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

Tuberculosis is a difficult disease to treat, a process made more harder as tools to monitor treatment response only provide a result long after the patient has provided a sample. The mycobacterial load assay (MBLA) provides a simple molecular test to quantify and determine the viability of M. tuberculosis in human or other samples.

Mycobacterial Load Assay.

Tuberculosis (TB) diagnostics are centralised, requiring long-distance transportation of specimens in most resource-limited settings. We evaluated the ability of OMNIgene.SPUTUM (OM-S) to obviate cold-chain transport of TB specimens. A two-arm (same-day and after 5 days sample processing) study was conducted to assess contamination rates and  viability in OM-S-treated samples against the standard decontamination procedure (SDP) in Mozambique, using Lowenstein Jensen (LJ) and mycobacterial growth indicator tube (MGIT) culture and molecular bacterial load assay. 270 specimens were processed using OM-S and SDP in same-day and 5-day arms. Contamination was lower in OM-S-treated than SDP-treated cultures: 12%  15% and 2%  27% in the same-day and 5-day arms, respectively.  recovery in OM-S-treated LJ cultures was 10% and 56% higher in the same-day and 5-day arms, respectively, than SDP-treated cultures, but lower in MGIT (52% and 28% lower in the same-day and 5-day arms, respectively).  viable count was 1log estimated CFU·mL lower in 5-day OM-S-treated sputa. OM-S was more effective at liquefying sputum with a shorter sample processing time: 22 min for culture. OM-S is simple to use and has demonstrated a high potency to suppress contaminants, maintenance of viability at ambient temperatures and higher  recovery, particularly in the solid LJ cultures. Optimisation of OM-S to achieve higher MGIT culture positivity and shorter time to result will increase its application and utility in the clinical management of TB.

OMNIgene.SPUTUM suppresses contaminants while maintaining  viability and obviates cold-chain transport.

The World Health Organization End Tuberculosis (TB) strategy has called for the development of-and increased access to-effective tools for diagnosis and treatment of TB disease.  , the causative agent of TB, is categorized as a highly infectious agent. Consequently, diagnostic tests that involve comprehensive manipulation of specimens from presumed tuberculosis cases must be performed in a category 3 laboratory. We have evaluated the use of heat inactivation to render TB samples safe to work with while preserving RNA for downstream molecular tests. Using  bacillus Calmette-Guérin (BCG) cultures and TB-positive sputum samples, we show that boiling for 20 min at 80, 85, and 95°C inactivates all  bacilli. The efficiency of inactivation was verified by culturing heat-treated and untreated (live) fractions of BCG and TB sputum samples for 42 days. No growth was observed in the cultures of heat-treated samples. In contrast, the optical density of untreated BCG in Middlebrook 7H9 broth rose from 0.04 to 0.85, and the untreated sputum samples flagged positive at 3 days of incubation in mycobacterial growth indicator tubes. Quantification of reference genes 16S rRNA, transfer-messenger RNA (tmRNA), pre-16S rRNA, and  by reverse transcriptase quantitative PCR (RT-qPCR) showed minimal loss in estimated bacterial load. The loss was RNA species dependent, <1 log, 1.1 log, 1.3 log, and 2.4 log estimated CFU/ml for 16S rRNA, tmRNA, pre-16S rRNA, and , respectively. The RNA loss was independent of inactivation temperature. These findings show that heat inactivation could obviate the need for category 3 laboratories to perform RNA-based testing of TB samples.

Heat Inactivation Renders Sputum Safe and Preserves  RNA for Downstream Molecular Tests.

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.

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

The molecular bacterial load (MBL) assay is a new tuberculosis biomarker which provides results in ∼4 hours. The relationship between MBL and time-to-positivity (TTP) has not been thoroughly studied, and predictive models do not exist. We aimed to develop a model for MBL and identify the MBL-TTP relationship in patients. The model was developed on data from 105 tuberculosis patients from Malawi, Mozambique, and Tanzania with joint MBL and TTP observations quantified from patient sputum collected for 12 weeks. MBL was quantified using PCR of mycobacterial RNA and TTP using the mycobacterial growth indicator tube (MGIT) 960 system. Treatment consisted of isoniazid, pyrazinamide, and ethambutol in standard doses together with rifampin 10 or 35 mg/kg of body weight. The developed MBL-TTP model included several linked submodels, a component describing decline of bacterial load in sputum, another component describing growth in liquid culture, and a hazard model translating bacterial growth into a TTP signal. Additional components for contaminated and negative TTP samples were included. Visual predictive checks performed using the developed model gave good description of the observed data. The model predicted greater total sample loss for TTP than MBL due to contamination and negative samples. The model detected an increase in bacterial killing for 35 versus 10 mg/kg rifampin ( = 0.002). In conclusion, a combined model for MBL and TTP was developed that described the MBL-TTP relationship. The full MBL-TTP model or each submodel was used separately. Second, the model can be used to predict biomarker response for MBL given TTP data or  in historical or future trials.

Wilber Sabiiti

Division of Infection and Global Health,
School of Medicine,
Division of Infection and Global Health, School of Medicine

University of St Andrews

A Tuberculosis Molecular Bacterial Load Assay (TB-MBLA)

Wilber Sabiiti

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Division of Infection and Global Health,School of Medicine,Division of Infection and Global Health, School of Medicine

University of St Andrews

A Tuberculosis Molecular Bacterial Load Assay (TB-MBLA)

Division of Infection and Global Health,
School of Medicine,
Division of Infection and Global Health, School of Medicine