Visceral Leishmaniasis (VL), or Kala-azar is a vector-borne neglected tropical disease caused by parasites, Leishmania spp resulting in enlargement of spleen, liver and low blood cell counts in affected individuals with fatality rate of 95% if untreated. Diagnosis of VL through the detection of its causative agent is traditionally based on immunochromatographic tests, microscopy of bone marrow, spleen aspirates, liver or lymph node. While the first process has low specificity, the later one carries the risk of fatal hemorrhage while sampling the specimen of interest.
Over the last decade, multiple Polymerase Chain Reaction (PCR) based diagnosis has been developed using invasive specimens. Since the presence of Leishmania bodies in kidney is well evidenced, we attempted to detect the causative agents using urine samples, and applied RT-PCR conditions to make the diagnosis rapid and quantitative. Diagnosis of VL was carried out using urine samples collected from clinically diagnosed VL patients of Bangladesh in Real Time PCR. Test results were validated by comparing blood samples from the same set of patients. Sensitivity and specificity of this diagnosis was analyzed using retrospective bone marrow samples, collected earlier from confirmed VL patients. The method showed 100% sensitivity in detecting L. donovani in urine and corresponding blood and retrospective bone marrow samples, as well as 100% specificity in control groups.
Figure: Ct values in different specimens, as indicated, of Kala-azar patients in RT-PCR analyses, each point indicates data collected from individual patient.
The developed Molecular Diagnostic is a patient-friendly, non-invasive approach for VL detection with absolute precision and perfection, so that treatment regimen if administered as soon as the detection is confirmed could be effective, thereby progressing one step closer towards eliminating this neglected tropical disease by 2030 – a goal set by WHO for global public health. Due to its commendable sensitivity and specificity for urine samples, the method can replace blood or more invasive specimens: bone marrow or spleen. This method can also be used in assessing the response to therapy, monitoring parasite kinetics, infection dynamics and epidemiological survey.
The method was developed in the Fermentation and Enzyme Biotechnology Lab, Department of Microbiology, University of Dhaka by an MS student Mr. Samiur Rahim under the mentorship of Professor Muhammad Manjurul Karim in collaboration with Prof. Md. Robed Amin and Dr Md. Mohiuddin Sharif of Dhaka Medical College Hospital, and Professor Mohammad Tariqur Rahman of University of Malaya, Malaysia. Their work was published in PLoS Global Public Health, linked below. The team hopes the impact of the research in eradicating VL in Bangladesh and abroad would be significant.