Safety and immunogenicity of the H56:IC31 tuberculosis vaccine candidate in adults successfully treated for drug-susceptible pulmonary TB: a phase 1 randomized trial.

BACKGROUND: H56:IC31 is a candidate vaccine against tuberculosis (TB) with the potential to reduce TB recurrence rate. It is thus important for future clinical trials to demonstrate safety and immunogenicity of H56:IC31 in individuals treated for TB. METHODS: 22 adults confirmed to be Mtb negative (by 2 GeneXpert tests or 2 sputum cultures) after four-five months of TB treatment, and not more than 28 days after completion of TB treatment, were randomized to receive two doses of H56:IC31 (5 mg H56:500 nmol IC31; N=16) or placebo (N=6) 56 days apart. Participants were followed for 420 days for safety and immunogenicity. RESULTS: H56:IC31 vaccination was associated with an acceptable safety profile, consisting mostly of mild self-limited injection site reactions. No serious adverse events, and no vaccine-related severe adverse events, were reported. H56:IC31 induced a CD4+ T-cell response for Ag85B and ESAT-6, with ESAT-6 being immunodominant, which persisted through six months after the last vaccination. There was some evidence of CD8+ T-cell responses for both Ag85B and ESAT-6, but to a lesser extent than CD4+ responses. CONCLUSIONS: H56:IC31 was associated with an acceptable safety profile, and induced a predominant CD4+ T-cell response, in adults recently treated for drug-susceptible, uncomplicated pulmonary TB. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02375698.

Tuberculosis Treatment Monitoring and Outcome Measures: New Interest and New Strategies.

Despite the advent of new diagnostics, drugs and regimens, tuberculosis (TB) remains a global public health threat. A significant challenge for TB control efforts has been the monitoring of TB therapy and determination of TB treatment success. Current recommendations for TB treatment monitoring rely on sputum and culture conversion, which have low sensitivity and long turnaround times, present biohazard risk, and are prone to contamination, undermining their usefulness as clinical treatment monitoring tools and for drug development. We review the pipeline of molecular technologies and assays that serve as suitable substitutes for current culture-based readouts for treatment response and outcome with the potential to change TB therapy monitoring and accelerate drug development.

Target product profiles: tests for tuberculosis treatment monitoring and optimization.

The World Health Organization has developed target product profiles containing minimum and optimum targets for key characteristics for tests for tuberculosis treatment monitoring and optimization. Tuberculosis treatment optimization refers to initiating or switching to an effective tuberculosis treatment regimen that results in a high likelihood of a good treatment outcome. The target product profiles also cover tests of cure conducted at the end of treatment. The development of the target product profiles was informed by a stakeholder survey, a cost-effectiveness analysis and a patient-care pathway analysis. Additional feedback from stakeholders was obtained by means of a Delphi-like process, a technical consultation and a call for public comment on a draft document. A scientific development group agreed on the final targets in a consensus meeting. For characteristics rated of highest importance, the document lists: (i) high diagnostic accuracy (sensitivity and specificity); (ii) time to result of optimally ≤ 2 hours and no more than 1 day; (iii) required sample type to be minimally invasive, easily obtainable, such as urine, breath, or capillary blood, or a respiratory sample that goes beyond sputum; (iv) ideally the test could be placed at a peripheral-level health facility without a laboratory; and (v) the test should be affordable to low- and middle-income countries, and allow wide and equitable access and scale-up. Use of these target product profiles should facilitate the development of new tuberculosis treatment monitoring and optimization tests that are accurate and accessible for all people being treated for tuberculosis.

