Evaluation of Novel Diagnostic Tools for Direct Detection of Leishmania donovani Markers in Clinical Samples to Sustain Visceral Leishmaniasis Elimination in Bangladesh

Background: With reduced prevalence of visceral leishmaniasis (VL) in the Indian subcontinent (ISC), direct and field deployable diagnostic tests are needed to implement an effective diagnostic and surveillance algorithm for post-elimination VL control. In this regard, here we investigated the diagnostic efficacies of a loop-mediated isothermal amplification (LAMP) assay (Loopamp™ Leishmania Detection Kit, Eiken Chemical CO., Ltd, Japan), a real-time quantitative PCR assay (qPCR) and the Leishmania antigen ELISA (CLIN-TECH, UK) with different sampling techniques. Method: VL and healthy controls (80 each) were enrolled in the study. Peripheral blood and dried blood spots (DBS) were collected from all the participants at the time of diagnosis. DNA was extracted from whole blood (WB) and DBS via silica columns (QIAGEN) and boil & spin (B&S) methods and tested with qPCR and LAMP. Urine was collected at the time of diagnosis and 30 days and 180 days after treatment from VL patients and was directly subjected to the Leishmania antigen ELISA. Findings: The sensitivities of the LAMP-WB(B&S) and LAMP-WB(QIA) were 96.2% and 95% respectively. The sensitivity of LAMP-DBS(QIA) was 85%. The sensitivities of the qPCR-WB(QIA) and qPCR-DBS(QIA) were 93.8% and 72.5% respectively. The specificity of all molecular assays was 100%. The sensitivity and specificity of the Leishmania antigen ELISA were 97.5% and 91.95% respectively. The Leishmania antigen ELISA depicted clinical cure at day 180 in the followed-up cases. Interpretation: Efficacy and sustainability identify the LAMP-WB(B&S) and the Leishmania antigen ELISA as minimally invasive VL diagnostic tools. Funding Statement: Federal Ministry of Education and Research, Germany. Declaration of Interests: AP is an employee of the Foundation for Innovative New Diagnostics (FIND). FIND contributed to the development of the Loopamp Leishmania Detection Kit. However, FIND does not have any financial interest in the product. The other authors declare that they have no conflict of interest. Ethics Approval Statement: The study was conducted in accordance with the Declaration of Helsinki and approved by the International Centre for Diarrheal Disease Research, Bangladesh (icddr,b) ethical review committee. Written informed consent was obtained from each adult participant or guardian of any participant aged less than 18 years.