Analytical and clinical performance of in-house and commercial real-time PCR assays for diagnosing

Molecular methods are increasingly used to diagnose visceral leishmaniasis (VL), a life-threatening disease caused by IMPORTANCE: Visceral leishmaniasis is a life-threatening disease, rendering early and accurate diagnosis essential for patient survival. However, highly sensitive diagnostic tools are lacking. In this study, we compared the analytical and clinical performances of three real-time PCR assays routinely used for diagnosis of visceral leishmaniasis in a referral center in Northern Italy. All three assays exhibited robust diagnostic performance, with the PCR targeting the conserved region of the kinetoplast minicircle DNA exhibiting higher sensitivity than the assays targeting the small subunit of the 18S ribosomal RNA gene. This enhanced sensitivity is crucial for detecting visceral leishmaniasis in patients with low concentration of parasitic DNA in peripheral blood, as misdiagnosis in these patients can lead to severe consequences. Our findings highlight the need for the development of commercial, automated assays targeting the kinetoplast minicircle DNA to enhance the accuracy of the diagnosis of this potentially lethal disease.