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

Abstract

Effective methods to detect viable Mycobacterium tuberculosis, the main causative agent of tuberculosis (TB), are urgently needed. To date, cultivation of M. tuberculosis is the gold standard, which depends on initial sample processing with N-acetyl-l-cysteine-sodium hydroxide (NALC-NaOH), chemicals that compromise M. tuberculosis viability and, consequently, the performance of downstream tests. We applied culture and the novel molecular bacterial load assay (MBLA) to measure the loss of M. tuberculosis viability following NALC-NaOH treatment of M. tuberculosis H37Rv pure culture and clinical sputum samples from pulmonary TB patients. Compared to the bacterial loads of untreated controls, NALC-NaOH treatment of M. tuberculosis reduced the MBLA-detectable bacillary load (estimated number of CFU [eCFU] per milliliter) by 0.66 ± 0.21 log₁₀ at 23°C (P = 0.018) and 0.72 ± 0.08 log₁₀ at 30°C (P = 0.013). Likewise, NALC-NaOH treatment reduced the viable count on solid culture by 0.84 ± 0.02 log₁₀ CFU/ml at 23°C (P < 0.001) and 0.85 ± 0.01 log₁₀ CFU/ml at 30°C (P < 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 (P < 0.001) and 1.1 days at 30°C (P < 0.001). This NaOH-induced M. tuberculosis 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 M. tuberculosis and treatment response monitoring.

Keywords: Mycobacterium tuberculosis; NALC-NaOH; culture; decontamination; molecular bacterial load assay; sputum.

FIG 1

Flow diagram of laboratory experiments, conditions, and downstream tests performed for M. tuberculosis cultures and sputum specimens. (A) In vitro experimental procedure employed for each decontaminated M. tuberculosis (Mtb) culture. (B) Experimental flow for pooled patient sputum specimens. Note that 5 biological replicates of experiments were performed for in vitro M. tuberculosis cultures (culture 1 to culture 5) and 7 replicates were performed for sputum aliquots. ZN, Ziehl-Neelsen stain.

FIG 2

NALC-NaOH decontamination reduces the viable M. tuberculosis counts measured by MBLA and on solid culture. The reduction in the number of M. tuberculosis eCFU per milliliter measured by MBLA (A) and the reduction in the number of CFU per milliliter on Middlebrook (7H11) (B) at 23°C and 30°C. The bars and the error bars represent the mean value with the standard error of the mean (SEM). Data are for 5 independent biological replicates on different M. tuberculosis cultures.

FIG 3

NALC-NaOH decontamination compromises test positivity and the detection limit. The effect of NALC-NaOH decontamination on MBLA positivity (A), positivity on solid culture (Middlebrook 7H11) (B), and the positivity of MGIT liquid culture (C) is shown. Data are for 5 independent biological replicates on different M. tuberculosis cultures.

FIG 4

NALC-NaOH decontamination of sputum reduces the viable M. tuberculosis count measured by MBLA. P was <0.0001 between fresh sputum and treated pellet, as determined by an independent t test. Each dot represents the value of each sputum aliquot (n = 7 aliquots).