Analysis of the molecular diagnosis by polymerase chain reaction (pcr) of asymptomatic canine infection by Leishmania infantum

Abstract

Visceral leishmaniasis is a zoonotic disease of high relevance to public health, with widespread geographical distribution and increasing impact in urban and periurban areas. The domestic dog plays a central role in maintaining the transmission cycle of Leishmania infantum, particularly in endemic contexts, where asymptomatic infections are frequent and epidemiologically relevant. In this scenario, accurate diagnosis of these animals represents one of the main challenges for disease surveillance and control strategies. Serological methods, widely used in official programs, present important limitations related to sensitivity in early phases of infection, variability of immune response, and the occurrence of cross-reactions, especially in asymptomatic dogs. Given these limitations, molecular techniques based on polymerase chain reaction (PCR) have been extensively investigated as more robust diagnostic tools for canine visceral leishmaniasis. The present work aimed to consolidate evidence on the true diagnostic accuracy of PCR methods for identifying asymptomatic dogs with visceral leishmaniasis through a systematic review and meta-analysis, according to established methodological guidelines. Specifically, this study sought to: (1) establish a theoretical framework addressing epidemiological aspects of visceral leishmaniasis, the importance of dogs as reservoirs, and the main limitations of current diagnostic methods; (2) conduct a meta-analysis of the diagnostic accuracy of PCR techniques in asymptomatic dogs; and (3) design and validate in silico primers specific for L. infantum targeting the HSP60, CPB, and G3PD genes. For the systematic review and meta-analysis, 13 studies were included. The analysis showed that qPCR presented sensitivity of 90.1% and specificity of 65.6%, while conventional PCR demonstrated specificity of 92.5% with sensitivity of 75.3%. Overall diagnostic accuracy was 77.3% (95% CI: 65.1- 86.2) with substantial heterogeneity. Sample type and primers used did not significantly influence performance. Risk of bias analysis revealed relevant methodological weaknesses among included studies. Regarding primer design, genomic sequences were selected from TriTrypDB. Thermodynamic analyses by OligoAnalyzer and in silico specificity assessments by Primer-BLAST confirmed preferential alignments with L. infantum and multiple polymorphisms in non-target species, indicating low probability of cross-amplification. The results underscore that PCR techniques constitute relevant tools for diagnosing asymptomatic canine infection by L. infantum. However, the incorporation of these techniques into control programs depends on methodological standardization, validation in different epidemiological scenarios, and integration of molecular, serological, and analytical approaches.