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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.

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.

Tuberculosis is the most serious infectious disease caused by a single organism, Mycobacterium tuberculosis (Mtb). The standard of care is a protracted and complex drug treatment regimen made more complicated and of longer duration by the incidence of multiple and extensively drug resistant disease. Pulmonary delivery of aerosols as a supplement to the existing regimen offers the advantage of delivering high local drug doses to the initial site of infection and most prominent organ system involved in disease. Pyrazinamide is used in combination with other drugs to treat tuberculosis. It is postulated that the action of pyrazinoic acid (POA), the active moiety of pyrazinamide, may be enhanced by local pH adjustment, when presented as a salt form. POA was prepared as leucine (POA-leu) and ammonium salts (POA-NH4), spray dried, and characterized in terms of physicochemical properties (melting point, crystallinity, moisture content), aerodynamic performance (aerodynamic particle size distribution, emitted dose), and in vitro inhibitory effect on two mycobacteria (Mtb and Mycobacterium bovis). Particles were prepared in sizes suitable for inhalation (3.3 and 5.4 μm mass median aerodynamic diameter and 61 and 40% of the aerodynamic particle size distribution less than 4.46 μm, as measured by inertial impaction, for POA-leu and POA-NH4, respectively) and with properties (stoichiometric 1:1 ratio of salt to drug, melting points at ∼180 °C, with water content of1%) that would support further development as an inhaled dosage form. In addition, POA salts demonstrated greater potency in inhibiting mycobacterial growth compared with POA alone, which is promising for therapy.

Studies in the mouse and in humans suggest that use of moxifloxacin and gatifloxacin may shorten the duration of treatment of pulmonary tuberculosis. We describe here the in vitro findings with gatifloxacin and moxifloxacin in regimens similar to those that might be used in the treatment of tuberculosis. The bactericidal activities of moxifloxacin and gatifloxacin were measured alone and in different combinations with isoniazid, rifampicin and pyrazinamide against a 30-day, stationary phase culture, at a pH of 5.9. There was a rapid, irregular fall in colony counts during the first 4 days followed by a slower consistent kill during days 4-21 with a mean kill of -0.36 (SD=2.74) and -0.106 (SD=0.011) log(10)CFU/ml/day, respectively. The 4-21-day kill is considered the best assessment of bactericidal activity against persisting bacilli that prolong treatment. The substitution of either of the quinolones for isoniazid in the control regimen of rifampicin, pyrazinamide and isoniazid did not increase bactericidal activity with log CFU of 5.00 and 4.88, but did result in increased bactericidal action with the log CFU of 4.11 and 4.10 for moxifloxacin and gatifloxacin respectively. Moxifloxacin and gatifloxacin had closely similar activities in all drug combinations. Adding moxifloxacin or gatifloxacin to the control regimen resulted in a significant increase in bactericidal action, considered sufficient to reduce the treatment duration.

A randomized comparison has been made of three times weekly rifampin plus isoniazid (HR3) with rifapentine plus isoniazid given once weekly (HRp1) or on 2 of 3 wk (HRp1.2/3) in the continuation phase of 6-mo regimens (each starting with an initial 2 mo of 4-drug therapy) for the treatment of pulmonary tuberculosis in 672 Chinese patients in Hong Kong. Because of poor bioavailability of the rifapentine used (produced in China), its dose size was increased from 600 mg initially to about 750 mg in the last third of patients to obtain serum concentrations similar to those with rifapentine of Western origin; all doses were given after a meal promoting absorption. After initial exclusions, an intent to treat analysis, done on the remaining 592 patients, showed 45 adverse treatment events in 7 of 190 HR3 patients, in 17 of 199 HRp1 patients, and in 21 of 203 HRp1.2/3 patients; of these, 42 were bacteriological or radiographic relapses after the end of treatment (HR3 versus HRp1, p = 0.04; HR3 versus HRp1.2/3, p = 0.01). Patients with organisms initially sensitive or resistant to isoniazid or streptomycin had similar relapse rates. The high relapse rate in the HRp1 regimen suggests that the rifapentine dose should be increased. Similarity of relapse rates, 8.9% and 10.4%, after the HRp1 and HRp1.2/3 regimens, respectively, indicates that irregularity in taking rifapentine/isoniazid could be tolerated. The few adverse side effects in the continuation phase in the rifapentine regimens were less frequent than in the HR3 regimen.

