Analytical and clinical performance of in-house and commercial real-time PCR assays for diagnosing L.infantum visceral leishmaniasis: a study from a hub center in northern Italy

Molecular methods are increasingly used to diagnose visceral leishmaniasis (VL), a life-threatening disease caused by Leishmania infantum in the Mediterranean basin. In this study, we compared the analytical and clinical performances of three real-time polymerase chain reaction (PCR) assays used for VL diagnosis at the University Hospital of Bologna, Northern Italy. The first test, which is commercially available, targets the small subunit of the 18S ribosomal RNA gene of Leishmania (rDNA). The second and third methods, developed in-house, target the rDNA and the kinetoplast minicircle conserved region (kDNA). The three PCR assays were performed on standard samples prepared using a L. infantum reference strain, as well as on 90 peripheral blood samples from patients with clinical suspicion of VL. Among these, 33 were confirmed as VL cases. The in-house kDNA PCR exhibited the highest observed analytical and clinical sensitivity. Conversely, kDNA PCR showed lower specificity (95%) on clinical samples when compared to rDNA amplification assays (100%). The three methods exhibited excellent concordance in VL identification (Cohen's Kappa >= 0.9). The higher sensitivity of the kDNA target compared to the rDNA target was confirmed by analyzing the cycle threshold values obtained from parasitic DNA amplification using the three real-time PCR assays in VL-positive samples. In summary, all three molecular assays exhibited good analytical and clinical performance, with the kDNA-based PCR showing the highest sensitivity. These findings support kDNA as the most suitable target for detecting low levels of Leishmania DNA in peripheral blood samples from VL patients.IMPORTANCEVisceral 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.