L'Organisation mondiale de la santé a élaboré des profils de produits cibles contenant des cibles minimales et optimales pour les caractéristiques principales des essais destinés au suivi et à l'optimisation du traitement de la tuberculose. L'optimisation du traitement de la tuberculose fait référence à l'instauration d'un régime de traitement efficace de la tuberculose ou à l'adoption d'un tel régime, avec une probabilité élevée d'obtenir de bons résultats thérapeutiques. Les profils de produits cibles couvrent également les essais de guérison effectués à l'issue du traitement. Les profils de produits cibles ont été élaborés sur la base d'un sondage auprès des parties prenantes, d'une analyse coût-efficacité et d'une analyse du parcours de soins du patient. Des retours supplémentaires des parties prenantes ont été obtenus au moyen d'un processus créé selon la méthode Delphi, d'une consultation technique et d'un appel à commentaires publics sur un projet de document. Un groupe d'élaboration scientifique s'est mis d'accord sur les objectifs finaux lors d'une réunion de concertation. En ce qui concerne les caractéristiques jugées les plus importantes, le document énumère ce qui suit: (i) une grande précision diagnostique (sensibilité et spécificité); (ii) un délai idéal d'obtention des résultats ≤ 2 heures et au maximum de 1 jour; (iii) le type d'échantillon requis doit être peu invasif et facile à obtenir, comme l'urine, l'haleine ou le sang capillaire, ou bien un échantillon respiratoire au-delà des expectorations; (iv) idéalement, l'essai pourrait avoir lieu dans un établissement de santé périphérique sans laboratoire ; et (v) l'essai devrait être abordable pour les pays à revenu faible et intermédiaire et permettre un accès large et équitable ainsi qu'une mise à l'échelle. L'utilisation de ces profils de produits cibles devrait faciliter la mise au point de nouveaux essais de surveillance et d'optimisation du traitement de la tuberculose qui soient précis et accessibles à toutes les personnes suivant un traitement pour la tuberculose.

La Organización Mundial de la Salud ha elaborado perfiles de productos objetivo que contienen objetivos mínimos y óptimos para las características principales de las pruebas de seguimiento y optimización del tratamiento de la tuberculosis. La optimización del tratamiento de la tuberculosis consiste en iniciar o cambiar a un régimen eficaz de tratamiento de la tuberculosis que ofrezca una alta probabilidad de un buen resultado terapéutico. Los perfiles de productos objetivo también abarcan las pruebas de curación realizadas al final del tratamiento. La elaboración de los perfiles de los productos objetivo se basó en una encuesta a las partes interesadas, un análisis de rentabilidad y un análisis de la vía de atención al paciente. Se obtuvo información adicional de las partes interesadas mediante un proceso tipo Delphi, una consulta técnica y una convocatoria de comentarios públicos sobre un borrador del documento. Un grupo de desarrollo científico acordó los objetivos finales en una reunión de consenso. Para las características clasificadas de mayor importancia, el documento enumera: (i) alta precisión diagnóstica (sensibilidad y especificidad); (ii) tiempo hasta el resultado de óptimamente ≤ 2 horas y no más de 1 día; (iii) el tipo de muestra requerida debe ser mínimamente invasiva, fácil de obtener, como orina, aliento o sangre capilar, o una muestra respiratoria que vaya más allá del esputo; (iv) idealmente la prueba podría realizarse en un centro sanitario periférico sin laboratorio; y (v) la prueba debe ser asequible para los países de ingresos bajos y medios y permitir un acceso amplio y equitativo y su expansión. El uso de estos perfiles de producto objetivo debería facilitar el desarrollo de pruebas nuevas de seguimiento y optimización del tratamiento de la tuberculosis que sean precisas y accesibles para todas las personas que reciben tratamiento antituberculoso.

GeneXpert Ultra in Urine Samples for Diagnosis of Extra-Pulmonary Tuberculosis.

Extra-pulmonary tuberculosis (EPTB) continues to be difficult to diagnose. Novel biomarkers in biological specimens offer promise. Detection of Mycobacterium tuberculosis (Mtb) DNA in urine could prove useful in diagnosis of EPTB, possibly due to disseminated disease or micro-abscesses reported in kidneys. The current study was designed to detect Mtb DNA in stored urine samples from patients with EPTB. Diagnosis of EPTB was reached using Microbiological Reference Standards (MRS) on samples from the disease site using WHO Recommended Diagnostics (WRD), [smear microscopy, liquid culture (MGIT-960)] and GX (molecular WRD, mWRD) and Comprehensive reference standards [CRS, clinical presentation, microbiological reference standards, radiology, histopathology]. GX-Ultra was performed on urine samples stored in -80oC deep freezer, retrospectively. Of 70 patients, 51 (72.9%) were classified as confirmed TB, 11 (15.7%) unconfirmed TB, and 8 (11.4%) unlikely TB. GX-Ultra in urine samples demonstrated sensitivity of 52.9% and specificity of 57.9% against MRS, and higher sensitivity of 56.5% and specificity of 100% against CRS. The sensitivity and specificity of GX-Ultra in urine was 53.6% and 75% for pus sample subset and 52.2% and 53.3% for fluid sample subset. Urine being non-invasive and easy to collect, detection of Mtb DNA using mWRD in urine samples is promising for diagnosis of EPTB.

