Bacterial Factors That Predict Relapse after Tuberculosis Therapy.

BACKGROUND: Approximately 5% of patients with drug-susceptible tuberculosis have a relapse after 6 months of first-line therapy, as do approximately 20% of patients after 4 months of short-course therapy. We postulated that by analyzing pretreatment isolates of Mycobacterium tuberculosis obtained from patients who subsequently had a relapse or were cured, we could determine any correlations between the minimum inhibitory concentration (MIC) of a drug below the standard resistance breakpoint and the relapse risk after treatment. METHODS: Using data from the Tuberculosis Trials Consortium Study 22 (development cohort), we assessed relapse and cure isolates to determine the MIC values of isoniazid and rifampin that were below the standard resistance breakpoint (0.1 µg per milliliter for isoniazid and 1.0 µg per milliliter for rifampin). We combined this analysis with clinical, radiologic, and laboratory data to generate predictive relapse models, which we validated by analyzing data from the DMID 01-009 study (validation cohort). RESULTS: In the development cohort, the mean (±SD) MIC of isoniazid below the breakpoint was 0.0334±0.0085 µg per milliliter in the relapse group and 0.0286±0.0092 µg per milliliter in the cure group, which represented a higher value in the relapse group by a factor of 1.17 (P=0.02). The corresponding MIC values of rifampin were 0.0695±0.0276 and 0.0453±0.0223 µg per milliliter, respectively, which represented a higher value in the relapse group by a factor of 1.53 (P<0.001). Higher MIC values remained associated with relapse in a multivariable analysis that included other significant between-group differences. In an analysis of receiver-operating-characteristic curves of relapse based on these MIC values, the area under the curve (AUC) was 0.779. In the development cohort, the AUC in a multivariable model that included MIC values was 0.875. In the validation cohort, the MIC values either alone or combined with other patient characteristics were also predictive of relapse, with AUC values of 0.964 and 0.929, respectively. The use of a model score for the MIC values of isoniazid and rifampin to achieve 75.0% sensitivity in cross-validation analysis predicted relapse with a specificity of 76.5% in the development cohort and a sensitivity of 70.0% and a specificity of 100% in the validation cohort. CONCLUSIONS: In pretreatment isolates of M. tuberculosis with decrements of MIC values of isoniazid or rifampin below standard resistance breakpoints, higher MIC values were associated with a greater risk of relapse than lower MIC values. (Funded by the National Institute of Allergy and Infectious Diseases.).

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.

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 GLC's 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.

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.

Relapse associated with active disease caused by Beijing strain of Mycobacterium tuberculosis.

The role of microbial factors in outcomes of tuberculosis treatment has not been well studied. We performed a case-control study to evaluate the association between a Beijing strain and tuberculosis treatment outcomes. Isolates from patients with culture-positive treatment failure (n = 8) or relapse (n = 54) were compared with isolates from randomly selected controls (n = 296) by using spoligotyping. Patients with Beijing strains had a higher risk for relapse (odds ratio [OR] 2.0, 95% confidence interval [CI] 1.0-4.0, p = 0.04) but not for treatment failure. Adjustment for factors previously associated with relapse had little effect on the association between Beijing strains and relapse. Beijing strains were strongly associated with relapse among Asian-Pacific Islanders (OR 11, 95% CI 1.1-108, p = 0.04). Active disease caused by a Beijing strain was associated with increased risk for relapse, particularly among Asian-Pacific Islanders.

Diagnostic performance and costs of Capilia TB for Mycobacterium tuberculosis complex identification from broth-based culture in Bangkok, Thailand.

OBJECTIVES: Broth-based culture (BBC) systems are increasingly being used to detect Mycobacterium tuberculosis complex (MTBC) in resource-limited. We evaluated the performance, time to detection and cost of the Capilia TB identification test from broth cultures positive for acid-fast bacilli (AFB) in Thailand. METHODS: From October-December 2007, broth cultures that grew AFB from specimens submitted by district TB clinics to the Bangkok city laboratory were tested for MTBC using Capilia TB and standard biochemical tests. Isolates that were identified as MTBC by biochemical tests but not by Capilia TB underwent repeat testing using Capilia TB, Accuprobe (Gen-Probe, San Diego, CA, USA) and sequencing. Costs of time, labour, infrastructure and consumables for all procedures were measured. RESULTS: Of 247 isolates evaluated, the sensitivity of Capilia TB was 97% and its true specificity 100% compared with biochemical testing. The median time from specimen receipt to confirmed MTBC identification was 20 days (range 7-53 days) for Capilia TB and 45 days (range 35-79 days) for biochemical testing (P < 0.01). Six isolates that were Capilia TB negative but positive by biochemical testing were confirmed as MTBC and mutations in the mpb64 gene were detected in all. The unit cost of using Capilia TB was 2.67 USD that of biochemical testing was 8.78 USD. CONCLUSIONS: In Thailand, Capilia TB had acceptable sensitivity and specificity, was lower in cost and had shorter turn-around times. Laboratories investing in BBC should consider Capilia TB for identification of MTBC, after validation of performance in their setting.

