Simplified molecular diagnosis of visceral leishmaniasis: Laboratory evaluation of miniature direct-on-blood PCR nucleic acid lateral flow immunoassay.

Background: Diagnosis of visceral leishmaniasis (VL) in resource-limited endemic regions is currently based on serological testing with rK39 immunochromatographic tests (ICTs). However, rK39 ICT frequently has suboptimal diagnostic accuracy. Furthermore, treatment monitoring and detection of VL relapses is reliant on insensitive and highly invasive tissue aspirate microscopy. Miniature direct-on-blood PCR nucleic acid lateral flow immunoassay (mini-dbPCR-NALFIA) is an innovative and user-friendly molecular tool which does not require DNA extraction and uses a lateral flow strip for result read-out. This assay could be an interesting candidate for more reliable VL diagnosis and safer test of cure at the point of care. Methodology/Principle findings: The performance of mini-dbPCR-NALFIA for diagnosis of VL in blood was assessed in a laboratory evaluation and compared with the accuracy of rK39 ICTs Kalazar Detect in Spain and IT LEISH in East Africa. Limit of detection of mini-dbPCR-NALFIA was 650 and 500 parasites per mL of blood for Leishmania donovani and Leishmania infantum, respectively. In 146 blood samples from VL-suspected patients from Spain, mini-dbPCR-NALFIA had a sensitivity of 95.8% and specificity 97.2%, while Kalazar Detect had a sensitivity of 71.2% and specificity of 94.5%, compared to a nested PCR reference. For a sample set from 58 VL patients, 10 malaria patients and 68 healthy controls from Ethiopia and Kenya, mini-dbPCR-NALFIA had a pooled sensitivity of 87.9% and pooled specificity of 100% using quantitative PCR as reference standard. IT LEISH sensitivity and specificity in the East African samples were 87.9% and 97.4%, respectively. Conclusions/Significance: Mini-dbPCR-NALFIA is a promising tool for simplified molecular diagnosis of VL and follow-up of treated patients in blood samples. Future studies should evaluate its use in endemic, resource-limited settings, where mini-dbPCR-NALFIA may provide an accurate and versatile alternative to rK39 ICTs and aspirate microscopy. Author summary: Visceral leishmaniasis (VL) is a severe infectious disease caused by unicellular parasites of the Leishmania donovani complex. As VL is fatal when left untreated, early and confirmatory laboratory diagnosis is crucial for adequate patient management. However, currently available serological assays, such as the rK39 rapid diagnostic test, fall short in terms of sensitivity and cannot be used for detection of relapsing infections. Therefore, we evaluated an innovative and user-friendly diagnostic assay called mini-dbPCR-NALFIA, which is based on the direct detection of Leishmania kinetoplast DNA in human blood samples. We found that mini-dbPCR-NALFIA can detect down to 500 parasites per millilitre of blood, which is lower than the parasite loads generally seen in untreated VL patients. Furthermore, we assessed the accuracy of mini-dbPCR-NALFIA using a set of Spanish and East-African samples from VL patients and control groups. In the Spanish samples, sensitivity of mini-dbPCR-NALFIA was very high and somewhat better than in the East-African samples. The specificity of mini-dbPCR-NALFIA was excellent in both sample sets. In conclusion, considering its robust performance and simplicity, the mini-dbPCR-NALFIA is a promising diagnostic assay that has the potential to improve VL diagnosis and follow-up of treated patients, especially in resource-limited situations. [ABSTRACT FROM AUTHOR]