Diagnostic accuracy of tongue swab testing on two automated tuberculosis diagnostic platforms, Cepheid Xpert MTB/RIF Ultra and Molbio Truenat MTB Ultima.

Tongue dorsum swabbing is a potential alternative to sputum collection for tuberculosis (TB) testing. Previous studies showed that Cepheid Xpert MTB/RIF Ultra (Xpert Ultra) can detect Mycobacterium tuberculosis DNA on tongue swabs stored in buffer, with 72% sensitivity and 100% specificity relative to a sputum microbiological reference standard (sputum MRS). The present study evaluated a more convenient sample collection protocol (dry swab storage), combined with streamlined sample processing protocols, for evaluating two commercial TB diagnostic tests: Xpert Ultra and Molbio Truenat MTB Ultima (MTB Ultima). Copan FLOQSwabs were self-collected or collected by study workers from 321 participants in Western Cape, South Africa. All participants had symptoms suggestive of TB, and 245 of them had sputum MRS-confirmed TB (by sputum MGIT culture and/or Xpert Ultra). One tongue swab per participant was tested on Xpert Ultra, and another tongue swab was tested with MTB Ultima. Xpert Ultra was 75.5% sensitive and 100% specific relative to sputum MRS, similar to previous methods that used swabs stored in buffer. MTB Ultima was 71.6% sensitive and 96.9% specific relative to sputum MRS. When sample lysates that were false-negative or invalid by MTB Ultima were frozen, thawed, and re-tested, MTB Ultima sensitivity rose to 79.1%. Both tests were more sensitive with swabs from participants with higher sputum Xpert Ultra semi-quantitative results. Although additional development could improve diagnostic accuracy, these results further support tongue swabs as easy-to-collect samples for TB testing. IMPORTANCE: Tongue dorsum swabbing is a promising alternative to sputum collection for tuberculosis (TB) testing. Our results lend further support for tongue swabs as exceptionally easy-to-collect samples for high-throughput TB testing.

Molecular Detection of Airborne Mycobacterium tuberculosis in South African High Schools.

Rationale: South African adolescents carry a high tuberculosis disease burden. It is not known if schools are high-risk settings for Mycobacterium tuberculosis (MTB) transmission. Objectives: To detect airborne MTB genomic DNA in classrooms. Methods: We studied 72 classrooms occupied by 2,262 students in two South African schools. High-volume air filtration was performed for median 40 (interquartile range [IQR], 35-54) minutes and assayed by droplet digital PCR (ddPCR)-targeting MTB region of difference 9 (RD9), with concurrent CO2 concentration measurement. Classroom data were benchmarked against public health clinics. Students who consented to individual tuberculosis screening completed a questionnaire and sputum collection (Xpert MTB/RIF Ultra) if symptom positive. Poisson statistics were used for MTB RD9 copy quantification. Measurements and Main Results: ddPCR assays were positive in 13/72 (18.1%) classrooms and 4/39 (10.3%) clinic measurements (P = 0.276). Median ambient CO2 concentration was 886 (IQR, 747-1223) ppm in classrooms versus 490 (IQR, 405-587) ppm in clinics (P < 0.001). Average airborne concentration of MTB RD9 was 3.61 copies per 180,000 liters in classrooms versus 1.74 copies per 180,000 liters in clinics (P = 0.280). Across all classrooms, the average risk of an occupant inhaling one MTB RD9 copy was estimated as 0.71% during one standard lesson of 35 minutes. Among 1,836/2,262 (81.2%) students who consented to screening, 21/90 (23.3%) symptomatic students produced a sputum sample, of which one was Xpert MTB/RIF Ultra positive. Conclusions: Airborne MTB genomic DNA was detected frequently in high school classrooms. Instantaneous risk of classroom exposure was similar to the risk in public health clinics.

Longitudinal Dynamics of a Blood Transcriptomic Signature of Tuberculosis.

