Christian Lienhardt and Payam Nahid launch the Collection on Advances in Clinical Trial Design for Development of New Tuberculosis Treatments.

To tackle the global TB epidemic effectively, novel treatment strategies are critically needed to shorten the duration of TB therapy and treat drug-resistant TB. Drug development for TB, stymied for decades, has enjoyed a renaissance over the past several years. However, the development of new TB regimens is hindered by the limitations in our understanding and use of preclinical models; the paucity of accurate, early surrogate markers of cure, and challenges in untangling the individual contributions of drugs to multidrug regimens in a complex, multi-compartment disease. Lack of profit motive, advocacy, and imagination has contributed mightily to the dearth of drugs we have on the shelf to treat this ancient disease. Areas that will speed the development of new regimens for TB include novel murine and in vitro pharmacodynamics models, clinical endpoints that are not culture-based, innovative clinical trial designs, and an infusion of much-needed funding.

Andrew Vernon and co-authors discuss adherence to therapy and its measurement in tuberculosis treatment trials.

Christian Lienhardt and colleagues discuss the importance of communication and coordination between regulators, researchers, and policy makers to ensure tuberculosis trials provide high-quality evidence for policy decisions.

Background

Mycobacterium tuberculosis (Mtb) is the causative agent of Tuberculosis (TB), the number one cause of death due to an infectious disease. TB diagnosis is performed by microscopy, culture or PCR amplification of bacterial DNA, all of which require patient sputum or the biopsy of infected tissue. Detection of mycobacterial products in serum, as biomarkers of diagnosis or disease status would provide an improvement over current methods. Due to the low-abundance of mycobacterial products in serum, we have explored exosome enrichment to improve sensitivity. Mtb resides intracellularly where its secreted proteins have been shown to be packaged into host exosomes and released into the bloodstream. Exosomes can be readily purified assuring an enrichment of mycobacterial analytes from the complex mix of host serum proteins.

Methods

Multiple reaction monitoring assays were optimized for the enhanced detection of 41 Mtb peptides in exosomes purified from the serum of individuals with TB. Exosomes isolated from the serum of healthy individuals was used to create and validate a unique data analysis algorithm and identify filters to reduce the rate of false positives, attributed to host m/z interference. The final optimized method was tested in 40 exosome samples from TB positive patients.

Results

Our enhanced methods provide limit of detection and quantification averaging in the low femtomolar range for detection of mycobacterial products in serum. At least one mycobacterial peptide was identified in 92.5% of the TB positive patients. Four peptides from the Mtb proteins, Cfp2, Mpt32, Mpt64 and BfrB, show normalized total peak areas significantly higher in individuals with active TB as compared to healthy controls; three of the peptides from these proteins have not previously been associated with serum exosomes from individuals with active TB disease. Some of the detected peptides were significantly associated with specific geographical locations, highlighting potential markers that can be linked to the Mtb strains circulating within each given region.

Conclusions

An enhanced MRM method to detect ultra-low abundance Mtb peptides in human serum exosomes is demonstrated, highlighting the potential of this methodology for TB diagnostic biomarker development.

Rationale: No evidence-based tools exist to enhance precision in the selection of patient-specific optimal treatment durations to study in tuberculosis clinical trials. Objectives: To develop risk stratification tools that assign patients with tuberculosis into risk groups of unfavorable outcome and inform selection of optimal treatment duration for each patient strata to study in clinical trials. Methods: Publicly available data from four phase 3 trials, each evaluating treatment duration shortening from 6 to 4 months, were used to develop parametric time-to-event models that describe unfavorable outcomes. Regimen, baseline, and on-treatment characteristics were evaluated as predictors of outcomes. Exact regression coefficients of predictors were used to assign risk groups and predict optimal treatment durations. Measurements and Main Results: The parametric model had an area under the receiver operating characteristic curve of 0.72. A six-item risk score (HIV status, smear grade, sex, cavitary disease status, body mass index, and Month 2 culture status) successfully grouped participants into low (1,060/3,791; 28%), moderate (1,740/3,791; 46%), and high (991/3,791; 26%) risk, requiring treatment durations of 4, 6, and greater than 6 months, respectively, to reach a target cure rate of 93% when receiving standard-dose rifamycin-containing regimens. With current one-duration-fits-all approaches, high-risk groups have a 3.7-fold (95% confidence interval, 2.7-5.1) and 2.4-fold (1.9-2.9) higher hazard risk of unfavorable outcomes compared with low- and moderate-risk groups, respectively. Four-month regimens were noninferior to the standard 6-month regimen in the low-risk group. Conclusions: Our model discrimination was modest but consistent with current models of unfavorable outcomes. Our results showed that stratified medicine approaches are feasible and may achieve high cure rates in all patients with tuberculosis. An interactive risk stratification tool is provided to facilitate decision-making in the regimen development pathway.

