Evaluation of high-dose rifampin in patients with new, smear-positive tuberculosis (HIRIF): study protocol for a randomized controlled trial.

BACKGROUND: Evidence has existed for decades that higher doses of rifampin may be more effective, but potentially more toxic, than standard doses used in tuberculosis treatment. Whether increased doses of rifampin could safely shorten treatment remains an open question. METHODS/DESIGN: The HIRIF study is a phase II randomized trial comparing rifampin doses of 20 and 15 mg/kg/day to the standard 10 mg/kg/day for the first 2 months of tuberculosis treatment. All participants receive standard doses of companion drugs and a standard continuation-phase treatment (4 months, 2 drugs). They are followed for 6 months post treatment. Study participants are adults with newly diagnosed, previously untreated, smear positive (≥2+) pulmonary tuberculosis. The primary outcome is rifampin area under the plasma concentration-time curve (AUC0-24) after at least 14 days of study treatment/minimum inhibitory concentration. 180 randomized participants affords 90 % statistical power to detect a difference of at least 14 mcg/mL*hr between the 20 mg/kg group and the 10 mg/kg group, assuming a loss to follow-up of up to 17 %. DISCUSSION: Extant evidence suggests the potential for increased doses of rifampin to shorten tuberculosis treatment duration. Early studies that explored this potential using intermittent, higher dosing were derailed by toxicity. Given the continued large, global burden of tuberculosis with nearly 10 million new cases annually, shortened regimens with existing drugs would offer an important advantage to patients and health systems. TRIAL REGISTRATION: This trial was registered with clinicaltrials.gov (registration number: NCT01408914 ) on 2 August 2011.

High-dose rifampicin kills persisters, shortens treatment duration, and reduces relapse rate in vitro and in vivo.

Although high-dose rifampicin holds promise for improving tuberculosis control by potentially shortening treatment duration, these effects attributed to eradication of persistent bacteria are unclear. The presence of persistent Mycobacterium tuberculosis was examined using resuscitation promoting factors (RPFs) in both in vitro hypoxia and in vivo murine tuberculosis models before and after treatment with incremental doses of rifampicin. Pharmacokinetic parameters and dose-dependent profile of rifampicin in the murine model were determined. The Cornell mouse model was used to test efficacy of high-dose rifampicin in combination with isoniazid and pyrazinamide and to measure relapse rate. There were large numbers of RPF-dependent persisters in vitro and in vivo. Stationary phase cultures were tolerant to rifampicin while higher concentrations of rifampicin eradicated plate count positive but not RPF-dependent persistent bacteria. In murine infection model, incremental doses of rifampicin exhibited a dose-dependent eradication of RPF-dependent persisters. Increasing the dose of rifampicin significantly reduced the risk of antibiotic resistance emergence. In Cornell model, mice treated with high-dose rifampicin regimen resulted in faster visceral clearance; organs were M. tuberculosis free 8 weeks post-treatment compared to 14 weeks with standard-dose rifampicin regimen. Organ sterility, plate count and RPF-dependent persister negative, was achieved. There was no disease relapse compared to the standard dose regimen (87.5%). High-dose rifampicin therapy results in eradication of RPF-dependent persisters, allowing shorter treatment duration without disease relapse. Optimizing rifampicin to its maximal efficacy with acceptable side-effect profiles will provide valuable information in human studies and can potentially improve current tuberculosis chemotherapy.

HspX knock-out in Mycobacterium tuberculosis leads to shorter antibiotic treatment and lower relapse rate in a mouse model--a potential novel therapeutic target.

Effective global tuberculosis control is hindered by the need for prolonged chemotherapy which leads to poor patient compliance. Therefore novel drug targets that shorten the duration of chemotherapy and reduce disease relapse rates are highly desirable. We have previously shown that HspX, an alpha-crystallin-like protein, is associated with growth suppression of Mycobacterium tuberculosis in mouse models. We determined to evaluate hspX as a novel target for controlling M. tuberculosis growth in combination with traditional antibiotic therapy in the Cornell mouse model. The hspX deletion mutant (ΔhspX) was used as a model of potential hspX inhibition. Normal BALB/c mice were infected with ΔhspX or the wild type (WT) strain. Three weeks after infection, the mice were treated with rifampicin, isoniazid and pyrazinamide for 14 weeks followed by 8 weeks of hydrocortisone. The effect of chemotherapy was measured by organ bacterial counts and the relapse rate. Antibiotic treatment of mice infected with ΔhspX resulted in faster visceral clearance; organs were disease free 8 weeks post-treatment for ΔhspX infection compared to 14 weeks for the WT strain. Disease relapse rate was significantly lower in ΔhspX infection (60.7%) compared to WT infection (92.6%). HspX may be a promising therapeutic target in combination with traditional antibiotic therapy to shorten the length of treatment and reduce disease relapse.

