| Summary: | 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 <i>Leishmania donovani</i>) 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 (10<sup>3</sup> to 10<sup>−2</sup>) of the DNA extract of a cultured <i>L. donovani</i> DD8 strain. Patients (<i>n</i> = 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%; <i>p</i> < 0.01). The RFLP pattern was <i>L. donovani</i> in all cases. The modified nested PCR performed well in clinically doubtful lesions (95% by PCR vs. 60% by SSS; <i>p</i> < 0.01), ulcerated nodules (91% vs. 71.8%; <i>p</i> < 0.01) and plaques (100% vs. 66.7%; <i>p</i> < 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.
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