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More efficacious treatment regimens are needed for tuberculosis, however, drug development is impeded by a lack of reliable biomarkers of disease severity and of treatment effect. We conducted a directed screen of host biomarkers in participants enrolled in a tuberculosis clinical trial to address this need. Serum samples from 319 protocol-correct, culture-confirmed pulmonary tuberculosis patients treated under direct observation as part of an international, phase 2 trial were screened for 70 markers of infection, inflammation, and metabolism. Biomarker assays were specifically developed for this study and quantified using a novel, multiplexed electrochemiluminescence assay. We evaluated the association of biomarkers with baseline characteristics, as well as with detailed microbiologic data, using Bonferroni-adjusted, linear regression models. Across numerous analyses, seven proteins, SAA1, PCT, IL-1β, IL-6, CRP, PTX-3 and MMP-8, showed recurring strong associations with markers of baseline disease severity, smear grade and cavitation; were strongly modulated by tuberculosis treatment; and had responses that were greater for patients who culture-converted at 8weeks. With treatment, all proteins decreased, except for osteocalcin, MCP-1 and MCP-4, which significantly increased. Several previously reported putative tuberculosis-associated biomarkers (HOMX1, neopterin, and cathelicidin) were not significantly associated with treatment response. In conclusion, across a geographically diverse and large population of tuberculosis patients enrolled in a clinical trial, several previously reported putative biomarkers were not significantly associated with treatment response, however, seven proteins had recurring strong associations with baseline radiographic and microbiologic measures of disease severity, as well as with early treatment response, deserving additional study.

Rifapentine is a highly active antituberculosis antibiotic with treatment-shortening potential; however, exposure-response relations and the dose needed for maximal bactericidal activity have not been established. We used pharmacokinetic/pharmacodynamic data from 657 adults with pulmonary tuberculosis participating in treatment trials to compare rifapentine (n = 405) with rifampin (n = 252) as part of intensive-phase therapy. Population pharmacokinetic/pharmacodynamic analyses were performed with nonlinear mixed-effects modeling. Time to stable culture conversion of sputum to negative was determined in cultures obtained over 4 months of therapy. Rifapentine exposures were lower in participants who were coinfected with human immunodeficiency virus, black, male, or fasting when taking drug. Rifapentine exposure, large lung cavity size, and geographic region were independently associated with time to culture conversion in liquid media. Maximal treatment efficacy is likely achieved with rifapentine at 1,200 mg daily. Patients with large lung cavities appear less responsive to treatment, even at high rifapentine doses.

Rationale: Optimizing pyrazinamide dosing is critical to improve treatment efficacy while minimizing toxicity during tuberculosis treatment. Study 31/AIDS Clinical Trials Group A5349 represents the largest phase 3 randomized controlled therapeutic trial to date for such an investigation. Objectives: We sought to report pyrazinamide pharmacokinetic parameters, risk factors for lower pyrazinamide exposure, and relationships between pyrazinamide exposure and efficacy and safety outcomes. We aimed to determine pyrazinamide dosing strategies that optimize risks and benefits. Methods: We analyzed pyrazinamide steady-state pharmacokinetic data using population nonlinear mixed-effects models. We evaluated the contribution of pyrazinamide exposure to long-term efficacy using parametric time-to-event models and safety outcomes using logistic regression. We evaluated optimal dosing with therapeutic windows targeting ≥95% durable cure and safety within the observed proportion of the primary safety outcome. Measurements and Main Results: Among 2,255 participants with 6,978 plasma samples, pyrazinamide displayed sevenfold exposure variability (151-1,053 mg·h/L). Body weight was not a clinically relevant predictor of drug clearance and thus did not justify the need for weight-banded dosing. Both clinical and safety outcomes were associated with pyrazinamide exposure, resulting in therapeutic windows of 231-355 mg · h/L for the control and 226-349 mg·h/L for the rifapentine-moxifloxacin regimen. Flat dosing of pyrazinamide at 1,000 mg would have permitted an additional 13.1% ( n  = 96) of participants allocated to the control and 9.2% ( n  = 70) to the rifapentine-moxifloxacin regimen dosed within the therapeutic window, compared with the current weight-banded dosing. Conclusions: Flat dosing of pyrazinamide at 1,000 mg/d would be readily implementable and could optimize treatment outcomes in drug-susceptible tuberculosis. Clinical trial registered with www.clinicaltrials.gov (NCT02410772).