Biomarkers That Correlate with Active Pulmonary Tuberculosis Treatment Response: a Systematic Review and Meta-analysis.

Current WHO recommendations for monitoring treatment response in adult pulmonary tuberculosis (TB) are sputum smear microscopy and/or culture conversion at the end of the intensive phase of treatment. These methods either have suboptimal accuracy or a long turnaround time. There is a need to identify alternative biomarkers to monitor TB treatment response. We conducted a systematic review of active pulmonary TB treatment monitoring biomarkers. We screened 9,739 articles published between 1 January 2008 and 31 December 2020, of which 77 met the inclusion criteria. When studies quantitatively reported biomarker levels, we meta-analyzed the average fold change in biomarkers from pretreatment to week 8 of treatment. We also performed a meta-analysis pooling the fold change since the previous time point collected. A total of 81 biomarkers were identified from 77 studies. Overall, these studies exhibited extensive heterogeneity with regard to TB treatment monitoring study design and data reporting. Among the biomarkers identified, C-reactive protein (CRP), interleukin-6 (IL-6), interferon gamma-induced protein 10 (IP-10), and tumor necrosis factor alpha (TNF-α) had sufficient data to analyze fold changes. All four biomarker levels decreased during the first 8 weeks of treatment relative to baseline and relative to previous time points collected. Based on limited data available, CRP, IL-6, IP-10, and TNF-α have been identified as biomarkers that should be further explored in the context of TB treatment monitoring. The extensive heterogeneity in TB treatment monitoring study design and reporting is a major barrier to evaluating the performance of novel biomarkers and tools for this use case. Guidance for designing and reporting treatment monitoring studies is urgently needed.

Antigen-Specific T-Cell Activation Distinguishes between Recent and Remote Tuberculosis Infection.

Rationale: Current diagnostic tests fail to identify individuals at higher risk of progression to tuberculosis disease, such as those with recent Mycobacterium tuberculosis infection, who should be prioritized for targeted preventive treatment. Objectives: To define a blood-based biomarker, measured with a simple flow cytometry assay, that can stratify different stages of tuberculosis infection to infer risk of disease. Methods: South African adolescents were serially tested with QuantiFERON-TB Gold to define recent (QuantiFERON-TB conversion <6 mo) and persistent (QuantiFERON-TB+ for >1 yr) infection. We defined the ΔHLA-DR median fluorescence intensity biomarker as the difference in HLA-DR expression between IFN-γ+ TNF+Mycobacterium tuberculosis-specific T cells and total CD3+ T cells. Biomarker performance was assessed by blinded prediction in untouched test cohorts with recent versus persistent infection or tuberculosis disease and by unblinded analysis of asymptomatic adolescents with tuberculosis infection who remained healthy (nonprogressors) or who progressed to microbiologically confirmed disease (progressors). Measurements and Main Results: In the test cohorts, frequencies of Mycobacterium tuberculosis-specific T cells differentiated between QuantiFERON-TB- (n = 25) and QuantiFERON-TB+ (n = 47) individuals (area under the receiver operating characteristic curve, 0.94; 95% confidence interval, 0.87-1.00). ΔHLA-DR significantly discriminated between recent (n = 20) and persistent (n = 22) QuantiFERON-TB+ (0.91; 0.83-1.00); persistent QuantiFERON-TB+ and newly diagnosed tuberculosis (n = 19; 0.99; 0.96-1.00); and tuberculosis progressors (n = 22) and nonprogressors (n = 34; 0.75; 0.63-0.87). However, ΔHLA-DR median fluorescent intensity could not discriminate between recent QuantiFERON-TB+ and tuberculosis (0.67; 0.50-0.84). Conclusions: The ΔHLA-DR biomarker can identify individuals with recent QuantiFERON-TB conversion and those with disease progression, allowing targeted provision of preventive treatment to those at highest risk of tuberculosis. Further validation studies of this novel immune biomarker in various settings and populations at risk are warranted.

