In silico selection and analytical evaluation of oligonucleotides for specific molecular diagnosis for Leishmania (Leishmania) infantum by real-time PCR with hydrolysis probe

Abstract

A relevant problem for the control of Visceral Leishmaniasis (VL) is the absence of laboratory tests with good accuracy to diagnose the infection, particularly in dogs. Dogs are the main reservoirs and sources of parasite infection in urban areas in Brazil, so the control of VL in urban areas is based on actions taken regarding these hosts.To identify dogs infected with Leishmania infantum, the causative agent of VL in the country, serological methods are the most employed due to their low cost and ease of execution. However, these tests have insufficient diagnostic sensitivity and do not necessarily allow for the detection of animals that are transmitting the agent. Thus, tests based on the direct detection of the agent have been widely indicated, and among these tests, molecular tests such as PCR and its variations stand out.Real-time PCR (qPCR) has become very popular as it is a fast method and less susceptible to cross-contamination than conventional PCR because it eliminates the need to open reaction tubes for post-PCR analysis. qPCR relies on the capture and analysis of fluorescent signals produced during amplification. Fluorescence can be generated using intercalating fluorescent dyes or fluorescent probes. The use of fluorescent probes is more costly than intercalating dyes, but it allows for multiplex assays and, because it depends on the successful hybridization of a probe, tends to produce results with higher specificity. Therefore, this approach is less prone to generating false positives than the method based on the use of intercalating dye.An important point regarding diagnostic surveys for the detection of L. infantum infections concerns cross-reactions with infections by other species of the genus, since in most endemic areas for VL in Brazil, species of Leishmania associated with cutaneous leishmaniasis can also occur. Therefore, it is desirable that methods for the diagnosis of leishmaniases are capable of correctly identifying the species of the parasite involved in the infection, either for correct decision-making in therapies or for epidemiological knowledge necessary for the implementation of control measures.Although there is ample scientific documentation on molecular tests for the differential diagnosis between species of the genus Leishmania, the vast majority of them refer to methods based on conventional PCR associated with enzymatic restriction or nucleic acid sequencing. Few studies have been developed with qPCR capable of differentiating L. infantum from other species, with most based on detection systems using intercalating dyes and very few with fluorescent probe systems. Therefore, the project presented is dedicated to identifying molecular markers for qPCR using a fluorescent probe that has the ability to distinguish L. infantum from other species of the genus through research in scientific literature and the use of analysis software (in silico evaluation). Candidate sets of primers and probes identified in the in silico analyses will have their analytical and diagnostic performance evaluated in samples of Leishmania spp. culture and in samples from naturally infected dogs, respectively.