title: 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.
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.
authors.affiliation: 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.
authors.forename: Ibrahim
authors.initials: I
authors.lastname: Abbasi
authors.forename: Samar
authors.initials: S
authors.lastname: Aramin
authors.forename: Asrat
authors.initials: A
authors.lastname: Hailu
authors.forename: Welelta
authors.initials: W
authors.lastname: Shiferaw
authors.forename: Aysheshm
authors.initials: A
authors.lastname: Kassahun
authors.forename: Shewaye
authors.initials: S
authors.lastname: Belay
authors.forename: Charles
authors.initials: C
authors.lastname: Jaffe
authors.forename: Alon
authors.initials: A
authors.lastname: Warburg
mesh_terms: D000328:Adult; D000818:Animals; D001769:Blood / Q000469:parasitology*; D002648:Child; D015331:Cohort Studies; D018105:DNA, Kinetoplast / Q000097:blood / Q000235:genetics; D016054:DNA, Protozoan / Q000097:blood* / Q000235:genetics; D003890:Desiccation; D005002:Ethiopia; D005260:Female; D006801:Humans; D007893:Leishmania donovani / Q000235:genetics / Q000302:isolation & purification*; D007898:Leishmaniasis, Visceral / Q000175:diagnosis* / Q000469:parasitology; D008297:Male; D011446:Prospective Studies; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013048:Specimen Handling / Q000379:methods*
chemical_list: D018105:DNA, Kinetoplast; D016054:DNA, Protozoan