A FRET-based real-time PCR assay to identify the main causal agents of New World tegumentary leishmaniasis

Abstract

In South America, various species of Leishmania are endemic and cause New World tegumentary leishmaniasis (NWTL). The correct identification of these species is critical for adequate clinical management and surveillance activities. We developed a real-time polymerase chain reaction (PCR) assay and evaluated its diagnostic performance using 64 archived parasite isolates and 192 prospectively identified samples collected from individuals with suspected leishmaniasis enrolled at two reference clinics in Lima, Peru. The real-time PCR assay was able to detect a single parasite and provided unambiguous melting peaks for five Leishmania species of the Viannia subgenus that are highly prevalent in South America: L. (V.) braziliensis, L. (V.) panamensis, L. (V.) guyanensis, L. (V.) peruviana and L. (V.) lainsoni. Using kinetoplastid DNA-based PCR as a gold standard, the real-time PCR had sensitivity and specificity values of 92% and 77%, respectively, which were significantly higher than those of conventional tests such as microscopy, culture and the leishmanin skin test (LST). In addition, the real-time PCR identified 147 different clinical samples at the species level, providing an overall agreement of 100% when compared to multilocus sequence typing (MLST) data performed on a subset of these samples. Furthermore, the real-time PCR was three times faster and five times less expensive when compared to PCR - MLST for species identification from clinical specimens. In summary, this new assay represents a cost-effective and reliable alternative for the identification of the main species causing NWTL in South America.

Figure 1. Melting curve analysis for the MPI-based real-time PCR assay on Leishmania reference strains.

Five nanograms of DNA (references) or 5 ul of nested-PCR products (samples) were used as template to run the assay. The species designation is given based on the Tm calculated for each melting peak (point in the x axis for the peak of the curve). FRET probes on L. (L.) amazonensis (ama) or L. (L.) mexicana (mex) DNA are melted at lower temperature (53°C) while on L. (V.) braziliensis (bra) DNA are melted at the highest temperature (74°C). This assay could not differentiate between L. (V.) guyanensis (guy) and L. (V.) panamensis (pan) due to their overlapping Tm (72°C). All assessed Leishmania species yielded a melting curve. A straight dotted line corresponds to the negative controls (neg), DNA free or parasite-free human DNA. inf = L. infantum; lain = L. (V.) lainsoni; per = L. (V.) peruviana.

Figure 2. Melting curve analysis for the 6PGD-based real-time PCR assay on Leishmania reference strains.

Five nanograms of DNA (references) or 5 ul of nested-PCR products (samples) were used as template to run the assay. The species designation is given based on the Tm calculated for each melting peak (point in the x axis for the peak of the curve). FRET probes on L. (V.) panamensis (pan) DNA are melted at the lowest temperature (56°C) while on L. (V.) braziliensis (bra) or L. (V.) peruviana (per) DNA are melted at the highest temperature (68°C). Due to their overlapping Tm, this assay could not differentiate these two species. L. (L.) amazonensis, L. (L.) mexicana and L. infantum did not yield a melting curve. A straight dotted line corresponds to the negative controls (neg), DNA free or parasite-free human DNA. guy = L. (V.) guyanensis; lain = L. (V.) lainsoni.