Collections of sputum from 105 patients with newly diagnosed pulmonary tuberculosis were made before and at 1 and 2 d after the start of chemotherapy with isoniazid (INH) alone given to groups of patients in doses of 600 mg, 300 mg, 150 mg, 75 mg, 37.5 mg, 18.75 mg, and 9 mg daily, as well as from an untreated group. Counts of colony forming units (cfu) of Mycobacterium tuberculosis in the collections were set up on plates of selective 7H10 medium. The early bactericidal activity (EBA) of INH was defined as the decrease in log10 cfu/ml sputum/day during the first 2 d of treatment. A smooth curve relating EBA to log dose was obtained, with 600 mg INH yielding the highest mean EBA of 0.539, and 18.75 mg INH yielding the lowest EBA (0.111) that could be distinguished from the bactericidal activity of the untreated group. The ratio of the usual dose of 300 mg INH to the lowest dose, of 18.75 mg, that produced a detectable EBA, termed the therapeutic margin, was therefore 16, in contrast to the lower therapeutic margin of 4 for rifampin. The EBA was related to the INH acetylator genotype of patients treated with 600 mg or 9 mg INH.

The bactericidal actions of ofloxacin and sulbactam-ampicillin, alone and in combination with rifampin and isoniazid, on exponential-phase and stationary-phase cultures of a drug-susceptible isolate of Mycobacterium tuberculosis were studied in vitro. In exponential-phase cultures, all drugs were bactericidal, with the higher concentrations of ofloxacin (5 micrograms/ml) and sulbactam-ampicillin (15 micrograms of ampicillin per ml) being as bactericidal as 1 microgram of isoniazid per ml or 1 microgram of rifampin per ml. In two-drug combinations, both drugs increased the levels of activity of isoniazid and rifampin and were almost as bactericidal as isoniazid-rifampin; they also appeared to increase the level of activity of isoniazid-rifampin in three-drug combinations. In contrast, ofloxacin and sulbactam-ampicillin had little bactericidal activity against stationary-phase cultures and were less active than isoniazid or rifampin alone. Furthermore, in two-drug or three-drug combinations, they did not increase the level of activity of isoniazid, rifampin, or isoniazid-rifampin. These findings suggest that ofloxacin and sulbactam-ampicillin are likely to be most useful in the early stages of treatment and in preventing the emergence of resistance to other drugs but are unlikely to be effective as sterilizing drugs helping to kill persisting lesional bacilli.

The early bactericidal activity of the aminoglycoside paromomycin (aminosidine) in doses of 7.5 and 15 mg/kg of body weight was measured in 22 patients with previously untreated smear-positive pulmonary tuberculosis. The fall in log 10 CFU per milliliter of sputum per day during the first 2 days of treatment for 7 patients receiving a paromomycin dosage of 7.5 mg/kg/day was 0.066, with a standard deviation (SD) of 0.216 and confidence limits from −0.134 to 0.266, and that for 15 patients receiving 15 mg/kg/day was 0.0924, with an SD of 0.140 and confidence limits from 0.015 to 0.170. The difference between the mean and zero was not significant for the 7.5-mg/kg dose group but was significant for the 15-mg/kg dose group ( t = 2.55, P = 0.023). Since paromomycin has no cross-resistance with streptomycin and has no greater toxicity than other aminoglycosides, these results suggest that it has the potential to substitute for streptomycin in antituberculosis regimens and may be a particularly valuable addition to the drug armamentarium for the management of multidrug-resistant tuberculosis.

The bactericidal activity of moxifloxacin, alone and in combination with isoniazid and rifampin, was studied on exponential and stationary phase cultures of Mycobacterium tuberculosis H37 Rv strain, the standard strain which is a wild type of M. tuberculosis strain, not exposed to any environment, susceptible to all anti-tuberculosis drugs. Moxifloxacin alone was highly bactericidal, being intermediate in activity between isoniazid and rifampin on both types of culture. The speed of activity was slow with the stationary phase culture, causing a reduction from 6.41 log(10)cfu/ml to 2.70 log(10)cfu/ml on day 6 with the higher moxifloxacin concentration of 4 microg/ml and to 4.08 log(10)cfu/ml with the lower concentration of 0.25 microg/ml. When added to isoniazid, its activity against both exponential and stationary phase cultures was increased. However, when it was added to rifampin, no increase in activity was found with either type of culture. Addition of moxifloxacin to isoniazid and rifampin resulted in a slight increase in activity against the exponential culture but a considerable increase against the stationary culture with counts below the limit of detection at 4 and 6 days with both moxifloxacin concentrations. The synergism found with isoniazid, but not with rifampin, supports the view that isoniazid should be included in combinations with moxifloxacin during the therapy of pulmonary tuberculosis.