Diagnostic accuracy of a novel point-of-care urine lipoarabinomannan assay for the detection of tuberculosis among adult outpatients in Zambia: a prospective cross-sectional study.

BACKGROUND: A novel, rapid, point-of-care urine-based lipoarabinomannan assay (Fujifilm SILVAMP TB LAM ("FujiLAM")) has previously demonstrated substantially higher sensitivity for tuberculosis (TB) compared with the commercially available Determine TB LAM assay using biobanked specimens. However, FujiLAM has not been prospectively evaluated using fresh urine specimens. Therefore, we determined the diagnostic accuracy of FujiLAM among HIV-positive and HIV-negative outpatients with presumptive TB in Zambia. METHODS: Adult (≥18 years old) presumptive TB patients presenting to two outpatient public health facilities in Lusaka were included. All patients submitted sputa samples for smear microscopy, Xpert MTB/RIF and mycobacterial culture, and urine samples for the FujiLAM assay. Microbiologically confirmed TB was defined by the detection of Mycobacterium tuberculosis in sputum using culture; this served as the reference standard to assess the diagnostic accuracy of FujiLAM. RESULTS: 151 adults with paired sputum microbiological tests and urine FujiLAM results were included; 45% were HIV-positive. Overall, 34 out of 151 (23%) patients had culture-confirmed pulmonary TB. The overall sensitivity and specificity of FujiLAM was 77% (95% CI 59-89%) and 92% (95% CI 86-96%), respectively. FujiLAM's sensitivity among HIV-positive patients was 75% (95% CI 43-95%) compared with 75% (95% CI 51-91%) among HIV-negative patients. The sensitivity of FujiLAM in patients with smear-positive, confirmed pulmonary TB was 87% (95% CI 60-98%) compared with 68% (95% CI 43-87%) among patients with smear-negative, confirmed pulmonary TB. CONCLUSIONS: FujiLAM demonstrated high sensitivity for the detection of TB among both HIV-positive and HIV-negative adults, and also demonstrated good specificity despite the lack of systematic extrapulmonary sampling to inform a comprehensive microbiological reference standard.

Tests for tuberculosis infection: landscape analysis.

BACKGROUND: Only the tuberculin skin test (TST) and two interferon-γ release assays (IGRAs), QuantiFERON-TB Gold In-Tube and T-SPOT.TB, are currently endorsed by the World Health Organization as tests for tuberculosis (TB) infection. While IGRAs are more specific than the TST, they require sophisticated laboratory infrastructure and are costly to perform. However, both types of tests have limited performance to predict development of active TB. Tests with improved predictive performance and operational characteristics are needed. METHODS: We reviewed the current landscape of tests for TB infection identified through a web-based survey targeting diagnostic manufacturers globally. RESULTS: We identified 20 tests for TB infection: 15 in vitro tests and five skin tests. 13 of the in vitro tests are whole-blood IGRAs and 14 use early secreted antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10), with or without additional antigens. 10 of the tests are based on assays other than an ELISA, such as a fluorescent lateral flow assay that requires less manual operation and shorter assay time and hence is more suitable for decentralisation compared with the existing IGRAs. Four of the five skin tests use ESAT-6 and CFP-10 proteins, while the remaining test uses a new antigen that is specific to Mycobacterium tuberculosis complex. CONCLUSIONS: New tests have the potential to improve accuracy, operational characteristics and end-user access to tests for TB infection. However, published data in various populations and settings are limited for most new tests. Evaluation of these new tests in a standardised design would facilitate their endorsement and programmatic scale-up.

Framework for the evaluation of new tests for tuberculosis infection.

