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

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.

Quantifying Viable M. tuberculosis Safely Obviating the Need for High Containment Facilities.

Mycobacterium tuberculosis is an infectious pathogen that requires biosafety level-3 laboratory for handling. The risk of transmission is high to laboratory staff, and to manage the organism safely, it is necessary to construct high containment laboratory facilities at great expense. This limits the application of tuberculosis diagnostics to areas where there is insufficient capital to invest in laboratory infrastructure. In this method, we describe a process of inactivating sputum samples by either heat or guanidine thiocyanate (GTC) that renders them safe without affecting the quantification of viable bacteria. This method eliminates the need for level 3 containment laboratory for the tuberculosis molecular bacterial load assay (TB-MBLA) and is applicable in low- and middle-income countries.

A practical approach to render tuberculosis samples safe for application of tuberculosis molecular bacterial load assay in clinical settings without a biosafety level 3 laboratory.

BACKGROUND: Mycobacterium tuberculosis is a category B infectious pathogen requiring level-3-containment laboratories for handling. We assessed the efficacy of heat and Guanidine thiocyanate (GTC) to inactivate M. tuberculosis prior to performance of tuberculosis Molecular Bacterial Load Assay (TB-MBLA). METHOD: We performed in vitro experiments using M.tb, H37Rv reference strain and replicated in sputum specimens. A 0.5 MacFarland standard of M. tuberculosis was serially diluted to 1x101 CFU/mL and pooled sputum was homogenised prior to serial dilutions and Xpert MTB/RIF Ultra. Three replicates for each containing 1 mL for M. tuberculosis and sputum were inactivated at 80 °C for 20 min and with GTC for 15 min. Inactivated samples were processed for culture and TB-MBLA. RESULTS: No M. tuberculosis growth was observed in MGIT for GTC or heat treated H37Rv cultures. All untreated H37Rv dilutions were MGIT positive except the most diluted specimens. Heat and GTC treatment of H37Rv reduced TB-MBLA load by 2.1log10 (P = 0.7) and 1.8log10 (P = 0.7) respectively, compared to controls. In contrast, heat treated sputum had TB-MBLA bacterial load of 3.47 ± 3.53 log10 compared to 5.4 ± 3.1 log10 eCFU/mL for GTC (p = 0.57). All heat and GTC treated sputum were culture negative. CONCLUSION: Heat or GTC renders M. tuberculosis non-viable and eliminates the need for BSL3 laboratory for performing TB-MBLA in routine healthcare settings.

Systematic assessment of clinical and bacteriological markers for tuberculosis reveals discordance and inaccuracy of symptom-based diagnosis for treatment response monitoring.

Background: Clinical symptoms are the benchmark of tuberculosis (TB) diagnosis and monitoring of treatment response but are not clear how they relate to TB bacteriology, particularly the novel tuberculosis-molecular bacterial load assay (TB-MBLA). Methods: Presumptive cases were bacteriologically confirmed for TB and assessed for symptoms and bacteriological resolution using smear microscopy (SM), culture, and TB-MBLA over 6-month treatment course. Kaplan-Meier and Kappa statistics were used to test the relationship between symptoms and bacteriological positivity. Results: A cohort of 46 bacteriologically confirmed TB cases were analyzed for treatment response over a 6-month treatment course. Pre-treatment symptoms and bacteriological positivity concurred in over 70% of the cases. This agreement was lost in over 50% of cases whose chest pain, night sweat, and loss of appetite had resolved by week 2 of treatment. Cough resolved at a 3.2% rate weekly and was 0.3% slower than the combined bacteriological (average of MGIT and TB-MBLA positivity) resolution rate, 3.5% per week. A decrease in TB-MBLA positivity reflected a fall in bacillary load, 5.7 ± 1.3- at baseline to 0.30 ± 1.0- log10 eCFU/ml at month 6, and closer to cough resolution than other bacteriological measures, accounting for the only one bacteriologically positive case out of seven still coughing at month 6. Low baseline bacillary load patients were more likely to be bacteriologically negative, HR 5.6, p = 0.003 and HR 3.2, p = 0.014 by months 2 and 6 of treatment, respectively. Conclusion: The probability of clinical symptoms reflecting bacteriological positivity weakens as the patient progresses on anti-TB therapy, making the symptom-based diagnosis a less reliable marker of treatment response.