Current treatment for pulmonary tuberculosis (TB) might be shortened by the incorporation of fluoroquinolones (FQs).A Phase II study aimed to assess the sterilising activities of three novel regimens containing FQs before a Phase III trial of a 4-month regimen containing gatifloxacin (GFX).A total of 217 newly diagnosed smear-positive patients were randomly allocated to one of four regimens: isoniazid (INH), pyrazinamide and rifampicin (RMP) with either ethambutol, GFX, moxifloxacin (MFX) or ofloxacin (OFX) for 2 months. At the end of the study, RMP and INH were given for 4 months. The rates of elimination of Mycobacterium tuberculosis were compared in the regimens using non-linear mixed effects modelling of the serial sputum colony counts (SSCC) during the first 8 weeks.After adjustment for covariates, MFX substitution appeared superior during the early phase of a bi-exponential fall in colony counts, but significant and similar acceleration of bacillary elimination during the late phase occurred with both GFX and MFX (P = 0.002). Substitution of OFX had no effect. These findings were supported by estimates of time to conversion, using Cox regression, but there were no significant differences in proportions culture-negative at 8 weeks.GFX and MFX improve the sterilising activity of regimens and might shorten treatment; their progression into Phase III trials therefore seems warranted.

To measure the bactericidal activity of the nitroimidazopyran PA-824 against Mycobacterium tuberculosis, strain H37Rv, in the three Hu/Coates models of bacterial persistence, in comparison with the activity of moxifloxacin (MXF), a drug shown to be likely to shorten treatment in current clinical trials.The bactericidal activity of a wide range of PA-824 and MXF concentrations was tested against a 100-day static, starved, anaerobically-adapted culture in Model 1. In Models 2 and 3, rifampicin (RMP) 100 mg/ml was added to the 100-day culture for 5-7 days and bactericidal activities against surviving tolerant bacilli were tested by addition of the test drugs after removal of RMP in Model 2, and during exposure to RMP in Model 3.PA-824 exhibited little bactericidal activity at low concentrations up to 1.25 microg/ml in each of these models, but high concentrations ofor=10 microg/ml showed considerable bactericidal activity, sufficient to kill all bacilli in Model 3, and appreciably greater than with MXF. However, as PA-824 is 94% plasma bound, concentrations of free drug sufficient to reach the zone of high bactericidal activity may not be obtained in cavities of pulmonary tuberculosis.

Pyrazinamide (PZA) is an effective sterilising drug in tuberculosis, but its mode of action is controversial.To test the bactericidal activity of 1.56-100 microg/ml PZA in Hu/Coates models of dormant and rifampicin (RMP) tolerant Mycobacterium tuberculosis.In model 1, bactericidal activity was tested in pH 5.5 medium against 4-day, 30-day or 100-day static, hypoxic cultures. In models 2 and 3, 100 microg/ml RMP was added to a 100-day culture and PZA was added either during incubation with RMP in model 3, or after resuspension in RMP-free medium in model 2.Model 1: cfu counts on the 100-day and 30-day cultures fell by a maximum of about 1.6 log cfu/ml with increasing culture age, PZA concentration and incubation period, while counts on the 4-day culture showed little change. Model 2: cfu counts at the end of 7 days of recovery showed little bactericidal activity. Model 3: viable bacilli were almost completely eliminated. Bactericidal activity in these models increased with decreasing metabolic bactericidal activity, as measured by the uptake of [3H] uridine into bacterial RNA.PZA differs from other anti-tuberculosis drugs in showing greater bactericidal activity the slower the bacillary metabolic activity, hence its great value as a sterilising drug, likely to remain as an effective companion drug with newer sterilising drugs.

The bactericidal activity of gatifloxacin, alone and in combination with isoniazid and rifampin, was studied on both exponential- and stationary-phase cultures of Mycobacterium tuberculosis strain H37Rv. On log-phase cultures, the bactericidal activity of gatifloxacin at 4 μg/ml was rapid and was very similar to that of isoniazid. At concentrations of 0.25 and 4 μg/ml, gatifloxacin enhanced the activity of isoniazid. Killing of the stationary-phase culture was biphasic. During the first 2 days, gatifloxacin at 4 μg/ml slightly increased the limited bactericidal activities of isoniazid and rifampin. However, no further additional bactericidal activity was found during further incubation with isoniazid alone or when gatifloxacin was added to either isoniazid or rifampin. This suggested that the stationary-phase culture contained a mixture of occasionally dividing bacilli that were killed during the first 2 days and true static persisters in the residual population that mimicked those in human lesions. In view of the failure of gatifloxacin to add to the sterilizing activity of isoniazid or rifampin during days 2 to 6 of exposure in the stationary-phase culture, it is unlikely to be a sterilizing drug that can be used to shorten the duration of treatment appreciably when it is added to present treatment regimens.