The scale-up of tuberculosis (TB) preventive treatment (TPT) must be accelerated to achieve the targets set by the United Nations High-level Meeting on TB and the End TB Strategy. The scale-up of effective TPT is hampered by concerns about operational challenges to implement the existing tests for TB infection. New simpler tests could facilitate the scale-up of testing for TB infection. We present a framework for evaluation of new immunodiagnostic tests for the detection of TB infection, with an aim to facilitate their standardised evaluation and accelerate adoption into global and national policies and subsequent scale-up. The framework describes the principles to be considered when evaluating new tests for TB infection and provides guidance to manufacturers, researchers, regulators and other users on study designs, populations, reference standards, sample size calculation and data analysis and it is also aligned with the Global Strategy for TB Research and Innovation adopted by the World Health Assembly in 2020. In addition, we briefly describe technical issues that should be considered when evaluating new tests, including the safety for skin tests, costs incurred by patients and the health system, and operational characteristics.

A prospective multicentre diagnostic accuracy study for the Truenat tuberculosis assays.

BACKGROUND: Bringing reliable and accurate tuberculosis (TB) diagnosis closer to patients is a key priority for global TB control. Molbio Diagnostics have developed the Truenat point-of-care molecular assays for detection of TB and rifampicin (RIF) resistance. METHODS: We conducted a prospective multicentre diagnostic accuracy study at 19 primary healthcare centres and seven reference laboratories in Peru, India, Ethiopia and Papua New Guinea to estimate the diagnostic accuracy of the point-of-care Truenat MTB, MTB Plus and MTB-RIF Dx assays for pulmonary TB using culture and phenotypic drug susceptibility testing as the reference standard, compared with Xpert MTB/RIF or Ultra. RESULTS: Of 1807 enrolled participants with TB signs/symptoms, 24% were culture-positive for Mycobacterium tuberculosis, of which 15% were RIF-resistant. In microscopy centres, the pooled sensitivity of Truenat MTB and Truenat MTB Plus was 73% (95% CI 67-78%) and 80% (95% CI 75-84%), respectively. Among smear-negative specimens, sensitivities were 36% (95% CI 27-47%) and 47% (95% CI 37-58%), respectively. Sensitivity of Truenat MTB-RIF was 84% (95% CI 62-95%). Truenat assays showed high specificity. Head-to-head comparison in the central reference laboratories suggested that the Truenat assays have similar performance to Xpert MTB/RIF. CONCLUSION: We found the performance of Molbio's Truenat MTB, MTB Plus and MTB-RIF Dx assays to be comparable to that of the Xpert MTB/RIF assay. Performing the Truenat tests in primary healthcare centres with very limited infrastructure was feasible. These data supported the development of a World Health Organization policy recommendation of the Molbio assays.

Comparative Analytical Evaluation of Four Centralized Platforms for the Detection of Mycobacterium tuberculosis Complex and Resistance to Rifampicin and Isoniazid.

Failure to rapidly identify drug-resistant tuberculosis (TB) increases the risk of patient mismanagement, the amplification of drug resistance, and ongoing transmission. We generated comparative analytical data for four automated assays for the detection of TB and multidrug-resistant TB (MDR-TB): Abbott RealTime MTB and MTB RIF/INH (Abbott), Hain Lifescience FluoroType MTBDR (Hain), BD Max MDR-TB (BD), and Roche cobas MTB and MTB-RIF/INH (Roche). We included Xpert MTB/RIF (Xpert) and GenoType MTBDRplus as comparators for TB and drug resistance detection, respectively. We assessed analytical sensitivity for the detection of the Mycobacterium tuberculosis complex using inactivated strains (M. tuberculosis H37Rv and M. bovis) spiked into TB-negative sputa and computed the 95% limits of detection (LOD95). We assessed the accuracy of rifampicin and isoniazid resistance detection using well-characterized M. tuberculosis strains with high-confidence mutations accounting for >85% of first-line resistance mechanisms globally. For H37Rv and M. bovis, we measured LOD95 values of 3,781 and 2,926 (Xpert), 322 and 2,182 (Abbott), 826 and 4,301 (BD), 10,398 and 23,139 (Hain), and 2,416 and 2,136 (Roche) genomes/ml, respectively. Assays targeting multicopy genes or targets (Abbott, BD, and Roche) showed increased analytical sensitivity compared to Xpert. Quantification of the panel by quantitative real-time PCR prevents the determination of absolute values, and results reported here can be interpreted for comparison purposes only. All assays showed accuracy comparable to that of Genotype MTBDRplus for the detection of rifampicin and isoniazid resistance. The data from this analytical study suggest that the assays may have clinical performances similar to those of WHO-recommended molecular TB and MDR-TB assays.

