Comparative study of a reference PCR assay targeting kinetoplast DNA and a commercial kit targeting ribosomal DNA for the molecular diagnosis of visceral leishmaniasis.

Visceral leishmaniasis (VL) is a severe parasitic disease that requires accurate and timely diagnosis to prevent fatal outcomes. Molecular methods, particularly quantitative PCR (qPCR), have become central to VL diagnosis because of their high sensitivity and specificity. However, assay performance depends on several factors, including the choice of DNA target. We compared the analytical and clinical performance of an in-house reference qPCR targeting kinetoplast DNA with the Viasure Leishmania Real-Time PCR Detection Kit targeting 18S ribosomal DNA. Analytical evaluation included PCR efficiency, linearity ranges, and detection limits. Clinical performance was assessed using 30 blood samples representative of routine diagnostic practice. Both assays showed high efficiency and broad linearity, but the kinetoplast DNA assay achieved a lower limit of detection. Clinical comparison performed on a cohort representative of the routine activity revealed substantial agreement, although the Viasure method produced several false-negative results and non-reproducible positives (amplification in only one of two replicate wells), mainly in samples with very low parasite levels. This likely reflected differences in target copy number, as kinetoplast DNA is present in thousands of copies per parasite, whereas ribosomal DNA occurs in only a few dozen copies. High analytical sensitivity is essential for diagnosis and follow-up because minimal parasitemia is common, and missed diagnoses can have severe consequences. Our findings indicate that assays targeting kinetoplast DNA should be preferred over ribosomal DNA-based alternatives for blood-based diagnosis and monitoring of visceral leishmaniasis.IMPORTANCEAccurate diagnosis of visceral leishmaniasis (VL) is essential to prevent delayed treatment and fatal outcomes. Molecular assays have become central to diagnosis, but their performance varies with the genetic target used. This study shows that assays targeting kinetoplast DNA provide superior sensitivity compared to ribosomal DNA-based methods, particularly in cases with very low parasite burdens-a common scenario in clinical practice. The difference likely reflects the much higher copy number of kinetoplast DNA, which enhances detection when parasite levels approach the limit of detection. These findings underscore the need for assay developers to prioritize high-copy-number targets to ensure reliable diagnosis and effective monitoring of treatment response, reducing the risk of relapse and improving patient outcomes.