Tracy Dalton

Molecular Detection of Mutations Associated with First- and Second-Line Drug Resistance Compared with Conventional Drug Susceptibility Testing of Mycobacterium tuberculosis

ABSTRACT The emergence of multi- and extensively drug-resistant tuberculosis is a significant impediment to the control of this disease because treatment becomes more complex and costly. Reliable and timely drug susceptibility testing is critical to ensure that patients receive effective treatment and become noninfectious. Molecular methods can provide accurate and rapid drug susceptibility results. We used DNA sequencing to detect resistance to the first-line antituberculosis drugs isoniazid (INH), rifampin (RIF), pyrazinamide (PZA), and ethambutol (EMB) and the second-line drugs amikacin (AMK), capreomycin (CAP), kanamycin (KAN), ciprofloxacin (CIP), and ofloxacin (OFX). Nine loci were sequenced: rpoB (for resistance to RIF), katG and inhA (INH), pncA (PZA), embB (EMB), gyrA (CIP and OFX), and rrs, eis, and tlyA (KAN, AMK, and CAP). A total of 314 clinical Mycobacterium tuberculosis complex isolates representing a variety of antibiotic resistance patterns, genotypes, and geographical origins were analyzed. The molecular data were compared to the phenotypic data and the accuracy values were calculated. Sensitivity and specificity values for the first-line drug loci were 97.1% and 93.6% for rpoB, 85.4% and 100% for katG, 16.5% and 100% for inhA, 90.6% and 100% for katG and inhA together, 84.6% and 85.8% for pncA, and 78.6% and 93.1% for embB. The values for the second-line drugs were also calculated. The size and scope of this study, in numbers of loci and isolates examined, and the phenotypic diversity of those isolates support the use of DNA sequencing to detect drug resistance in the M. tuberculosis complex. Further, the results can be used to design diagnostic tests utilizing other mutation detection technologies.

Prevalence of and risk factors for resistance to second-line drugs in people with multidrug-resistant tuberculosis in eight countries: a prospective cohort study

BACKGROUND The prevalence of extensively drug-resistant (XDR) tuberculosis is increasing due to the expanded use of second-line drugs in people with multidrug-resistant (MDR) disease. We prospectively assessed resistance to second-line antituberculosis drugs in eight countries. METHODS From Jan 1, 2005, to Dec 31, 2008, we enrolled consecutive adults with locally confirmed pulmonary MDR tuberculosis at the start of second-line treatment in Estonia, Latvia, Peru, Philippines, Russia, South Africa, South Korea, and Thailand. Drug-susceptibility testing for study purposes was done centrally at the Centers for Disease Control and Prevention for 11 first-line and second-line drugs. We compared the results with clinical and epidemiological data to identify risk factors for resistance to second-line drugs and XDR tuberculosis. FINDINGS Among 1278 patients, 43·7% showed resistance to at least one second-line drug, 20·0% to at least one second-line injectable drug, and 12·9% to at least one fluoroquinolone. 6·7% of patients had XDR tuberculosis (range across study sites 0·8-15·2%). Previous treatment with second-line drugs was consistently the strongest risk factor for resistance to these drugs, which increased the risk of XDR tuberculosis by more than four times. Fluoroquinolone resistance and XDR tuberculosis were more frequent in women than in men. Unemployment, alcohol abuse, and smoking were associated with resistance to second-line injectable drugs across countries. Other risk factors differed between drugs and countries. INTERPRETATION Previous treatment with second-line drugs is a strong, consistent risk factor for resistance to these drugs, including XDR tuberculosis. Representative drug-susceptibility results could guide in-country policies for laboratory capacity and diagnostic strategies. FUNDING US Agency for International Development, Centers for Disease Control and Prevention, National Institutes of Health/National Institute of Allergy and Infectious Diseases, and Korean Ministry of Health and Welfare.

Extensive drug resistance acquired during treatment of multidrug-resistant tuberculosis.

