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Open Access 17-06-2024 | Rifampicin | Research

Accurate and affordable detection of rifampicin and isoniazid resistance in Tuberculosis sputum specimens by multiplex PCR-multiple probes melting analysis

Authors: Long Xie, Xiao-Ya Zhu, Li Xu, Xiao-Xie Xu, Ze-Fan Ruan, Ming-Xiang Huang, Li Chen, Xi-Wen Jiang

Published in: Infection

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Abstract

Background

Escalating cases of multidrug-resistant tuberculosis (MDR-TB) pose a major challenge to global TB control efforts, necessitating innovative diagnostics to empower decentralized detection of gene mutations associated with resistance to rifampicin (RIF) and isoniazid (INH) in Mycobacterium tuberculosis (M. tuberculosis) in resource-constrained settings.

Methods

Combining multiplex fluorescent PCR and Multiple Probes Melting Analysis, we identified mutations in the rpoB, katG, ahpC and inhA genes from sputum specimens. We first constructed a reference plasmid library comprising 40 prevalent mutations in the target genes’ resistance determining regions and promoters, serving as positive controls. Our assay utilizes a four-tube asymmetric PCR method with specifically designed molecular beacon probes, enabling simultaneous detection of all 40 mutations. We evaluated the assay’s effectiveness using DNA isolated from 50 clinically confirmed M. tuberculosis sputum specimens, comparing our results with those obtained from Sanger sequencing and retrospective validation involving bacteriological culture and phenotypic drug susceptibility testing (pDST). We also included the commercial Xpert MTB/RIF assay for accuracy comparison.

Results

Our data demonstrated remarkable sensitivity in detecting resistance to RIF and INH, achieving values of 93.33% and 95.24%, respectively, with a specificity of 100%. The concordance between our assay and pDST was 98.00%. Furthermore, the accuracy of our assay was comparable to both Sanger sequencing and the Xpert assay. Importantly, our assay boasts a 4.2-h turnaround time and costs only $10 per test, making it an optimal choice for peripheral healthcare settings.

Conclusion

These findings highlight our assay’s potential as a promising tool for rapidly, accurately, and affordably detecting MDR-TB.
Appendix
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Metadata
Title
Accurate and affordable detection of rifampicin and isoniazid resistance in Tuberculosis sputum specimens by multiplex PCR-multiple probes melting analysis
Authors
Long Xie
Xiao-Ya Zhu
Li Xu
Xiao-Xie Xu
Ze-Fan Ruan
Ming-Xiang Huang
Li Chen
Xi-Wen Jiang
Publication date
17-06-2024
Publisher
Springer Berlin Heidelberg
Published in
Infection
Print ISSN: 0300-8126
Electronic ISSN: 1439-0973
DOI
https://doi.org/10.1007/s15010-024-02295-w
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