Background: The optimal dosing strategy for rifampicin in treating drug-susceptible tuberculosis (TB) is still highly debated. In the phase 3 clinical trial Study 31/ACTG 5349 (NCT02410772), all participants in the control regimen arm received 600 mg rifampicin daily as a flat dose. Here, we evaluated relationships between rifampicin exposure and efficacy and safety outcomes., Methods: We analyzed rifampicin concentration time profiles using population nonlinear mixed-effects models. We compared simulated rifampicin exposure from flat- and weight-banded dosing. We evaluated the effect of rifampicin exposure on stable culture conversion at 6 months; TB-related unfavorable outcomes at 9, 12, and 18 months using Cox proportional hazard models; and all trial-defined safety outcomes using logistic regression., Results: Our model-derived rifampicin exposure ranged from 4.57 mg · h/L to 140.0 mg · h/L with a median of 41.8 mg · h/L. Pharmacokinetic simulations demonstrated that flat-dosed rifampicin provided exposure coverage similar to the weight-banded dose. Exposure-efficacy analysis (n = 680) showed that participants with rifampicin exposure below the median experienced similar hazards of stable culture conversion and TB-related unfavorable outcomes compared with those with exposure above the median. Exposure-safety analysis (n = 722) showed that increased rifampicin exposure was not associated with increased grade 3 or higher adverse events or serious adverse events., Conclusions: Flat-dosing of rifampicin at 600 mg daily may be a reasonable alternative to the incumbent weight-banded dosing strategy for the standard-of-care 6-month regimen. Future research should assess the optimal dosing strategy for rifampicin, at doses higher than the current recommendation., Competing Interests: Potential conflicts of interest. The authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Background: A 4-month regimen containing rifapentine and moxifloxacin has noninferior efficacy compared to the standard 6-month regimen for drug-sensitive tuberculosis. We evaluated the effect of regimens containing daily, high-dose rifapentine on efavirenz pharmacokinetics and viral suppression in patients with human immunodeficiency virus (HIV)-associated tuberculosis (TB)., Methods: In the context of a Phase 3 randomized controlled trial, HIV-positive individuals already virally suppressed on efavirenz--containing antiretroviral therapy (ART) (EFV1), or newly initiating efavirenz (EFV2) received TB treatment containing rifapentine (1200 mg), isoniazid, pyrazinamide, and either ethambutol or moxifloxacin. Mid-interval efavirenz concentrations were measured (a) during ART and TB cotreatment (Weeks 4, 8, 12, and 17, different by EFV group) and (b) when ART was taken alone (pre- or post-TB treatment, Weeks 0 and 22). Apparent oral clearance (CL/F) was estimated and compared. Target mid-interval efavirenz concentrations were > 1 mg/L. Co-treatment was considered acceptable if > 80% of participants had mid-interval efavirenz concentrations meeting this target., Results: EFV1 and EFV2 included 70 and 41 evaluable participants, respectively. The geometric mean ratio comparing efavirenz CL/F with vs without TB drugs was 0.79 (90% confidence interval [CI] .72-.85) in EFV1 and 0.84 [90% CI .69-.97] in EFV2. The percent of participants with mid-interval efavirenz concentrations > 1mg/L in EFV1 at Weeks 0, 4, 8, and 17 was 96%, 96%, 88%, and 89%, respectively. In EFV2, at approximately 4 and 8 weeks post efavirenz initiation, the value was 98%., Conclusions: TB treatment containing high-dose daily rifapentine modestly decreased (rather than increased) efavirenz clearance and therapeutic targets were met supporting the use of efavirenz with these regimens, without dose adjustment., Clinical Trials Registration: NCT02410772., Competing Interests: Potential conflicts of interest. The authorship team members have declared (below or attached) any potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Sanofi commercial interests did not influence the study design; the collection, analysis, or interpretation of data; the preparation of this manuscript; or the decision to submit this manuscript for publication. A Sanofi technical expert served on the protocol team. N. M. reports a grant to the institution for pneumonia research from Pfizer and a subaward to collect specimens to validate a TB diagnostic from Roche. L. M. reports grants or contracts from Merck Sharpe & Dohme (institution received clinical trial fees), ViiV Healthcare (institution received clinical trial fees), Kowa Pharmaceuticals America (pharmaceutical support on protocol), and Sanofi-Aventis (pharmaceutical support on protocol) outside of the submitted work. S. S. reports research grant support to the institution from ViiV Healthcare outside of the submitted work and support for attending meetings and/or travel from the National Institutes of Health (NIH). C. V. F. reports being a member of the Safety Monitoring Committee for Division of Microbiology and Infectious Diseases (DMID), NIAID, NIH, for 2 protocols on (1) an influenza virus challenge study and (2) a safety and pharmacokinetics of single doses of an antifungal, VT-1598 (no payments are made to institution or personally for service on this SMC). R. E. C. reports consulting fees from Sanofi. U. L. reports funding support for travel to ACTG Annual Network Meeting from AIDS Clinical Trials Group. A. A. V. reports working with a group at CDC that perform TB trials. The group has collaborated with pharmaceutical partners. Sanofi collaborated in Study 31/A5349 and provided support for testing PK of TB drugs, and provided drugs for the trial. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2021. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