BACKGROUND Increasing access to drugs for the treatment of multidrug-resistant (MDR) tuberculosis is crucial but could lead to increasing resistance to these same drugs. In 2000, the international Green Light Committee (GLC) initiative began to increase access while attempting to prevent acquired resistance. METHODS To assess the GLCs impact, we followed adults with pulmonary MDR tuberculosis from the start to the end of treatment with monthly sputum cultures, drug susceptibility testing, and genotyping. We compared the frequency and predictors of acquired resistance to second-line drugs (SLDs) in 9 countries that volunteered to participate, 5 countries that met GLC criteria, and 4 countries that did not apply to the GLC. RESULTS In total, 832 subjects were enrolled. Of those without baseline resistance to specific SLDs, 68 (8.9%) acquired extensively drug-resistant (XDR) tuberculosis, 79 (11.2%) acquired fluoroquinolone (FQ) resistance, and 56 (7.8%) acquired resistance to second-line injectable drugs (SLIs). The relative risk (95% confidence interval [CI]) of acquired resistance was lower at GLC-approved sites: 0.27 (.16-.47) for XDR tuberculosis, 0.28 (.17-.45) for FQ, and 0.15 (.06-.39) to 0.60 (.34-1.05) for 3 different SLIs. The risk increased as the number of potentially effective drugs decreased. Controlling for baseline drug resistance and differences between sites, the odds ratios (95% CIs) were 0.21 (.07-.62) for acquired XDR tuberculosis and 0.23 (.09-.59) for acquired FQ resistance. CONCLUSIONS Treatment of MDR tuberculosis involves substantial risk of acquired resistance to SLDs, increasing as baseline drug resistance increases. The risk was significantly lower in programs documented by the GLC to meet specific standards.

Sputum culture conversion as a prognostic marker for end-of-treatment outcome in patients with multidrug-resistant tuberculosis: a secondary analysis of data from two observational cohort studies

BACKGROUND Sputum culture conversion is often used as an early microbiological endpoint in phase 2 clinical trials of tuberculosis treatment on the basis of its assumed predictive value for end-of-treatment outcome, particularly in patients with drug-susceptible tuberculosis. We aimed to assess the validity of sputum culture conversion on solid media at varying timepoints, and the time to conversion, as prognostic markers for end-of-treatment outcome in patients with multidrug-resistant (MDR) tuberculosis. METHODS We analysed data from two large cohort studies of patients with MDR tuberculosis. We defined sputum culture conversion as two or more consecutive negative cultures from sputum samples obtained at least 30 days apart. To estimate the association of 2 month and 6 month conversion with successful treatment outcome, we calculated odds ratios (ORs) and 95% CIs with random-effects multivariable logistic regression. We calculated predictive values with bivariate random-effects generalised linear mixed modelling. FINDINGS We assessed data for 1712 patients who had treatment success, treatment failure, or who died. Among patients with treatment success, median time to sputum culture conversion was significantly shorter than in those who had poor outcomes (2 months [IQR 1-3] vs 7 months [3 to ≥24]; log-rank p<0·0001). Furthermore, conversion status at 6 months (adjusted OR 14·07 [95% CI 10·05-19·71]) was significantly associated with treatment success compared with failure or death. Sputum culture conversion status at 2 months was significantly associated with treatment success only in patients who were HIV negative (adjusted OR 4·12 [95% CI 2·25-7·54]) or who had unknown HIV infection (3·59 [1·96-6·58]), but not in those who were HIV positive (0·38 [0·12-1·18]). Thus, the overall association of sputum culture conversion with a successful outcome was substantially greater at 6 months than at 2 months. 2 month conversion had low sensitivity (27·3% [95% confidence limit 16·6-41·4]) and high specificity (89·8% [82·3-94·4]) for prediction of treatment success. Conversely, 6 month sputum culture conversion status had high sensitivity (91·8% [85·9-95·4]), but moderate specificity (57·8% [42·5-71·6]). The maximum combined sensitivity and specificity for sputum culture conversion was reached between month 6 and month 10 of treatment. INTERPRETATION Time to sputum culture conversion, conversion status at 6 months, and conversion status at 2 months in patients without known HIV infection can be considered as proxy markers of end-of-treatment outcome in patients with MDR tuberculosis, although the overall association with treatment success is substantially stronger for 6 month than for 2 month conversion status. Investigators should consider these results regarding the validity of sputum culture conversion at various timepoints as an early predictor of treatment efficacy when designing phase 2 studies before investing substantial resources in large, long-term, phase 3 trials of new treatments for MDR tuberculosis. FUNDING US Agency for International Development, US Centers for Disease Control and Prevention, Division of Intramural Research of the US National Institute of Allergy and Infectious Diseases, Korea Centers for Disease Control and Prevention.