Patients with sputum smear-positive, newly diagnosed pulmonary tuberculosis studied at Tygerburg Hospital, Cape Town, for their early response to streptomycin (SM).To determine the standard early bactericidal activity (EBA), namely the fall in viable counts of tubercle bacilli in 16-hour sputum collections during the first 2 days of treatment with SM.Patients were randomised to logarithmically spaced daily doses of 7.5, 15 or 30 mg/kg SM. A comparison by standard biological assay methods was then made with previous estimations of the EBA of paromomycin in doses of 7.5 and 15 mg/kg.An EBA of 0.133 obtained with 30 mg/kg SM differed significantly from zero (P = 0.0009), while the EBAs of 0.043 with 15 mg/kg and -0.025 with 7.5 mg/kg did not so differ. A linear regression equation of EBA = -0.2587 + 0.2627 log10 dose was obtained with significant slope (P = 0.007). Paromomycin was estimated to be 1.745 more potent than SM with wide 95% confidence limits (0.6-28.6), indicating that it cannot be considered more potent than SM.The low EBAs show that SM has low, dose-related, bactericidal activity in cavities, consistent with results from clinical trials. If streptomycin-resistant bacilli are present, paromomycin is probably the aminoglycoside of choice.

Clinical trial in 672 patients with newly diagnosed pulmonary tuberculosis in Hong Kong. After an initial 2 months of a four-drug intensive phase consisting of thrice-weekly streptomycin, isoniazid, rifampicin and pyrazinamide (SHRZ), a random allocation was made to a continuation phase of once-weekly 600 mg rifapentine + 15 mg/kg isoniazid (HRp1), HRp1 given in 2 of every 3 weeks (HRp1.2/3), or to thrice-weekly isoniazid + rifampicin (HR3), the standard treatment in Hong Kong.Final report evaluating adverse events (46 relapses and one failure) after 5 years and the prognostic influence of various factors.Kaplan-Meier analysis of adverse events and Cox proportional hazards analysis of prognostic factors.The two rifapentine regimens, HRp1 and HRp1.2/3 had similar final rates of adverse events of 10.8% and 11.7%, respectively, compared to 4.2% for the HR3 regimen (P = 0.02 and 0.009, respectively). In the initial univariate proportional hazards analysis, adverse events were significantly related to the regimen, age, sex, pretreatment radiographic extent of disease and cavitation, and also to sputum culture at 2 months. In the final multivariate analysis, after step-wise removal of non-significant factors, adverse events were related only to the regimen, patients' sex and pretreatment radiographic extent of disease. Elderly male patients were more at risk of an adverse event, as were those with more severe disease. Adverse events occurred at life table rates of 9.0% in patients with drug-sensitive strains and in 8.9% of those with initially isoniazid-resistant organisms at 5 years.The two rifapentine regimens were unsatisfactory because of their high incidence of adverse events. Isoniazid appeared not to contribute to preventing relapse. Further studies with increased rifapentine dosage are necessary.

Monocytes from purified protein derivative S Mantoux-negative children and young adults inhibited intracellular growth of Mycobacterium microti more in Chingleput than in London. Mycobacterium bovis BCG vaccination did not enhance bacteriostasis with the Indians but did so with the Londoners. No evidence was found for involvement of cytokines such as macrophage-activating factor and granulocyte macrophage colony-stimulating factor in the differences.

Stellenbosch University, a tertiary care hospital in Cape Town, South Africa.To determine the early bactericidal activity (EBA) of amikacin in dosages of 5 mg/kg, 10 mg/kg and 15 mg/kg body weight in comparison to that of isoniazid 6 mg/kg body weight or no drug.An open, randomised trial.Patients with previously untreated, sputum smear-positive pulmonary tuberculosis.Patients received amikacin 5 mg/kg (12 patients), 10 mg/kg (13 patients) or 15 mg/kg (15 patients), isoniazid 6 mg/kg (9 patients) or no drug (10 patients).The rate of decrease in log viable colony forming units of Mycobacterium tuberculosis per ml of sputum per day during the first 2 days of treatment with amikacin 5 mg/kg, 10 mg/kg and 15 mg/kg was 0.041 (SD 0.100), 0.045 (SD 0.144) and 0.052 (SD 0.096), respectively, 0.515 (SD 0.173) in the patients receiving isoniazid 6 mg/kg, and 0.041 (SD 0.113) in those receiving no drug. The EBA found in patients receiving amikacin did not differ significantly from that of the no drug group. However, as the EBA in the no drug group was the highest ever encountered at Stellenbosch University, the mean in patients receiving drug was tested against 0 and found to differ significantly (P = 0.03), suggesting minimal activity. Mean amikacin serum concentrations 1 hour after intramuscular drug administration were 13.5 microg/ml, 26.7 microg/ml and 39.2 microg/ml in the patients receiving 5 mg, 10 mg and 15 mg per kg body weight, respectively.Despite serum concentrations well in excess of the minimal inhibitory concentration of 2-4 microg/ml, the EBA of amikacin in patients with smear-positive pulmonary tuberculosis was only just detectable.