The identification of sensitive, specific, and reliable biomarkers that can be quantified in the early phases of tuberculosis treatment and predictive of long-term outcome is key for the development of an effective short-course treatment regimen. Time to positivity (TTP), a biomarker of treatment outcome against Mycobacterium tuberculosis, measures longitudinal bacterial growth in mycobacterial growth indicator tube broth culture and may be predictive of standard time to stable culture conversion (TSCC). In two randomized phase 2b trials investigating dose-ranging rifapentine (Studies 29 and 29X), 662 participants had sputum collected over 6 months where TTP, TSCC, and time to culture conversion were quantified. The goals of this post hoc study were to characterize longitudinal TTP profiles and to identify individual patient characteristics associated with delayed time to culture conversion. In order to do so, a nonlinear mixed-effects model describing longitudinal TTP was built. Independent variables associated with increased bacterial clearance (increased TTP), assessed by subject-specific and population-level trajectories, were higher rifapentine exposure, lower baseline grade of sputum acid-fast bacillus smear, absence of productive cough, and lower extent of lung infiltrates on radiographs. Importantly, sensitivity analysis revealed that major learning milestones in phase 2b trials, such as significant exposure-response and covariate relationships, could be detected using truncated TTP data as early as 6 weeks from start of treatment, suggesting alternative phase 2b study designs. The TTP model built depicts a novel phase 2b surrogate endpoint that can inform early assessment of experimental treatment efficacy and treatment failure or relapse in patients treated with shorter and novel TB treatment regimens, improving efficiency of phase 2 clinical trials. (The studies discussed in this paper have been registered at ClinicalTrials.gov under identifiers NCT00694629 and NCT01043575.).

Introduction: With the growing use of online study management systems and rapid availability of data, timely data review and quality assessments are necessary to ensure proper clinical trial implementation. In this report we describe central monitoring used to ensure protocol compliance and accurate data reporting, implemented during a large phase 3 clinical trial., Material and Methods: The Tuberculosis Trials Consortium (TBTC) Study 31/AIDS Clinical Trials Group (ACTG) study A5349 (S31) is an international, multi-site, randomized, open-label, controlled, non-inferiority phase 3 clinical trial comparing two 4-month regimens to a standard 6 month regimen for treatment of drug-susceptible tuberculosis (TB) among adolescents and adults with a sample size of 2500 participants., Results: Central monitoring utilized primary study data in a five-tiered approach, including (1) real-time data checks & topic-specific intervention reports, (2) missing forms reports, (3) quality assurance metrics, (4) critical data reports and (5) protocol deviation identification, aimed to detect and resolve quality challenges. Over the course of the study, 240 data checks and reports were programed across the five tiers used., Discussion: This use of primary study data to identify issues rapidly allowed the study sponsor to focus quality assurance and data cleaning activities on prioritized data, related to protocol compliance and accurate reporting of study results. Our approach enabled us to become more efficient and effective as we informed sites about deviations, resolved missing or inconsistent data, provided targeted guidance, and gained a deeper understanding of challenges experienced at clinical trial sites., Trial Registration: This trial was registered with ClinicalTrials.gov (Identifier: NCT02410772) on April 8, 2015., (Published by Elsevier Inc.)