The role of microbial factors in outcomes of tuberculosis treatment has not been well studied. We performed a case-control study to evaluate the association between a Beijing strain and tuberculosis treatment outcomes. Isolates from patients with culture-positive treatment failure (n = 8) or relapse (n = 54) were compared with isolates from randomly selected controls (n = 296) by using spoligotyping. Patients with Beijing strains had a higher risk for relapse (odds ratio [OR] 2.0, 95% confidence interval [CI] 1.0-4.0, p = 0.04) but not for treatment failure. Adjustment for factors previously associated with relapse had little effect on the association between Beijing strains and relapse. Beijing strains were strongly associated with relapse among Asian-Pacific Islanders (OR 11, 95% CI 1.1-108, p = 0.04). Active disease caused by a Beijing strain was associated with increased risk for relapse, particularly among Asian-Pacific Islanders.

BACKGROUND: A better understanding of treatment progression and recovery in pulmonary tuberculosis (TB) infectious disease is crucial. This study analyzed longitudinal serum samples from pulmonary TB patients undergoing interventional treatment to identify surrogate markers for TB-related outcomes. METHODS: Serum that was collected at baseline and 8, 17, 26, and 52 weeks from 30 TB patients experiencing durable cure were evaluated and compared using a sensitive LC-MS/MS proteomic platform for the detection and quantification of differential host protein signatures relative to timepoint. The global proteome signature was analyzed for statistical differences across the time course and between disease severity and treatment groups. RESULTS: A total of 676 proteins showed differential expression in the serum over these timepoints relative to baseline. Comparisons to understand serum protein dynamics at 8 weeks, treatment endpoints at 17 and 26 weeks, and post-treatment at 52 weeks were performed. The largest protein abundance changes were observed at 8 weeks as the initial effects of antibiotic treatment strongly impacted inflammatory and immune modulated responses. However, the largest number of proteome changes was observed at the end of treatment time points 17 and 26 weeks respectively. Post-treatment 52-week results showed an abatement of differential proteome signatures from end of treatment, though interestingly those proteins uniquely significant at post-treatment were almost exclusively downregulated. Patients were additionally stratified based upon disease severity and compared across all timepoints, identifying 461 discriminating proteome signatures. These proteome signatures collapsed into discrete expression profiles with distinct pathways across immune activation and signaling, hemostasis, and metabolism annotations. Insulin-like growth factor (IGF) and Integrin signaling maintained a severity signature through 52 weeks, implying an intrinsic disease severity signature well into the post-treatment timeframe. CONCLUSION: Previous proteome studies have primarily focused on the 8-week timepoint in relation to culture conversion status. While this study confirms previous observations, it also highlights some differences. The inclusion of additional end of treatment and post-treatment time points offers a more comprehensive assessment of treatment progression within the serum proteome. Examining the expression dynamics at these later time periods will help in the investigation of relapse patients and has provided indicative markers of response and recovery.

BACKGROUND: Pyrazinamide (PZA) is a first line agent for the treatment of active tuberculosis. PZA is also considered a potent companion drug for newer regimens under development. There are limited data on the demographic, clinical, and pathogen characteristics of PZA resistant tuberculosis. METHODS: Using a retrospective cohort study design, we evaluated all PZA resistant M. tuberculosis (M.tb) and M. bovis cases reported in San Francisco from 1991 to 2011. Demographic, clinical, and molecular data were analyzed. M.tb lineage was determined for all PZA resistant strains and compared to PZA susceptible strains. RESULTS: PZA resistance was identified in 1.8% (50 of 2,842) of mycobacterial isolates tested, corresponding to a case rate of 0.3 per 100,000 in the population. Monoresistant PZA infection was associated with the Hispanic population ([OR], 6.3; 95% [CI], 1.97-20.16) and 48% of cases were due to M. bovis. Infection with monoresistant PZA was also associated with extrapulmonary disease ([OR], 6.0; 95% [CI], 2.70-13.26). There was no statistically significant difference between treatment failure and mortality rates in patients infected with PZA monoresistance compared to pansusceptible controls (4% vs. 8%, p = 0.51), or those with PZA and MDR resistance (PZA-MDR) compared to MDR controls (18% vs. 29%, p = 0.40). PZA resistance was not associated with M.tb lineage. CONCLUSIONS: Across two decades of comprehensive epidemiologic data on tuberculosis in San Francisco County, PZA resistance was uncommon. PZA resistance caused predominantly extrapulmonary disease and was more common in Hispanics compared to other ethnicities, with nearly half the cases attributed to M. bovis. No association was found between PZA monoresistance and M.tb lineage. Treatment outcomes were not adversely influenced by the presence of PZA resistance.