Evaluation of urine for Leishmania infantum DNA detection by real-time quantitative PCR.

The availability of some sorts of biological samples which require noninvasive collection methods has led to an even greater interest in applying molecular biology on visceral leishmaniasis (VL) diagnosis, since these samples increase the safety and comfort of both patients and health professionals. In this context, this work aimed to evaluate the suitability of the urine as a specimen for Leishmania infantum kinetoplast DNA detection by real-time quantitative PCR (qPCR). Subsequent to the reproducibility analysis, the detection limit of the qPCR assay was set at 5fg (~0.025 parasites) per µL of urine. From the comparative analysis performed with a set of diagnostic criteria (serological and molecular reference tests), concordance value of 96.08% was obtained (VL-suspected and HIV/AIDS patients, n=51) (P>0.05). Kappa coefficient (95% CI) indicated a good agreement between the test and the set of diagnostic criteria (k=0.778±0.151). The detection of Leishmania DNA in urine by qPCR was possible in untreated individuals, and in those with or without suggestive renal impairment. Fast depletion of the parasite's DNA in urine after treatment (from one dose of meglumine antimoniate) was suggested by negative qPCR results, thus indicating it as a potential alternative specimen to follow up the efficacy of therapeutic approaches. Even when evaluated in a clinically heterogeneous set of patients, the urine showed good prospect as sample for VL diagnosis by qPCR, also indicating a good negative predictive value for untreated suspected patients.

Quantitative real time PCR assays for the detection of Leishmania (Viannia) braziliensis in animals and humans.

American cutaneous leishmaniasis (ACL) caused by Leishmania (Viannia) braziliensis is a neglected disease of humans in the New World that may also cause irreversible skin and eventually mucocutaneous lesions. This parasite can also infect dogs and represents a diagnostic challenge for veterinarians. Methods currently available for the diagnosis of ACL have a low sensitivity and may be time-consuming, representing a limit for treatment expedition of ACL. Quantitative real time PCR assays (qPCR) for the detection of L. (V.) braziliensis in canine blood samples were developed herein, and the detection limit and specificity of different molecular targets (kDNA and rDNA) evaluated. Of the protocols assessed, two qPCR assays, one targeting the kDNA and other the SSU rDNA of L. (V.) braziliensis, performed better, with detection limits of 100 fg and 10 pg, respectively. These assays were also used to test skin samples from humans with suspected ACL. The results indicate that the qPCR protocols developed represent an advance for the diagnosis of ACL in dogs and humans from this region, and provide a rapid and non-invasive diagnosis of the infection by L. (V.) braziliensis. Considering the quantitative nature of the assays, they will also be useful for monitoring treatment efficacy and preventing relapses in human patients in Brazil, although further studies are needed to critically evaluate the specificity of the qPCRs for their capacity to distinguish different Leishmania species and subspecies (represented by zymodemes) in other countries. Finally, molecular assays established may represent new tools for future basic and applied research focused on species identification, host-parasite associations, and infection dynamics in host and vector populations.