Background: Rifapentine exposure is associated with bactericidal activity against Mycobacterium tuberculosis, but high interindividual variation in plasma concentrations is encountered., Objectives: To investigate a genomic association with interindividual variation of rifapentine exposure, SNPs of six human genes involving rifamycin metabolism (AADAC, CES2), drug transport (SLCO1B1, SLCO1B3) and gene regulation (HNF4A, PXR) were evaluated., Methods: We characterized these genes in 173 adult participants in treatment trials of the Tuberculosis Trials Consortium. Participants were stratified by self-identified race (black or non-black), and rifapentine AUC from 0 to 24 h (AUC0-24) was adjusted by analysis of covariance for SNPs, rifapentine dose, sex, food and HIV coinfection. This study was registered at ClinicalTrials.gov under identifier NCT01043575., Results: The effect on rifapentine least squares mean AUC0-24 in black participants overall decreased by -10.2% for AADAC rs1803155 G versus A allele (Wald test: P = 0.03; false discovery rate, 0.10). Black participants with one G allele in AADAC rs1803155 were three times as likely to have below target bactericidal rifapentine exposure than black participants with the A allele (OR, 2.97; 95% CI: 1.16, 7.58). With two G alleles, the OR was greater. In non-black participants, AADAC rs1803155 SNP was not associated with rifapentine exposure. In both black and non-black participants, other evaluated genes were not associated with rifapentine exposure (P > 0.05; false discovery rate > 0.10)., Conclusions: Rifapentine exposure in black participants varied with AADAC rs1803155 genotype and the G allele was more likely to be associated with below bactericidal target rifapentine exposure. Further pharmacogenomic study is needed to characterize the association of the AADAC rs1803155 with inadequate rifapentine exposure in different patient groups., (Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy 2020. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

More efficacious treatment regimens are needed for tuberculosis, however, drug development is impeded by a lack of reliable biomarkers of disease severity and of treatment effect. We conducted a directed screen of host biomarkers in participants enrolled in a tuberculosis clinical trial to address this need. Serum samples from 319 protocol-correct, culture-confirmed pulmonary tuberculosis patients treated under direct observation as part of an international, phase 2 trial were screened for 70 markers of infection, inflammation, and metabolism. Biomarker assays were specifically developed for this study and quantified using a novel, multiplexed electrochemiluminescence assay. We evaluated the association of biomarkers with baseline characteristics, as well as with detailed microbiologic data, using Bonferroni-adjusted, linear regression models. Across numerous analyses, seven proteins, SAA1, PCT, IL-1β, IL-6, CRP, PTX-3 and MMP-8, showed recurring strong associations with markers of baseline disease severity, smear grade and cavitation; were strongly modulated by tuberculosis treatment; and had responses that were greater for patients who culture-converted at 8weeks. With treatment, all proteins decreased, except for osteocalcin, MCP-1 and MCP-4, which significantly increased. Several previously reported putative tuberculosis-associated biomarkers (HOMX1, neopterin, and cathelicidin) were not significantly associated with treatment response. In conclusion, across a geographically diverse and large population of tuberculosis patients enrolled in a clinical trial, several previously reported putative biomarkers were not significantly associated with treatment response, however, seven proteins had recurring strong associations with baseline radiographic and microbiologic measures of disease severity, as well as with early treatment response, deserving additional study., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

The Xpert MTB/RIF assay is both sensitive and specific as a diagnostic test. Xpert also reports quantitative output in cycle threshold (C T ) values, which may provide a dynamic measure of sputum bacillary burden when used longitudinally. We evaluated the relationship between Xpert C T trajectory and drug exposure during tuberculosis (TB) treatment to assess the potential utility of Xpert C T for treatment monitoring. We obtained serial sputum samples from patients with smear-positive pulmonary TB who were consecutively enrolled at 10 international clinical trial sites participating in study 29X, a CDC-sponsored Tuberculosis Trials Consortium study evaluating the tolerability, safety, and antimicrobial activity of rifapentine at daily doses of up to 20 mg/kg of body weight. Xpert was performed at weeks 0, 2, 4, 6, 8, and 12. Longitudinal C T data were modeled using a nonlinear mixed effects model in relation to rifapentine exposure (area under the concentration-time curve [AUC]). The rate of change of C T was higher in subjects receiving rifapentine than in subjects receiving standard-dose rifampin. Moreover, rifapentine exposure, but not assigned dose, was significantly associated with rate of change in C T (P = 0.02). The estimated increase in C T slope for every additional 100 μg · h/ml of rifapentine drug exposure (as measured by AUC) was 0.11 C T /week (95% confidence interval [CI], 0.05 to 0.17). Increasing rifapentine exposure is associated with a higher rate of change of Xpert C T , indicating faster clearance of Mycobacterium tuberculosis DNA. These data suggest that the quantitative outputs of the Xpert MTB/RIF assay may be useful as a dynamic measure of TB treatment response., (Copyright © 2016 Jayakumar et al.)