During the course of chemotherapy, certain drugs are predominant in their bactericidal activities. Isoniazid is responsible for an initial kill of about 95% of organisms during the first 2 days of treatment. Its bactericidal role is then replaced by rifampicin and pyrazinamide during the intensive phase. In the continuation phase with an isoniazid/rifampicin regimen, rifampicin is the only effective drug against persisters, as shown by the similarity of response by patients with initially isoniazid-resistant or sensitive strains. If the continuation phase regimen does not contain rifampicin but does contain isoniazid, the dominant bactericidal drug is isoniazid. In this case, the response of patients with initial isoniazid resistance is appreciably less good than in those with sensitive organisms. The review suggests exploration in randomised control trials of a continuation phase of rifampicin (or rifapentine) alone. It also suggests the importance of the dose size of rifampicin and the need for exploring a higher dose. Finally, it emphasises the importance of finding drugs that act on persisting organisms that are phenotypically but not genetically resistant to rifampicin.

Clinical trial amongst 762 patients with newly diagnosed pulmonary tuberculosis in Hong Kong. After an initial 2 months of a four-drug intensive phase consisting of streptomycin, isoniazid, rifampicin and pyrazinamide (SHRZ), a random allocation in continuation to once-weekly rifapentine + isoniazid (HRp1), HRp1 given in 2 of every 3 weeks (HRp1.2/3), or to three times weekly isoniazid + rifampicin (HR3).Interim report evaluating progress of study and the role of isoniazid in the continuation phase.Kaplan-Meier analysis and response of patients related to susceptibility of pretreatment organisms to isoniazid and to rate of isoniazid acetylation determined by NAT2 genotyping.In the 30-month follow-up, rates for adverse treatment events (failure and relapse) were 4.2% in the HR3, 10.2% in the HRp1 and 11.2% in the HRp1.2/3 series (P = 0.02 for HR3 vs HRp1 and P = 0.01 for HR3 vs HRp1.2/3). Occurrence of adverse events was not related to initial susceptibility to isoniazid nor to the rate of acetylation of isoniazid.The two rifapentine regimens had similar final rates of adverse events which were unsatisfactory. Isoniazid had little or no activity in the continuation phase, indicating that no improvement of the continuation regimen is likely to be obtained by alteration of the isoniazid dosage.

Mycobacterium tuberculosis converts pyrazinamide to its active form by using the enzyme pyrazinamidase. This enzyme is coded for on the pncA gene, and mutations in the pncA gene result in absence of active enzyme, conferring resistance to the drug pyrazinamide. We investigated 27 strains of Mycobacterium tuberculosis suspected of being multidrug resistant. Each isolate was tested for susceptibility to pyrazinamide by the BACTEC 460TB method, and 19 were pyrazinamide resistant. The presence of active pyrazinamidase enzyme was sought by using the Wayne assay, which was positive in all of the sensitive isolates and four of the resistant isolates. The pncA gene was amplified by PCR, and mutations were sought by single-strand conformation polymorphism (SSCP) analysis. We identified four isolates which were phenotypically resistant to pyrazinamide, but which had active pyrazinamide enzyme on the Wayne assay and normal pncA gene SSCP. MICs measured by BACTEC 460TB and susceptibility testing at a lower pH of 5.5 confirmed genuine resistance. The pncA gene was sequenced in these four isolates and found not to have any mutations. This implies that an alternative mechanism of resistance exists in these strains. We conclude that genotypic assessment of pyrazinamide resistance is unreliable, because it depends on the identification of a single resistance mechanism. Phenotypic methods such as the BACTEC 460TB technique remain the best methods for pyrazinamide susceptibility testing.