Diagnostic Accuracy of Interferon Gamma-Induced Protein 10 mRNA Release Assay for Tuberculosis.

Interferon gamma (IFN-γ) release assays (IGRAs) are increasingly used to test for latent tuberculosis (TB) infection. Although highly specific, IGRAs have a relatively high false-negative rate in active TB patients. A more sensitive assay is needed. IFN-γ-induced protein 10 (IP-10) is an alternative biomarker with a 100-fold-higher expression level than IFN-γ, allowing for different analysis platforms, including molecular detection. The PCR technique is already an integrated tool in most TB laboratories and, thus, an obvious platform to turn to. In this case-control study, we investigated the diagnostic sensitivity and specificity of a molecular assay detecting IP-10 mRNA expression following antigen stimulation of a blood sample. We included 89 TB patients and 99 healthy controls. Blood was drawn in QuantiFeron-TB gold in-tube (QFT) assay tubes. Eight hours poststimulation, IP-10 mRNA expression was analyzed, and 20 h poststimulation, IP-10 and IFN-γ protein plasma levels were analyzed using an in-house IP-10 enzyme-linked immunosorbent assay (ELISA) and the official QFT ELISA, respectively. The IP-10 mRNA assay provided high specificity (98%), sensitivity (80%), and area under the concentration-time curve (AUC) (0.97); however, the QFT assay provided a higher overall diagnostic potential, with specificity of 100%, sensitivity of 90%, and AUC of 0.99. The IP-10 protein assay performed on par with the QFT assay, with specificity of 98%, sensitivity of 87%, and AUC of 0.98. We have provided proof of high technical performance of a molecular assay detecting IP-10 mRNA expression. As a diagnostic tool, this assay would gain from further optimization, especially on the kinetics of IP-10 mRNA expression.

Dose Optimization of H56:IC31 Vaccine for Tuberculosis-Endemic Populations. A Double-Blind, Placebo-controlled, Dose-Selection Trial.

RATIONALE: Global tuberculosis (TB) control requires effective vaccines in TB-endemic countries, where most adults are infected with Mycobacterium tuberculosis (M.tb). OBJECTIVES: We sought to define optimal dose and schedule of H56:IC31, an experimental TB vaccine comprising Ag85B, ESAT-6, and Rv2660c, for M.tb-infected and M.tb-uninfected adults. METHODS: We enrolled 98 healthy, HIV-uninfected, bacillus Calmette-Guérin-vaccinated, South African adults. M.tb infection was defined by QuantiFERON-TB (QFT) assay. QFT-negative participants received two vaccinations of different concentrations of H56 in 500 nmol of IC31 to enable dose selection for further vaccine development. Subsequently, QFT-positive and QFT-negative participants were randomized to receive two or three vaccinations to compare potential schedules. Participants were followed for safety and immunogenicity for 292 days. MEASUREMENTS AND MAIN RESULTS: H56:IC31 showed acceptable reactogenicity profiles irrespective of dose, number of vaccinations, or M.tb infection. No vaccine-related severe or serious adverse events were observed. The three H56 concentrations tested induced equivalent frequencies and functional profiles of antigen-specific CD4 T cells. ESAT-6 was only immunogenic in QFT-negative participants who received three vaccinations. CONCLUSIONS: Two or three H56:IC31 vaccinations at the lowest dose induced durable antigen-specific CD4 T-cell responses with acceptable safety and tolerability profiles in M.tb-infected and M.tb-uninfected adults. Additional studies should validate applicability of vaccine doses and regimens to both QFT-positive and QFT-negative individuals. Clinical trial registered with www.clinicaltrials.gov (NCT01865487).