Setting: Two-month solid medium culture conversion is a commonly used, if suboptimal, endpoint for phase 2 anti-tuberculosis treatment trials., Objective and Design: To model the effect of the performance characteristics (sensitivity and contamination rate) of solid medium on required sample size for a two-arm clinical trial with 85% true (gold standard) culture conversion in the control and 95% in the experimental arm., Results: Increasing sensitivity and decreasing contamination reduced the sample size from 239 subjects/arm (60% sensitivity, 30% contamination) to 138 subjects/arm (95% sensitivity, 1% contamination)., Conclusion: Optimizing solid medium has significant potential to reduce sample size and increase the efficiency of tuberculosis clinical trials.

Background: The QuantiFERON®-TB Gold In-Tube test (QFT-GIT) detects Mycobacterium tuberculosis (Mtb) infection by measuring release of interferon gamma (IFN-γ) when T-cells (in heparinized whole blood) are stimulated with specific Mtb antigens. The amount of IFN-γ is determined by enzyme-linked immunosorbent assay (ELISA). Automation of the ELISA method may reduce variability. To assess the impact of ELISA automation, we compared QFT-GIT results and variability when ELISAs were performed manually and with automation., Methods: Blood was collected into two sets of QFT-GIT tubes and processed at the same time. For each set, IFN-γ was measured in automated and manual ELISAs. Variability in interpretations and IFN-γ measurements was assessed between automated (A1 vs. A2) and manual (M1 vs. M2) ELISAs. Variability in IFN-γ measurements was also assessed on separate groups stratified by the mean of the four ELISAs., Results: Subjects (N = 146) had two automated and two manual ELISAs completed. Overall, interpretations were discordant for 16 (11%) subjects. Excluding one subject with indeterminate results, 7 (4.8%) subjects had discordant automated interpretations and 10 (6.9%) subjects had discordant manual interpretations (p = 0.17). Quantitative variability was not uniform; within-subject variability was greater with higher IFN-γ measurements and with manual ELISAs. For subjects with mean TB Responses ±0.25 IU/mL of the 0.35 IU/mL cutoff, the within-subject standard deviation for two manual tests was 0.27 (CI95 = 0.22-0.37) IU/mL vs. 0.09 (CI95 = 0.07-0.12) IU/mL for two automated tests., Conclusion: QFT-GIT ELISA automation may reduce variability near the test cutoff. Methodological differences should be considered when interpreting and using IFN-γ release assays (IGRAs).

Objective. Use of tuberculin skin tests (TSTs) and interferon gamma release assays (IGRAs) as part of tuberculosis (TB) screening among immigrants from high TB-burden countries has not been fully evaluated. Methods. Prevalence of Mycobacterium tuberculosis infection (MTBI) based on TST, or the QuantiFERON-TB Gold test (QFT-G), was determined among immigrant applicants in Vietnam bound for the United States (US); factors associated with test results and discordance were assessed; predictive values of TST and QFT-G for identifying chest radiographs (CXRs) consistent with TB were calculated. Results. Of 1,246 immigrant visa applicants studied, 57.9% were TST positive, 28.3% were QFT-G positive, and test agreement was 59.4%. Increasing age was associated with positive TST results, positive QFT-G results, TST-positive but QFT-G-negative discordance, and abnormal CXRs consistent with TB. Positive predictive values of TST and QFT-G for an abnormal CXR were 25.9% and 25.6%, respectively. Conclusion. The estimated prevalence of MTBI among US-bound visa applicants in Vietnam based on TST was twice that based on QFT-G, and 14 times higher than a TST-based estimate of MTBI prevalence reported for the general US population in 2000. QFT-G was not better than TST at predicting abnormal CXRs consistent with TB.

