Molecular Bacterial Load Assay Concurs with Culture on NaOH-Induced Loss of Mycobacterium tuberculosis Viability

Abstract

Effective methods to detect viable <i>Mycobacterium tuberculosis</i>, the main causative agent of tuberculosis (TB), are urgently needed. To date, cultivation of <i>M. tuberculosis</i> is the gold standard, which depends on initial sample processing with <i>N</i>-acetyl-l-cysteine-sodium hydroxide (NALC-NaOH), chemicals that compromise <i>M. tuberculosis</i> viability and, consequently, the performance of downstream tests. We applied culture and the novel molecular bacterial load assay (MBLA) to measure the loss of <i>M. tuberculosis</i> viability following NALC-NaOH treatment of <i>M. tuberculosis</i> H37Rv pure culture and clinical sputum samples from pulmonary TB patients. Compared to the bacterial loads of untreated controls, NALC-NaOH treatment of <i>M. tuberculosis</i> reduced the MBLA-detectable bacillary load (estimated number of CFU [eCFU] per milliliter) by 0.66 ± 0.21 log₁₀ at 23°C (<i>P</i> = 0.018) and 0.72 ± 0.08 log₁₀ at 30°C (<i>P</i> = 0.013). Likewise, NALC-NaOH treatment reduced the viable count on solid culture by 0.84 ± 0.02 log₁₀ CFU/ml at 23°C (<i>P</i> < 0.001) and 0.85 ± 0.01 log₁₀ CFU/ml at 30°C (<i>P</i> < 0.001), respectively. The reduction in the viable count was reflected by a corresponding increase in the time to positivity of the mycobacterial growth indicator tube (MGIT) liquid culture: 1.2 days at 23°C (<i>P</i> < 0.001) and 1.1 days at 30°C (<i>P</i> < 0.001). This NaOH-induced <i>M. tuberculosis</i> viability loss was replicated in clinical sputum samples, with the bacterial load dropping by 0.65 ± 0.17 log₁₀ from 5.36 ± 0.24 log₁₀ eCFU/ml to 4.71 ± 0.16 log₁₀ eCFU/ml for untreated and treated sputa, respectively. Applying the model of Bowness et al. (R. Bowness, M. J. Boeree, R. Aarnoutse, R. Dawson, et al., J Antimicrob Chemother 70:448-455, 2015, https://doi.org/10.1093/jac/dku415) revealed that the treated MGIT time to culture positivity of 142 ± 7.02 h was equivalent to 4.86 ± 0.28 log₁₀ CFU, consistent with the MBLA-measured bacterial load. Our study confirms the contribution of NALC-NaOH treatment to the loss of viable bacterial counts. Tests that obviate the need for decontamination may offer an alternative option for the accurate detection of viable <i>M. tuberculosis</i> and treatment response monitoring.