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Clinical evaluation of tuberculosis viability microscopy for assessing treatment response.

ABSTRACT: BACKGROUND:It is difficult to determine whether early tuberculosis treatment is effective in reducing the infectiousness of patients' sputum, because culture takes weeks and conventional acid-fast sputum microscopy and molecular tests cannot differentiate live from dead tuberculosis. METHODS:To assess treatment response, sputum samples (n=124) from unselected patients (n=35) with sputum microscopy-positive tuberculosis were tested pretreatment and after 3, 6, and 9 days of empiric first-line therapy. Tuberculosis quantitative viability microscopy with fluorescein diacetate, quantitative culture, and acid-fast auramine microscopy were all performed in triplicate. RESULTS:Tuberculosis quantitative viability microscopy predicted quantitative culture results such that 76% of results agreed within ±1 logarithm (rS=0.85; P<.0001). In 31 patients with non-multidrug-resistant (MDR) tuberculosis, viability and quantitative culture results approximately halved (both 0.27 log reduction, P<.001) daily. For patients with non-MDR tuberculosis and available data, by treatment day 9 there was a >10-fold reduction in viability in 100% (24/24) of cases and quantitative culture in 95% (19/20) of cases. Four other patients subsequently found to have MDR tuberculosis had no significant changes in viability (P=.4) or quantitative culture (P=.6) results during early treatment. The change in viability and quantitative culture results during early treatment differed significantly between patients with non-MDR tuberculosis and those with MDR tuberculosis (both P<.001). Acid-fast microscopy results changed little during early treatment, and this change was similar for non-MDR tuberculosis vs MDR tuberculosis (P=.6). CONCLUSIONS:Tuberculosis quantitative viability microscopy is a simple test that within 1 hour predicted quantitative culture results that became available weeks later, rapidly indicating whether patients were responding to tuberculosis therapy.

SUBMITTER: Datta S

PROVIDER: S-EPMC4370166 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature

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Clinical evaluation of tuberculosis viability microscopy for assessing treatment response.

Datta Sumona S Sherman Jonathan M JM Bravard Marjory A MA Valencia Teresa T Gilman Robert H RH Evans Carlton A CA

Clinical infectious diseases : an official publication of the Infectious Diseases Society of America 20141223 8

<h4>Background</h4>It is difficult to determine whether early tuberculosis treatment is effective in reducing the infectiousness of patients' sputum, because culture takes weeks and conventional acid-fast sputum microscopy and molecular tests cannot differentiate live from dead tuberculosis.<h4>Methods</h4>To assess treatment response, sputum samples (n=124) from unselected patients (n=35) with sputum microscopy-positive tuberculosis were tested pretreatment and after 3, 6, and 9 days of empiric ...[more]

PMID: 25537870

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Project description:BACKGROUND:The DevR(DosR) regulon is implicated in hypoxic adaptation and virulence of Mycobacterium tuberculosis. The present study was designed to decipher the impact of perturbation in DevR-mediated signaling on these properties. METHODOLOGY/PRINCIPAL FINDINGS:M. tb complemented (Comp) strains expressing different levels of DevR were constructed in Mut1* background (expressing DevR N-terminal domain in fusion with AphI (DevR(N)-Kan) and in Mut2?devR background (deletion mutant). They were compared for their hypoxia adaptation and virulence properties. Diverse phenotypes were noted; basal level expression (?5.3±2.3 µM) when induced to levels equivalent to WT levels (?25.8±9.3 µM) was associated with robust DevR regulon induction and hypoxic adaptation (Comp 9* and 10*), whereas low-level expression (detectable at transcript level) as in Comp 11* and Comp15 was associated with an adaptation defect. Intermediate-level expression (?3.3±1.2 µM) partially restored hypoxic adaptation functions in Comp2, but not in Comp1* bacteria that co-expressed DevR(N)-Kan. Comp* strains in Mut1* background also exhibited diverse virulence phenotypes; high/very low-level DevR expression was associated with virulence whereas intermediate-level expression was associated with low virulence. Transcription profiling and gene expression analysis revealed up-regulation of the phosphate starvation response (PSR) in Mut1* and Comp11* bacteria, but not in WT/Mut2?devR/other Comp strains, indicating a plasticity in expression pathways that is determined by the magnitude of signaling perturbation through DevR(N)-Kan. CONCLUSIONS/SIGNIFICANCE:A minimum DevR concentration of ?3.3±1.2 µM (as in Comp2 bacteria) is required to support HspX expression in the standing culture hypoxia model. The relative intracellular concentrations of DevR and DevR(N)-Kan appear to be critical for determining dormancy regulon induction, hypoxic adaptation and virulence. Dysregulated DevR(N)-Kan-mediated signaling selectively triggers the PSR in bacteria expressing no/very low level of DevR. Our findings illustrate the important role of appropriate two-component-mediated signaling in pathogen physiology and the resilience of bacteria when such signaling is perturbed.

