Abstract
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 and evaluated their prospect to incorporate into post-elimination VL control strategies. Eighty clinically and rK39 rapid diagnostic test confirmed VL cases and 80 endemic healthy controls 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 Loopamp. Urine was collected from all participants at the time of diagnosis and was directly subjected to the Leishmania antigen ELISA. 41 patients were followed up and urine samples were collected at day 30 and day 180 after treatment and ELISA was performed. The sensitivities of the Loopamp-WB(B&S) and Loopamp-WB(QIA) were 96.2% (95% CI 89·43-99·22) and 95% (95% CI 87·69-98·62) respectively. The sensitivity of Loopamp-DBS(QIA) was 85% (95% CI 75·26- 92·00). The sensitivities of the qPCR-WB(QIA) and qPCR-DBS(QIA) were 93.8% (95% CI 86·01-97·94) and 72.5% (95% CI 61·38-81·90) respectively. The specificity of all molecular assays was 100%. The sensitivity and specificity of the Leishmania antigen ELISA were 97.5% (95% CI 91·47-99·70) and 91.95% (95% CI 84·12-96·70) respectively. The Leishmania antigen ELISA depicted clinical cure at day 180 in all the followed-up cases. Efficacy and sustainability identify the Loopamp-WB(B&S) and the Leishmania antigen ELISA as promising and minimally invasive VL diagnostic tools to support VL diagnostic and surveillance activities respectively in the post-elimination era.
Highlights
Visceral leishmaniasis (VL), known as kala-azar, is a vectorborne parasitic disease caused by protozoans of the Leishmania donovani complex, which are transmitted by phlebotomine sandflies (Chappuis et al, 2007)
We performed the Loopamp assay with whole blood and dried blood spots and evaluated the effect on the diagnostic efficacies of two different extraction methods, commercial silica-based spin-columns and an in-house boil &
For the quantitative polymerase chain reaction (qPCR) assay, the sensitivity of WB DNA extracted by QIAGEN extraction method was 93·8%, whereas Dried blood spots (DBS) DNA extracted by QIAGEN extraction method achieved a sensitivity of 72·5%
Summary
Visceral leishmaniasis (VL), known as kala-azar, is a vectorborne parasitic disease caused by protozoans of the Leishmania donovani complex, which are transmitted by phlebotomine sandflies (Chappuis et al, 2007). The estimated global VL incidence ranged between 50,000-90,000 cases per year (Bi et al, 2018), and three countries, Bangladesh, India, and Nepal, contributed to 60-80% of the reported incidences. The program aimed to attain less than 1 case per 10,000 people at the intervention unit level in three phases: achieving the target incidence in the attack phase, retaining the incidence for 3 consecutive years in the consolidation phase, and following validation by the World Health Organization (WHO), sustaining the target incidence in the maintenance phase (Le Rutte et al, 2018). The attainment of the target incidence led to a drastic reduction in the reported case numbers in the three countries, from 41,158 in 2005 to 3,105 cases that contributed to a 77% decrease in global VL incidence in 20191
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