Salleh, Nur Eyuni Mohd
(2022)
Development of recombinase polymerase amplification assay for the detection of mycobacterium tuberculosis.
PhD thesis, Universiti Sains Malaysia.
Abstract
The World Health Organization (WHO) has incorporated various strategies to
strengthen tuberculosis (TB) control programmes. The current diagnostic tools for TB
detection require different levels of laboratory sophistication due to technical
complexities, expertise and biosafety requirements in nucleic acid (NA) based TB
testing. Therefore, it is challenging to use NA-based assays in resource-constrained
settings. With such limitations, there is a need to develop new approaches that can
facilitate point-of-care (POC) diagnostics. Recombinase amplification assay (RPA) is
an NA-based amplification platform that requires an optimal heat source for accurate
diagnosis of a particular disease in a short time. The method is capable of amplifying
specific NA at a single, low and constant temperature with minimal amount of sample
preparation. In this study, a rapid assay for the detection of Mycobacterium
tuberculosis (Mtb) based on the RPA targeting the B9 sequence was developed. The
RPA-based detection of Mtb DNA was achieved within 20 minutes at 39°C. The
analytical sensitivity of the test was one pg when tested using purified Mtb genomic
DNA. The clinical performance of the RPA was evaluated using 387 sputum samples
with the culture method as the gold standard. RPA and microscopy were compared to
the gold standard in terms of sensitivity, specificity, positive predictive value, negative
predictive value, false positive value and false negative value. The results showed that
the sensitivity, specificity, positive predictive value, negative predictive value, false
positive value and false negative value of RPA were 97.2% (95% CI: 93.5, 99.0), 92.2% (95% CI: 87.6, 95.2), 91.7% (95% CI: 87.8, 95.6), 97.4% (95% CI: 95.2, 99.7),
7.8% (4.2, 11.4), 2.8% (95% CI:0.4, 5.1), respectively, while microscopy had a
sensitivity, specificity, positive predictive value, negative predictive value, false
positive value and false negative value of 90.6% (95% CI: 85.4, 94.1), 88.8% (95%
CI: 83.7, 92.5), 87.7% (95% CI: 83.0, 92.4), 91.5% (95% CI:87.6, 95.4), 11.2% (95%
CI:6.9, 15.4) and 9.4% (95% CI:5.2, 13.6), respectively. RPA was found to be more
sensitive and specific compared to microscopy, suggesting that the method has the
potential to be used as a point-of-care (POC) TB diagnostic tool.
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