Verma, Renu

Abstract Background Estimating bacterial load in clinical samples has important applications in tuberculosis (TB) management, including assessment of disease severity, treatment response, and baseline genome copies required for sequencing. However, rapid and affordable tools for quantifying Mycobacterium tuberculosis ( M. tuberculosis ) remain limited. Methods We developed a high-resolution melt (HRM) based quantitative PCR assay using molecular beacon chemistry targeting the single-copy RD9 region of M. tuberculosis . Analytical performance was assessed using 10-fold serial dilutions of H37Rv DNA. Clinical validation included DNA from 100 M. tuberculosis culture isolates and 40 sputum samples from Xpert MTB/RIF- and culture-positive pulmonary TB patients. To evaluate specificity, we tested 30 non-tuberculous mycobacterium (NTM) culture isolates from patients infected with Mycobacterium abscessus (n=25) and Mycobacterium fortuitum (n=5) and DNA from saliva samples of 10 healthy controls. Results The HRM-qPCR assay showed a linear dynamic range from 10 1 to 10 6 genome copies per reaction, with a lower limit of detection of 10 copies. Standardized melt-curve analysis yielded a single target-specific peak at 73.7±0.12°C across dilutions, confirming specific amplification. Sensitivity for M. tuberculosis detection was 100% in culture isolates and 95.0% (38/40) in sputum, with no false positives among M. tuberculosis -negative controls. Assay specificity was 100% in both culture isolates and sputum, with no additional melt peaks. We did not observe any peaks indicative of non-specific binding in NTMs. Amplification was observed in two NTM samples whose Tm matched M. tuberculosis RD9 (median M. tuberculosis copies=678.5), suggesting possible co-infection or contamination. No amplification or specific melt peaks were observed in saliva samples, indicating high probe specificity. Conclusions Molecular beacon-based HRM-qPCR assay enables rapid, highly specific quantification of M. tuberculosis genome copies in clinical samples and has potential utility for treatment monitoring, triaging specimens for sequencing, and assessing transmission risk in high-burden settings.

Abstract Background Direct whole genome sequencing (WGS) of Mycobacterium tuberculosis (Mtb) can be used as a tool to study drug resistance, mixed infections, and within-host diversity. However, WGS is challenging to obtain from clinical samples due to low number of bacilli against a high background. Methods We prospectively collected 34 samples (sputum, n = 17; bronchoalveolar lavage, n = 13; and pus, n = 4) from patients with active tuberculosis (TB). Prior to DNA extraction, we used a ligand-mediated magnetic bead method to enrich Mtb from clinical samples and performed WGS on Illumina platform. Results Mtb was definitively identified based on WGS from 88.2% (30/34) of the samples, of which 35.3% (12/34) were smear negative. The overall median genome coverage was 15.2% (interquartile range [IQR], 7.7%–28.2%). There was a positive correlation between load of bacilli on smears and genome coverage (P < .001). We detected 58 genes listed in the World Health Organization mutation catalogue in each positive sample (median coverage, 85% [IQR, 61%–94%]), enabling the identification of mutations missed by routine diagnostics. Mutations causing resistance to rifampicin, isoniazid, streptomycin, and ethambutol were detected in 5 of 34 (14.7%) samples, including the rpoB S441A mutation that confers resistance to rifampicin, which is not covered by Xpert MTB/RIF. Conclusions We demonstrate the feasibility of magnetic bead–based enrichment for culture-free WGS of Mtb from clinical specimens, including smear-negative samples. This approach can also be integrated with low-cost sequencing workflows such as targeted sequencing for rapid detection of Mtb and drug resistance.