In African countries with a high tuberculosis burden, the 2-month sputum culture conversion rate is only 50%–70%, and acquired drug resistance (ADR) continues to be a major problem [1–4]. In the laboratory, the hollow-fiber model of tuberculosis has demonstrated that the microbial kill and ADR prevention of first-line anti-tuberculosis agents are driven by such drug concentration measures as the 0–24 hour area under the concentration–time curve (AUC) to minimum inhibitory concentration (MIC) ratio and the peak concentration to MIC ratio [5–7]. These data have been used in computer-aided clinical trial simulations in the face of 100% adherence, which led us to hypothesize that between-patient pharmacokinetic variability could explain a large proportion of therapy failure and that adherence plays a relatively minor role [8, 9]. Here, we investigated whether such pharmacokinetic variability would indeed lead to a large proportion of patients failing to attain adequate concentrations and then failing therapy. We also sought to identify the drug concentrations that are predictive of clinical outcome.

Several attempts to relate drug concentrations to tuberculosis outcomes have been made with conflicting results [10–16]. The reasons are unclear but could be one of several. First, in some studies, a single measure such as the 2-hour drug concentration (peak) was used to dichotomize patients into those with poor vs good outcomes. However, since drug AUCs are strongly associated with efficacy of first-line anti-tuberculosis agents in preclinical models [5–7, 17], a more intensive multisample schedule that allows AUC identification may be more informative.

Second, several studies utilized predetermined peak concentration drug cutoff values to classify patients as having either low or high drug concentrations. These are peak concentrations of 3–5 mg/L for isoniazid, 8–24 mg/L for rifampin, and 20–50 mg/L for pyrazinamide [18]. These concentrations need further validation with regard to clinical outcomes.

Third, noncompartmental pharmacokinetic analysis was utilized in some studies; however, pharmacokinetics of some anti-tuberculosis drugs may be best described using multiple compartments. A fourth possible reason may be the type of statistical analysis used. Biological systems such as anti-tuberculosis drug pharmacokinetics and the tuberculosis disease are best analyzed using nonlinear statistical approaches since they and most natural phenomena are nonlinear systems [19–23]. In linear analysis, complex problems are broken into smaller components that are then solved, after which the solutions are put together (superimposed) and added up to a solution of the whole problem. Nonlinear systems are characterized by discontinuities and relationships of higher-order complexity between components; the total function of the whole system is often more than the linear sum of its components. Therefore, components need to be analyzed in the context of all parameters interacting within the whole system. Here, we utilized classification and regression tree analysis (CART) to examine the role of several clinical factors, including drug concentrations, in toto, as predictors of clinical outcome in our cohort [24–29]. CART uses nonparametric techniques that examine both linear and nonlinear interactions simultaneously in the whole dataset and creates a hierarchy of predictors, starting with the most predictive to the least predictive.

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Serum Drug Concentrations Predictive of Pulmonary Tuberculosis Outcomes

Finding new treatment-shortening antibiotics to improve cure rates and curb the alarming emergence of drug resistance is the major objective of tuberculosis (TB) drug development Using a matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging suite in a biosafety containment facility, we show that the key sterilizing drugs rifampicin and pyrazinamide efficiently penetrate the sites of TB infection in lung lesions Rifampicin even accumulates in necrotic caseum, a critical lesion site where persisting tubercle bacilli reside In contrast, moxifloxacin, which is active in vitro against a subpopulation of Mycobacterium tuberculosis that persists in specific niches under drug pressure and has achieved treatment shortening in mice, does not diffuse well in caseum, concordant with its failure to shorten therapy in recent clinical trials We suggest that such differential spatial distribution and kinetics of accumulation in lesions may create temporal and spatial windows of monotherapy in specific niches, allowing the gradual development of multidrug-resistant TB We propose an alternative working model to prioritize new antibiotic regimens based on quantitative and spatial distribution of TB drugs in the major lesion types found in human lungs The finding that lesion penetration may contribute to treatment outcome has wide implications for TB

The association between sterilizing activity and drug distribution into tuberculosis lesions