Background: The QuantiFERON®-TB Gold In-Tube test (QFT-GIT) is a viable alternative to the tuberculin skin test (TST) for detecting Mycobacterium tuberculosis infection. However, within-subject variability may limit test utility. To assess variability, we compared results from the same subjects when QFT-GIT enzyme-linked immunosorbent assays (ELISAs) were performed in different laboratories., Methods: Subjects were recruited at two sites and blood was tested in three labs. Two labs used the same type of automated ELISA workstation, 8-point calibration curves, and electronic data transfer. The third lab used a different automated ELISA workstation, 4-point calibration curves, and manual data entry. Variability was assessed by interpretation agreement and comparison of interferon-γ (IFN-γ) measurements. Data for subjects with discordant interpretations or discrepancies in TB Response >0.05 IU/mL were verified or corrected, and variability was reassessed using a reconciled dataset., Results: Ninety-seven subjects had results from three labs. Eleven (11.3%) had discordant interpretations and 72 (74.2%) had discrepancies >0.05 IU/mL using unreconciled results. After correction of manual data entry errors for 9 subjects, and exclusion of 6 subjects due to methodological errors, 7 (7.7%) subjects were discordant. Of these, 6 (85.7%) had all TB Responses within 0.25 IU/mL of the manufacturer's recommended cutoff. Non-uniform error of measurement was observed, with greater variation in higher IFN-γ measurements. Within-subject standard deviation for TB Response was as high as 0.16 IU/mL, and limits of agreement ranged from -0.46 to 0.43 IU/mL for subjects with mean TB Response within 0.25 IU/mL of the cutoff., Conclusion: Greater interlaboratory variability was associated with manual data entry and higher IFN-γ measurements. Manual data entry should be avoided. Because variability in measuring TB Response may affect interpretation, especially near the cutoff, consideration should be given to developing a range of values near the cutoff to be interpreted as "borderline," rather than negative or positive.

Introduction: Interferon gamma (IFN-γ) release assays, such as QuantiFERON®-TB Gold test (QFT-G) and QuantiFERON®-TB Gold In-Tube test (QFT-GIT) are designed to detect M. tuberculosis (Mtb) infection. Recognition of unusual IFN-γ measurements may help indicate inaccurate results., Methods: We examined QFT-G and QFT-GIT results from subjects who had two or more tests completed. We classified unusual IFN-γ measurements as: 1) High Nil Concentration (HNC) when IFN-γ concentration in plasma from unstimulated blood exceeded 0.7 IU/mL; 2) Low Mitogen Response (LMR) when Mitogen Response was <0.5 IU/mL; 3) Very Low Mitogen Response (VLMR) when Mitogen Response was ≤-0.5 IU/mL; and 4) Very Low Antigen Response (VLAR) when the response to a Mtb antigen was ≤-0.35 IU/mL and ≤-0.5 times the IFN-γ concentration in plasma from unstimulated blood., Results: Among 5,309 results from 1,728 subjects, HNC occurred in 234 (4.4%) tests for 162 subjects, LMR in 108 (2.0%) tests for 85 subjects, VLMR in 22 (0.4%) tests for 21 subjects, and VLAR in 41 (0.8%) tests for 39 subjects. QFT-GIT had fewer HNC, VLMR, and VLAR (p = 0.042, 0.004, and 0.067 respectively); QFT-G had fewer LMR (p = 0.005). Twenty-four (51.6%) of 47 subjects with positive results and HNC were negative or indeterminate by all other tests. Thirteen (61.9%) of 21 subjects with positive results and LMR were negative or indeterminate by all other tests., Conclusion: Unusual IFN-γ measurements including HNC, LMR, VLMR, and VLAR were encountered in small numbers, and in most instances were not seen on simultaneously or subsequently performed tests. To avoid erroneous diagnosis of Mtb infection, IGRAs with unusual IFN-γ measurements should be repeated with another blood sample and interpreted with caution if they recur.

