Comparison of PCR-based diagnoses for visceral leishmaniasis in Bangladesh

Khan, Md. Gulam Musawwir and Bhaskar, Khondaker Rifat Hasan and Kikuchi, Mihoko and Salam, Md. Abdus and Akther, Tania and Haque, Rashidul and Mondal, Dinesh and Hamano, Shinjiro (2014) Comparison of PCR-based diagnoses for visceral leishmaniasis in Bangladesh. Parasitology International, 63 (2). pp. 327-331. ISSN 13835769

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Abstract

The diagnosis of visceral leishmaniasis (VL) is performed using multiple methods encompassing parasitological, serological and nucleic acid-based diagnostic tools, each method with its own unique advantages and disadvantages. Conventional parasitologicalmethods are risky for the patient and require skilled personnel to collect specimens from spleen or bone marrow, and hence they are not generally available in impoverished areas. Polymerase chain reaction (PCR) has been validated as an excellent alternative to microscopy in terms of sensitivity and specificity. Here, we evaluate four different PCR assays targeting ITS1, ITS2, mini-exon and small subunit-rRNA (SSUrRNA) using DNA extracted from peripheral blood buffy coat in order to avoid more invasive processes. A total of 61 VL patients and 75 non-VL infected control individuals were enrolled. The VL patients were confirmed to be positive for Leishmania amastigotes in splenic smears by microscopy. Sensitivities of the PCR targeting ITS1, ITS2, SSUrRNA and mini-exon were 96.7%, 91.8%, 88.5% and 34.4%, respectively, while the specificity was 98.7% for all methods. Nested PCR for ITS1 resulted in 100% sensitivity. The efficacy of each PCR was evaluated with various Leishmania amastigote parasite loads in each spleen smear, graded from 1+ to 5+. The PCR targeting ITS1 showed 100% sensitivity for the detection of Leishmania donovani in all samples from grades ≥3, ≥4, and ≥5, respectively. The restriction fragment length polymorphism observed in ITS1 amplicons digested by HaeIII classified the parasite into L. donovani complex. The ITS1 PCR was found to be equal to conventional, but very invasive and risky parasitological diagnoses and superior to other PCR based methods in sensitivity and examination of genetic heterogeneity. We recommend the PCR targeting ITS1 using peripheral blood buffy coat DNA as an alternate, less invasive diagnostic choice for the confirmation of L. donovani infection.

Item Type:	Article (Journal)
Additional Information:	9217/87013
Uncontrolled Keywords:	Visceral leishmaniasis Leishmania donovani Diagnosis Buffy coat PCR ITS1 ITS2 Mini-exon Small subunit-rRNA (SSUrRNA)
Subjects:	R Medicine > RC Internal medicine > RC111 Infectious and Parasitic Diseases
Kulliyyahs/Centres/Divisions/Institutes (Can select more than one option. Press CONTROL button):	Kulliyyah of Medicine > Department of Basic Medical Kulliyyah of Medicine
Depositing User:	Prof. Dr. Md. Abdus Salam
Date Deposited:	28 Dec 2020 13:37
Last Modified:	28 Dec 2020 13:37
URI:	http://irep.iium.edu.my/id/eprint/87013

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