Analytical Performance of Four Polymerase Chain Reaction (PCR) and Real Time PCR (qPCR) Assays for the Detection of Six Leishmania Species DNA in Colombia

Abstract

Leishmaniasis comprises a spectrum of parasitic diseases caused by protozoans of the genus Leishmania. Molecular tools have been widely employed for the detection of Leishmania due to its high sensitivity and specificity. However, the analytical performance of molecular platforms as PCR and real time PCR (qPCR) including a wide variety of molecular markers has never been evaluated. Herein, the aim was to evaluate the analytical performance of 4 PCR-based assays (designed on four different targets) and applied on conventional and real-time PCR platforms. We evaluated the analytical performance of conventional PCR and real time PCR, determining exclusivity and inclusivity, Anticipated Reportable Range (ARR), limit of detection (LoD) and accuracy using primers directed to kDNA, HSP70, 18S and ITS-1 targets. We observed that the kDNA was the most sensitive but does not meet the criterion of exclusivity. The HSP70 presented a higher LoD in conventional PCR and qPCR in comparison with the other markers (1 × 101 and 1 × 10-1 equivalent parasites/mL respectively) and had a higher coefficient of variation in qPCR. No statistically significant differences were found between the days of the test with the four molecular markers. The present study revealed that the 18S marker presented the best performance in terms of analytical sensitivity and specificity for the qPCR in the species tested (species circulating in Colombia). Therefore, we recommend to explore the analytical and diagnostic performance in future studies using a broader number of species across America.

Semi-Quantitative, Duplexed qPCR Assay for the Detection of Leishmania spp. Using Bisulphite Conversion Technology

“…It achieves sensitivities and specificities of up to 100% [26,27]. The novel qPCR achieved a lower LOD than cPCR, an outcome seen in other Leishmania assays utilising various targets [14,28,29]. Our future studies will determine the clinical sensitivity of samples previously tested positive for visceral leishmaniasis, to complement the clinical data obtained here.…”

“…The assay is based on the gene coding for the small subunit rRNA, a highly conserved region of the ribosomal DNA, located on chromosome 27. This gene was used for the detection of Leishmania in other assays, due to its excellent sensitivity, attributed to the fact that it is a multicopy gene, which is transcribed into abundant rRNA found in the cytoplasm, where it is predicted to be present at 10 4 copies [7,[14][15][16].…”

Analytical Performance of a Loop-Mediated Isothermal Amplification Assay for Leishmania DNA Detection in Sandflies and Direct Smears of Patients with Cutaneous Leishmaniasis

“…These findings differ from our previous results that revealed better analytical performance for LAMP than PCR-based tests. 61 In conclusion, LAMP is a valuable diagnostic tool that offers advantages compared with qPCR and microscopy such as a LoD of 1 × 10 −2 parasites/mL, exclusivity for Leishmania amplification, lower cost, and ease to perform and interpret. One of the most important advantages is its ability to detect Leishmania DNA from direct smears and sandflies, which indicate its potential application in primary care centers and in the diagnostic and entomological surveillance of leishmaniasis in endemic countries.…”

Novel PCR primers for improved detection of Mycobacterium leprae and diagnosis of leprosy

“…Many studies have shown that PCR is an important methodology for leprosy’s diagnosis and cure control (Azevedo et al ; Soto et al ). Some authors have demonstrated that primers for the RLEP repeating sequence are successfully used to amplify M. leprae ’s DNA from leprosy patients at different stages of the disease, demonstrating that they had a higher positivity than primers from the 16SrRNA ribosomal region (León et al ).…”