Buruli Ulcer Pathogen Detected by Isothermal RPA Assay

Buruli ulcer (BU) is a neglected tropical disease caused by Mycobacterium ulcerans. The pathogenesis of BU is linked to the production of a polyketide toxin known as mycolactone, which is cytotoxic and has immunomodulatory properties and presents as nodules, plaques, ulcers and edema.

Nucleic acid amplification of insertion sequence IS2404 by polymerase chain reaction (PCR) is the most sensitive and specific method to detect M. ulcerans, the causative agent of BU. However, PCR is not always available in endemic communities in Africa due to its cost and technological sophistication. Microscopy for acid fast bacilli and culture for M. ulcerans have low sensitivity and histopathology is rarely available in endemic areas.

A team of scientists associated with the Kwame Nkrumah University of Science and Technology (Kumasi, Ghana) evaluated the clinical performance of the M. ulcerans (Mu-RPA) assay was evaluated using DNA extracted from fine needle aspirates or swabs taken from 67 patients in whom BU was suspected and 12 patients with clinically confirmed non-BU lesions. The team developed an isothermal DNA amplification system using the recombinase polymerase amplification (RPA) method.

All samples were tested with both the real-time PCR and the Mu-RPA assay to determine the clinical sensitivity and specificity of the assay using real-time PCR as the reference test. In the case of real-time RPA detection, TwistAmp Exo “Improved Formulation” kit was used. Fluorescence detection at 570 nm for FAM channel was measured and a threshold set by increasing the fluorescence above the three standard deviations over the background detected in the first minute of incubation. The team programmed the T8- fluorometer using the T8-ISO Desktop application to detect the lowest dilutions that met criteria for distinguishing positive samples from negative controls based on serial dilutions of the molecular standard.

The scientists tested all samples by both RPA and real-time PCR and 58 of these samples were confirmed by PCR as BU. Of the 58 confirmed cases, 51 were correctly identified by the RPA assay with seven false negative results giving a sensitivity of 88%. The 21 PCR negative samples were all negative by RPA, specificity of 100% and a 100% positive predictive value (PPV) with a Youden’s index of 88%. When the analysis was stratified by type of sample, the sensitivity and specificity of the RPA for swabs in comparison to PCR were 92% and 100% respectively with a 100% PPV. Similarly, the sensitivity and specificity of FNA samples were 82% and 100% respectively.

The authors concluded that the developed real-time RPA assay for the rapid and accurate detection of M. ulcerans DNA with high sensitivity, specificity and reproducibility was comparable to real-time PCR. It was significantly faster than available real-time PCR methods for detecting M. ulcerans with a run time of 15 minutes, compared to almost two hours for real-time PCR. Potentially the Mu-RPA can be used in a low resource setting closer to the patients when combined with a fast DNA extraction method. The study was published on February 1, 2019, in the journal PLOS NTD.

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Kwame Nkrumah University of Science and Technology