Polymerase Chain Reaction-Based Diagnosis of Leishmaniasis Targeting Kinetoplast Minicircle DNA in Infants with Hemophagocytic Lymphohistiocytosis.

Abstract

In this study, we aimed to assess the efficacy of polymerase chain reaction (PCR) targeting Leishmania kinetoplast minicircle DNA (kDNA) for diagnosing visceral leishmaniasis-associated hemophagocytic lymphohistiocytosis (VL-HLH). Three pediatric patients diagnosed with VL-HLH via the microscopic examination of bone marrow smears were selected to explore the diagnostic value of kDNA PCR in Leishmania species identification and parasite load quantification in three cases of visceral leishmaniasis. Total DNA was extracted from the bone marrow samples, and PCR testing targeting the Leishmania kDNA was conducted. The amplified PCR products were sequenced and analyzed by using National Center for Biotechnology Information Basic Local Alignment Search Tool for Leishmania species identification and homology cluster analysis. Furthermore, universal primers and probes were designed to quantify the kDNA load in patient samples through real-time quantitative PCR (qPCR) testing. Polymerase chain reaction testing targeting the kDNA gene yielded a specific 139 base pairs band in all samples. Real-time qPCR testing targeting the kDNA gene revealed significant amplification in all patients. The kDNA copy numbers for the three patients were 3,830/µL, 150/µL, and 77,168/µL, respectively. Homology analysis revealed 97.8% similarity between Patients 1 and 2, as well as 87.1% and 87.8% similarity between Patients 1 and 3 and Patients 2 and 3, respectively. All sequences were identified as belonging to the genus Leishmania, with Patients 1 and 2 clustering with Leishmania donovani sequences and Patient 3 clustering with Leishmania infantum. Polymerase chain reaction testing targeting the kDNA gene is a rapid, accurate, and sensitive method for detecting and quantifying Leishmania in VL-HLH patients. It also facilitates species identification through sequencing, demonstrating its potential as a valuable tool in clinical diagnostics.