Rationale: Performance of blood transcriptomic tuberculosis (TB) signatures in longitudinal studies and effects of TB-preventive therapy and coinfection with HIV or respiratory organisms on transcriptomic signatures has not been systematically studied. Objectives: We evaluated longitudinal kinetics of an 11-gene blood transcriptomic TB signature, RISK11, and effects of TB-preventive therapy (TPT) and respiratory organisms on RISK11 signature score, in HIV-uninfected and HIV-infected individuals. Methods: RISK11 was measured in a longitudinal study of RISK11-guided TPT in HIV-uninfected adults, a cross-sectional respiratory organisms cohort, or a longitudinal study in people living with HIV (PLHIV). HIV-uninfected RISK11+ participants were randomized to TPT or no TPT; RISK11- participants received no TPT. PLHIV received standard-of-care antiretroviral therapy and TPT. In the cross-sectional respiratory organisms cohort, viruses and bacteria in nasopharyngeal and oropharyngeal swabs were quantified by real-time quantitative PCR. Measurements and Main Results: RISK11+ status was transient in most of the 128 HIV-negative participants with longitudinal samples; more than 70% of RISK11+ participants reverted to RISK11- by 3 months, irrespective of TPT. By comparison, reversion from a RISK11+ state was less common in 645 PLHIV (42.1%). Non-HIV viral and nontuberculous bacterial organisms were detected in 7.2% and 38.9% of the 1,000 respiratory organisms cohort participants, respectively, and among those investigated for TB, 3.8% had prevalent disease. Median RISK11 scores (%) were higher in participants with viral organisms alone (46.7%), viral and bacterial organisms (42.8%), or prevalent TB (85.7%) than those with bacterial organisms other than TB (13.4%) or no organisms (14.2%). RISK11 could not discriminate between prevalent TB and viral organisms. Conclusions: Positive RISK11 signature status is often transient, possibly due to intercurrent viral infection, highlighting potentially important challenges for implementation of these biomarkers as new tools for TB control.

Immune Profiling Enables Stratification of Patients With Active Tuberculosis Disease or Mycobacterium tuberculosis Infection.

BACKGROUND: Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mtb) infection and is a major public health problem. Clinical challenges include the lack of a blood-based test for active disease. Current blood-based tests, such as QuantiFERON (QFT) do not distinguish active TB disease from asymptomatic Mtb infection. METHODS: We hypothesized that TruCulture, an immunomonitoring method for whole-blood stimulation, could discriminate active disease from latent Mtb infection (LTBI). We stimulated whole blood from patients with active TB and compared with LTBI donors. Mtb-specific antigens and live bacillus Calmette-Guérin (BCG) were used as stimuli, with direct comparison to QFT. Protein analyses were performed using conventional and digital enzyme-linked immunosorbent assay (ELISA), as well as Luminex. RESULTS: TruCulture showed discrimination of active TB cases from LTBI (P < .0001, AUC = .81) compared with QFT (P = .45, AUC = .56), based on an interferon γ (IFNγ) readout after Mtb antigen (Ag) stimulation. This result was replicated in an independent cohort (AUC = .89). In exploratory analyses, TB stratification could be further improved by the Mtb antigen to BCG IFNγ ratio (P < .0001, AUC = .91). Finally, the combination of digital ELISA and transcriptional analysis showed that LTBI donors with high IFNγ clustered with patients with TB, suggesting the possibility to identify subclinical disease. CONCLUSIONS: TruCulture offers a next-generation solution for whole-blood stimulation and immunomonitoring with the possibility to discriminate active and latent infection.

Prevention of M. tuberculosis Infection with H4:IC31 Vaccine or BCG Revaccination.

BACKGROUND: Recent Mycobacterium tuberculosis infection confers a predisposition to the development of tuberculosis disease, the leading killer among global infectious diseases. H4:IC31, a candidate subunit vaccine, has shown protection against tuberculosis disease in preclinical models, and observational studies have indicated that primary bacille Calmette-Guérin (BCG) vaccination may offer partial protection against infection. METHODS: In this phase 2 trial, we randomly assigned 990 adolescents in a high-risk setting who had undergone neonatal BCG vaccination to receive the H4:IC31 vaccine, BCG revaccination, or placebo. All the participants had negative results on testing for M. tuberculosis infection on the QuantiFERON-TB Gold In-tube assay (QFT) and for the human immunodeficiency virus. The primary outcomes were safety and acquisition of M. tuberculosis infection, as defined by initial conversion on QFT that was performed every 6 months during a 2-year period. Secondary outcomes were immunogenicity and sustained QFT conversion to a positive test without reversion to negative status at 3 months and 6 months after conversion. Estimates of vaccine efficacy are based on hazard ratios from Cox regression models and compare each vaccine with placebo. RESULTS: Both the BCG and H4:IC31 vaccines were immunogenic. QFT conversion occurred in 44 of 308 participants (14.3%) in the H4:IC31 group and in 41 of 312 participants (13.1%) in the BCG group, as compared with 49 of 310 participants (15.8%) in the placebo group; the rate of sustained conversion was 8.1% in the H4:IC31 group and 6.7% in the BCG group, as compared with 11.6% in the placebo group. Neither the H4:IC31 vaccine nor the BCG vaccine prevented initial QFT conversion, with efficacy point estimates of 9.4% (P=0.63) and 20.1% (P=0.29), respectively. However, the BCG vaccine reduced the rate of sustained QFT conversion, with an efficacy of 45.4% (P=0.03); the efficacy of the H4:IC31 vaccine was 30.5% (P=0.16). There were no clinically significant between-group differences in the rates of serious adverse events, although mild-to-moderate injection-site reactions were more common with BCG revaccination. CONCLUSIONS: In this trial, the rate of sustained QFT conversion, which may reflect sustained M. tuberculosis infection, was reduced by vaccination in a high-transmission setting. This finding may inform clinical development of new vaccine candidates. (Funded by Aeras and others; C-040-404 ClinicalTrials.gov number, NCT02075203 .).

