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

Ibrahim Abbasi, Samar Aramin, Asrat Hailu, Welelta Shiferaw, Aysheshm Kassahun, Shewaye Belay, Charles Jaffe, Alon Warburg*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

47 Scopus citations

Abstract

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.

Original languageAmerican English
Article number153
JournalBMC Infectious Diseases
Volume13
Issue number1
DOIs
StatePublished - 27 Mar 2013

Bibliographical note

Funding Information:
The authors acknowledge the invaluable assistance of research assistants at Faculty of Medicine, Addis Ababa University: Asrat Bezuneh, Habtamu Belay, Tedla Zegeye, Mulugeta Gichile, Kedir Ali, Hagos Teka, Hadas Gebreyesus, and Siltan Gebre-Selasssie. Amit Huppert, Ezer Miller. Abdelmajeed Nasereddin and Petr Volf provided invaluable assistance. This study was supported by the Bill and Melinda Gates Foundation Global Health Program [grant number OPPGH5336].

Keywords

  • Asymptomatic infections
  • Cohort study
  • DNA extraction
  • Ethiopia
  • Leishmania donovani
  • Visceral Leishmaniasis
  • kDNA-PCR

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