Inclusion of Quality Controls on Leishmaniases Molecular Tests to Increase Diagnostic Accuracy in Research and Reference Laboratories

Abstract

Early detection of leishmaniases and prompt institution of treatment are paramount for individuals and communities affected by these diseases. To overcome the remaining limitations inherent to molecular methods currently used and to ensure the accuracy of results in leishmaniases diagnosis, two triplex polymerase chain reaction (PCR) assays with quality controls for the reactions were developed. Validity indicators were assessed in 186 dog blood samples from endemic areas in Brazil. The level of agreement between the new tools and their singleplex protocols was assessed by kappa analysis. The triplex PCR for visceral leishmaniasis showed sensitivity (S) = 78.68 %, specificity (E) = 85.29 %, and efficiency (e) = 81.05 %. The cutaneous leishmaniasis protocol showed S = 97.29 %, E = 79.16 %, and e = 90.16 %. Both protocols showed good agreement with gold standards. These new tools enable, in a single reaction, the diagnosis of the diseases and the evaluation of the sample quality and DNA extraction process, thus reducing the cost of reagents and avoiding the eventual need for collecting a second sample.

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

“…The novel assay includes an exogenous control, which controls for extraction and PCR performance, and an external positive control, controlling for PCR performance. The inclusion of quality controls, both internal and external, was highlighted as important in Leishmania detection assays [13]. The turn-around time is less than 2.5 hours from sample to result, and the system has a small laboratory footprint (the area required in the laboratory for instrumentation) of 75cm by 75cm.…”

Standardization and evaluation of a duplex real-time quantitative PCR for the detection of Leishmania infantum DNA: a sample quality control approach

“…As evidenced in Figure 2, the amplification of the parasite DNA is favored, and this is associated with the design of the probes, as well as with the chosen targets. The large amount of the host's genetic material that is simultaneously purified in the extraction step could impair the detection of the etiological agent DNA, mainly because of competition between the primer sets for the PCR reagents 9,25 . In larger parasite DNA concentrations, there is no amplification of the G3PD gene (as from the concentration of 2x10 3 fg per µL of sample), but this does not affect the validation of the results, since the intention of the reaction is to favor the target DNA appearance.…”

“…Gonçalves et al 8 used the same sample quality control (G3PD gene) in multiplex reactions to detect VL through conventional PCR (cPCR) reactions, and demonstrated that it was possible to detect potential false negative results in 33% of the samples tested (no amplification of the G3PD gene). Gonçalves-de-Albuquerque et al 25 standardized multiplex cPCR reactions for the diagnosis of VL in dogs, also using the G3PD gene as a quality control, and more than 15% of the samples were considered unsuitable for diagnostic definition because of no quality control amplification in the negative samples. In this study, all samples with negative results in the duplex qPCR protocol presented amplification of the quality control.…”