotes favourable breeding of sand flies (Ghatee et al 2018a).…”

The Geographical Distribution of Cutaneous Leishmaniasis Causative Agents in Iran and Its Neighboring Countries, A Review

“…CL is widely distributed in Iran and has been reported in 25 of 31 Iran provinces in central, west, southwest, south, southeast, east, and northeast regions (23)(24)(25)(26)(27)(28)(29).…”

“…city has long been recognized as endemic for ACL but no DNAbased molecular data have characterized the species until 2016. In one study, ACL was detected in the old and central parts of the city while L. major was reported in suburbs (27). Bushehr province is a known focus of L. major causing ZCL in southwest Iran (39), but a small study in Bushehr, the provincial capital, identified L. tropica in 6/8 CL patients (40).…”