title: Comparison of PCR-based diagnoses for visceral leishmaniasis in Bangladesh.
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 parasitological methods 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.
authors: International Centre for Diarrheal Disease Research, Bangladesh (ICDDR,B), Mohakhali, Dhaka 1212, Bangladesh.
authors: Md Gulam Musawwir
authors: MG
authors: Khan
authors: International Centre for Diarrheal Disease Research, Bangladesh (ICDDR,B), Mohakhali, Dhaka 1212, Bangladesh.
authors: Khondaker Rifat Hasan
authors: KR
authors: Bhaskar
authors: Department of Immunogenetics, Institute of Tropical Medicine (NEKKEN), Nagasaki University, Japan.
authors: Mihoko
authors: M
authors: Kikuchi
authors: Rajshahi Medical College, Rajshahi 6000, Bangladesh.
authors: Md Abdus
authors: MA
authors: Salam
authors: International Centre for Diarrheal Disease Research, Bangladesh (ICDDR,B), Mohakhali, Dhaka 1212, Bangladesh.
authors: Tania
authors: T
authors: Akther
authors: International Centre for Diarrheal Disease Research, Bangladesh (ICDDR,B), Mohakhali, Dhaka 1212, Bangladesh.
authors: Rashidul
authors: R
authors: Haque
authors: International Centre for Diarrheal Disease Research, Bangladesh (ICDDR,B), Mohakhali, Dhaka 1212, Bangladesh.
authors: Dinesh
authors: D
authors: Mondal
authors: Department of Parasitology, Institute of Tropical Medicine (NEKKEN), Nagasaki University, Japan; Global COE Program, Nagasaki University, Japan. Electronic address: shinjiro@nagasaki-u.ac.jp.
authors: Shinjiro
authors: S
authors: Hamano
mesh_terms: D016022:Case-Control Studies; D005260:Female; D049668:History, 15th Century; D049669:History, 16th Century; D006801:Humans; D007898:Leishmaniasis, Visceral / Q000175:diagnosis*; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods*; D012335:RNA, Ribosomal / Q000145:classification / Q000235:genetics; D012680:Sensitivity and Specificity
chemical_list: D012335:RNA, Ribosomal
keywords: Buffy coat; Diagnosis; ITS1; ITS2; Leishmania donovani; Mini-exon; PCR; Small subunit-rRNA (SSUrRNA); Visceral leishmaniasis