In the study of the activity of PA-824 in mice performed by Nuermberger et al. ([3][1]), the authors observe that “regimens were not statistically different on the primary outcome” and propose that new studies using shorter regimens are needed “to determine conclusively whether substitution of

Experiments in vitro on the bactericidal activity of metronidazole and in the Cornell model of murine tuberculosis.To assess the sterilising activity of maximal metronidazole dosage and its activity against bacilli held dormant by immunity in the mouse.In vitro experiments showed that metronidazole was only bactericidal at attainable concentrations (50-100 microg/ml) under anaerobic conditions. In the Cornell model, isoniazid 25 mg/kg and high dosage pyrazinamide 1000 mg/kg was given in the diet with and without 1500 mg/kg metronidazole for the initial 14 weeks of sterilising chemotherapy. In the subsequent sterile state, metronidazole at 0, 100 and 250 mg/kg was given by daily gavage for 6 weeks. Finally, the mice were given 3 weeks of high dosage steroids and their organs were cultured in selective liquid medium.Metronidazole had no activity either in the initial sterilising phase or in the subsequent sterile state.The O2 tension in the cellular lesions of murine tuberculosis is unlikely to be sufficiently low to allow metronidazole to act. Its activity should be assessed in caseous lesions.

To explore mechanisms by which drug resistance might arise as a result of poor compliance during short course chemotherapy.Four theoretical mechanisms are first described.Examples of the way the mechanisms probably operate are taken from: 1) a study of once-weekly chemotherapy with streptomycin and isoniazid, and 2) the pattern of drug susceptibility in cultures from patients who relapsed after the end of treatment.Good compliance is vitally important. The value of a fourth drug in the initial phase of chemotherapy in preventing resistance is questioned. An explanation for mono-resistance to rifampicin in patients with the acquired immune deficiency syndrome (AIDS) is suggested.

Oxygen-limiting conditions are critical to the survival of the bacteria in tuberculosis. Mycobacterium tuberculosis can survive anaerobiosis in vitro for long periods of time only after a gradual transition to a microaerophilic stationary phase. The underlying mechanism behind stationary phase adaption needs to be elucidated. The protein profiles of Mycobacterium tuberculosis during long-term stationary phase growth and under strict anaerobic incubation were monitored by [35S]methionine labelling, SDS-PAGE and fluorography. These experiments have established that protein synthesis gradually decreased over 50 days in the long-term stationary phase cultures which were considered to be microaerophilic. There was an 80% linear decrease in the level of total protein synthesis during the first 40 days of microaerophilic growth and then the rate of protein synthesis faded quickly. For the first time we have shown that total protein synthesis shutdown occurred when bacilli were incubated under further anaerobic conditions. Viability, estimated by cfu counts, remained constant during stationary phase growth and under anaerobic incubation. Furthermore, when oxygen was introduced into the anaerobic culture, protein synthesis restarted. Also heat shock at 45 degrees C, 48 degrees C and 50 degrees C rapidly induced protein synthesis in stationary and anaerobic cultures. These data indicate that dormant bacteria shut down protein synthesis but remain responsive to specific stimuli which restore protein synthesis. In addition the dormant bacilli induced by anaerobiosis developed more heat resistance than nondormant organisms.

A clinical trial of rifapentine in Hong Kong.Assessment of the bioavailability of the Chinese rifapentine used in the trial.The content of rifapentine in serum samples taken from 287 patients during the administration of four batches of the drug was measured by microbiological assay.An initial comparison of areas under curve obtained in a random allocation to 40 patients of rifapentine either of Western or Chinese origin indicated that the bioavailability of the Chinese drug was 74% of the Western drug. The bioavailability of the second batch was found to be about 66% of the Western drug. The dose of the last two batches of rifapentine was therefore increased from the planned 600 mg to 750 mg, or briefly to 900 mg; serum concentrations were then similar to those obtained with the Western drug. Bioavailability did not change during the use of each drug batch.A comparison of the results obtained in the trial with the initial two batches and the final batches will estimate the effects of rifapentine dose size on its efficacy and toxicity.

This chapter deals with the broad issue of the efficacy of the drugs used in chemotherapy. It starts with a brief history of how the randomized controlled clinical trial was used in the development of effective drug treatment, as controlled trials provided the most direct evidence of the efficacy of individual drugs and also indicated the manner in which they should be used. There follow sections dealing with intermittent treatment, the bactericidal activity of drugs, their grading according to different criteria of activity, and their action during different phases of chemotherapy. The molecular mode of action of drugs is only mentioned when knowledge of the mechanism has a direct bearing on the use of the drug in treatment. Even though dosage of the drug is determined by the balance between antibacterial efficacy and toxicity, little will be said about mechanisms of toxicity. Abbreviations for the names of the major antituberculosis drugs will be used as follows: isoniazid (H), rifampin (R), streptomycin (S), pyrazinamide (Z), ethambutol (M), p-aminosalicylic acid (P), and thiacetazone (T).