Persister populations of Mycobacterium tuberculosis in sputum that grow in liquid but not on solid culture media.

OBJECTIVES: Can the characteristics of persisters in cultures of Mycobacterium tuberculosis also be found in bacilli from the sputum of pulmonary tuberculosis patients? The objective of this study was to explore whether the ability of persisters to grow in liquid but not on solid culture media, as in 100 day static cultures, can also be found in bacilli in sputum. METHODS: Serial dilutions of homogenized sputum obtained from patients before or during the first week of treatment were inoculated into broths to estimate the probable number of organisms and onto plates to give colony counts. RESULTS: Cultures in broths grew slowly to reach a maximal count at 12 weeks of probable numbers about 10-fold higher than the colony counts on plates, which did not grow beyond the initial count at 3-4 weeks. No such excess growth in liquid medium was found with control log-phase cultures. CONCLUSIONS: About 90% of the bacilli in sputum are persisters that can grow in liquid media but not on solid plates.

Mechanisms of Pyrazinamide Action and Resistance.

Pyrazinamide (PZA) is a unique antituberculosis (anti-TB) drug that plays a key role in shortening TB therapy. PZA kills nonreplicating persisters that other TB drugs fail to kill, which makes it an essential drug for inclusion in any drug combinations for treating drug-susceptible and drug-resistant TB such as multidrug-resistant TB. PZA acts differently from common antibiotics by inhibiting multiple targets such as energy production, trans-translation, and perhaps pantothenate/coenzyme A required for persister survival. Resistance to PZA is mostly caused by mutations in the pncA gene encoding pyrazinamidase, which is involved in conversion of the prodrug PZA to the active form pyrazinoic acid. Mutations in the drug target ribosomal protein S1 (RpsA) are also found in some PZA-resistant strains. The recent finding that panD mutations are found in some PZA-resistant strains without pncA or rpsA mutations may suggest a third PZA resistance gene and a potential new target of PZA. Current phenotype-based PZA susceptibility testing is not reliable due to false resistance; sequencing of the pncA gene represents a more rapid, cost-effective, and reliable molecular test for PZA susceptibility testing and should be used for guiding improved treatment of multidrug-resistant and extensively multidrug-resistant TB. Finally, the story of PZA has important implications for not only TB therapy but also chemotherapy in general. PZA serves as a model prototype persister drug and hopefully a "tipping point" that inspires new efforts at developing a new type of antibiotic or drug that targets nonreplicating persisters for improved treatment of not only TB but also other persistent bacterial infections.

Mechanisms of Pyrazinamide Action and Resistance.

PZA is a unique anti-tuberculosis drug that plays a key role in shortening the TB therapy. PZA kills non-replicating persisters that other TB drugs fail to kill, and thus making it an essential drug for inclusion in any drug combinations for treating drug susceptible and drug-resistant TB such as MDR-TB. PZA acts differently from common antibiotics by inhibiting multiple targets such as energy production, trans-translation and perhaps pantothenate /coenzyme A required for persister survival. Resistance to PZA is mostly caused by mutations in the pncA gene encoding pyrazinamidase involved in conversion of the prodrug PZA to the active form POA. Mutations in the drug target RpsA are also found in some PZA-resistant strains. The recent finding that panD mutations are found in some PZA-resistant strains without pncA or rpsA mutations may suggest a third PZA resistance gene and a potential new target of PZA. Current phenotype based PZA susceptibility testing is not reliable due to false resistance, and sequencing of the pncA gene represents a more rapid, cost-effective and more reliable molecular test for PZA susceptibility testing and should be used for guiding improved treatment of MDR/XDR-TB. Finally, the story of PZA has important implications for not only TB therapy but also chemotherapy in general. PZA serves as a model prototype persister drug and hopefully a 'tipping point' that inspires new efforts at developing a new type of antibiotics or drugs that target non-replicating persisters for improved treatment of not only TB but also other persistent bacterial infections.

Prevention of drug resistance by combined drug treatment of tuberculosis.

