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

Alon Warburg²  Charles Jaffe²  Shewaye Belay³  Aysheshm Kassahun¹  Welelta Shiferaw¹  Asrat Hailu¹  Samar Aramin²  Ibrahim Abbasi² 

[1]Department of Microbiology, Immunology & Parasitology, Faculty of Medicine, Addis Ababa University, PO Box 9086, Addis Ababa, Ethiopia

[2]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, Jerusalem 91120, Israel

[3]Department of Microbiology, Immunology & Parasitology, College of Health Sciences, Mekele University, Mekele, Ethiopia

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

Others  :  1149102
DOI  :  10.1186/1471-2334-13-153

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

【 摘 要 】

Background

Visceral 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.

Methods

The 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.

Results

Of 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.

Conclusions

Although 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】

   
2013 Abbasi et al.; licensee BioMed Central Ltd.

【 图 表 】

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