Propidium monoazide and Xpert MTB/RIF to quantify Mycobacterium tuberculosis cells

Kayigire, Xavier A.; Friedrich, Sven O.; Karinja, Miriam N.; Merwe, Lize van der; Martinson, Neil; Diacon, Andreas H.

doi:10.1016/j.tube.2016.08.006

Cited by 17 publications

(18 citation statements)

References 22 publications

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“…A DNA-positive test result in treatment follow-up specimens does not necessarily indicate the presence of viable bacilli and could mislead the assessment of treatment progress (4,(6)(7)(8). Unsuccessful attempts have been made to use propidium monoazide, a dye which penetrates and inactivates DNA from dead cells, so that tests like the Xpert/MTB RIF assay can detect viable M. tuberculosis bacilli and be used for treatment monitoring (9,10).…”

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confidence: 99%

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

et al. 2019

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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.

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confidence: 99%

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

et al. 2019

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“…The proportion with a positive sputum Xpert result declined gradually, from 100% at baseline to 80% at four weeks to 50% at eight weeks. The previously-published replication cohort included 19 South African patients with drug-susceptible TB and serial Xpert testing performed without PMA pre-treatment [ 16 ].…”

Section: Resultsmentioning

confidence: 99%

“…To determine whether our results were reproducible, we selected a previously-published study in South Africa as a replication cohort. The South African study evaluated the effect of pre-treatment with propidium monoazide (PMA) on GeneXpert MTB/RIF (also Version G4) [ 16 ]. Our analysis used only data from 19 patients with non-PMA treated (control) samples obtained at day 0 and after 3, 7, 14, 28, 35, and 56 days of treatment.…”

Section: Methodsmentioning

confidence: 99%

Does discovery of differentially culturable M tuberculosis really demand a new treatment paradigm? Longitudinal analysis of DNA clearance from sputum

et al. 2018

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BackgroundAccording to the traditional tuberculosis (TB) treatment paradigm, the initial doses of treatment rapidly kill most Mycobacterium tuberculosis (Mtb) bacilli in sputum, yet many more months of daily treatment are required to eliminate a small, residual subpopulation of drug-tolerant bacilli. This paradigm has recently been challenged following the discovery that up to 90% of Mtb bacilli in sputum are culturable only with growth-factor supplementation. These “differentially culturable” bacilli are hypothesized to be more drug-tolerant than routinely culturable bacilli. This hypothesis implies an alternative paradigm in which TB treatment does not rapidly reduce the total Mtb population but only the small, routinely culturable subpopulation. To evaluate these competing paradigms, we developed a culture-independent method for quantifying the viable fraction of Mtb bacilli in sputum during treatment.MethodsWe used GeneXpert MTB/RIF to quantify Mtb DNA in sputa collected longitudinally from Ugandan adults taking standard 4-drug treatment for drug-susceptible pulmonary TB. We modeled GeneXpert cycle thresholds over time using nonlinear mixed-effects regression. We adjusted these models for clearance of DNA from killed-but-not-yet-degraded bacilli, assuming clearance half-lives ranging from 0 to 1.25 days. We used a convolution integral to quantify DNA from viable bacilli only, and converted cycle thresholds to Mtb genomic equivalents. We replicated our results in a South African cohort.ResultsWe enrolled 41 TB patients in Uganda. Assuming a DNA-clearance half-life of 0 days, genomic equivalents of viable sputum bacilli decreased by 0.22 log/day until 8.8 days, then by 0.07 log/day afterwards. Assuming a DNA-clearance half-life of 1.25 days, genomic equivalents of viable bacilli decreased by 0.36 log/day until 5.0 days, then by 0.06 log/day afterwards. By day 7, viable Mtb had decreased by 97.2–98.8%. We found similar results for 19 TB patients in South Africa.DiscussionUsing a culture-independent method, we found that TB treatment rapidly eliminates most viable Mtb in sputum. These findings are incompatible with the hypothesis that differentially culturable bacilli are drug-tolerant.ConclusionsA culture-independent method for measuring viable Mtb in sputum during treatment corroborates the traditional TB treatment paradigm in which a rapid bactericidal phase precedes slow, elimination of a small, residual bacillary subpopulation.Electronic supplementary materialThe online version of this article (10.1186/s12879-018-3213-7) contains supplementary material, which is available to authorized users.

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“…Of these DNA‐binding chemicals, propidium monoazide (PMA) is considered as an optimized agent, which penetrates only dead bacterial cells rather than living cells with intact membrane, thus resulting in better specificity . The usefulness of PMA‐based assay to differentiate live bacilli from dead ones was proven in both clinical MTB isolates and sputum specimens from TB patients . However, the interpretation of results in previous studies on MTB was always reliant on the signal reduction of ~5 cycles by quantitative PCR, which was significantly lower than 10 cycles' signal reduction from other bacteria species .…”

Section: Introductionmentioning

confidence: 98%

“…15 The usefulness of PMA-based assay to differentiate live bacilli from dead ones was proven in both clinical MTB isolates and sputum specimens from TB patients. 7,16,17 However, the interpretation of results in previous studies on MTB was always reliant on the signal reduction of ~5 cycles by quantitative PCR, 18 which was significantly lower than 10 cycles' signal reduction from other bacteria species. 12,14 This difference indicated that the pretreatment condition of PMA was not optimal for blocking all the gene amplification from dead MTB, thereby possibly resulting in falsepositive results due to inadequate treatment of PMA.…”

mentioning

confidence: 94%

Direct detection from clinical sputum samples to differentiate live and dead Mycobacterium Tuberculosis

Chu

Han

et al. 2018

Clinical Laboratory Analysis

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Background In this study, we aimed to optimize the condition of propidium monoazide (PMA) treatment for direct detection of Mycobacterium tuberculosis (MTB) from clinical specimens. Methods The light exposure time, dark incubation time, bacterial load, and PMA concentration were varied to determine the optimal condition of PMA treatment. Results Overall, the maximum ΔCq value was observed in the group receiving a light exposure time of 20 minutes, which was significantly higher than the others (P < 0.05). The prolongation of dark incubation time seemed more likely to result in greater ΔCq value, and the ΔCq values were 2.0, 4.1, 6.5, 10.1, and 12.7 cycles under dark incubation time of 10, 20, 40, 60, and 120 minutes, respectively. Alternatively, the 4+ samples exhibited favorable detection results at the application of 104‐fold dilution by PMA assay with Cq values higher than 35 cycles. Further evaluation revealed that the PMA assay showed an accordance rate of 98.0% (98/100) among clinical sputa. Conclusions we develop an acceptable method to directly identify the live bacteria from sputum samples. Our data demonstrate that the dark incubation plays a crucial role in the efficacy of PMA treatment for MTB.

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Propidium monoazide and Xpert MTB/RIF to quantify Mycobacterium tuberculosis cells

Cited by 17 publications

References 22 publications

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

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

Does discovery of differentially culturable M tuberculosis really demand a new treatment paradigm? Longitudinal analysis of DNA clearance from sputum

Direct detection from clinical sputum samples to differentiate live and dead Mycobacterium Tuberculosis

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