Interest in geographic differences in response to TB chemotherapy dates back to 1956 when Fox et al. compared clinical, radiographic, and microbiologic outcomes in patients from Britain and Uganda 
. In TBTC study 28 during the first 8 weeks of therapy we found later sputum culture conversion and lower rates of conversion in liquid media in African patients compared to non-African patients, despite African patients having comparatively higher conversion rates on solid media. African patients had more severe disease when their therapy was begun. However when we evaluated conversion rates stratified by varying levels of disease severity it became clear that our measures of baseline severity of disease did not explain the lower and delayed culture conversion in liquid media in African patients. In fact, African patients with the lowest severity of disease had conversion rates in liquid media that were similar to or lower than non-African patients with the highest severity of disease.
Some studies suggest that smoking 
, diabetes 
, increasing age 
and HIV positive status 
are associated with delayed conversion. In Study 28, African patients had a lower prevalence of smoking and diabetes and were younger compared to non-African patients, so these conditions did not appear to contribute to the lower conversion rates in Africa. Patients with HIV infection actually had slightly higher conversion rates in liquid media compared to HIV-negative patients.
In Study 28 the administration of all doses of anti-TB drugs were directly observed. In an intensive pharmacokinetic sub-study performed on a convenience sample of 72 patients enrolled in TBTC Studies 27 and 28 (37 African and 35 non-African), the mean AUC0-24
for rifampin and moxifloxacin did not differ between African and non-African patients 
. Isoniazid levels were not examined. We did not find a pharmacokinetic reason for lower conversion rates among Africans.
A panel of experienced mycobacteriologists, clinicians, and epidemiologists reviewed laboratory processes in the context of the site-specific conversion rates and the number of patients represented. They determined that the laboratories were operating within accepted guidelines for conducting mycobacterial cultures
. We found some variation in laboratory processes (e.g., decontamination procedures) that might have led to differences in the rates of culture conversion. In particular, the concentration of NaOH used during specimen decontamination was generally lower at African sites (albeit with longer time for decontamination). Laboratory studies done on sputum seeded with bacteria and M. tuberculosis
H37Ra revealed that decontamination with NALC-NaOH with final concentrations of 1–2% NaOH and decontamination treatment times of up to 30 minutes did not affect the viability of M. tuberculosis
when grown on solid media 
. However a study of patient sputum specimens collected prior to initiation of TB therapy suggested that relatively small increases in the final NaOH concentration used in decontamination from 1% to 1.25% significantly decreased the recovery of M. tuberculosis
on a solid media 
. When we evaluated the effect of the NaOH concentration on liquid culture results after the initiation of TB therapy, stratified by geographic region, we found that the NaOH concentration did not explain delayed conversion in African patients compared to those at non-African sites.
At baseline, prior to the initiation of therapy, solid and liquid media performed equally well for isolating M. tuberculosis
from the sputa of the smear-positive patients enrolled in Study 28. As therapy progressed, at both African and non-African sites, patients had lower conversion rates in liquid media compared to solid media. This is expected, as liquid media allow for growth of some M. tuberculosis
organisms that are unable to grow on solid media
. Unexpectedly, the difference in conversion rates between solid and liquid media was much greater in Africa. This finding of large differences in the yield of solid and liquid media for M. tuberculosis
for African patients receiving therapy is not unique to TBTC Study 28. A similar clinical trial conducted in South Africa by the OFLOTUB group found that at 8 weeks of TB therapy the proportion of sputum cultures that were negative on solid media (7H11) was twice that found on liquid media (MGIT)
. Joloba et. al. studied sputum culture conversion on both solid (7H10 selective) and liquid media (BACTEC 460) during the course of TB therapy for both HIV infected and uninfected populations. They also found that conversion in liquid media was lower and substantially delayed compared to that of solid media for both populations
Regional differences in outcomes of TB therapy are not unique to TBTC studies. In a recent phase 3 trial conducted in Brazil, the Philippines and Uganda examining treatment shortening for HIV negative adults with non-cavitary TB and negative 2-month sputum culture on solid media, patients from Uganda were more likely to relapse than participants from Brazil or the Philippines
. This study was stopped early because patients in the 4-month treatment arm had significantly more relapse (13 relapses among 196 patients) than those in the 6-month treatment arm (3 of 198). Among the 16 relapses, 12 (75%) occurred in the Ugandan patients and 4 (25%) in Brazilian patients. Initial sputum smear grade and the number of lung zones involved by tuberculous lesions were greater among patients enrolled in Uganda. In the multivariate analysis, enrollment at the Uganda site was an independent risk factor for relapse after accounting for baseline sputum smear and radiographic extent of disease. Two-month sputum culture results in liquid media were not reported in that study.
Recent TB trials illustrate important advantages and disadvantages of different approaches to phase 2 clinical trials 
. Multicenter international studies are likely to enroll more rapidly and to be more representative and easier to generalize, they also allow examination for regional differences in response to therapy. However, the potential influence that variability in laboratory or other procedures might have on the interpretation of clinical trial results must be considered. While laboratories engaged in clinical trials should meet recognized clinical diagnostic standards for culturing mycobacteria, the degree of heterogeneity of procedures allowed to meet these routine clinical diagnostic standards may not be sufficient for early phase controlled clinical trials. Further research is needed to determine whether and how modest differences in processing and culturing of sputa influence microbiological outcomes over the course of TB therapy in phase 2 studies. Such studies should focus on determining the principle processes that contribute to variability in microbiological outcomes measured during clinical trials. Laboratory processes that diminish the sensitivity or specificity of microbial biomarkers of treatment outcomes (e.g., 8-week culture conversion) will influence the ability of both single site and multi-site studies to determine the relative efficacy of drug regimens. Efforts to optimize the performance characteristics of microbiological tests in predicting treatment failure and relapse are needed if we hope to make good decisions regarding which drugs and regimens to move forward into phase 3 clinical trials. If, after investigation, variations in laboratory practices are not found to explain geographic difference in time to culture conversion, then alternative explanations (e.g., differences in socioeconomic/nutritional status, host immunity, or microbial pathogenesis) should be explored.