Sensitive diagnosis of cutaneous leishmaniasis by lesion swab sampling coupled to qPCR

Abstract

Variation in clinical accuracy of molecular diagnostic methods for cutaneous leishmaniasis (CL) is commonly observed depending on the sample source, the method of DNA recovery and the molecular test. Few attempts have been made to compare these variables. Two swab and aspirate samples from lesions of patients with suspected CL (n = 105) were evaluated alongside standard diagnosis by microscopic detection of amastigotes or culture of parasites from lesion material. Three DNA extraction methods were compared: Qiagen on swab and aspirate specimens, Isohelix on swabs and Boil/Spin of lesion aspirates. Recovery of Leishmania DNA was evaluated for each sample type by real-time polymerase chain reaction detection of parasitic 18S rDNA, and the diagnostic accuracy of the molecular method determined. Swab sampling combined with Qiagen DNA extraction was the most efficient recovery method for Leishmania DNA, and was the most sensitive (98%; 95% CI: 91-100%) and specific (84%; 95% CI: 64-95%) approach. Aspirated material was less sensitive at 80% (95% CI: 70-88%) and 61% (95% CI: 50-72%) when coupled to Qiagen or Boil-Spin DNA extraction, respectively. Swab sampling of lesions was painless, simple to perform and coupled with standardized DNA extraction enhances the feasibility of molecular diagnosis of CL.

Fig. 1

Flow of samples in evaluation. Two lesion smears and 4 aspirates were obtained as part of the reference diagnostic procedure (microscopy and culture). Two lesion swab samples and 2 aspirates were obtained for evaluation by qPCR.

Fig. 2

Specimen collection by swab sampling of a cutaneous ulcer of a patient with suspected CL.

Fig. 3

Quantification of parasite loads from lesion swabs and aspirate samples. (A) True positive samples and (B) false positive samples. Parasite number was calculated from a defined Ct threshold of 125 CTU and extrapolated to a parasite DNA standard curve. Data represent number of parasites per reaction (1.25 μL DNA from a total of 50 μL of DNA extraction material) and expressed as median values. Whiskers in box plots show minimum and maximum values. Statistical significance was estimated using the Kruskal–Wallis 1-way ANOVA followed by the Dunn’s test for multiple comparisons. ***: P<0.01. Qiagen and Isohelix: DNA extractions with Qiagen and Isohelix commercial kits, respectively. B/S: Boil-Spin.