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.
Table 1.
RPA-LF detection of Leishmania Viannia species isolated from different countries in Latin America.
Table 2.
Agreement between RPA-LF and PCR to amplify Leishmania Viannia DNA purified from lesions of cutaneous leishmaniasis patients from Peru.
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.