Treatment with a combination of anti-tuberculosis drugs is thought to work by the first drug killing mutants resistant to the second drug, while the second drug kills those resistant to the first drug. Combined treatment has been remarkably successful in preventing the emergence of resistance during the treatment of tuberculosis. This success has led to the introduction of multi-drug treatment for leprosy, HIV infections and cancer. Its success in tuberculosis depends on a number of conditions such as the chromosomal nature of drug resistance in Mycobacterium tuberculosis and the absence of plasmids carrying resistance factors as well as the manner in which the bacterial population in tuberculosis does not come into contact with other potentially resistant bacteria. For multi-drug treatment to be effective in preventing resistance, the drugs must be sufficiently active so that each can inhibit all the bacteria in lesions. There must also be effective post-antibiotic lags in growth restarting to prevent growth between doses. Special bacterial populations that are drug tolerant or survive drug action unusually successfully are also a potential source of resistance.

Optimizing outpatient serial sputum colony counting for studies of tuberculosis treatment in resource-poor settings.

Serial Sputum Colony Counting (SSCC) is an important technique in clinical trials of new treatments for tuberculosis (TB). Quantitative cultures on selective Middlebrook agar are used to calculate the rate of bacillary elimination from sputum collected from patients at different time points during the first 2 months of therapy. However, the procedure can be complicated by high sample contamination rates. This study, conducted in a resource-poor setting in Malawi, assessed the ability of different antifungal drugs in selective agar to reduce contamination. Overall, 229 samples were studied and 15% to 27% were contaminated. Fungal organisms were particularly implicated, and samples collected later in treatment were at particular risk (P < 0.001). Amphotericin B (AmB) is the standard antifungal drug used on SSCC plates at a concentration of 10 mg/ml. On selective Middlebrook 7H10 plates, AmB at 30 mg/ml reduced sample contamination by 17% compared with AmB at 10 mg/ml. The relative risk of contamination using AmB at 10 mg/ml was 1.79 (95% confidence interval [CI], 1.25 to 3.55). On Middlebrook 7H11 plates, a combination of AmB at 10 mg/ml and carbendazim at 50 mg/ml was associated with 10% less contamination than AmB at 30 mg/ml. The relative risk of contamination with AmB at 30 mg/ml was 1.79 (95% CI, 1.01 to 3.17). Improved antifungal activity was accompanied by a small reduction in bacillary counts, but this did not affect modeling of bacillary elimination. In conclusion, a combination of AmB and carbendazim optimized the antifungal activity of selective media for growth of TB. We recommend this method to reduce contamination rates and improve SSCC studies in African countries where the burden of TB is highest.

Effects of low incubation temperatures on the bactericidal activity of anti-tuberculosis drugs.

OBJECTIVES: to explore the effect of low incubation temperatures and the consequent slowing of bacterial metabolism on the bactericidal action of anti-tuberculosis drugs against Mycobacterium tuberculosis. METHODS: counting of surviving bacteria during exposure of static cultures to 1 mg/L isoniazid, 2 mg/L rifampicin, 0.5 or 2 mg/L TMC207 and 40 or 160 mg/L pyrazinamide, usually for periods of 21 days at temperatures of 37, 25, 22, 19, 16 or 8°C. RESULTS: the bactericidal activities of isoniazid and rifampicin were progressively reduced at 25 and 22°C, and were minimal at lower temperatures. TMC207 was immediately bactericidal at 37°C, in contrast to the early static phase reported with log phase cultures, and showed less change in activity as incubation temperatures were reduced than did rifampicin or isoniazid. Pyrazinamide was more bactericidal when incubation temperatures were reduced below 37°C and when the static seed cultures were most dormant. CONCLUSIONS: these results can be explained by the surmise that at low temperatures bacterial energy is at a low level with only just sufficient ATP to maintain homeostasis, making the bacteria more susceptible to the blocking of ATP synthesis by TMC207. Insufficient ATP at low temperature would also hinder the export of pyrazinoic acid, the toxic product of the pro-drug pyrazinamide, from the mycobacterial cell by an inefficient efflux pump that requires energy.

Bactericidal activity of the diarylquinoline TMC207 against Mycobacterium tuberculosis outside and within cells.