The early bactericidal activity (EBA) of ciprofloxacin (CIP) was measured in 80 patients with previously untreated, smear-positive pulmonary tuberculosis by counting viable bacilli in sputum collections during the first 2 d of treatment. Groups of about 10 patients were treated daily with graded doses of CIP or with 300 mg isoniazid or with no drug. The mean EBA, defined as the fall in log CFU/ ml sputum/d, increased from -0.011 in the no drug group to 0.046, 0.092, 0.121, and 0.205 in the groups receiving 250, 500, 1,000, or 1,500 mg CIP, respectively, a highly significant trend. These results demonstrate the antimycobacterial activity of CIP in high dosage, though the mean EBAs of 0.55 and 0.66 in two groups receiving isoniazid were much higher.

The early bactericidal activity (EBA) of ethambutol, pyrazinamide and the fixed combination of isoniazid, rifampicin and pyrazinamide (Rifater: Mer National) was evaluated in patients with pulmonary tuberculosis who were sputum-positive on microscopy for acid-fast bacilli. Twenty-eight patients (mean age 33 years and weight 51 kg on average; range 40-59 kg) were studied. The fall in viable counts of Mycobacterium tuberculosis in sputum collections during the 2 days following the start of treatment was estimated from counts of colony-forming units (CFUs) of M. tuberculosis per ml of sputum cultured on selective 7H10 agar medium. The EBA for ethambutol determined in 9 patients was 0.245 +/- 0.046, log10 CFU/ml sputum/day, that for pyrazinamide was 0.003 +/- 0.014 log10 CFU/ml sputum/day and that for Rifater 0.558 +/- 0.054 log10 CFU/ml sputum/day. The results obtained are similar to those reported in a previous study of the first 2 days of treatment, but in smaller numbers of patients, and confirm the moderate EBA of ethambutol while pyrazinamide is again shown to have very little EBA. Rifater has a marked EBA which may be due mainly to the action of isoniazid. This methodology may be valuable in the rapid evaluation of the bactericidal activity of new antituberculosis agents and the comparison of different dose sizes of agents of the same class.

Mice that had been inoculated intravenously with 6.30 log10 Mycobacterium tuberculosis H37Rv 14 days earlier were administered one of three combinations of drugs, i.e., isoniazid (INH)-rifampin (RMP)-pyrazinamide (PZA), INH-RMP, and RMP-PZA, during an initial 2-month period to mimic the initial phase of chemotherapy for human tuberculosis and during a later 4-month period to mimic the continuation phase of chemotherapy. At the end of the initial phase, all three combined regimens were found to have been highly effective in terms of the number of CFUs in the spleens of infected mice. The bactericidal activities of INH-RMP-PZA and INH-RMP were similar, whereas that of RMP-PZA was significantly greater. The spleens of all of the mice that had been treated initially with INH-RMP-PZA were culture negative by the end of 6 months of treatment, regardless of the regimen employed during the continuation phase. However, after an additional period of 6 months without treatment, the proportion of spleen culture positivity, or relapse rate, was significantly smaller in the subgroup treated with RMP-PZA during the continuation phase than in the subgroups treated with INH-RMP-PZA or INH-RMP; the relapse rate did not differ significantly between the latter two subgroups. These results suggest that antagonism occurs between INH and the combination RMP-PZA during both the initial and continuation phases of chemotherapy, compromising the benefit conferred by the addition of PZA to the combined regimen. The preliminary pharmacokinetic analysis suggested that the pharmacological interaction between INH and RMP was very likely to be involved in the mechanism of antagonism, as concomitant treatment with INH had significantly reduced the peak serum level and the area under the serum concentration-time curve of RMP in mice.

Pairs of sputum specimens obtained pre-treatment from 166 smear-positive patients with pulmonary tuberculosis were examined by direct smear, culture on Löwenstein-Jensen medium after decontamination by the Petroff method, and by quantitative colony counting on selective 7H11 medium after digestion with dithiothreitol. The selective medium counts ranged from no growth to 8.3 log10 cfu/ml with the largest numbers in the range 3.5-7.0 log10 cfu/ml. Although there was overlap in counts between specimens with negative and positive direct smears, a specimen with a count of 4.0 log10 cfu/ml or more was likely to have a positive smear while a negative smear was likely if the count was lower. This demarcation value should be increased to 4.5 log10 cfu/ml to account for under-estimation in the selective medium counts. The corresponding demarcation estimate for LJ cultures was 80 colonies. In distinguishing between patients in the bacterial contents of their sputum, the selective medium counts were better than the gradings of either LJ cultures or direct smears.