Diagnostic Accuracy of Early Secretory Antigenic Target-6-Free Interferon-gamma Release Assay Compared to QuantiFERON-TB Gold In-tube.

BACKGROUND: Early secretory antigenic target-6 (ESAT-6) is an immunodominant Mycobacterium tuberculosis (M.tb) antigen included in novel vaccines against tuberculosis (TB) and in interferon-gamma (IFN-γ) release assays (IGRAs). Therefore, the availability of an ESAT-6-free IGRA is essential to determine M.tb infection status following vaccination with ESAT-6-containing vaccines. We aimed to qualify a recently developed ESAT-6-free IGRA and to assess its diagnostic performance in comparison to QuantiFERON-TB Gold In-tube (QFT). METHODS: Participants with different levels of M.tb exposure and TB disease were enrolled to determine the ESAT-6-free IGRA cutoff, test assay performance in independent cohorts compared to standard QFT, and perform a technical qualification of antigen-coated blood collection tubes. RESULTS: ESAT-6-free IGRA antigen recognition was evaluated in QFT-positive and QFT-negative South African adolescents. The ESAT-6-free IGRA cutoff was established at 0.61 IU/mL, based on receiver operating characteristic analysis in M.tb-unexposed controls and microbiologically confirmed pulmonary TB patients. In an independent cohort of healthy adolescents, levels of IFN-γ released in QFT and ESAT-6-free IGRA were highly correlated (P < .0001, r = 0.83) and yielded comparable positivity rates, 41.5% and 43.5%, respectively, with 91% concordance between the tests (kappa = 0.82; 95% confidence interval, 0.74-0.90; McNemar test P = .48). ESAT-6-free IGRA blood collection tubes had acceptable lot-to-lot variability, precision, and stability. CONCLUSIONS: The novel ESAT-6-free IGRA had diagnostic accuracy comparable to QFT and is suitable for use in clinical trials to assess efficacy of candidate TB vaccines to prevent established M.tb infection.

Optimization and Interpretation of Serial QuantiFERON Testing to Measure Acquisition of Mycobacterium tuberculosis Infection.

RATIONALE: Conversion from a negative to positive QuantiFERON-TB test is indicative of Mycobacterium tuberculosis (Mtb) infection, which predisposes individuals to tuberculosis disease. Interpretation of serial tests is confounded by immunological and technical variability. OBJECTIVES: To improve the consistency of serial QuantiFERON-TB testing algorithms and provide a data-driven definition of conversion. METHODS: Sources of QuantiFERON-TB variability were assessed, and optimal procedures were identified. Distributions of IFN-γ response levels were analyzed in healthy adolescents, Mtb-unexposed control subjects, and patients with pulmonary tuberculosis. MEASUREMENTS AND MAIN RESULTS: Individuals with no known Mtb exposure had IFN-γ values less than 0.2 IU/ml. Among individuals with IFN-γ values less than 0.2 IU/ml, 0.2-0.34 IU/ml, 0.35-0.7 IU/ml, and greater than 0.7 IU/ml, tuberculin skin test positivity results were 15%, 53%, 66%, and 91% (P < 0.005), respectively. Together, these findings suggest that values less than 0.2 IU/ml were true negatives. In short-term serial testing, "uncertain" conversions, with at least one value within the uncertainty zone (0.2-0.7 IU/ml), were partly explained by technical assay variability. Individuals who had a change in QuantiFERON-TB IFN-γ values from less than 0.2 to greater than 0.7 IU/ml had 10-fold higher tuberculosis incidence rates than those who maintained values less than 0.2 IU/ml over 2 years (P = 0.0003). By contrast, "uncertain" converters were not at higher risk than nonconverters (P = 0.229). Eighty-seven percent of patients with active tuberculosis had IFN-γ values greater than 0.7 IU/ml, suggesting that these values are consistent with established Mtb infection. CONCLUSIONS: Implementation of optimized procedures and a more rigorous QuantiFERON-TB conversion definition (an increase from IFN-γ <0.2 to >0.7 IU/ml) would allow more definitive detection of recent Mtb infection and potentially improve identification of those more likely to develop disease.

