Uncovering latent infections in kidneys: A novel molecular approach for differential Leptospira detection

Leptospirosis, a re-emerging zoonotic disease caused by <i>Leptospira</i> spp., poses significant global health and veterinary challenges. Long-term colonization of renal tubules by <i>Leptospira</i> in asymptomatic hosts highlights the need for sensitive detection methods. This study evaluates the chronic or latent <i>Leptospira</i> infections in kidneys using a novel molecular approach to examine individual immune responses differences. Digital PCR strategies employing newly developed primer-probe sets targeting the flagellar <i>fliG</i> gene were used to assess the presence of trace <i>Leptospira</i> in infected murine kidneys and urine samples from laboratory-confirmed leptospirosis patients. RNA-based digital PCR detected leptospires in 58 % (targeting <i>lipl32</i>) and 83 % (targeting <i>fliG</i>) of infected kidneys, demonstrating that the digital PCR strategy targeting the <i>fliG</i> gene offers superior sensitivity. Notably, the newly developed <i>fliG</i>-targeting assay detected as low as 20 fg of <i>Leptospira</i> DNA, offering ten-fold greater sensitivity than traditional qPCR for trace detection. This allows for differential detection of <i>Leptospira</i> species and facilitates monitoring of extremely low bacterial loads with greater sensitivity than conventional methods. We also observed regenerating renal tubules with mitosis and elevated cytokine expression in kidneys with transcriptionally active <i>Leptospira</i> during chronic infection. This approach aids in identifying latent infections and offers insights into individual variations. Our research provides a powerful molecular tool for epidemiological studies and public health surveillance, contributing valuable insights into the prevalence and transmission dynamics of this pervasive zoonotic disease.