The bactericidal activities of the diarylquinoline TMC207 in a liquid culture medium started with a bacteriostatic phase lasting about 7 days and then continued with a dose-related bactericidal phase. In comparison, its intra-cellular activity in primary mouse peritoneal macrophages (PM) and in the J774 macrophage-like cell line had little or no static phase so that the bactericidal kill was evident by 5-7 days presumably due to low bacterial ATP levels. Bactericidal activities in the three systems were compared by estimating the rate of bacterial killing (K) during exposure to 0.12-1.0 µg/ml TMC207 which were similar at, -0.35 in the J774 cells and -0.27 in mouse PM (p = 0.6) with each lower than -0.11 in extra-cellular cultures (p < 0.001) and [2] the TMC207 concentration at the intersection between the curve relating cfu count to TMC207 concentration and the cfu count at day-0, defined as the static concentration. Static concentrations were 0.22 µg/ml for extra-cellular cultures, 0.17 µg/ml for mouse PM and 0.06 µg/ml for J774 cells, significantly lower than the extra-cellular value (p < 0.001). Thus, the intra-cellular activity of TMC207 is clearly greater than its extra-cellular activity mainly because the preliminary static phase was a shorter or absent.

Effects of four different meal types on the population pharmacokinetics of single-dose rifapentine in healthy male volunteers.

Rifapentine and its primary metabolite, 25-desacetyl rifapentine, are active against mycobacterium tuberculosis. The objectives of this study were to describe the population pharmacokinetics of rifapentine and 25-desacetyl rifapentine in fasting and fed states. Thirty-five male healthy volunteers were enrolled in an open-label, randomized, sequential, five-way crossover study. Participants received a single 900-mg dose of rifapentine after meals with high fat (meal A), bulk and low fat (meal B), bulk and high fat (meal C), high fluid and low fat (meal D), or 200 ml of water (meal E). Venous blood samples were collected over 72 h after each rifapentine dose, and plasma was analyzed for rifapentine and 25-desacetyl rifapentine using high-performance liquid chromatography. Pharmacokinetic data were analyzed by nonlinear mixed-effect modeling using NONMEM. Compared with the fasting state, meal A had the greatest effect on rifapentine oral bioavailability, increasing it by 86%. Meals B, C, and D resulted in 33%, 46%, and 49% increases in rifapentine oral bioavailability, respectively. Similar trends were observed for 25-desacetyl rifapentine. As meal behavior has a substantial impact on rifapentine exposure, it should be considered in the evaluation of optimal dosing approaches.

Examining the basis of isoniazid tolerance in nonreplicating Mycobacterium tuberculosis using transcriptional profiling.

BACKGROUND: Understanding how growth state influences Mycobacterium tuberculosis responses to antibiotic exposure provides a window into drug action during patient chemotherapy. In this article, we describe the transcriptional programs mediated by isoniazid (INH) during the transition from log-phase to nonreplicating bacilli, from INH-sensitive to INH-tolerant bacilli respectively, using the Wayne model. RESULTS: INH treatment did not elicit a transcriptional response from nonreplicating bacteria under microarophilic conditions (NRP2), unlike the induction of a robust and well-characterized INH signature in log-phase bacilli. CONCLUSION: The differential regulation (between drug-free NRP2 and log-phase bacilli) of genes directly implicated in INH resistance could not account for the abrogation of INH killing in nongrowing bacilli. Thus, factors affecting the requirement for mycolic acids and the redox status of bacilli are likely responsible for the reduction in INH efficacy. We speculate on additional mechanisms revealed by transcriptome analysis that might account for INH tolerance.

Tuberculosis diagnosis and multidrug resistance testing by direct sputum culture in selective broth without decontamination or centrifugation.

Tuberculosis culture usually requires sputum decontamination and centrifugation to prevent cultures from being overgrown by contaminating bacteria and fungi. However, decontamination destroys many tuberculous bacilli, and centrifugation often is not possible in resource-poor settings. We therefore assessed the performance of Mycobacterium tuberculosis culture with unprocessed samples plated directly by using tuberculosis-selective media and compared this procedure to conventional culture using centrifuge decontamination. Quadruplicate aliquots of strain H37RV were cultured in 7H9 broth with and without selective antimicrobials and after centrifuge decontamination. The subsequent comparison was made with 715 sputum samples. Split paired sputum samples were cultured conventionally with centrifuge decontamination and by direct culture in tuberculosis-selective media containing antibiotics. Centrifuge decontamination reduced tuberculosis H37RV colonies by 78% (P < 0.001), whereas direct culture in tuberculosis-selective media had no inhibitory effect. Similarly, in sputum cultures that were not overgrown by contaminants, conventional culture yielded fewer tuberculosis colonies than direct culture (P < 0.001). However, the sensitivity of conventional culture was greater than that of direct culture, because samples were less affected by contamination. Thus, of the 340 sputum samples that were tuberculosis culture positive, conventional culture detected 97%, whereas direct culture detected 81% (P < 0.001). Conventional and direct cultures both took a median of 8.0 days to diagnose tuberculosis (P = 0.8). In those direct cultures that detected drug resistance or susceptibility, there was a 97% agreement with the results of conventional culture (Kappa agreement statistic, 0.84; P < 0.001). Direct culture is a simple, low-technology, and rapid technique for diagnosing tuberculosis and determining drug susceptibility. Compared to that of conventional culture, direct culture has reduced sensitivity because of bacterial overgrowth, but in basic laboratories this deficit may be outweighed by the ease of use.