Epidemiology of Pyrazinamide-Resistant Tuberculosis in the United States, 1999–2009

BACKGROUND Pyrazinamide (PZA) is essential in tuberculosis treatment. We describe the prevalence, trends, and predictors of PZA resistance in Mycobacterium tuberculosis complex (MTBC) in the United States. METHODS We analyzed culture-positive MTBC cases with reported drug susceptibility tests for PZA in 38 jurisdictions routinely testing for PZA susceptibility from 1999 to 2009. National Tuberculosis Genotyping Service data for 2004-2009 were used to distinguish M. tuberculosis from Mycobacterium bovis and determine phylogenetic lineage. RESULTS Overall 2.7% (2167/79 321) of MTBC cases had PZA resistance, increasing annually from 2.0% to 3.3% during 1999-2009 (P < .001), largely because of an increase in PZA monoresistance. PZA-monoresistant MTBC (vs drug-susceptible) was associated with an age of 0-24 years (adjusted prevalence ratio [aPR],1.50; 95% confidence interval [CI], 1.31-1.71), Hispanic ethnicity (aPR, 3.52; 95% CI, 2.96-4.18), human immunodeficiency virus infection (aPR, 1.43; 95% CI, 1.15-1.77), extrapulmonary disease (aPR, 3.02; 95% CI, 2.60-3.52), and normal chest radiograph (aPR, 1.88; 95% CI, 1.63-2.16) and was inversely associated with Asian (aPR, 0.59; 95% CI, .47-.73) and black (aPR, 0.37; 95% CI, .29-.49) race. Among multidrug-resistant (MDR) cases, 38.0% were PZA-resistant; PZA resistance in MDR MTBC was associated with female sex (aPR, 1.25; 95% CI, 1.08-1.46) and previous tuberculosis diagnosis (aPR, 1.37; 95% CI, 1.16-1.62). Of 28 080 cases with genotyping data, 925 (3.3%) had PZA resistance; 465 of 925 (50.3%) were M. bovis. In non-MDR M. tuberculosis cases, PZA resistance was higher in the Indo-Oceanic than the East Asian lineage (2.2% vs 0.9%, respectively; aPR, 2.26; 95% CI, 1.53-3.36), but in MDR cases it was lower in the Indo-Oceanic lineage (22.0% vs 43.4%, respectively; aPR, 0.54; 95% CI, .32-.90). CONCLUSIONS Specific human and mycobacterial characteristics were associated with PZA-resistant MTBC, reflecting both specific subgroups of the population and phylogenetic lineages of the mycobacteria.

Multidrug-Resistant Tuberculosis Treatment Outcomes in Relation to Treatment and Initial Versus Acquired Second-Line Drug Resistance

