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Development of Quantitative Rapid Isothermal Amplification Assay for Leishmania donovani.

ABSTRACT: Quantification of pathogen load, although challenging, is of paramount importance for accurate diagnosis and clinical management of a range of infectious diseases in a point-of-need testing (PONT) scenario such as in resource-limited settings. We formulated a quantification approach to test the standard-curve based absolute quantification ability of isothermal recombinase polymerase amplification (RPA) assay. As a test of principle, a 10-fold dilution series of Leishmania donovani (LD) genomic DNA prepared in nuclease-free-water (NFW), and from culture-spiked-blood (CSB) were tested, and a 15 min assay was performed. A modified algorithm was formulated to derive the detection outcome. The threshold-record times (Tr) in seconds thus obtained were plotted against the initial load of parasite genomes for log-linear regression analysis. The quantitative RPA (Q-RPA) assay was further evaluated against a LD quantitative (q)-PCR assay with DNA extracted from visceral and post-Kala-azar dermal leishmaniasis case specimens and stratified into different ranges of threshold cycle (Ct). The best-fitted regression models were found linear with mean r²/root mean square error (RMSE) values of residual points (in seconds) estimated as 0.996/8.063 and 0.992/7.46 for replicated series of NFW and CSB, respectively. In both series, the lower limit of detection reached less than 0.1 parasite genome equivalent DNA. Absolute agreement between Q-RPA and LD-qPCR was found for test positivity, and strong positive correlations were observed between the Tr and Ct values (r = 0.89; p < 0.0001) as well as between the absolute parasite loads (r = 0.87; p < 0.0001) quantified by respective assays. The findings in this very first Q-RPA assay for leishmaniasis are suggestive of its potential in monitoring LD load in clinical specimens, and the development of rapid Q-RPA assays for other infectious diseases.

SUBMITTER: Khan MAA

PROVIDER: S-EPMC8625035 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Leishmaniasis is a spectrum of diseases with great relevance to public health. Conventional diagnostic methods are time consuming, needing trained personnel. A robust, rapid and cost effective diagnostic test is warranted for on-time diagnosis and field application.We have developed a loop mediated isothermal amplification (LAMP) assay with primers (n?=?6) based on Leishmania donovani kDNA for detection of Leishmania infection, using a closed tube to prevent cross-contamination. The assay was used to detect Leishmania infection in biological samples obtained from patients of visceral leishmaniasis (VL), post kala-azar dermal leishmaniasis (PKDL) and cutaneous leishmaniasis (CL).The assay was positive for L. donovani, L. tropica and L. major parasites, with the highest sensitivity towards L. donovani (1 fg DNA). The high sensitivity of the assay for detection of L. donovani was reflected in its ability to detect parasite DNA within 30 min of amplification time with a threshold detection limit of ?25 copies per reaction. The assay detected parasite in 64 of 66 VL blood samples (sensitivity, 96.9%; 95% CI: 89.6-99.2%), 15 of 15 VL bone marrow aspirate samples (sensitivity, 100%; 95% CI:79.6-100%), 65 of 67 PKDL tissue biopsy samples (sensitivity, 97%; 95% CI:89.7-99.2%). The assay was evaluated in a few cases of CL wherein it was found positive in 8 of 10 tissue biopsies (sensitivity, 80%; 95% CI: 49-94.3%). The assay was negative in all control blood (n?=?76) and tissue biopsy (n?=?24) samples (specificity, 100%; 95% CI: 96.3-100%). Further, the assay was evaluated for its utility in assessment of cure in treated VL and PKDL patients. The assay detected parasite DNA in 2 of 20VL blood samples and 2 of 21 PKDL tissue samples. Out of 4 cases that were positive for parasite DNA at post treatment stage, 2 patients (1VL and 1 PKDL) returned with relapse.The study demonstrated a Leishmania genus specific closed tube LAMP assay for reliable and rapid molecular diagnosis of VL and PKDL with potential for application in assessment of cure.

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Development of Quantitative Rapid Isothermal Amplification Assay for Leishmania donovani.

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