Multidrug-resistant tuberculosis outbreak among US-bound Hmong refugees, Thailand, 2005.

In January 2005, tuberculosis (TB), including multidrug-resistant TB (MDR TB), was reported among Hmong refugees who were living in or had recently immigrated to the United States from a camp in Thailand. We investigated TB and drug resistance, enhanced TB screenings, and expanded treatment capacity in the camp. In February 2005, 272 patients with TB (24 MDR TB) remained in the camp. Among 17 MDR TB patients interviewed, 13 were found to be linked socially. Of 23 MDR TB isolates genotyped, 20 were similar according to 3 molecular typing methods. Before enhanced screening was implemented, 46 TB cases (6 MDR TB) were diagnosed in the United States among 9,455 resettled refugees. After enhanced screening had begun, only 4 TB cases (1 MDR TB), were found among 5,705 resettled refugees. An MDR TB outbreak among US-bound refugees led to importation of disease; enhanced pre-immigration TB screening and treatment decreased subsequent importation.

Tuberculosis genotyping in six low-incidence States, 2000-2003.

BACKGROUND: As tuberculosis incidence declines in the United States, a new tool for TB control efforts is Mycobacterium tuberculosis genotyping. Colorado, Iowa, Montana, New Hampshire, West Virginia, and Wisconsin began routine genotyping of all culture-confirmed TB cases in October 2000. METHODS: M. tuberculosis isolates from cases reported October 2000 through December 2003 were genotyped by spoligotyping, mycobacterial interspersed repetitive units, and IS6110-based restriction fragment length polymorphism methods. Genotyping results were linked to demographic variables from national surveillance records. Patients who were in genotype clusters were interviewed and their records reviewed to determine possible transmission links among clustered patients. Final analysis was completed during April 2004 through June 2005. RESULTS: Of 971 reported TB cases, 774 (80%) were culture-confirmed, of which 728 (94%) were genotyped. Most genotyped isolates (634 [87%]) were unique. Within 36 clusters linking 94 individuals, four clusters involved both U.S.- and foreign-born individuals. For eight clusters, genotyping results led to the discovery of previously unsuspected transmission. Transmission links between individuals were established in 21 (58%) of the 36 clusters. CONCLUSIONS: In these six low-incidence states, most isolates had unique genotypes, suggesting that most cases arose from activation of latent infection. Few TB clusters involved the foreign-born. For 58% of genotype clusters, epidemiologic investigation ascertained that clustering represented recent M. tuberculosis transmission.

Transmission network analysis to complement routine tuberculosis contact investigations.

OBJECTIVE: We examined the feasibility and value of network analysis to complement routine tuberculosis (TB) contact investigation procedures during an outbreak. METHODS: We reviewed hospital, health department, and jail records and interviewed TB patients. Mycobacterium tuberculosis isolates were genotyped. We evaluated contacts of TB patients for latent TB infection (LTBI) and TB, and analyzed routine contact investigation data, including tuberculin skin test (TST) results. Outcomes included number of contacts identified, number of contacts evaluated, and their TST status. We used network analysis visualizations and metrics (reach, degree, betweenness) to characterize the outbreak. RESULTS: secondary TB patients and more than 1200 contacts. Genotyping detected a 21-band pattern of a strain W variant. No HIV-infected patients were diagnosed. Contacts prioritized by network analysis were more likely to have LTBI than nonprioritized contacts (odds ratio=7.8; 95% confidence interval=1.6, 36.6). Network visualizations and metrics highlighted patients central to sustaining the outbreak and helped prioritize contacts for evaluation. CONCLUSIONS: A network-informed approach to TB contact investigations provided a novel means to examine large quantities of data and helped focus TB control.

Unsuspected recent transmission of tuberculosis among high-risk groups: implications of universal tuberculosis genotyping in its detection.

BACKGROUND: The initiation of universal genotyping revealed 3 clusters of 19 patients with tuberculosis (TB) in Wisconsin, with no apparent epidemiologic links among most of them. An epidemiologic investigation was conducted to determine whether genotype clustering resulted from recent transmission. METHODS: We conducted additional interviews with patients and reviewed medical records. Places frequented by the patients while they were infectious were visited to identify contacts. RESULTS: Our investigation revealed several previously unrecognized possible sites of TB transmission: a single-room occupancy hotel, 2 homeless shelters, 1 bar, and 2 crack houses. Seven patients with previously diagnosed TB were added to the clusters. Of 26 patients, we identified epidemiologic links for all but 1. Common risk factors among patients included alcohol abuse, crack cocaine use, homelessness, and unemployment. Additionally, 98 contacts missed during routine contact investigation were identified. CONCLUSIONS: Transmission of TB, particularly among high-risk groups, may go undetected for years. Our investigation demonstrated the value of universal genotyping in revealing unsuspected recent TB transmission and previously unrecognized sites of transmission, which can be targeted for specific TB interventions.

