Evaluation of PCR procedures for detecting and quantifying Leishmania donovani DNA in large numbers of dried human blood samples from a visceral leishmaniasis focus in Northern Ethiopia

BMC Infectious Diseases

Technical Advance

Charles Jaffe¹  Alon Warburg¹  Samar Aramin¹  Ibrahim Abbasi¹  Shewaye Belay²  Asrat Hailu³  Welelta Shiferaw³  Aysheshm Kassahun³ 

[1] Department of Microbiology and Molecular Genetics, The Institute for Medical Research Israel-Canada, The Kuvin Centre for the Study of Infectious and Tropical Diseases, The Hebrew University - Hadassah Medical School, The Hebrew University of Jerusalem, 91120, Jerusalem, Israel;Department of Microbiology, Immunology & Parasitology, College of Health Sciences, Mekele University, Mekele, Ethiopia;Department of Microbiology, Immunology & Parasitology, Faculty of Medicine, Addis Ababa University, PO Box 9086, Addis Ababa, Ethiopia;

关键词: Asymptomatic infections;    Cohort study;    DNA extraction;    Ethiopia;    Visceral Leishmaniasis;    Leishmania donovani;    kDNA-PCR;   

DOI  :  10.1186/1471-2334-13-153

 received in 2012-09-26, accepted in 2013-03-18,  发布年份 2013

来源: Springer

【 摘 要 】

BackgroundVisceral Leishmaniasis (VL) is a disseminated protozoan infection caused by Leishmania donovani parasites which affects almost half a million persons annually. Most of these are from the Indian sub-continent, East Africa and Brazil. Our study was designed to elucidate the role of symptomatic and asymptomatic Leishmania donovani infected persons in the epidemiology of VL in Northern Ethiopia.MethodsThe efficacy of quantitative real-time kinetoplast DNA/PCR (qRT-kDNA PCR) for detecting Leishmania donovani in dried-blood samples was assessed in volunteers living in an endemic focus.ResultsOf 4,757 samples, 680 (14.3%) were found positive for Leishmania k-DNA but most of those (69%) had less than 10 parasites/ml of blood. Samples were re-tested using identical protocols and only 59.3% of the samples with 10 parasite/ml or less were qRT-kDNA PCR positive the second time. Furthermore, 10.8% of the PCR negative samples were positive in the second test. Most samples with higher parasitemias remained positive upon re-examination (55/59 =93%). We also compared three different methods for DNA preparation. Phenol-chloroform was more efficient than sodium hydroxide or potassium acetate. DNA sequencing of ITS1 PCR products showed that 20/22 samples were Leishmania donovani while two had ITS1 sequences homologous to Leishmania major.ConclusionsAlthough qRT-kDNA PCR is a highly sensitive test, the dependability of low positives remains questionable. It is crucial to correlate between PCR parasitemia and infectivity to sand flies. While optimal sensitivity is achieved by targeting k-DNA, it is important to validate the causative species of VL by DNA sequencing.

【 授权许可】

Unknown   
© Abbasi et al.; licensee BioMed Central Ltd. 2013. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.