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.

Laboratory diagnostics for human Leishmania infections: a polymerase chain reaction-focussed review of detection and identification methods

“…Two sequential PCRs are used: first, an outer set of primers is used to amplify the target gene (first round), then the amplicons of this PCR are re-amplified with a set of inner primers (second round) [ 173 ]. The disadvantages of this approach are that the use of two PCRs is more time consuming and requires more reagent, and since the amplicon tube is opened for the second PCR, it is an open system, which poses a contamination risk [ 174 ]. Encouragingly, however, 100% sensitivity and 100% specificity were reported for a recently developed Leishmania spp.-specific modified version of a nested PCR that was created to reduce carryover and cross-contamination [ 175 ].…”

A novel detection method based on MIRA-CRISPR/Cas13a-LFD targeting the repeated DNA sequence of Trichomonas vaginalis

“…It involves multiple amplification steps, increasing the risk of contamination and necessitating meticulous laboratory conditions. Additionally, the method typically requires skilled personnel and advanced laboratory infrastructure, limiting its applicability in resource-limited settings and at the point of care [ 33 ]. IAT, although slightly inferior in specificity, represents a significant advancement from nested PCR.…”

Diagnostic Tools for Cutaneous Leishmaniasis Caused by Leishmania donovani: A Narrative Review

“…That includes L. donovani , L. infantum , L. major , L. tropica and L. aethiopica . Hence, PCR amplification of the ITS region followed by restriction digestion by Hae III can be considered as a suitable method for identification and differentiation between all medically important Leishmania parasite species [ 75 , 76 , 77 ] ( Table 4 ).…”

“…A modified method of this nested PCR was developed and tested on samples from 194 CL patients, and it proved to be successful with patients with atypical lesions and with low parasite loads ( Table 4 ) [ 75 ]. In India, a case was identified through the examination of both blood and skin samples using polymerase chain reaction (PCR).…”