Sensitivity of C-Tb: a novel RD-1-specific skin test for the diagnosis of tuberculosis infection.

C-Tb, a novel Mycobacterium tuberculosis and 6-kDa early secretory antigenic target/10-kDa culture filtrate protein (ESAT-6/CFP-10)-specific skin test, has high specificity in bacille Calmette-Guerin-vaccinated healthy controls. However, the sensitivity of C-Tb has hitherto not been determined. The objective was to determine the sensitivity of C-Tb in patients with active tuberculosis (TB) in comparison with the tuberculin skin test (TST) and QuantiFERON-TB Gold In-Tube (QFT-GIT).C-Tb and TST were randomly administered in a double-blinded fashion to one or the other forearm in 253 patients with active TB with or without HIV co-infection. QFT-GIT testing was performed prior to skin testing.Using a receiver operating characteristic curve-derived cut-point of 5 mm, C-Tb sensitivity was similar to QFT-GIT (73.9 (95% CI 67.8-79.3) versus 75.1 (95% CI 69.3-80.2)), and similar in HIV-infected and HIV-uninfected patients (76.7 (95% CI 69.0-83.3) versus 69.5 (95% CI 59.2-78.5)). However, sensitivity was significantly diminished in HIV-infected patients with CD4 counts <100 cells·mm(-3). C-Tb and QFT-GIT combined had significantly higher sensitivity than C-Tb alone (p<0.0001). C-Tb was safe with no significant adverse events. The 5 mm cut-point corresponded to that found in the previously published specificity study (TESEC-04).C-Tb has similar sensitivity compared with QFT-GIT for the diagnosis of M. tuberculosis infection. Sensitivity was reduced only in HIV-infected patients with severe immunosuppression. Further studies in different settings are required to validate the proposed 5 mm cut-point.

Getting personal perspectives on individualized treatment duration in multidrug-resistant and extensively drug-resistant tuberculosis.

Tuberculosis (TB) differs from most other bacterial infectious diseases by a very long duration of combination antibiotic therapy required to achieve relapse-free cure. Although the standard recommended "short-course" treatment length for TB is 6 months, the World Health Organization recommends a duration of 20 months for the treatment of patients with multidrug-resistant and extensively drug-resistant TB (M/XDR-TB). Apart from the long duration of anti-TB therapy, treatment of M/XDR-TB is very expensive and often associated with adverse drug events. The optimal duration for treatment of TB likely differs between individuals and depends on a variety of variables, such as the extent of the disease, the immune status of the host, and the virulence and the drug resistance of the causative strain of Mycobacterium tuberculosis. Some patients with M/XDR-TB may have to be treated with currently available antituberculosis drug regimens for more than 20 months, whereas much shorter treatment durations may be possible to achieve cure for the majority of patients with M/XDR-TB. Personalization of the duration of treatment for TB, especially for patients with M/XDR-TB, would be highly desired. Until recently there has been little interest in the identification of biosignatures that could eventually lead to individual recommendations for the duration of anti-TB therapy. This pulmonary perspective reviews the knowledge on clinical and radiological scores, host- and pathogen disease-related profiles, molecules, and signatures that are currently explored as biomarkers to personalize the duration of therapy in TB.

Beyond the IFN-γ horizon: biomarkers for immunodiagnosis of infection with Mycobacterium tuberculosis.

Latent infection with Mycobacterium tuberculosis (LTBI) is defined by the presence of M. tuberculosis-specific immunity in the absence of active tuberculosis. LTBI is detected using interferon-γ release assays (IGRAs) or the tuberculin-skin-test (TST). In clinical practice, IGRAs and the TSTs have failed to distinguish between active tuberculosis and LTBI and their predictive value to identify individuals at risk for the future development of tuberculosis is limited. There is an urgent need to identify biomarkers that improve the clinical performance of current immunodiagnostic methods for tuberculosis prevention, diagnosis and treatment monitoring. Here, we review the landscape of potential alternative biomarkers useful for detection of infection with M. tuberculosis. We describe what individual markers add in terms of specificity for active/latent infection, prediction of progression to active tuberculosis and immunodiagnostic potential in high-risk groups' such as HIV-infected individuals and children.