Background: Mycobacterium tuberculosis (Mtb) infection may cause overt disease or remain latent. Interferon gamma release assays (IGRAs) detect Mtb infection, both latent infection and infection manifesting as overt disease, by measuring whole-blood interferon gamma (IFN-γ) responses to Mtb antigens such as early secreted antigenic target-6 (ESAT-6), culture filtrate protein 10 (CFP-10), and TB7.7. Due to a lack of adequate diagnostic standards for confirming latent Mtb infection, IGRA sensitivity for detecting Mtb infection has been estimated using patients with culture-confirmed tuberculosis (CCTB) for whom recovery of Mtb confirms the infection. In this study, cytokines in addition to IFN-γ were assessed for potential to provide robust measures of Mtb infection., Methods: Cytokine responses to ESAT-6, CFP-10, TB7.7, or combinations of these Mtb antigens, for patients with CCTB were compared with responses for subjects at low risk for Mtb infection (controls). Three different multiplexed immunoassays were used to measure concentrations of 9 to 20 different cytokines. Responses were calculated by subtracting background cytokine concentrations from cytokine concentrations in plasma from blood stimulated with Mtb antigens., Results: Two assays demonstrated that ESAT-6, CFP-10, ESAT-6+CFP-10, and ESAT-6+CFP-10+TB7.7 stimulated the release of significantly greater amounts of IFN-γ, IL-2, IL-8, MCP-1 and MIP-1β for CCTB patients than for controls. Responses to combination antigens were, or tended to be, greater than responses to individual antigens. A third assay, using whole blood stimulation with ESAT-6+CFP-10+TB7.7, revealed significantly greater IFN-γ, IL-2, IL-6, IL-8, IP-10, MCP-1, MIP-1β, and TNF-α responses among patients compared with controls. One CCTB patient with a falsely negative IFN-γ response had elevated responses with other cytokines., Conclusions: Multiple cytokines are released when whole blood from patients with CCTB is stimulated with Mtb antigens. Measurement of multiple cytokine responses may improve diagnostic sensitivity for Mtb infection compared with assessment of IFN-γ alone.

Purpose: Assessing familial risk for early-onset coronary heart disease (CHD) is typically limited to first-degree relatives with early-onset CHD. To evaluate the impact of additional family history, we examined the associations between various family history definitions and early-onset CHD., Methods: By using the national HealthStyles 2003 survey data, we assessed associations between self-reported family history and personal history of early-onset CHD (diagnosed at or before age 60 years), adjusting for demographics, hypercholesterolemia, hypertension, and obesity., Results: Of 4,035 respondents, 60% were female and 72% were white, with a mean age of 48.8 years; 4.4% had early-onset CHD. In addition to having at least one first-degree relative with early-onset CHD, other significant associations included having at least one first-degree relative with late-onset CHD, at least one second-degree relative with early-onset CHD, and two or more affected second-degree relatives regardless of age of onset of CHD. Early-onset stroke in at least one first-degree relative and, in women, having at least one first-degree relative with diabetes were also significantly associated with early-onset CHD., Conclusions: Family history beyond early-onset CHD in first-degree relatives is significantly associated with prevalent CHD diagnosed at or before age 60 years.

Purpose: We examined the performance of a familial risk assessment method that stratifies risk for early-onset coronary heart disease by considering the number of relatives with coronary disease, degree of relationship, lineage, and age at diagnosis., Methods: By using data from the HealthStyles 2003 survey, we assessed the associations between familial risk and early-onset coronary heart disease, diabetes, hypercholesterolemia, hypertension, and obesity. By using area under the curve statistics, we evaluated the discriminatory ability of various risk assessment models., Results: Of 4,035 respondents, 60% were female and 72% were white, with a mean age of 48.8 years. After adjustment for demographics, strong and moderate risk were significantly associated with approximately a five- and twofold risk of early-onset coronary disease, respectively. After adjustment for demographics and personal history of cardiovascular disease, strong familial risk was also significantly associated with diabetes, hypercholesterolemia, hypertension, and obesity. A risk assessment model that included familial risk, demographics, and personal history of diabetes, hypercholesterolemia, hypertension, and obesity was most optimal with an area under the curve statistic of 87.2%, Conclusions: Familial risk assessment can stratify risk for early-onset coronary heart disease. Several conditions associated with increased familial risk can be prevented. These results have important implications for risk assessment and risk-reducing interventions.

