The Canadian journal of infectious diseases & medical microbiology = Journal canadien des maladies infectieuses et de la microbiologie medicale · 2026
Rapid Extensively Drug-Resistant (XDR) TB Diagnosis: An In-House DNA Biochip for Drug Resistance Detection and Mutation Profiling of Mycobacterium tuberculosis.
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Jain Bharti, Kulkarni Savita, Baghel Nawab Singh
Аннотация
BACKGROUND: Emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis (M. tuberculosis) poses a major public health threat, especially in high burden countries. Current diagnostic modalities are slow, expensive, or inaccessible in low-resource setting. We designed, fabricated, and validated an in-house DNA biochip capable of detecting resistance-associated mutations in key M. tuberculosis genes linked to resistance against first- (rifampicin and isoniazid) and second-line (fluoroquinolones and second-line injectables) antituberculosis drugs.
METHODS: The biochip allowed the detection of 20 drug resistance-determining mutations in the rpoB, katG, inhA, gyrA, rrs, and eis genes in the M. tuberculosis genome. Biochip consists of 33 probes spotted in duplicate including probe for M. tuberculosis detection. The biochip assay is based on the amplification of 7 fragments of the genome using two sets of multiplex PCRs. The biochip assay was optimized, and enhanced chemiluminescence was used for signal detection on biochip. Performance evaluation of the biochip was done using 175 clinical isolates. Culture-based drug susceptibility test (DST) was used as the gold standard to compare biochip results, and sequencing was used to resolve the discordance.
RESULTS: Out of 59 culture sensitive isolates, 53 were sensitive to all drugs by biochip, while 6 isolates showed different mutations. The biochip showed high concordance with culture DST. The diagnostic sensitivity of the biochip assay for all the drugs ranged from 75% to 100%. The specificities of the biochip for all the 7 drugs were over 97%. The developed biochip demonstrated analytical sensitivity of 103 genome copies per assay and showed high reproducibility, with intra-assay coefficient of variation (CV) < 10% and interassay CV < 19% across all probes on the biochip.
CONCLUSIONS: The biochip enables simultaneous analysis of multiple resistance-associated mutation in a single assay providing a powerful tool for early detection, personalized therapy, and effective containment of both MDR and XDR-TB. The biochip is useful for clinical microbiology studies and surveillance programs.
METHODS: The biochip allowed the detection of 20 drug resistance-determining mutations in the rpoB, katG, inhA, gyrA, rrs, and eis genes in the M. tuberculosis genome. Biochip consists of 33 probes spotted in duplicate including probe for M. tuberculosis detection. The biochip assay is based on the amplification of 7 fragments of the genome using two sets of multiplex PCRs. The biochip assay was optimized, and enhanced chemiluminescence was used for signal detection on biochip. Performance evaluation of the biochip was done using 175 clinical isolates. Culture-based drug susceptibility test (DST) was used as the gold standard to compare biochip results, and sequencing was used to resolve the discordance.
RESULTS: Out of 59 culture sensitive isolates, 53 were sensitive to all drugs by biochip, while 6 isolates showed different mutations. The biochip showed high concordance with culture DST. The diagnostic sensitivity of the biochip assay for all the drugs ranged from 75% to 100%. The specificities of the biochip for all the 7 drugs were over 97%. The developed biochip demonstrated analytical sensitivity of 103 genome copies per assay and showed high reproducibility, with intra-assay coefficient of variation (CV) < 10% and interassay CV < 19% across all probes on the biochip.
CONCLUSIONS: The biochip enables simultaneous analysis of multiple resistance-associated mutation in a single assay providing a powerful tool for early detection, personalized therapy, and effective containment of both MDR and XDR-TB. The biochip is useful for clinical microbiology studies and surveillance programs.