Safety and Immunogenicity of Newborn MVA85A Vaccination and Selective, Delayed Bacille Calmette-Guerin for Infants of Human Immunodeficiency Virus-Infected Mothers: A Phase 2 Randomized, Controlled Trial.

Background: Vaccination of human immunodeficiency virus (HIV)-infected infants with bacille Calmette-Guérin (BCG) is contraindicated. HIV-exposed newborns need a new tuberculosis vaccination strategy that protects against tuberculosis early in life and avoids the potential risk of BCG disease until after HIV infection has been excluded. Methods: This double-blind, randomized, controlled trial compared newborn MVA85A prime vaccination (1 × 108 PFU) vs Candin® control, followed by selective, deferred BCG vaccination at age 8 weeks for HIV-uninfected infants and 12 months follow-up for safety and immunogenicity. Results: A total of 248 HIV-exposed infants were enrolled. More frequent mild-moderate reactogenicity events were seen after newborn MVA85A vaccination. However, no significant difference was observed in the rate of severe or serious adverse events, HIV acquisition (n = 1 per arm), or incident tuberculosis disease (n = 5 MVA85A; n = 3 control) compared to the control arm. MVA85A vaccination induced modest but significantly higher Ag85A-specific interferon gamma (IFNγ)+ CD4+ T cells compared to control at weeks 4 and 8 (P < .0001). BCG did not further boost this response in MVA85A vaccinees. The BCG-induced Ag85A-specific IFNγ+ CD4+ T-cell response at weeks 16 and 52 was of similar magnitude in the control arm compared to the MVA85A arm at all time points. Proliferative capacity, functional profiles, and memory phenotype of BCG-specific CD4 responses were similar across study arms. Conclusions: MVA85A prime vaccination of HIV-exposed newborns was safe and induced an early modest antigen-specific immune response that did not interfere with, or enhance, immunogenicity of subsequent BCG vaccination. New protein-subunit and viral-vectored tuberculosis vaccine candidates should be tested in HIV-exposed newborns. Clinical Trials Registration: NCT01650389.

Predictive value of recent QuantiFERON conversion for tuberculosis disease in adolescents.

RATIONALE: Conversions and reversions occur with IFN-γ release assay (IGRA) serial testing, as with the tuberculin skin test (TST). Recent TST conversion is associated with an established risk of developing tuberculosis (TB) disease, but the risk associated with recent IGRA conversions is unknown. OBJECTIVES: To compare the incidence rate of TB disease after recent QuantiFERON TB Gold In-Tube (QFT) conversion compared with nonconverters. METHODS: Adolescents with converted IGRA status (QFT converters [n = 534]) and randomly chosen adolescents whose IGRA status had remained negative over a period of 2 years (QFT nonconverters [n = 629]) were identified in a cohort study of TB infection and disease. Subsequent TB disease incidence was compared between the two groups. MEASUREMENTS AND MAIN RESULTS: For QFT converters, the TB incidence rate (all cases) was 1.46 cases per 100 person-years (95% confidence interval [CI], 0.82-2.39), and the cumulative incidence was 2.8% (95% CI, 1.58-4.59). A significantly lower TB incidence rate (0.17 cases per 100 person-yr [95% CI, 0.02-0.62]) and cumulative incidence (0.32% [95% CI, 0.03-1.14]) was observed for QFT nonconverters. The incidence rate ratio was 8.54 (95% CI, 2.51-29.13) for all cases of TB and 9.1 (95% CI, 1.65-50.36) for protocol-defined TB. CONCLUSIONS: Recent QFT conversion was indicative of an approximately eight fold higher risk of progression to TB disease (compared with nonconverters) within 2 years of conversion in a cohort of adolescents in a high-TB burden population.