Outbreak of tuberculosis among homeless persons coinfected with human immunodeficiency virus.

We investigated a cluster of patients with tuberculosis (TB) in North Carolina and determined the extent of transmission of 1 strain of Mycobacterium tuberculosis. A retrospective cohort study was conducted. Homeless shelter attendance and medical records for 1999 and 2000 were reviewed. The period of exposure to M. tuberculosis was determined, and shelter residents were offered TB screening. DNA fingerprinting was performed on 72 M. tuberculosis isolates. In addition to the initial index cluster of 9 patients, another 16 patients were identified. Isolates of M. tuberculosis from all 25 patients shared a matching DNA fingerprint pattern. All but 1 patient was male, 22 (88%) were African American, and 14 (56%) were human immunodeficiency virus-infected. An epidemiological link to a single shelter was identified for all but 1 patient. Earlier recognition of this shelter as a site of M. tuberculosis transmission could have been facilitated through innovative approaches to contact investigation and through genetic typing of isolates.

Geographic differences in time to culture conversion in liquid media: Tuberculosis Trials Consortium study 28. Culture conversion is delayed in Africa.

BACKGROUND: Tuberculosis Trials Consortium Study 28, was a double blind, randomized, placebo-controlled, phase 2 clinical trial examining smear positive pulmonary Mycobacterium tuberculosis. Over the course of intensive phase therapy, patients from African sites had substantially delayed and lower rates of culture conversion to negative in liquid media compared to non-African patients. We explored potential explanations of this finding. METHODS: In TBTC Study 28, protocol-correct patients (n = 328) provided spot sputum specimens for M. tuberculosis culture in liquid media, at baseline and weeks 2, 4, 6 and 8 of study therapy. We compared sputum culture conversion for African and non-African patients stratified by four baseline measures of disease severity: AFB smear quantification, extent of disease on chest radiograph, cavity size and the number of days to detection of M. tuberculosis in liquid media using the Kaplan-Meier product-limit method. We evaluated specimen processing and culture procedures used at 29 study laboratories serving 27 sites. RESULTS: African TB patients had more extensive disease at enrollment than non-African patients. However, African patients with the least disease by the 4 measures of disease severity had conversion rates on liquid media that were substantially lower than conversion rates in non-African patients with the greatest extent of disease. HIV infection, smoking and diabetes did not explain delayed conversion in Africa. Some inter-site variation in laboratory processing and culture procedures within accepted practice for clinical diagnostic laboratories was found. CONCLUSIONS: Compared with patients from non-African sites, African patients being treated for TB had delayed sputum culture conversion and lower sputum conversion rates in liquid media that were not explained by baseline severity of disease, HIV status, age, smoking, diabetes or race. Further investigation is warranted into whether modest variation in laboratory processes substantially influences the efficacy outcomes of phase 2 TB treatment trials or if other factors (e.g., nutrition, host response) are involved. TRIAL REGISTRATION: ClinicalTrials.gov NCT00144417.

Influence of M. tuberculosis lineage variability within a clinical trial for pulmonary tuberculosis.

Recent studies suggest that M. tuberculosis lineage and host genetics interact to impact how active tuberculosis presents clinically. We determined the phylogenetic lineages of M. tuberculosis isolates from participants enrolled in the Tuberculosis Trials Consortium Study 28, conducted in Brazil, Canada, South Africa, Spain, Uganda and the United States, and secondarily explored the relationship between lineage, clinical presentation and response to treatment. Large sequence polymorphisms and single nucleotide polymorphisms were analyzed to determine lineage and sublineage of isolates. Of 306 isolates genotyped, 246 (80.4%) belonged to the Euro-American lineage, with sublineage 724 predominating at African sites (99/192, 51.5%), and the Euro-American strains other than 724 predominating at non-African sites (89/114, 78.1%). Uneven distribution of lineages across regions limited our ability to discern significant associations, nonetheless, in univariate analyses, Euro-American sublineage 724 was associated with more severe disease at baseline, and along with the East Asian lineage was associated with lower bacteriologic conversion after 8 weeks of treatment. Disease presentation and response to drug treatment varied by lineage, but these associations were no longer statistically significant after adjustment for other variables associated with week-8 culture status.

Evaluation of a two-step approach for large-scale, prospective genotyping of Mycobacterium tuberculosis isolates in the United States.

