Evaluation of new antituberculosis drugs in mouse models.
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Animals↗
Biomedical subjects
D A Mitchison
Publications and source records attributed to D A Mitchison.
Clinical development of anti-tuberculosis drugs.
In the clinical development of new anti-tuberculosis drugs, the most important step is efficient Phase II studies to show whether the drug is likely to be able to shorten treatment and with what other drugs it has the greatest sterilizing activity. The use of non-linear mixed effects modelling applied to serial sputum cfu counts appears to be the most effective technique, but we know little about the optimal design of such novel studies. A paper in the current journal reports on the relative efficiencies of various timing patterns in sampling sputum.
Antitubercular Agents↗
Sterilising action of pyrazinamide in models of dormant and rifampicin-tolerant Mycobacterium tuberculosis.
SETTING: Pyrazinamide (PZA) is an effective sterilising drug in tuberculosis, but its mode of action is controversial. OBJECTIVE: To test the bactericidal activity of 1.56-100 microg/ml PZA in Hu/Coates models of dormant and rifampicin (RMP) tolerant Mycobacterium tuberculosis. METHODS: 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. RESULTS: 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. CONCLUSION: 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.
Antibiotics, Antitubercular↗
Bactericidal action of gatifloxacin, rifampin, and isoniazid on logarithmic- and stationary-phase cultures of Mycobacterium tuberculosis.
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 microg/ml was rapid and was very similar to that of isoniazid. At concentrations of 0.25 and 4 microg/ml, gatifloxacin enhanced the activity of isoniazid. Killing of the stationary-phase culture was biphasic. During the first 2 days, gatifloxacin at 4 microg/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.
Anti-Bacterial Agents↗
Drug resistance in tuberculosis.
A drug-resistant strain of Mycobacterium tuberculosis is defined as one differing from the tight distribution of wild strains that have not come into contact with the drug concerned. Sensitivity tests are performed by the absolute concentration method, the resistance ratio method or the proportion method. The hypothesis underlying the proportion method is that there are appreciable differences in inoculum size so that there should be an association between the proportion on drug-free medium and the proportion on drug-containing medium. This hypothesis was not supported by a study on ethionamide-resistant strains. It indicated that variation in the proportion on drug-free medium was due to clumping of the bacilli in the inoculum rather than to differences in the number of bacilli. Hence, the use of the proportion method introduces errors in susceptibility testing. While the method can produce reliable results, it is more time consuming than a minimal inhibitory concentration determination, and should not be adopted as a standard method.
Antitubercular Agents↗
The control of tuberculosis: progress & prospect.
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Animals↗
Split-drug regimens for the treatment of patients with sputum smear-positive pulmonary tuberculosis--a unique approach.
OBJECTIVE: To evaluate the efficacy of split-drug regimens for treatment of patients with sputum smear-positive pulmonary tuberculosis in south India. DESIGN: Randomized controlled clinical trial where eligible patients were randomly allocated to: (i) 2RE(3)HZ(3)(alt)/4RH(2) (split I): rifampicin plus ethambutol given on one day and isoniazid plus pyrazinamide the next day for first 2 months followed by rifampicin plus isoniazid twice weekly for 4 months, or (ii) 3RE(3)HZ(3)(alt)/3RH(2) (split II): similar to regimen 1, except duration was 3 months in each phase, or (iii) 2REHZ(3)/4RH(2) (control): rifampicin, isoniazid, ethambutol and pyrazinamide, given thrice weekly for 2 months followed by isoniazid and rifampicin twice weekly for 4 months. All patients were followed up clinically and bacteriologically every month up to 2 years and every 6 months for up to 5 years. RESULTS: A favourable response (cultures negative for Mycobacterium tuberculosis during the last 2 months of treatment) was observed in 91% of 407 patients in split I, 94% of 415 in split II and 89% of 418 in the control regimen. Ninety-one per cent of 370 patients in split I, 93% of 389 in split II and 90% of 370 in control regimens had quiescent disease at the end of 60 months. Gastrointestinal symptoms were more frequent under the control regimen (P = 0.01). CONCLUSION: Split-drug regimens were as effective as the control regimen in terms of favourable response at the end of treatment and quiescent disease at 5 years, and caused fewer gastrointestinal side-effects.
Adult↗
A reiterative method for calculating bactericidal activity.
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Antitubercular Agents↗
Rifapentine and isoniazid in the continuation phase of a 6-month regimen. Final report at 5 years: prognostic value of various measures.
SETTING: 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. OBJECTIVE: Final report evaluating adverse events (46 relapses and one failure) after 5 years and the prognostic influence of various factors. METHODS: Kaplan-Meier analysis of adverse events and Cox proportional hazards analysis of prognostic factors. RESULTS: 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. CONCLUSIONS: 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.
Adolescent↗
The early bactericidal activity of streptomycin.
