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. 2014 Jun;90(6):1082-6.
doi: 10.4269/ajtmh.13-0329. Epub 2014 Apr 21.

A new molecular surveillance system for leishmaniasis

Kishor Pandey  1 , Basu Dev Pandey  1 , Arun Kumar Mallik  1 , Jyoti Acharya  1 , Kentaro Kato  1 , Osamu Kaneko  1 , Pedro Eduardo Ferreira  2
Affiliations

A new molecular surveillance system for leishmaniasis

Kishor Pandey et al. Am J Trop Med Hyg. 2014 Jun.

Abstract

Presently, global efforts are being made to control and eradicate the deadliest tropical diseases through the improvement of adequate interventions. A critical point for programs to succeed is the prompt and accurate diagnosis in endemic regions. Rapid diagnostic tests (RDTs) are being massively deployed and used to improve diagnosis in tropical countries. In the present report, we evaluated the hypothesis of, after use for diagnosis, the reuse of the Leishmania RDT kit as a DNA source, which can be used downstream as a molecular surveillance and/or quality control tool. As a proof of principle, a polymerase chain reaction-based method was used to detect Leishmania spp. minicircle kinetoplast DNA from leishmaniasis RDT kits. Our results show that Leishmania spp. DNA can be extracted from used RDTs and may constitute an important, reliable, and affordable tool to assist in future leishmaniasis molecular surveillance methods.

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Figures

Figure 1.
Figure 1.
Sensitivity of the PCR and extraction methods to detect Leishmania parasite in RDT or FP. In A (Hs = Homo sapiens), a human-specific cytb PCR product (330-bp band) is shown to detect human DNA in eight patients (lanes 1–8), L. major(Lm) limiting diluted samples (lanes 9–12), mouse blood (lanes 13 and 14), and distilled water (D2W, lane 15). In B, detection of L. major kDNA (620-bp band) in limiting dilution solutions of L. major plotted in the RDT kits or FP with a parasite density (parasites per microliter) of 6.2 × 103 (lane 1, RDT; lane 2, FP), 2 × 103 (lane 3, RDT; lane 4, FP), 1.8 × 102 (lane 5, RDT; lane 6, FP), 26 (lane 7, RDT; lane 8, FP), 0 (lane 9, RDT; lane 10, FP), and 0 (lane 11, RDT; lane 12, FP). Lanes 13 and 14 correspond for RDT- and FP-negative controls, respectively. Note that 0 parasites/μL means that parasites were not detected under Giemsa staining microscopy. The M lanes stand for 100-bp molecular marker.
Figure 2.
Figure 2.
kDNA diversity of Leishmania parasites in Nepal. Patient's origin and unrooted dendrogram of Leishmania sequences obtained in Nepal based on the minicircle kDNA nucleotide sequence are shown. Numbers on branches indicate bootstrap values based on 1,000 pseudoreplicates, and only values above 70 are shown.
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