Log-phase cultures of Mycobacterium tuberculosis in Tween-albumin medium were exposed to streptomycin, isoniazid, rifampin, ethambutol, and pyrazinamide in concentrations in the range likely to be present in serum during treatment of patients. The bactericidal activity of the drugs was measured as the decrease in viable counts at 4 and 7 days. The activity of single drugs was highest for streptomycin and next highest for rifampin and isoniazid, but ethambutol only started to kill after 4 days. When exposed to 2 drugs, bactericidal synergism was found with streptomycin/isoniazid and isoniazid/ethambutol; additivity, with streptomycin/rifampin; indifference, with isoniazid rifampin and streptomycin/ethambutol; and antagonism, with rifampin/ethambutol and isoniazid/pyrazinamide. When cultures were exposed to the 3 drugs, isoniazid, rifampin, and ethambutol, marked antagonism was found between isoniazid and rifampin, whereas the addition of isoniazid or an increase in its concentration increased the bactericidal activity.

Operation specimens from 52 Hong Kong patients considered to have tuberculosis of the spine were sent at -78 degrees C to London where they were cultured on two Löwenstein-Jensen medium slopes, a slope of 7H11 medium made selective with antibiotics and in two bottles of selective liquid Kirchner medium. Cultures of M tuberculosis, usually with only scanty colonies, were obtained from 37 of the patients. Pus and caseous matter yielded more positive cultures and more numerous colonies than other specimens, but eight of the 37 patients yielded positive cultures only from tissues lining abscess walls or from bone or intervertebral disc specimens. Cultures in Kirchner medium were never contaminated and twice as many were positive as cultures on the slopes; nine patients had cultures positive only on Kirchner medium. It is recommended that (i) a variety of operation specimens, always including pus or caseous material in volumes of at least 1 ml, should be sent for bacteriology; (ii) specimens should be cultured in selective Kirchner medium, with or without Löwenstein-Jensen slopes; (iii) cultures should be incubated for a minimum of 8-9 wk.

Pretreatment, isoniazid-sensitive cultures ofM. tuberculosis were obtained from 36 patients at each of 9 centres (Britain, Teheran, Afghan-Pakistan border, East Africa, Madras, Rangoon, Djakarta, Hong Kong). In the guinea-pig, the virulence of the cultures was uniformly high. The mean virulence of cultures was lower in East Africa, lower still in Madras, but increased again in Rangoon and Bangkok, to reach a level only slightly lower than in British cultures at Singapore, Djakarta and Hong Kong. Thiacetazone resistance was rare in British cultures, very common in cultures from Madras, and fairly common in those from East Africa, Singapore, Djakarta and Hong Kong. These findings suggest the existence of 3 main types of bacilli, with independent evolution of factors affecting virulence and thiacetazone sensitivity.

SUMMARY: Streptomycin resistant strains of Bacterium coli were obtained by plating numerous 1 or 1.5 ml. volume cultures of a sensitive strain on plates containing 100 μg. streptomycin/ml. The growth rate of these strains was measured by mixing them with the sensitive strain and measuring the fall in the numbers of resistant organisms during several periods of daily subculture in streptomycin free broth. Their resistance to streptomycin and stability during successive subcultures in broth were also studied. They could be divided into three main types. All type I strains had identical slow growth rates, were completely resistant, though growing poorly at high streptomycin concentrations and were quickly replaced by rapidly growing slightly resistant variants when subcultured in broth. All type II strains had identical slightly more rapid growth rates, were partially resistant and gradually decreased their resistance in a stepwise manner during subculture. Type III strains had growth rates slightly less than that of the sensitive strain and of a degree of variability similar to those found in clones of the sensitive strain. They were completely resistant and were stable during subculture. They could be subdivided on the basis of one characteristic. Type III a strains grew as well in the presence or absence of streptomycin but type III b strains grew much more slowly in the presence of 1600 μg. ml. streptomycin or over. Since all the strains from any one plate were usually of the same type they were considered to have arisen from a single mutant cell. Since each mutation occurred several times in independent cultures and the number of tubes in which multiple mutations occurred was not greater than would be expected by chance, each type is considered to have arisen by an independent mutation.