Diagnosis Of Indian Visceral Leishmaniasis By Nucleic Acid Detection Using Pcr

Abstract: Background: PCR based diagnosis for visceral leishmaniasis is far from being applied in field, despite numerous published primers. Although the technique is simple, selection of appropriate primer is crucial for the success of the investigative technique. The present study was planned to design a Leishmania specific diagnostic assay and evaluate its sensitivity and specificity on a sample size, which according to us is the largest ever screened. Methods: Two pairs of Leishmania specific primers were designed using the 18S rRNA gene and sensitivity was evaluated on 500 parasitologically confirmed VL patients and 25 PKDL patients. Specificity was calculated on 250 healthy non endemic controls, 250 healthy endemic controls and 250 non leishmanial diseases like malaria. To check whether amplified products correspond well to Leishmania, amplicons were sequenced. Results: Of the 500 KA samples and 25 PKDL samples 439 (87.8%) and 19(76%) were respectively PCR positive with BHUL18S primer. While using BHUSSU primer PCR positivity in same no. of KA and PKDL samples are 436(87.2%) and 19(76%) respectively. The test provided successful diagnosis of VL of 87.2% sensitivity using patient"s whole-blood with BHUL18S primer and 86.7% sensitivity with BHUSSU primer. None of 250 non endemic controls was positive and thus, specificity in this group was 100% using both primers. The data from different diseases group also showed 100% specificity of both primers as none of the 250 nonleishmanial disease samples were PCR positive. In endemic controls, using BHUL18S primer specificity was 84.0% and 90.4% specificity using BHUSSU primer set. Overall specificity of BHUL18S primer was 94.6% while BHUSSU primer was 96.8%. Conclusion: With Indian strains and isolates of L. donovani, the PCR assay developed by us is sensitive enough to detect 18S rRNA gene in an amount equivalent to a single parasite or less in a one million human cell environment. High sensitivity of PCR in relatively non-invasive peripheral blood opens up the possibility of its deployment in field for the routine diagnosis of VL, and it is possible to eliminate the risky method of splenic aspiration. References: 1. Sundar S, Rai M. Laboratory diagnosis of visceral leishmaniasis. Clin Diagn Lab Immunol 2002;9:951-8 2. 2.  Zijlstra EE, Nur Y, Desjeux P, Khalil EA, El-Hassan AM and Groen J. Diagnosing visceral leishmaniasis with the recombinant K39 strip test: experience from the Sudan. Trop Med Int Health 2001;6:108-13 3.  Minodier P, Piarroux R, Gambarelli F, Joblet C and Dumon H. Rapid identification of causative species in patients with Old World leishmaniasis. 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