title: SYBR green-based detection of Leishmania infantum DNA using peripheral blood samples.
abstract: Parasitological methods for the diagnosis of visceral leishmaniasis (VL) require invasive sampling procedures. The aim of this study was to detect Leishmania infantum (L. infantum) DNA by real time-PCR method in peripheral blood of symptomatic VL patient and compared its performance with nested PCR, an established molecular method with very high diagnostic indices. 47 parasitologically confirmed VL patients diagnosed by direct agglutination test (DAT > 3200), bone marrow aspiration and presented characteristic clinical features (fever, hepatosplenomegaly, and anemia) and 40 controls (non-endemic healthy control-30, Malaria-2, Toxoplasma gondii-2, Mycobacterium tuberculosis-2, HBV-1, HCV-1, HSV-1 and CMV-1) were enrolled in this study. SYBR-green based real time-PCR and nested PCR was performed to amplify the Kinetoplast DNA minicircle gene using the DNA extracted from Buffy coat. From among 47 patients, 45 (95.7 %) were positive by both nested-PCR and real time-PCR. These results indicate that real time-PCR was not only as sensitive as a nested-PCR assay for detection of Leishmania kDNA in clinical sample, but also more rapid. The advantage of real time-PCR based methods over nested-PCR is simple to perform, more faster in which nested-PCR requires post-PCR processing and reducing contamination risk.
authors.affiliation: Department of Parasitology and Mycology, Faculty of Medicine, Iran University of Medical Sciences, Tehran, Iran.
authors.forename: Mehrdad
authors.initials: M
authors.lastname: Ghasemian
authors.affiliation: Department of Parasitology, Faculty of Allied Medicine, Iran University of Medical Sciences, Tehran, Iran.
authors.forename: Mohammad Javad
authors.initials: MJ
authors.lastname: Gharavi
authors.affiliation: Department of Parasitology and Mycology, Faculty of Medicine, Iran University of Medical Sciences, Tehran, Iran.
authors.forename: Lame
authors.initials: L
authors.lastname: Akhlaghi
authors.affiliation: Department of Parasitology, Faculty of Public Health, Tehran University of Medical Sciences, Tehran, Iran.
authors.forename: Mehdi
authors.initials: M
authors.lastname: Mohebali
authors.affiliation: Department of Parasitology and Mycology, Faculty of Medicine, Iran University of Medical Sciences, Tehran, Iran.
authors.forename: Ahmad Reza
authors.initials: AR
authors.lastname: Meamar
authors.affiliation: Antimicrobial Resistance Research Center, Department of Medical Microbiology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
authors.forename: Ehsan
authors.initials: E
authors.lastname: Aryan
authors.affiliation: Department of Parasitology and Mycology, Faculty of Medicine, Iran University of Medical Sciences, Tehran, Iran.
authors.forename: Hormozd
authors.initials: H
authors.lastname: Oormazdi
authors.affiliation: Department of Parasitology and Mycology, Faculty of Medicine, Iran University of Medical Sciences, Tehran, Iran.
authors.forename: Zahra
authors.initials: Z
authors.lastname: Ghayour
keywords: Human; Iran; Nested-PCR; Peripheral blood; Real time-PCR; Visceral leishmaniasis