An Innovative Field-Applicable Molecular Test to Diagnose Cutaneous Leishmania Viannia spp. Infections

Abstract

Cutaneous and mucosal leishmaniasis is widely distributed in Central and South America. Leishmania of the Viannia subgenus are the most frequent species infecting humans. L. (V.) braziliensis, L. (V.) panamensis are also responsible for metastatic mucosal leishmaniasis. Conventional or real time PCR is a more sensitive diagnostic test than microscopy, but the cost and requirement for infrastructure and trained personnel makes it impractical in most endemic regions. Primary health systems need a sensitive and specific point of care (POC) diagnostic tool. We developed a novel POC molecular diagnostic test for cutaneous leishmaniasis caused by Leishmania (Viannia) spp. Parasite DNA was amplified using isothermal Recombinase Polymerase Amplification (RPA) with primers and probes that targeted the kinetoplast DNA. The amplification product was detected by naked eye with a lateral flow (LF) immunochromatographic strip. The RPA-LF had an analytical sensitivity equivalent to 0.1 parasites per reaction. The test amplified the principal L. Viannia species from multiple countries: L. (V.) braziliensis (n = 33), L. (V.) guyanensis (n = 17), L. (V.) panamensis (n = 9). The less common L. (V.) lainsoni, L. (V.) shawi, and L. (V.) naiffi were also amplified. No amplification was observed in parasites of the L. (Leishmania) subgenus. In a small number of clinical samples (n = 13) we found 100% agreement between PCR and RPA-LF. The high analytical sensitivity and clinical validation indicate the test could improve the efficiency of diagnosis, especially in chronic lesions with submicroscopic parasite burdens. Field implementation of the RPA-LF test could contribute to management and control of cutaneous and mucosal leishmaniasis.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1. Sensitivity of RPA-LF to detect L. viannia spp. compared with real-time PCR (the current gold standard).

Ten-fold serial dilutions of parasite DNA, extracted with Qiagen DNeasy blood and tissue kit, were amplified by qPCR (SYBRgreen) (A) or RPA-LF (B). W = water. The control band is the upper band, while the test band is the lower band.

Fig 2. Specificity of RPA-LF to amplify species of the Viannia subgenus.

A) The most relevant L. Viannia species (L. braziliensis, L. guyanensis, L. panamensis) produced stronger bands in the lateral flow strip than other less common species of this subgenus. B) Species of the Leishmania subgenus, Trypanosoma cruzi, and human DNA were not amplified by the RPA-LF test. NTC = no template control.

Fig 3. RPA-LF amplification of clinical samples using a simplified DNA extraction method.

Samples from patients suspected of having cutaneous leishmaniasis were obtained by pressing Whatman FTA filter paper (two 6 mm diameter discs) over the dermal lesions. Two, 3 mm diameter papers were cut from the original samples using a punch, washed thrice with FTA washing reagent and twice with TE buffer pH 8. A 2.5 μL aliquot was amplified by RPA and subsequently read using a lateral flow strip. Patients infected with Leishmania Viannia spp. (L. lainsoni, L. braziliensis) were readily detected. The test did not amplify a strain originally labeled as Leishmania sp. in NAMRU-6, Peru. Presumably, it did not belong to the Viannia subgenus as confirmed by real-time PCR at UTMB. There was agreement between NAMRU-6 and UTMB labs with regard to the negative clinical samples. L. lainsoni was used as positive control; the negative control was run without template.