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      Molecular technique for detection of Leishmania infantum isolates in Iran

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          Abstract

          Introduction:

          Leishmania infantum is the causative agent of autochthonous cutaneous and visceral cases of leishmaniasis and transmitted by female sandflies. The dogs are considered the main reservoir hosts; however, there are the reports on Leishmania infection in other animals. In this study, occurrence types of L. infantum isolates have been analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique.

          Materials and Methods:

          In this experimental study, 77 samples were cultured and prepared for microscopic study and examined through PCR-RFLP. The samples were used for both deoxyribonucleic acid (DNA) and smear-slide preparations. The DNAs were amplified by PCR for the detection of Leishmania subgenus and PCR products were restricted with HaeIII for the species differentiation.

          Results:

          The visceral Leishmania parasites were genotyped as L. infantum. It was also determined sensitivity in PCR (100%) was higher than microscopic examination.

          Conclusion:

          PCR-RFLP technique appears to be most sensitive for the detection and differentiation of L. infantum. There exists a relationship between genetic heterogeneousness and clinical manifestation and geographical regions of this disease in human.

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          Identification and differentiation of Leishmania species in clinical samples by PCR amplification of the miniexon sequence and subsequent restriction fragment length polymorphism analysis.

          Jutta Marfurt, Hans-Peter Beck, Charles L. Jaffe … (2003)
          We recently developed a new PCR-restriction fragment length polymorphism (RFLP)-based assay using the miniexon sequence from the genus Leishmania. Here we report the application of this new genotyping method to naturally infected clinical samples for the differentiation of New and Old World Leishmania species. Of the newly developed assay and four currently applied diagnostic tests (i.e., in vitro cultivation, serology, and two other molecular assays using either the small subunit-internal transcribed spacer sequence or a repetitive genomic sequence), the miniexon assay showed the highest sensitivity, 89.7%, compared to 70.6, 57.1, 51.7, and 79.3%, respectively. Species differentiation was robust and reliable compared with that by two other Leishmania genotyping techniques. The assay provides a valuable tool for the identification of Leishmania directly from clinical samples and enables determination of the infecting species by a facile technique with high discrimination power. Since Leishmania causes a broad spectrum of diseases distinguished by different parasite and host factors, detection and characterization of the infecting species is crucial for the confirmation of a diagnosis as well as the establishment of the clinical prognosis and the initiation of an adequate therapeutic approach. The miniexon PCR-RFLP assay will facilitate such determination and might improve diagnosis and treatment of leishmaniasis.
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            Molecular methods for diagnosis and epidemiological studies of parasitic infections.

            Balbir Bagicha Singh (1997)
            Direct microscopy is widely used for the diagnosis of parasitic infections although it often requires an experienced microscopist for accurate diagnosis, is labour intensive and not very sensitive. In order to overcome some of these shortcomings, molecular or nucleic acid-based diagnostic methods for parasitic infections have been developed over the past 12 years. The parasites which have been studied with these techniques include the human Plasmodia, Leishmania, the trypanosomes, Toxoplasma gondii, Entamoeba histolytica, Giardia, Trichomonas vaginalis, Cryptosporidium parvum, Taenia, Echinococcus, Brugia malayi, Wuchereria bancrofti, Loa loa and Onchocerca volvulus. Early methods, which involved hybridisation of specific probes (radiolabelled and non-radiolabelled) to target deoxyribonucleic acid (DNA), have been replaced by more sensitive polymerase chain reaction (PCR)-based assays. Other methods, such as PCR-hybridisation assays, PCR-restriction fragment length polymorphism (PCR-RFLP) assays and random amplified polymorphic DNA (RAPD) analysis have also proved valuable for epidemiological studies of parasites. The general principles and development of DNA-based methods for diagnosis and epidemiological studies will be described, with particular reference to malaria. These methods will probably not replace current methods for routine diagnosis of parasitic infections in developing countries where parasitic diseases are endemic, due to high costs. However, they will be extremely useful for genotyping parasite strains and vectors, and for accurate parasite detection in both humans and vectors during epidemiological studies.
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              Usefulness of PCR in the diagnosis of cutaneous leishmaniasis in Tunisia.

              L Crobu, H Babba, N Chargui … (2005)
              We assessed the efficiency of a PCR method in establishing the diagnosis of cutaneous leishmaniasis (CL) in Tunisian patients. Four hundred and thirty specimens collected passively from patients with cutaneous ulcers suggestive of leishmaniasis attending health centres for diagnosis were included in the study. Dermal scrapings were analysed both by parasitological (examination of Giemsa-stained smears and in vitro cultivation) methods and by a genus-specific PCR detecting a fragment of the 18S rRNA gene. Microscopy revealed amastigotes in 245 samples (57.0%) and in vitro cultivation gave positive results in 88 cases (20.5%), whereas PCR detected Leishmania in 301 samples (70%). The sensitivities inferred from our results were 99.3%, 80.8% and 29% for PCR, microscopic examination and in vitro cultivation, respectively. The different forms of CL in this country are caused by three species of Leishmania and are treated with the same protocol. Of 303 well-documented cases in our study, 99% were probably caused by Leishmania major and 1% by Leishmania infantum. The lack of species-specific diagnosis is not known to affect treatment or prognosis in Tunisia. These data support the incorporation of PCR into diagnostic strategies for CL, particularly in Tunisia.
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                Author and article information

                Journal
                Journal ID (nlm-ta): Trop Parasitol
                Journal ID (iso-abbrev): Trop Parasitol
                Journal ID (publisher-id): TP
                Title: Tropical Parasitology
                Publisher: Medknow Publications & Media Pvt Ltd (India )
                ISSN (Print): 2229-5070
                ISSN (Electronic): 2229-7758
                Publication date (Print): Jan-Jun 2014
                Volume: 4
                Issue: 1
                Pages: 35-37
                Affiliations
                [1] Department of Molecular, Reference Health Laboratory, Ministry of Health and Medical Education, Tehran, Iran
                [1 ] Department of Immunolgy School of Allied Health, University of Medical Sciences, Tehran, Iran
                Author notes
                Address for correspondence: Dr. Alireza Dolatyar Dehkharghani, #48, Shahrokh Alley, Zartoshtian Ave, Hafez Street, Tehran, Iran. E-mail: ardltyr@ 123456ymail.com
                Article
                Publisher ID: TP-4-35
                DOI: 10.4103/2229-5070.129160
                PMC ID: 3992800
                PubMed ID: 24754025
                SO-VID: 2225f42b-b8be-4eb4-83b2-a73dfc1ed9ee
                Copyright © Copyright: © Tropical Parasitology
                License:

                This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-Share Alike 3.0 Unported, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                Date received : 01 January 2014
                Date accepted : 20 March 2014
                Categories
                Subject: Original Article

                Keywords: detection,leishmania infantum,polymerase chain reaction-restriction fragment length polymorphism,visceral leishmaniasis
                Data availability:
                Keywords: detection, leishmania infantum, polymerase chain reaction-restriction fragment length polymorphism, visceral leishmaniasis

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