Project description:Cell viability, an essential measurement for cell therapy products, lacks traceability. One of the most common cell viability tests is trypan blue dye exclusion where blue-stained cells are counted via brightfield imaging. Typically, live and dead cells are classified based on their pixel intensities which may vary arbitrarily making it difficult to compare results. Herein, a traceable absorbance microscopy method to determine the intracellular uptake of trypan blue is demonstrated. The intensity pixels of the brightfield images are converted to absorbance images which are used to calculate moles of trypan blue per cell. Trypan blue cell viability measurements, where trypan blue content in each cell is quantified, enable traceable live-dead classifications. To implement the absorbance microscopy method, we developed an open-source AbsorbanceQ application that generates quantitative absorbance images. The validation of absorbance microscopy is demonstrated using neutral density filters. Results from four different microscopes demonstrate a mean absolute deviation of 3% from the expected optical density values. When assessing trypan blue-stained Jurkat cells, the difference in intracellular uptake of trypan blue in heat-shock-killed cells using two different microscopes is 3.8%. Cells killed with formaldehyde take up ~50% less trypan blue as compared to the heat-shock-killed cells, suggesting that the killing mechanism affects trypan blue uptake. In a test mixture of approximately 50% live and 50% dead cells, 53% of cells were identified as dead (±6% standard deviation). Finally, to mimic batches of low-viability cells that may be encountered during a cell manufacturing process, viability was assessed for cells that were 1) overgrown in the cell culture incubator for five days or 2) incubated in DPBS at room temperature for five days. Instead of making live-dead classifications using arbitrary intensity values, absorbance imaging yields traceable units of moles that can be compared, which is useful for assuring quality for biomanufacturing processes.

Project description:BackgroundThe diagnosis of tuberculosis (TB) in resource-limited settings relies on Ziehl-Neelsen (ZN) smear microscopy. LED fluorescence microscopy (LED-FM) has many potential advantages over ZN smear microscopy, but requires evaluation in the field. The aim of this study was to assess the sensitivity/specificity of LED-FM for the diagnosis of pulmonary TB and whether its performance varies with the timing of specimen collection.Methods and findingsAdults with cough ?2 wk were enrolled consecutively in Ethiopia, Nepal, Nigeria, and Yemen. Sputum specimens were examined by ZN smear microscopy and LED-FM and compared with culture as the reference standard. Specimens were collected using a spot-morning-spot (SMS) or spot-spot-morning (SSM) scheme to explore whether the collection of the first two smears at the health care facility (i.e., "on the spot") the first day of consultation followed by a morning sample the next day (SSM) would identify similar numbers of smear-positive patients as smears collected via the SMS scheme (i.e., one on-the-spot-smear the first day, followed by a morning specimen collected at home and a second on-the-spot sample the second day). In total, 529 (21.6%) culture-positive and 1,826 (74.6%) culture-negative patients were enrolled, of which 1,156 (49%) submitted SSM specimens and 1,199 (51%) submitted SMS specimens. Single LED-FM smears had higher sensitivity but lower specificity than single ZN smears. Using two LED-FM or two ZN smears per patient was 72.8% (385/529, 95% CI 68.8%-76.5%) and 65.8% (348/529, 95% CI 61.6%-69.8%) sensitive (p<0.001) and 90.9% (1,660/1,826, 95% CI 89.5%-92.2%) and 98% (1,790/1,826, 95% CI 97.3%-98.6%) specific (p<0.001). Using three LED-FM or three ZN smears per patient was 77% (408/529, 95% CI 73.3%-80.6%) and 70.5% (373/529, 95% CI 66.4%-74.4%, p<0.001) sensitive and 88.1% (95% CI 86.5%-89.6%) and 96.5% (95% CI 96.8%-98.2%, p<0.001) specific. The sensitivity/specificity of ZN smear microscopy and LED-FM did not vary between SMS and SSM.ConclusionsLED-FM had higher sensitivity but, in this study, lower specificity than ZN smear microscopy for diagnosis of pulmonary TB. Performance was independent of the scheme used for collecting specimens. The introduction of LED-FM needs to be accompanied by appropriate training, quality management, and monitoring of performance in the field.Trial registrationCurrent Controlled Trials ISRCTN53339491. Please see later in the article for the Editors' Summary.

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Clinical evaluation of tuberculosis viability microscopy for assessing treatment response.

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Clinical evaluation of tuberculosis viability microscopy for assessing treatment response.

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