A Multi-Country, Single-Blinded, Phase 2 Study to Evaluate a Point-of-Need System for Rapid Detection of Leishmaniasis and Its Implementation in Endemic Settings.

Anik Ashfaq Khan ,Monica Sooriyaarachchi,Shalindra Ranasinghe,Rasel Uddin ,Keshav Rai,Basudha Khanal,Amresh Kumar,Withanage Lakma Kumari Abhayarathna,Pradeep Das,Md Utba Rashid ,Khaledul Faisal,Kumar Abhishek,Faria Hossain,Satyabrata Routray,Prahlad Karki,Prakash Ghosh,Susanne Böhlken-Fascher,Rajashree Chowdhury,Abhijit Sharma,Narayan Bhattarai ,Sachin Kumar ,Dinesh Mondal,Ahmed Abd El Wahed,Thilini Nisansala,Shomik Maruf

doi:10.3390/microorganisms9030588

Abstract

With the advancement of isothermal nucleic acid amplification techniques, detection of the pathogenic DNA in clinical samples at point-of-need is no longer a dream. The newly developed recombinase polymerase amplification (RPA) assay incorporated in a suitcase laboratory has shown promising diagnostic efficacy over real-time PCR in detection of leishmania DNA from clinical samples. For broader application of this point-of-need system, we undertook a current multi-country diagnostic evaluation study towards establishing this technique in different endemic settings which would be beneficial for the ongoing elimination programs for leishmaniasis. For this study purpose, clinical samples from confirmed visceral leishmaniasis (VL) and post-kala-azar dermal leishmaniasis (PKDL) patients were subjected to both real-time PCR and RPA assay in Bangladesh, India, and Nepal. Further skin samples from confirmed cutaneous leishmaniasis (CL) patients were also included from Sri Lanka. A total of 450 clinical samples from VL patients, 429 from PKDL patients, 47 from CL patients, and 322 from endemic healthy/healthy controls were under investigation to determine the diagnostic efficacy of RPA assay in comparison to real-time PCR. A comparative sensitivity of both methods was found where real-time PCR and RPA assay showed 96.86% (95% CI: 94.45–98.42) and 88.85% (95% CI: 85.08–91.96) sensitivity respectively in the diagnosis of VL cases. This new isothermal method also exhibited promising diagnostic sensitivity (93.50%) for PKDL cases, when a skin sample was used. Due to variation in the sequence of target amplicons, RPA assay showed comparatively lower sensitivity (55.32%) than that of real-time PCR in Sri Lanka for the diagnosis of CL cases. Except for India, the assay presented absolute specificity in the rest of the sites. Excellent concordance between the two molecular methods towards detection of leishmania DNA in clinical samples substantiates the application of RPA assay incorporated in a suitcase laboratory for point-of-need diagnosis of VL and PKDL in low resource endemic settings. However, further improvisation of the method is necessary for diagnosis of CL.

Highlights

With the auspices of holistic efforts followed by the London Declaration, strides are ongoing to alleviate the scourge driven by (Neglected Tropical Diseases) NTDs in poverty stricken endemic tropics
This vector-borne disease is comprised of three major forms including visceral leishmaniasis or kala-azar (VL), cutaneous leishmaniasis (CL), and mucocutaneous leishmaniasis affecting disproportionately both the new and old worlds, where more than 20 leishmania species are associated with disease pathogenesis [3]
Real-time PCR was found to be highly sensitive in detecting leishmania DNA in clinical samples from VL patients

Summary

Introduction

With the auspices of holistic efforts followed by the London Declaration, strides are ongoing to alleviate the scourge driven by (Neglected Tropical Diseases) NTDs in poverty stricken endemic tropics. Among 17 parasite-borne NTDs recognized by the WHO, leishmaniasis ranks next to malaria as the second worst in the age-standardized disability-adjusted life years (DALYs), and requires control mechanisms and tools including drugs, vaccine, diagnostics and vector control agents as well as strategies towards absolute elimination [1,2] This vector-borne disease is comprised of three major forms including visceral leishmaniasis or kala-azar (VL), cutaneous leishmaniasis (CL), and mucocutaneous leishmaniasis affecting disproportionately both the new and old worlds, where more than 20 leishmania species are associated with disease pathogenesis [3]. The microsatellite analysis found that LD isolates from Sri Lanka are clustered together and close to a group containing LD isolates from Bangladesh, India, and Nepal [7] These three countries have been jointly working to eliminate VL through implementing the kala-azar elimination program since 2005 [8]. An accurate, rapid, and simple diagnostic tool is imperative, which can be implemented at decentralized facilities for mass surveillance, diagnosis, and to cure assessment as well, if possible

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