Novel highly sensitive and specific multiplex real-time pcr assay for detection of Leishmania donovani and human immunodeficiency virus-1 co-infection.

The co-infection of Leishmania donovani the etiological agent of visceral leishmaniasis (VL) and Human Immunodeficiency Virus-1 (HIV-1) poses a significant public health challenge, especially in regions where these pathogens have geographical overlaps. Individuals with this coinfection often have severe morbidity and mortality. Diagnosis of VL-HIV coinfections can be challenging due to atypical clinical presentations and overlapping manifestations with other infections. Serological diagnosis of VL is often poor, due to low humoral response in HIV-infected individuals characterized by reduced antibody production making antibody tests difficult hence the need to develop a more sensitive and specific diagnosis assay for both pathogens. In this study, oligonucleotides were designed to target the long terminal repeat (LTR) and internal transcribed spacer 1 (ITS1) in the HIV-1and L. donovani genomes respectively. The human beta-actin gene was used as the internal control. The analytical sensitivity and specificity were tested using ATCC DNA standards and L. major culture DNA. Diagnostic performance was tested using conventional PCR characterized clinical samples. The limit of detection of the multiplex assay was 1 fg/μL and 100 fg/μL for HIV-1 and L. donovani targets respectively. No cross-reactivity was observed, and the assay diagnostic performance was comparable to the conventional PCR (kappa = 1). With the advantages of high efficiency and rapid and accurate diagnosis of multiplex real-time RT-PCR, the data obtained shows that this assay is very feasible for the diagnosis of VL-HIV-1 co-infection and would be crucial in proper treatment, and monitoring and would lead to reduced treatment failure and recurrent VL episodes.