SETTING: Patients with sputum smear-positive, newly diagnosed pulmonary tuberculosis studied at Tygerburg Hospital, Cape Town, for their early response to streptomycin (SM). OBJECTIVE: 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. DESIGN: 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. RESULTS: 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. CONCLUSIONS: 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.
Adult↗
The early bactericidal activity of a low-clearance liposomal amikacin in pulmonary tuberculosis.
The early bactericidal activity (EBA) of a liposomal preparation of amikacin (MiKasome) with a long plasma half-life of 120-200 h was examined in seven patients with newly diagnosed, smear-positive pulmonary tuberculosis. Liposomal amikacin was given in slow iv infusions of 30 mg total amikacin/kg body weight on three successive days. Cfu counts were set up on 16 h sputum collections preceding the first dose and following each dose and were used for calculating the EBA. Despite the high concentrations of total amikacin, >1000 mg/L, obtainable in plasma, no evidence of EBA was obtained. In view of the considerable activity of liposomal amikacin in experimental murine tuberculosis, this finding indicates that liberation of amikacin from the long-life liposomes occurs only in macrophages that are not usually present in the vicinity of the large extracellular clumps of bacilli in the cavity caseum.
Adult↗
The early bactericidal activity of amikacin in pulmonary tuberculosis.
SETTING: Stellenbosch University, a tertiary care hospital in Cape Town, South Africa. OBJECTIVE: 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. DESIGN: An open, randomised trial. PATIENTS: Patients with previously untreated, sputum smear-positive pulmonary tuberculosis. INTERVENTION: 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). RESULTS: 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. CONCLUSION: 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.
Adolescent↗
A multicentre study of the early bactericidal activity of anti-tuberculosis drugs.
The early bactericidal activities (EBAs) of 300 mg isoniazid, 18.5 mg isoniazid, 600 mg rifampicin and 800 mg ofloxacin given daily to 262 patients with newly diagnosed pulmonary tuberculosis in Cape Town, Nairobi, Madras and Hong Kong were measured by counting cfu and total acid-fast bacilli in sputum collections taken pre-treatment (S1), at 2 days (S3) and at 5 days (S6). In Cape Town, Nairobi and Madras, the cfu findings suggested that isoniazid produced a massive kill, perhaps of actively growing organisms, during the first 2 days (mean S1-S3 EBAs of 0.636-1.006) but was almost inactive thereafter (mean S3-S6 EBAs of 0.000-0.081), whereas rifampicin maintained moderate activity against slowly growing organisms throughout the 5 days (mean S3-S6 EBAs of 0.242-0.305). This finding suggests that EBAs measured during the 2-5 day interval might be able to assess the sterilizing activity of drugs. Ofloxacin had moderately high mean S1-S3 EBAs of 0.130-0.391. However, in Hong Kong rifampicin appeared to be the most bactericidal drug from the start, possibly because patients had more chronic disease. A method of adjusting the cfu EBAs using total counts was devised which decreased the variability between patients within a treatment group without altering the mean cfu EBA. This resulted in a large gain in precision in Hong Kong, suggesting that their estimates were greatly affected by type II variation, due to dilution of pus by saliva and bronchial secretions, whereas small or no gains were obtained in the other three centres, suggesting that the main cause of variability was type I, due to other factors.
Adult↗
Early bactericidal activity of paromomycin (aminosidine) in patients with smear-positive pulmonary tuberculosis.
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.
Adolescent↗
Detection of mRNA transcripts and active transcription in persistent Mycobacterium tuberculosis induced by exposure to rifampin or pyrazinamide.
Mycobacterium tuberculosis can persist in an altered physiological state for many years after initial infection, and it may reactivate to cause active disease. An analogous persistent state, possibly consisting of several different subpopulations of bacteria, may arise during chemotherapy; this state is thought to be responsible for the prolonged period required for effective chemotherapy. Using two models of drug-induced persistence, we show that both microaerophilic stationary-phase M. tuberculosis treated with a high dose of rifampin in vitro and pyrazinamide-induced persistent bacteria in mice are nonculturable yet still contain 16S rRNA and mRNA transcripts. Also, the in vitro persistent, plate culture-negative bacteria incorporate radioactive uridine into their RNA in the presence of rifampin and can rapidly up-regulate gene transcription after the replacement of the drug with fresh medium and in response to heat shock. Our results show that persistent M. tuberculosis has transcriptional activity. This finding provides a molecular basis for the rational design of drugs targeted at persistent bacteria.
Animals↗
Comparison of phenotypic and genotypic methods for pyrazinamide susceptibility testing with Mycobacterium tuberculosis.
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.
Amidohydrolases↗
Rifapentine and isoniazid in the continuation phase of a 6-month regimen. Interim report: no activity of isoniazid in the continuation phase.
SETTING: 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). OBJECTIVE: Interim report evaluating progress of study and the role of isoniazid in the continuation phase. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.
Acetylation↗
Role of individual drugs in the chemotherapy of tuberculosis.
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.
Antibiotics, Antitubercular↗