In a recent article entitled, “Comparison of time to positive and colony counting in an early bactericidal activity study of anti-tuberculosis treatment” we found a relatively lower early bactericidal activity (EBA) of combination anti-TB treatment with isoniazid (INH), rifampicin, ethambutol and pyrazinamide between days 0 and 2 in HIV-uninfected Ugandan adults with newly-diagnosed, initial episodes of smear-positive pulmonary TB.1 The mean EBA 0-2, measured in colony forming units (CFU), from 40 patients receiving directly observed therapy (DOT) was 0.26 ± 0.24 log10 CFU/ml/day. This result was similar to an EBA 0-2 value of 0.28 ± 0.42 log10 CFU/ml/day we calculated using data from an earlier 2003 clinical trial conducted at the same site.2 In comparison, previous studies done mainly in South Africa have demonstrated a higher EBA 0-2 for standard 4-drug therapy of approximately 0.5 log10 CFU/ml/day, while the EBA 0-14 was similar to that found in our study.3,4 The reason for the difference in EBA during the first 2 days of therapy is not clear. Patients in our study received DOT on an inpatient basis with Good Manufacturing Practice grade medications. Medications were administered as loose tablets, and all patients received 300 mg of INH. The mean dose of INH was 5.8 ± 0.8 mg/kg (range 3.7 to 7.9), which should yield maximum EBA 0-2.5 CFU counts were done on 16-hour pooled overnight sputum collections using standardized methods. The mean baseline sputum colony count of 6.57 ± 0.58 log10 CFU/ml was similar to previous EBA studies. Baseline sputum colony count has been associated with EBA 0-2 in previous studies,5,6 and we did find a correlation in this study (rs = 0.34, P = 0.03), however the relatively high mean baseline count in our patients would again predict maximal EBA 0-2. Finally, recent exposure to INH was excluded by urine INH metabolite testing (BBL Taxo INH Test Strips, BD, Sparks, MD, USA) during screening and at the time of enrollment to the ward to begin the study. All Mycobacterium tuberculosis (MTB) isolates from the study subjects were susceptible to INH, rifampicin, ethambutol and pyrazinamide, and sputum collection and processing methods in our study were similar to those used in the earlier studies.
Given the unexplained finding, we performed further pharmacokinetic and pharmacogenomic investigations after appropriate protocol, IRB, and consent amendments. We focused on INH because most bactericidal activity during the first 2 days of combination therapy is believed to be due to INH.7 Variations in INH serum concentrations are directly determined by genetic differences in metabolism of the drug by the liver enzyme N-acetyltransferase 2 (NAT2). Individuals are either homozygous slow, heterozygous fast, or homozygous fast acetylators of INH.8 Donald and colleagues have previously shown that patients with fast acetylator genotypes have significantly lower EBA 0-2.9 We obtained NAT2 genotypes on 35 of the 41 patients included in our original study using TaqMan SNP Genotyping Assays (Applied Biosystems) and the Statistical Analysis for Genetic Epidemiology software (S.A.G.E., Release 6.3: http://darwin.cwru.edu). Only 3 (9%) were fast acetylators, and there were no significant differences by NAT2 status (table), excluding this as the cause for the low EBA 0-2. We also measured INH levels in the 7 patients who enrolled after the protocol amendment approval. Six of the seven patients had 2 hour INH levels greater than 2 (range 1.8-4.3), which has previously been shown to produce maximum INH EBA.9
Table.
EBA 0-2 and N-acetyltransferase 2 (NAT2) status during combination isoniazid, rifampin, pyrazinamide and ethambutol therapy
| NAT2 Status | n | Mean EBA 0-2* (SD) | P-value, t-test (NAT2 status vs slow) |
|---|---|---|---|
| Homozygous slow | 19 | 0.22 (0.27) | - |
| Heterozygous fast | 13 | 0.30 (0.18) | 0.32 |
| Homozygous fast | 3 | 0.32 (0.25) | 0.53 |
| Combined homozygous and heterozygous fast | 16 | 0.31 (0.19) | 0.27 |
log10 CFU/ml/day
The EBA 0-2 of 0.26 log10 CFU/ml/day in our study was lower than expected given the adequate INH exposure demonstrated by NAT2 genotype and plasma INH levels. We were unable to identify the cause of the lower EBA 0-2. Other microbial and patient factors such as slow bacillary multiplication in our patients due to more chronic disease, or differences in MTB strains in different geographic regions may have influenced the kinetics of initial mycobacterial killing.10-12
Acknowledgement
This work was supported by the Tuberculosis Research Unit at Case Western Reserve University, established with funds from the United States National Institutes of Allergy and Infectious Diseases, National Institutes of Health and Human Services, under Contract No. NO1-AI95383 and HHSN266200700022C/NO1-AI-70022. C.M.B is supported by the Clinical and Translational Science Collaborative of Cleveland under grant number KL2TR000440 from the National Center for Advancing Translational Sciences (NCATS) component of the NIH and NIH Roadmap for Medical Research.
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