BACKGROUND Resistance to second-line drugs develops during treatment of multidrug-resistant (MDR) tuberculosis, but the impact on treatment outcome has not been determined. METHODS Patients with MDR tuberculosis starting second-line drug treatment were enrolled in a prospective cohort study. Sputum cultures were analyzed at a central reference laboratory. We compared subjects with successful and poor treatment outcomes in terms of (1) initial and acquired resistance to fluoroquinolones and second-line injectable drugs (SLIs) and (2) treatment regimens. RESULTS Of 1244 patients with MDR tuberculosis, 973 (78.2%) had known outcomes and 232 (18.6%) were lost to follow-up. Among those with known outcomes, treatment succeeded in 85.8% with plain MDR tuberculosis, 69.7% with initial resistance to either a fluoroquinolone or an SLI, 37.5% with acquired resistance to a fluoroquinolone or SLI, 29.3% with initial and 13.0% with acquired extensively drug-resistant tuberculosis (P < .001 for trend). In contrast, among those with known outcomes, treatment success increased stepwise from 41.6% to 92.3% as the number of drugs proven effective increased from ≤1 to ≥5 (P < .001 for trend), while acquired drug resistance decreased from 12% to 16% range, depending on the drug, down to 0%-2% (P < .001 for trend). In multivariable analysis, the adjusted odds of treatment success decreased 0.62-fold (95% confidence interval, .56-.69) for each increment in drug resistance and increased 2.1-fold (1.40-3.18) for each additional effective drug, controlling for differences between programs and patients. Specific treatment, patient, and program variables were also associated with treatment outcome. CONCLUSIONS Increasing drug resistance was associated in a logical stepwise manner with poor treatment outcomes. Acquired resistance was worse than initial resistance to the same drugs. Increasing numbers of effective drugs, specific drugs, and specific program characteristics were associated with better outcomes and less acquired resistance.

Association between Regimen Composition and Treatment Response in Patients with Multidrug-Resistant Tuberculosis: A Prospective Cohort Study.

Background For treating multidrug-resistant tuberculosis (MDR TB), the World Health Organization (WHO) recommends a regimen of at least four second-line drugs that are likely to be effective as well as pyrazinamide. WHO guidelines indicate only marginal benefit for regimens based directly on drug susceptibility testing (DST) results. Recent evidence from isolated cohorts suggests that regimens containing more drugs may be beneficial, and that DST results are predictive of regimen effectiveness. The objective of our study was to gain insight into how regimen design affects treatment response by analyzing the association between time to sputum culture conversion and both the number of potentially effective drugs included in a regimen and the DST results of the drugs in the regimen. Methods and Findings We analyzed data from the Preserving Effective Tuberculosis Treatment Study (PETTS), a prospective observational study of 1,659 adults treated for MDR TB during 2005–2010 in nine countries: Estonia, Latvia, Peru, Philippines, Russian Federation, South Africa, South Korea, Thailand, and Taiwan. For all patients, monthly sputum samples were collected, and DST was performed on baseline isolates at the US Centers for Disease Control and Prevention. We included 1,137 patients in our analysis based on their having known baseline DST results for at least fluoroquinolones and second-line injectable drugs, and not having extensively drug-resistant TB. These patients were followed for a median of 20 mo (interquartile range 16–23 mo) after MDR TB treatment initiation. The primary outcome of interest was initial sputum culture conversion. We used Cox proportional hazards regression, stratifying by country to control for setting-associated confounders, and adjusting for the number of drugs to which patients’ baseline isolates were resistant, baseline resistance pattern, previous treatment history, sputum smear result, and extent of disease on chest radiograph. In multivariable analysis, receiving an average of at least six potentially effective drugs (defined as drugs without a DST result indicating resistance) per day was associated with a 36% greater likelihood of sputum culture conversion than receiving an average of at least five but fewer than six potentially effective drugs per day (adjusted hazard ratio [aHR] 1.36, 95% CI 1.09–1.69). Inclusion of pyrazinamide (aHR 2.00, 95% CI 1.65–2.41) or more drugs to which baseline DST indicated susceptibility (aHR 1.65, 95% CI 1.48–1.84, per drug) in regimens was associated with greater increases in the likelihood of sputum culture conversion than including more drugs to which baseline DST indicated resistance (aHR 1.33, 95% CI 1.18–1.51, per drug). Including in the regimen more drugs for which DST was not performed was beneficial only if a minimum of three effective drugs was present in the regimen (aHR 1.39, 95% CI 1.09–1.76, per drug when three effective drugs present in regimen). The main limitation of this analysis is that it is based on observational data, not a randomized trial, and drug regimens varied across sites. However, PETTS was a uniquely large and rigorous observational study in terms of both the number of patients enrolled and the standardization of laboratory testing. Other limitations include the assumption of equivalent efficacy across drugs in a category, incomplete data on adherence, and the fact that the analysis considers only initial sputum culture conversion, not reversion or long-term relapse. Conclusions MDR TB regimens including more potentially effective drugs than the minimum of five currently recommended by WHO may encourage improved response to treatment in patients with MDR TB. Rapid access to high-quality DST results could facilitate the design of more effective individualized regimens. Randomized controlled trials are necessary to confirm whether individualized regimens with more than five drugs can indeed achieve better cure rates than current recommended regimens.

