Development of a Highly Sensitive Nested PCR and Its Application for the Diagnosis of Cutaneous Leishmaniasis in Sri Lanka

Abstract

The recent surge in cutaneous leishmaniasis (CL) in Sri Lanka has rendered clinical diagnosis difficult; thus, laboratory confirmation is indispensable. A modified (two novel inner primers to detect CL caused by Leishmania donovani) nested Internal Transcribed Spacer-1 (ITS1) PCR-Restriction Fragment Length Polymorphism (RFLP) method was developed and tested. The sensitivity of the modified nested PCR was tested using serial dilutions (103 to 10−2) of the DNA extract of a cultured L. donovani DD8 strain. Patients (n = 194) from Southern Sri Lanka were examined clinically, microscopically (Slit Skin Smear-SSS) and using the modified nested PCR. The modified nested PCR detected 2.55 fg of parasite DNA compared to ITS1 PCR (25 fg) and detected more cases than SSS (94.3% vs. 77.3%; p < 0.01). The RFLP pattern was L. donovani in all cases. The modified nested PCR performed well in clinically doubtful lesions (95% by PCR vs. 60% by SSS; p < 0.01), ulcerated nodules (91% vs. 71.8%; p < 0.01) and plaques (100% vs. 66.7%; p < 0.01). SSS demonstrated sensitivity (80.9%), specificity (81.8%), PPV (98.7%) and NPV (20.5%) against modified PCR. Low parasite loads and atypical lesions can be diagnosed by the proposed method with higher accuracy.