Genotyping of Mycobacterium tuberculosis isolates is useful in tuberculosis control for confirming suspected transmission links, identifying unsuspected transmission, and detecting or confirming possible false-positive cultures. The value is greatly increased by reducing the turnaround time from positive culture to genotyping result and by increasing the proportion of cases for which results are available. Although IS6110 fingerprinting provides the highest discrimination, amplification-based methods allow rapid, high-throughput processing and yield digital results that can be readily analyzed and thus are better suited for large-scale genotyping. M. tuberculosis isolates (n = 259) representing 99% of culture-positive cases of tuberculosis diagnosed in Wisconsin in the years 2000 to 2003 were genotyped by using spoligotyping, mycobacterial interspersed repetitive unit (MIRU) typing, and IS6110 fingerprinting. Spoligotyping clustered 64.1% of the isolates, MIRU typing clustered 46.7% of the isolates, and IS6110 fingerprinting clustered 29.7% of the isolates. The combination of spoligotyping and MIRU typing yielded 184 unique isolates and 26 clusters containing 75 isolates (29.0%). The addition of IS6110 fingerprinting reduced the number of clustered isolates to 30 (11.6%) if an exact pattern match was required or to 44 (17.0%) if the definition of a matching IS6110 fingerprint was expanded to include patterns that differed by the addition of a single band. Regardless of the genotyping method chosen, the addition of a second or third method decreased clustering. Our results indicate that using spoligotyping and MIRU typing together provides adequate discrimination in most cases. IS6110 fingerprinting can then be used as a secondary typing method to type the clustered isolates when additional discrimination is needed.

Variable-number tandem repeat typing of Mycobacterium tuberculosis isolates with low copy numbers of IS6110 by using mycobacterial interspersed repetitive units.

A study set of 180 Mycobacterium tuberculosis and Mycobacterium bovis isolates having low copy numbers of IS6110 were genotyped using the recently introduced method based on the variable-number tandem repeats of mycobacterial interspersed repetitive units (MIRU-VNTR). The results were compared with results of the more commonly used methods, IS6110 restriction fragment length polymorphism (RFLP) and spoligotyping. The isolates were collected in Michigan from 1996 to 1999 as part of a project to genotype all isolates from new cases of tuberculosis in the state. Twelve MIRU loci were amplified, and the amplicons were analyzed by agarose gel electrophoresis to determine the copy number at each MIRU locus. MIRU-VNTR produced more distinct patterns (80 patterns) than did IS6110 RFLP (58 patterns), as would be expected in this study set. Spoligotyping identified 59 patterns. No single method defined all unique isolates, and the combination of all three typing methods generated 112 distinct patterns identifying 90 unique isolates and 90 isolates in 22 clusters. The results confirm the potential utility of MIRU-VNTR typing and show that typing with multiple methods is required to attain maximum specificity.

Transfer of a Mycobacterium tuberculosis genotyping method, Spoligotyping, from a reverse line-blot hybridization, membrane-based assay to the Luminex multianalyte profiling system.

Spoligotyping using Luminex technology was shown to be a highly reproducible method suitable for high-throughput analysis. Spoligotyping of 48 isolates using the traditional membrane-based assay and the Luminex assay yielded concordant results for all isolates. The Luminex platform provides greater flexibility and cost effectiveness than the membrane-based assay.

Evaluation of the epidemiologic utility of secondary typing methods for differentiation of Mycobacterium tuberculosis isolates.

Spoligotyping and mycobacterial interspersed repetitive unit-variable-number tandem repeat analysis (MIRU-VNTR) were evaluated for the ability to differentiate 64 Mycobacterium tuberculosis isolates from 10 IS6110-defined clusters. MIRU-VNTR performed slightly better than spoligotyping in reducing the number of clustered isolates and the sizes of the clusters. All epidemiologically related isolates remained clustered by MIRU-VNTR but not by spoligotyping.

A two-component direct fluorescent-antibody assay for rapid identification of Bacillus anthracis.

A two-component direct fluorescent-antibody (DFA) assay, using fluorescein-labeled monoclonal antibodies specific to the Bacillus anthracis cell wall (CW-DFA) and capsule (CAP-DFA) antigens, was evaluated and validated for rapid identification of B. anthracis. We analyzed 230 B. anthracis isolates; 228 and 229 were positive by CW-DFA and CAP-DFA assays, respectively. We also tested 56 non-B. anthracis strains; 10 B. cereus and 2 B. thuringiensis were positive by the CW-DFA assay, and 1 B. megaterium strain was positive by CAP-DFA. Analysis of the combined DFA results identified 227 of 230 B. anthracis isolates; all 56 strains of the other Bacillus spp. were negative. Both DFA assays tested positive on 14 of 26 aging clinical specimens from the 2001 anthrax outbreak investigation. The two-component DFA assay is a sensitive, specific, and rapid confirmatory test for B. anthracis in cultures and may be useful directly on clinical specimens.