Estimating the future burden of multidrug-resistant and extensively drug-resistant tuberculosis in India, the Philippines, Russia, and South Africa: a mathematical modelling study

BACKGROUND Multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis are emerging worldwide. The Green Light Committee initiative supported programmatic management of drug-resistant tuberculosis in 90 countries. We used estimates from the Preserving Effective TB Treatment Study to predict MDR and XDR tuberculosis trends in four countries with a high burden of MDR tuberculosis: India, the Philippines, Russia, and South Africa. METHODS We calibrated a compartmental model to data from drug resistance surveys and WHO tuberculosis reports to forecast estimates of incident MDR and XDR tuberculosis and the percentage of incident MDR and XDR tuberculosis caused by acquired drug resistance, assuming no fitness cost of resistance from 2000 to 2040 in India, the Philippines, Russia, and South Africa. FINDINGS The model forecasted the percentage of MDR tuberculosis among incident cases of tuberculosis to increase, reaching 12·4% (95% prediction interval 9·4-16·2) in India, 8·9% (4·5-11·7) in the Philippines, 32·5% (27·0-35·8) in Russia, and 5·7% (3·0-7·6) in South Africa in 2040. It also predicted the percentage of XDR tuberculosis among incident MDR tuberculosis to increase, reaching 8·9% (95% prediction interval 5·1-12·9) in India, 9·0% (4·0-14·7) in the Philippines, 9·0% (4·8-14·2) in Russia, and 8·5% (2·5-14·7) in South Africa in 2040. Acquired drug resistance would cause less than 30% of incident MDR tuberculosis during 2000-40. Acquired drug resistance caused 80% of incident XDR tuberculosis in 2000, but this estimate would decrease to less than 50% by 2040. INTERPRETATION MDR and XDR tuberculosis were forecast to increase in all four countries despite improvements in acquired drug resistance shown by the Green Light Committee-supported programmatic management of drug-resistant tuberculosis. Additional control efforts beyond improving acquired drug resistance rates are needed to stop the spread of MDR and XDR tuberculosis in countries with a high burden of MDR tuberculosis. FUNDING US Agency for International Development and US Centers for Disease Control and Prevention, Division of Tuberculosis Elimination.

Challenges and controversies in defining totally drug-resistant tuberculosis.

In March 2012, in response to reports of tuberculosis (TB) resistant to all anti-TB drugs, the World Health Organization convened an expert consultation that identified issues to be resolved before defining a new category of highly drug-resistant TB. Proposed definitions are ambiguous, and extensive drug resistance is encompassed by the already defined extensively drug-resistant (XDR) TB. There is no evidence that proposed totally resistant TB differs from strains encompassed by XDR TB. Susceptibility tests for several drugs are poorly reproducible. Few laboratories can test all drugs, and there is no consensus list of all anti-TB drugs. Many drugs are used off-label for highly drug resistant TB, and new drugs formulated to combat resistant strains would render the proposed category obsolete. Labeling TB strains as totally drug resistant might lead providers to think infected patients are untreatable. These challenges must be addressed before defining a new category for highly drug-resistant TB.

Additional Drug Resistance of Multidrug-Resistant Tuberculosis in Patients in 9 Countries

Less toxic and less expensive drugs and shorter treatment regimens are needed.