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.

Mycobactericidal Effects of Different Regimens Measured by Molecular Bacterial Load Assay among People Treated for Multidrug-Resistant Tuberculosis in Tanzania

“…In this study, approximately 1 and 4 out of 10 patients had, respectively, positive LJ culture and positive TB-MBLA at day 56. This supports the previous argument that TB-MBLA is more sensitive compared to agar-based Loewenstein-Jensen culture, in which the M. tuberculosis population gets lost due to contamination at later time points (18). Limitations of the study include the timing of endpoints, which were limited to 4 months, such that predicting long-term treatment success was beyond the scope of this study.…”

“…This bacterial load corresponds to a RT-qPCR quantification cycle (Cq) value of 30, the limit of quantification for positive M. tuberculosis (14). Therefore, TB-MBLA has the potential to replace or complement both smear microscopy and culture for monitoring TB treatment response (14,16,18).…”

A novel, rapid (within hours) culture-free diagnostic method for detecting live Mycobacterium tuberculosis with high sensitivity

“…More recently, a molecular bacterial load assay (MBLA) has been reported as a molecular test for detection of live M. tuberculosis bacilli. It is a reverse transcriptase quantitative PCR that quantifies the M. tuberculosis load from patient sputum using the 16S rRNA gene as a reference gene [52] . We have not yet attempted to use MBLA and thus still cannot compare the results between MBLA and our ultrasensitive ELISA.…”

“…We have not yet attempted to use MBLA and thus still cannot compare the results between MBLA and our ultrasensitive ELISA. However, it is noted that MBLA is a time-consuming method because they have two complicated processes: (1) RNA extraction and purification and (2) the use of real-time PCR [52] .…”

Deploying a novel tuberculosis molecular bacterial load assay to assess the elimination rate ofMycobacterium tuberculosisin patients with multidrug-resistant tuberculosis in Tanzania

“…rRNA from both viable replicating and dormant Mtb in patient's sputa during treatment [6] . Previously, TB-MBLA was assessed by the Pan-African Consortium for Evaluation of Anti-TB Antibiotics (PanACEA) group in patients treated for drug-sensitive (DS)-TB, and demonstrated considerable potential to replace both smear microscopy and culture for monitoring TB treatment response [6][7][8] . TB-MBLA was found to be consistently read as positive for samples with as low as 10 CFU/mL of M. tuberculosis and the cycle threshold for this read-out has been optimized at a value of 30 [6] .…”