Objective: To characterize allele frequencies of known single-nucleotide polymorphisms (SNPs) in tumor necrosis factor receptor (TNFR) genes in African Americans with rheumatoid arthritis (RA), healthy African Americans, and healthy Caucasians., Methods: One TNFRSF1B SNP (196 G/T) that influences susceptibility to familial RA in Caucasians and 3 SNPs in the 5' flanking region of the TNFRSF1A gene (-609G/T, -580A/G, and -383A/C) were genotyped in 108 African Americans with RA, 62 healthy African Americans, and 59 healthy Caucasians., Results: There were no differences in TNFRSF1A allele frequencies between African Americans with RA and healthy African Americans. Allele frequencies were strikingly different, however, between healthy African Americans and healthy Caucasians: 0.13 versus 0.42 for -609T, 0.49 versus 0 for -580G, and 0.14 versus 0 for -383C. We identified 4 novel haplotypes defined by the 3 TNFRSF1A SNPs, the distribution of which was markedly different in healthy Caucasians and healthy African Americans (P = 0.000001 by chi-square test-. The frequencies of the TNFRSF1B 196 genotypes were similar in African Americans with RA and healthy African Americans but differed between healthy African Americans and healthy Caucasians (P = 0.05)., Conclusion: Although we observed no associations between known TNFR SNPs or haplotypes and RA, significant racial differences were observed at both loci. Comparison of these data with other published frequencies of TNFRSF1A and TNFRSF1B genotypes according to race suggests that the distribution in African American, Caucasian, and Asian populations differs significantly. These striking racial/ethnic differences in TNFR SNP frequencies may influence the likelihood of familial RA, severe disease, or response to TNF inhibitors and may have important evolutionary implications.

HLA-DRB1, DRB3, DRB4, DRB5 and DQB1 polymorphisms were studied using molecular methods in a population of 100 unrelated healthy individuals from an area in north-west Colombia (Medellin) inhabited by the "Paisa", a community with features of a genetically isolated group. The most frequently observed specificities at the DRB1 locus were *07 (16.4%) and *15 (12%), and at the DQB1 locus *02 (18.8%) and *03 (33.6%), of which *0302 was the most prevalent allele (14.3%). The most polymorphic specificities were DRB1*04, 13 and 11, and DQB1*06. Both the HLA-DRB1 and DQB1 loci were in linkage disequilibrium. Haplotypes were estimated using maximum likelihood methods. The most frequent two locus haplotype was DRB1*07-DQB1*02 (6.6%) and these specificities were in linkage disequilibrium. Several unusual possible haplotypes were observed. Both the HLA-DRB1 and DQB1 locus were in Hardy-Weinberg equilibrium.

We identified functionally important regions of the DR(alpha, beta 1*0401) peptide binding site and present a model of bound peptide. DR(alpha, beta 1*0401)-restricted T cell recognition and peptide binding of Mycobacterium leprae (ML) peptide 38-50 and overlapping peptides from the 18-kDa heat-shock protein were analyzed. ML38-50 is unusual in its restricted binding pattern, binding to only one of five DR4 subtypes and no other DR molecules tested. Amino acid substitutions were introduced into ML38-50 and the DR(alpha, beta 1*0401) peptide binding site at positions likely to influence peptide-MHC or peptide- or MHC-TCR interactions. Peptide binding, T cell proliferation, and computer modeling studies suggest that residues 39F, 42E, and 44D of ML38-50 interact with pockets 1, 4, and 6, respectively, of the peptide binding site. Only DR(alpha, beta 1*0401) substitutions at residues in pockets 4 or 7 prevented binding of ML38-50, while multiple substitutions at other positions negatively affected its T cell recognition. In contrast, T cell recognition of some high affinity ML peptides that overlapped ML38-50, and contained N-terminal extensions, was only abolished with pocket 4 substitutions. An inverse correlation of peptide affinity for DR(alpha, beta 1*0401) with negative effects of MHC substitutions on T cell recognition of the overlapping ML peptides was observed. Thus, some regions, such as pocket 4, dominantly influence T cell recognition of multiple DR(alpha, beta 1*0401)-binding peptides. However, each DR(alpha, beta 1*0401)-binding peptide appears to have unique properties that determine the outcome of its MHC-peptide interactions and the relative importance of other polymorphic pockets.