DNA Sequencing of MDR TB in Eastern Siberia

The multidrug-resistant tuberculosis (MDR-TB) epidemic in the Irkutsk oblast of Eastern Siberia is arguably the most severe in the world. The investigators have found that in the Irkutsk TB Dispensary, the referral hospital for the oblast (state), patients with HIV and primary MDR-TB (no prior history of TB treatment) suffered poor outcome during the inpatient phase of treatment. It was also found that genotypic drug-resistance mutations only partially explain the extent of phenotypic resistance that is so widespread in Irkutsk. Suboptimal serum anti-TB drug concentrations amplify such phenotypic resistance and work in other settings has demonstrated the importance of optimizing pharmacokinetics to improve TB outcome. Such foundational work in anti-TB/antiretroviral drug concentrations is lacking for drugs used in the treatment of MDR-TB, and absent in the HIV-infected population. Furthermore, the investigators have found that HIV infection in Irkutsk was associated with the Beijing TB genotype similar to other high burden settings, and that overall mortality was more frequent in those with the M. tuberculosis sublineage Beijing MIT17. Thus taken together the investigators hypothesize that the high early TB mortality in Irkutsk is due to a combination of complex MDR and extensively drug resistant (XDR)-TB that is incompletely diagnosed and incompletely treated, poor anti-TB/antiretroviral pharmacokinetics that lead to low efficacy and increased circulation of hyper-transmissible and virulent M. tuberculosis sublineages.

For subjects suspected of TB, sputum samples or other leftover sputum/blood specimens will be screened by GeneXpert and if positive, cultured for TB and the cultured specimen subjected to drug-susceptibility testing by conventional qualitative resistance and minimum inhibitory concentration (MIC), sequencing for drug-resistance mutation and spoligotyping for sublineage identification. Primary analysis will include standard sensitivity/specificity of each drug-mutation compared to conventional qualitative resistance and then median/range MIC values among isolates with/without mutation for improved discrimination. Sublineage analysis will be by mycobacterial interspersed repetitive unit/variable-number tandem repeats (MIRU24-VNTR) and correlated with drug-resistance by conventional susceptibility, MIC and the sequencing results for drug-resistance mutations. The transmission capacity of sublineages will be evaluated by the level of their clustering via the 24-loci MIRU typing.

For subjects ultimately initiating TB treatment, the pharmacokinetic parameters to be tested against the MIC for each TB drug in the patient's regimen include peak (Cmax) and area under the concentration-time curve (AUC). Parameters will be compared to the expected ranges for each drug and expressed as both a population value and at the individual level (proportion of patients below the expected range for each drug). The clinical outcome of TB treatment failure will be compared to the proportion of subjects with a baseline (2 week) Cmax/MIC below the minimal target (lowest µg/ml concentration/ highest MIC of an isolate still considered susceptible) for 2 key drugs in the regimen. It is anticipated that TB treatment failure to be more frequent in subjects treated for drug-susceptible TB when Cmax/MIC values are lower than target for both rifampin and isoniazid, and in subjects treated for MDR-TB when values are below the minimal target for both ofloxacin and kanamycin. A binary logistic regression model will be used to determine the possible risk factors to TB treatment failure in addition to the pharmacokinetic parameters.

The investigators aim for drug-resistance and sublineage analysis to be performed on M. tuberculosis isolates from 250 subjects, 200 of whom will undergo pharmacokinetic study.

Between the Irkutsk Dispensary and the Irkutsk AIDS Center, it is estimated that more than 700 subjects would be eligible. The majority enrolled from the Irkutsk Dispensary will have M. tuberculosis positive specimens (anticipated ~175 total enrolled) and complete pharmacokinetic analysis. It is expected that fewer subjects (anticipated ~25) of those enrolled from the referral Irkutsk AIDS Center will commence TB treatment at Irkutsk Dispensary and complete pharmacokinetic analysis. Sample size is restricted by the exploratory/observational focus of the R21 funding source, and thus estimated upon subjects recruited/enrolled over a 12 month period.