A new antituberculosis drug that selectively kills nonmultiplying Mycobacterium tuberculosis.

An important report by Bryk et al. in this issue of Cell Host & Microbe describes the properties of a rhodanine prodrug active against nonmultiplying Mycobacterium tuberculosis (Mtb). Considering the tolerance of nonreplicating Mtb to most currently available agents, such a drug could be a major addition to our antituberculosis arsenal and would greatly benefit control of the disease.

Assessment of the Efficacy of New Anti-Tuberculosis Drugs.

The pathology of tuberculosis in humans starts with an initial Ghon focus in the lungs followed by transmission of bacilli though the blood and lymph to other regions in the lungs and to other organs. While these bacilli usually lie latent without causing further disease, some 10% start foci of adult type disease usually starting in the sub-apical regions of the lungs. Bacilli multiply, killing tissue by caseation and then forming colonies within the caseum. Cavities form connecting to the air in whose walls vigorous bacillary multiplication occurs. The history of the development of anti-tuberculosis chemotherapy is described, starting with the use of multi-drug regimens to prevent the emergence of drug resistance and continuing with the shortening of the treatment period to 6 months by the incorporation in the regimens of rifampicin and pyrazinamide, which are the two drug responsible for bactericidal activity during treatment. Prospects for further shortening of treatment rest with the introduction of higher dosage with rifamycins and with new anti-tuberculosis drugs. These new drugs include the 8 methoxyfluoroquinolones moxifloxacin and gatifloxacin which inhibit topoisomerases and protein formation, the diarylquinoline TM-207 which inhibits the mycobacterial ATP synthase and thus energy formation, the nitroimidazopyran PA-824 and the closely related OPC-676832 which are pro-drugs with uncertain modes of action and the pyrrole SQ-109, a cell wall inhibitor. Anti-tuberculosis drugs have widely variable pharmacokinetic characteristics but as they work efficiently together, it is unnecessary to match these when giving drug combinations. The effects of drug-drug interactions are usually small though the interactions with anti-retroviral drugs can pose problems. Dose sizes have usually been chosen to minimize side effects while retaining activity and thus tend to have low therapeutic margins, the exception being the margin of about 20 for isoniazid. The role of high plasma binding, important in limiting the efficacy of rifamycins, is uncertain for the newer drugs. Post antibiotic effects are vital to the prevention of drug resistance and need exploration for new drugs. The main aims of current drug development are (1) to shorten treatment, and (2) to make it more convenient, by for instance using widely intermittent regimens. The current techniques for measuring efficacy during drug development start with in vitro models, including the Hu/Coates models, which should contain bacterial populations resembling the bacterial persisters in lesions that are responsible for the long duration of treatment. The next stage is the mouse model of the chemotherapy of established tuberculosis, which has proved remarkably useful in assessing the value of the different drugs. The main problem in clinical assessment arises from the use of relapse after treatment as the main end-point, and the consequent need for very large numbers of patients required to provide measurable relapse rates in final phase III licensing studies. For this reason, surrogate studies are necessary in phase II which require much smaller numbers of patients. The first such investigations are phase IIA studies of early bactericidal activity which establish whether the drug given alone has bactericidal activity on cavitary bacilli and which can estimate the minimal effective dose of the drug, useful for decisions of dose size. The next step should be phase IIB studies which measure the rate of elimination of viable bacilli in sputum during the initial 8-weeks of treatment with various combinations of the new drug with established drugs. Measurement can be as (1) the proportion of patients with positive sputum at the end of the 8-weeks period, the easiest method but the least sensitive, or (2) as the speed with which sputum cultures become negative in a survival analysis, or (3) as the mean regression in modeling of serial sputum collections colony counts (SSCC). The relation between these surrogate estimates and the amoun of treatment shortening that can be obtained has still to be worked out.