What is the most reliable solid culture medium for tuberculosis treatment trials?

We conducted a prospective study to determine which solid medium is the most reliable overall and after two months of therapy to detect Mycobacterium tuberculosis complex (MTB). MTB isolation and contamination rates on LJ and Middlebrook 7H10 and 7H11 agar with and without selective antibiotics were examined in a single laboratory and compared against a constructed reference standard and MGIT 960 results. Of 50 smear positive adults with pulmonary TB enrolled, 45 successfully completed standard treatment. Two spot sputum specimens were collected before treatment and at week 8 and one spot specimen each at weeks 2, 4, 6, and 12. The MTB recovery rate among all solid media for pre-treatment specimens was similar. After 8 weeks, selective (S) 7H11 had the highest positivity rate. Latent class analysis was used to construct the primary reference standard. The 98.7% sensitivity of 7H11S (95% Wilson confidence interval 96.4%-99.6%) was highest among the 5 solid media (P = 0.003 by bootstrap); the 82.6% specificity of 7H10S (95% CI 75.7%-87.8%) was highest (P = 0.098). Our results support 7H11S as the medium of choice. Further studies in different areas where recovery and contamination are likely to vary, are recommended.

How we determined the most reliable solid medium for studying treatment of tuberculosis.

Phase 2 clinical trials for tuberculosis (TB) treatment require reliable culture methods to determine presence or absence of Mycobacterium tuberculosis (Mtb) over the course of therapy, as these trials are based primarily on bacteriological endpoints. We evaluate which of 5 solid media is most reliable: Lowenstein-Jensen (LJ) egg-base medium and 4 Middlebrook agar media (nonselective 7H10 and 7H11 and selective 7H10 and 7H11). We analyze 393 specimens from 50 HIV-negative Ugandan adults with newly-diagnosed, pulmonary TB and high acid-fast bacillus smear grade. Specimens were collected every 2-4 weeks during the first 12 weeks of therapy. We compare the results for each culture to 2 composite reference standards--one that was deemed positive if any solid culture was positive for Mtb and another based on latent-class analysis. Both reference standards established that the 2 selective Middlebrook media most reliably determine the presence or absence of Mtb (P < 0.003), largely because of their lower contamination rates. We also showed that results on Middlebrook media were similar to each other, while LJ was most frequently discordant. Contaminated results appeared more likely to be truly negative than to harbor undetected Mtb.

A metabolic biosignature of early response to anti-tuberculosis treatment.

BACKGROUND: The successful treatment of tuberculosis (TB) requires long-term multidrug chemotherapy. Clinical trials to evaluate new drugs and regimens for TB treatment are protracted due to the slow clearance of Mycobacterium tuberculosis (Mtb) infection and the lack of early biomarkers to predict treatment outcome. Advancements in the field of metabolomics make it possible to identify metabolic profiles that correlate with disease states or successful chemotherapy. However, proof-of-concept of this approach has not been provided for a TB-early treatment response biosignature (TB-ETRB). METHODS: Urine samples collected at baseline and during treatment from 48 Ugandan and 39 South African HIV-seronegative adults with pulmonary TB were divided into discovery and qualification sets, normalized to creatinine concentration, and analyzed by liquid chromatography-mass spectrometry to identify small molecule molecular features (MFs) in individual patient samples. A biosignature that distinguished baseline and 1 month treatment samples was selected by pairwise t-test using data from two discovery sample sets. Hierarchical clustering and repeated measures analysis were applied to additional sample data to down select molecular features that behaved consistently between the two clinical sites and these were evaluated by logistic regression analysis. RESULTS: Analysis of discovery samples identified 45 MFs that significantly changed in abundance at one month of treatment. Down selection using an extended set of discovery samples and qualification samples confirmed 23 MFs that consistently changed in abundance between baseline and 1, 2 and 6 months of therapy, with 12 MFs achieving statistical significance (p < 0.05). Six MFs classified the baseline and 1 month samples with an error rate of 11.8%. CONCLUSIONS: These results define a urine based TB-early treatment response biosignature (TB-ETRB) applicable to different parts of Africa, and provide proof-of-concept for further evaluation of this technology in monitoring clinical responses to TB therapy.

Mycobacterium tuberculosis specific CD8(+) T cells rapidly decline with antituberculosis treatment.

RATIONALE: Biomarkers associated with response to therapy in tuberculosis could have broad clinical utility. We postulated that the frequency of Mycobacterium tuberculosis (Mtb) specific CD8(+) T cells, by virtue of detecting intracellular infection, could be a surrogate marker of response to therapy and would decrease during effective antituberculosis treatment. OBJECTIVES: We sought to determine the relationship of Mtb specific CD4(+) T cells and CD8(+) T cells with duration of antituberculosis treatment. MATERIALS AND METHODS: We performed a prospective cohort study, enrolling between June 2008 and August 2010, of HIV-uninfected Ugandan adults (n = 50) with acid-fast bacillus smear-positive, culture confirmed pulmonary TB at the onset of antituberculosis treatment and the Mtb specific CD4(+) and CD8(+) T cell responses to ESAT-6 and CFP-10 were measured by IFN-γ ELISPOT at enrollment, week 8 and 24. RESULTS: There was a significant difference in the Mtb specific CD8(+) T response, but not the CD4(+) T cell response, over 24 weeks of antituberculosis treatment (p<0.0001), with an early difference observed at 8 weeks of therapy (p = 0.023). At 24 weeks, the estimated Mtb specific CD8(+) T cell response decreased by 58%. In contrast, there was no significant difference in the Mtb specific CD4(+) T cell during the treatment. The Mtb specific CD4(+) T cell response, but not the CD8(+) response, was negatively impacted by the body mass index. CONCLUSIONS: Our data provide evidence that the Mtb specific CD8(+) T cell response declines with antituberculosis treatment and could be a surrogate marker of response to therapy. Additional research is needed to determine if the Mtb specific CD8(+) T cell response can detect early treatment failure, relapse, or to predict disease progression.

Shortening treatment in adults with noncavitary tuberculosis and 2-month culture conversion.

RATIONALE: Cavitary disease and delayed culture conversion have been associated with relapse. Combining patient characteristics and measures of bacteriologic response might allow treatment shortening with current drugs in some patients. OBJECTIVES: To assess whether treatment could be shortened from 6 to 4 months in patients with noncavitary tuberculosis whose sputum cultures converted to negative after 2 months. METHODS: This study was a randomized, open-label equivalence trial. HIV-uninfected adults with noncavitary tuberculosis were treated daily with isoniazid, rifampin, pyrazinamide, and ethambutol for 2 months, followed by 2 months of isoniazid and rifampin. After 4 months, patients with drug-susceptible TB whose sputum cultures on solid media were negative after 8 weeks of treatment were randomly assigned to continue treatment for 2 more months or to stop treatment. Patients were followed for relapse for 30 months after beginning treatment. MEASUREMENTS AND MAIN RESULTS: Enrollment was stopped by the safety monitoring committee after 394 patients were enrolled due to apparent increased risk for relapse in the 4-month arm. A total of 370 patients were eligible for per protocol analysis. Thirteen patients in the 4-month arm relapsed, compared with three subjects in the 6-month arm (7.0 vs. 1.6%; risk difference, 0.054; 95% confidence interval with Hauck-Anderson correction, 0.01-0.10). CONCLUSION: Shortening treatment from 6 to 4 months in adults with noncavitary disease and culture conversion after 2 months using current drugs resulted in a greater relapse rate. The combination of noncavitary disease and 2-month culture conversion was insufficient to identify patients with decreased risk for relapse.