a-tuberculosis-molecular-bacterial-load-assay-tb-mbla

A Tuberculosis Molecular Bacterial Load Assay (TB-MBLA)

Tuberculosis is caused by Mycobacterium tuberculosis (Mtb), a pathogen classified by the United Nations (UN) as a dangerous category B biological ...

University of St Andrews

National Institute for Medical Research (NIMR)-Mbeya Medical Research Centre

The relationship between cfu and Mycobacterial Growth Indicator Tube (MGIT) time to positivity (TTP) is uncertain. We attempted to understand this relationship and create a mathematical model to relate these two methods of determining mycobacterial load.

The relationship between Mycobacterium tuberculosis MGIT time to positivity and cfu in sputum samples demonstrates changing bacterial phenotypes potentially reflecting the impact of chemotherapy on critical sub-populations.

The Xpert MTB/RIF assay detects Mycobacterium tuberculosis in unprocessed or NALC/NaOH- decontaminated sputum. The effect of repeated NALC/NaOH-decontamination on several Xpert performance parameters was assessed in this study. A second NALC/NaOH-decontamination had no effect on the binary Xpert-outcome but increased the value for the quantitative readout (C). Repeated decontamination was not associated with PCR-inhibition or invalid results. The C of M.tb positive samples was higher in inhibited Xpert runs. Our data indicate that NALC/NaOH-decontamination has an effect on the performance of the Xpert assay, and that C readouts of decontaminated sputum samples should be interpreted with caution.

The impact of repeated NALC/NaOH- decontamination on the performance of Xpert MTB/RIF assay.

 The analysis of phenotypic characteristics on  (MTB)-specific T cells is a promising approach for the diagnosis of active tuberculosis (aTB) and for monitoring treatment success. We therefore studied phenotypic changes on MTB-specific CD4 T cells upon anti-tuberculosis treatment initiation in relation to the treatment response as determined by sputum culture.  Peripheral blood mononuclear cells from subjects with latent MTB infection ( = 16) and aTB ( = 39) at baseline, weeks 9, 12, and 26 (end of treatment) were analyzed after intracellular interferon gamma staining and overnight stimulation with tuberculin. Liquid sputum cultures were performed weekly until week 12 and during 4 visits until week 26.  T cell activation marker expression on MTB-specific CD4 T cells differed significantly between subjects with aTB and latent MTB infection with no overlap for the frequencies of CD38 and Ki67 cells (both  < 0.0001). At 9 weeks after anti-TB treatment initiation the frequencies of activation marker (CD38, HLA-DR, Ki67) positive MTB-specific, but not total CD4 T cells, were significantly reduced ( < 0.0001). Treatment induced phenotypic changes from baseline until week 9 and until week 12 differed substantially between individual aTB patients and correlated with an individual's time to stable sputum culture conversion for expression of CD38 and HLA-DR (both  < 0.05). In contrast, the frequencies of maturation marker CD27 positive MTB-specific CD4 T cells remained largely unchanged until week 26 and significantly differed between subjects with treated TB disease and latent MTB infection ( = 0.0003).  Phenotypic changes of MTB-specific T cells are potential surrogate markers for tuberculosis treatment efficacy and can help to discriminate between aTB (profile: CD38, CD27), treated TB (CD38, CD27), and latent MTB infection (CD38, CD27).

Phenotypic Changes on Mycobacterium Tuberculosis-Specific CD4 T Cells as Surrogate Markers for Tuberculosis Treatment Efficacy.

Effective methods to detect viable , the main causative agent of tuberculosis (TB), are urgently needed. To date, cultivation of  is the gold standard, which depends on initial sample processing with -acetyl-l-cysteine-sodium hydroxide (NALC-NaOH), chemicals that compromise  viability and, consequently, the performance of downstream tests. We applied culture and the novel molecular bacterial load assay (MBLA) to measure the loss of  viability following NALC-NaOH treatment of  H37Rv pure culture and clinical sputum samples from pulmonary TB patients. Compared to the bacterial loads of untreated controls, NALC-NaOH treatment of  reduced the MBLA-detectable bacillary load (estimated number of CFU [eCFU] per milliliter) by 0.66 ± 0.21 log at 23°C ( = 0.018) and 0.72 ± 0.08 log at 30°C ( = 0.013). Likewise, NALC-NaOH treatment reduced the viable count on solid culture by 0.84 ± 0.02 log CFU/ml at 23°C ( < 0.001) and 0.85 ± 0.01 log CFU/ml at 30°C ( < 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 ( < 0.001) and 1.1 days at 30°C ( < 0.001). This NaOH-induced  viability loss was replicated in clinical sputum samples, with the bacterial load dropping by 0.65 ± 0.17 log from 5.36 ± 0.24 log eCFU/ml to 4.71 ± 0.16 log 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 log 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  and treatment response monitoring.

Article	Year
The relationship between Mycobacterium tuberculosis MGIT time to positivity and cfu in sputum samples demonstrates changing bacterial phenotypes potentially reflecting the impact of chemothe... The Journal of antimicrobial chemotherapy\| PubMed ID: 25344806	2015
The impact of repeated NALC/NaOH- decontamination on the performance of Xpert MTB/RIF assay. Tuberculosis (Edinburgh, Scotland)\| PubMed ID: 29779774	2018
Phenotypic Changes on Mycobacterium Tuberculosis-Specific CD4 T Cells as Surrogate Markers for Tuberculosis Treatment Efficacy. Frontiers in immunology\| PubMed ID: 30323818	2018
Molecular Bacterial Load Assay Concurs with Culture on NaOH-Induced Loss of Mycobacterium tuberculosis Viability. Journal of clinical microbiology\| PubMed ID: 31018981	2019

Bariki Mtafya

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