The 2-year follow-up results for a randomized placebo-controlled study of 47 patients with multidrug-resistant pulmonary tuberculosis treated with either the new diarylquinoline TMC207, recently renamed bedaquiline, or placebo, added to the first 8 weeks of a background regimen, are presented. Bedaquiline significantly reduced the time to culture conversion over 24 weeks (hazard ratio, 2.253; 95% confidence interval, 1.08 to 4.71; P = 0.031). With the exception of nausea reported in 26% of patients receiving bedaquiline and none receiving placebo, adverse events occurred at similar frequencies in both groups of patients: bilateral hearing impairment, extremity pain, acne, and noncardiac chest pain occurred in 13 and 21%, 17 and 13%, 9 and 17%, and 4 and 17% of patients, respectively, receiving bedaquiline or placebo. Excluding resistance to ethambutol and ethionamide, only one patient receiving bedaquiline acquired resistance to companion drugs, but five patients receiving placebo (4.8% versus 21.7%; P = 0.18) acquired resistance to companion drugs, and resistance to ofloxacin was acquired in four patients receiving placebo and none receiving bedaquiline (0% versus 22%; 0 = 0.066). In all, 23 patients (49%), including 13 receiving placebo (54%) and 10 receiving bedaquiline (44%), discontinued the study prior to its completion, 12 during the first 24 weeks of treatment. Eight subjects were withdrawn for noncompliance or default, and seven withdrew consent, citing the rigorous program of investigations for safety and pharmacokinetic monitoring. Bedaquiline may contribute to the management of multidrug-resistant tuberculosis by effecting more rapid sputum culture negativity and by preventing acquired resistance to companion drugs.

Randomized Pilot Trial of Eight Weeks of Bedaquiline (TMC207) Treatment for Multidrug-Resistant Tuberculosis: Long-Term Outcome, Tolerability, and Effect on Emergence of Drug Resistance

M. tuberculosis is evolving antibiotic resistance, threatening attempts at tuberculosis epidemic control. Mechanisms of resistance, including genetic changes favored by selection in resistant isolates, are incompletely understood. Using 116 newly sequenced and 7 previously sequenced M. tuberculosis whole genomes, we identified genome-wide signatures of positive selection specific to the 47 drug-resistant strains. By searching for convergent evolution--the independent fixation of mutations in the same nucleotide position or gene--we recovered 100% of a set of known resistance markers. We also found evidence of positive selection in an additional 39 genomic regions in resistant isolates. These regions encode components in cell wall biosynthesis, transcriptional regulation and DNA repair pathways. Mutations in these regions could directly confer resistance or compensate for fitness costs associated with resistance. Functional genetic analysis of mutations in one gene, ponA1, demonstrated an in vitro growth advantage in the presence of the drug rifampicin.

Genomic analysis identifies targets of convergent positive selection in drug-resistant Mycobacterium tuberculosis

Drug-susceptible tuberculosis is currently treated with isoniazid, rifampin, and pyrazinamide as part of a directly observed therapy–short-course strategy (DOTS). However, therapy success is threatened by the emergence of multidrug-resistant tuberculosis (MDR-tuberculosis), defined as simultaneous resistance to isoniazid and rifampin. It is believed that most MDR-tuberculosis arises due to poor adherence, hence the need for a DOTS. The World Health Organization (WHO) has built DOTS into the global approach for tuberculosis control. However, the level of nonadherence associated with the emergence of MDR-tuberculosis is unknown; thus the program requires that all tuberculosis patients be supervised by healthcare workers while swallowing antibiotics. The WHO Director-General has called DOTS the most important health breakthrough of past decades [1].

Most studies that established the utility of directly supervised therapy were retrospective and relied on examining rates of MDR-tuberculosis before and after implementation of DOTS programs [2, 3]. However, DOTS has 5 components, including provision of an adequate and standardized drug supply, which could lead to reduction of MDR-tuberculosis rates independent of adherence. Recently, a meta-analysis questioned the efficacy of direct supervision by a healthcare worker [4]. Moreover, despite >98% adherence, extensive emergence of MDR-tuberculosis still occurs [5]. We were interested in determining whether poor adherence leads to therapy failure and to MDR-tuberculosis emergence. Because DOTS is now accepted worldwide, it would be neither ethical nor desirable to perform a randomized clinical study to answer these questions. Therefore, we examined the questions in our hollow fiber system (HFS) model of tuberculosis.

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Multidrug-Resistant Tuberculosis Not Due to Noncompliance but to Between-Patient Pharmacokinetic Variability

We investigated the emergence and evolution of drug-resistant tuberculosis (TB) in an HIV co-infected population at a South African gold mine with a well-functioning TB control program. Of 128 patients with drug-resistant TB diagnosed during January 2003-November 2005, a total of 77 had multidrug-resistant (MDR) TB, 26 had pre-extensively drug-resistant TB (XDR TB), and 5 had XDR TB. Genotyping suggested ongoing transmission of drug-resistant TB, and contact tracing among case-patients in the largest cluster demonstrated multiple possible points of contact. Phylogenetic analysis demonstrated stepwise evolution of drug resistance, despite stringent treatment adherence. These findings suggested that existing TB control measures were inadequate to control the spread of drug-resistant TB in this HIV co-infected population. Diagnosis delay and inappropriate therapy facilitated disease transmission and drug-resistance. These data call for improved infection control measures, implementation of rapid diagnostics, enhanced active screening strategies, and pharmacokinetic studies to determine optimal dosages and treatment regimens.

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Odelia J. Strauss

Papers

Emergence of increased resistance and extensively drug-resistant tuberculosis despite treatment adherence, South Africa.