A phase IIa trial of the new tuberculosis vaccine, MVA85A, in HIV- and/or Mycobacterium tuberculosis-infected adults.

RATIONALE: Novel tuberculosis (TB) vaccines should be safe and effective in populations infected with Mycobacterium tuberculosis (M.tb) and/or HIV for effective TB control. OBJECTIVE: To determine the safety and immunogenicity of MVA85A, a novel TB vaccine, among M.tb- and/or HIV-infected persons in a setting where TB and HIV are endemic. METHODS: An open-label, phase IIa trial was conducted in 48 adults with M.tb and/or HIV infection. Safety and immunogenicity were analyzed up to 52 weeks after intradermal vaccination with 5 × 10(7) plaque-forming units of MVA85A. Specific T-cell responses were characterized by IFN-γ enzyme-linked immunospot and whole blood intracellular cytokine staining assays. MEASUREMENTS AND MAIN RESULTS: MVA85A was well tolerated and no vaccine-related serious adverse events were recorded. MVA85A induced robust and durable response of mostly polyfunctional CD4(+) T cells, coexpressing IFN-γ, tumor necrosis factor-α, and IL-2. Magnitudes of pre- and postvaccination T-cell responses were lower in HIV-infected, compared with HIV-uninfected, vaccinees. No significant effect of antiretroviral therapy on immunogenicity of MVA85A was observed. CONCLUSIONS: MVA85A was safe and immunogenic in persons with HIV and/or M.tb infection. These results support further evaluation of safety and efficacy of this vaccine for prevention of TB in these target populations.

Randomized trial of type 1 and type 3 oral monovalent poliovirus vaccines in newborns in Africa.

BACKGROUND: The Global Polio Eradication Initiative aims to eradicate wild poliovirus by the end of 2012. Therefore, more-immunogenic polio vaccines, including monovalent oral poliovirus vaccines (mOPVs), are needed for supplemental immunization activities. This trial assessed the immunogenicity of monovalent types 1 and 3, compared with that of trivalent oral poliovirus vaccine (tOPV), in South Africa. METHODS: We conducted a blinded, randomized, 4-arm controlled trial comparing the immunogenicity of a single dose of mOPV1 (from 2 manufacturers) and mOPV3 (from 1 manufacturer), given at birth, with the immunogenicity of tOPV. RESULTS: Eight hundred newborns were enrolled; 762 (95%) were included in the analysis. At 30 days after vaccine administration, seroconversion to poliovirus type 1 was 73.4% and 76.4% in the 2 mOPV1 arms, compared with 39.1% in the tOPV arm (P < .0000001), and seroconversion to poliovirus type 3 was 58.0% in the mOPV3 arm, compared with 21.2% in the tOPV arm (P < .0000001). The vaccines were well tolerated, and no adverse events were attributed to trial interventions. CONCLUSION: A dose of mOPV1 or mOPV3 at birth was superior to that of tOPV in inducing type-specific seroconversion in this sub-Saharan African population. Our results support continued use of mOPVs in supplemental immunization activities in countries where poliovirus types 1 or 3 circulate. Clinical Trials Registration. ISRCTN18107202.

Systematic review of diagnostic and prognostic host blood transcriptomic signatures of tuberculosis disease in people living with HIV.

Background HIV-associated tuberculosis (TB) has high mortality; however, current triage and prognostic tools offer poor sensitivity and specificity, respectively. We conducted a systematic review of diagnostic and prognostic host-blood transcriptomic signatures of TB in people living with HIV (PLHIV). Methods We systematically searched online databases for studies published in English between 1990-2020. Eligible studies included PLHIV of any age in test or validation cohorts, and used microbiological or composite reference standards for TB diagnosis. Inclusion was not restricted by setting or participant age. Study selection, quality appraisal using the QUADAS-2 tool, and data extraction were conducted independently by two reviewers. Thereafter, narrative synthesis of included studies, and comparison of signatures performance, was performed. Results We screened 1,580 records and included 12 studies evaluating 31 host-blood transcriptomic signatures in 10 test or validation cohorts of PLHIV that differentiated individuals with TB from those with HIV alone, latent Mycobacterium tuberculosis infection, or other diseases (OD). Two (2/10; 20%) cohorts were prospective (29 TB cases; 51 OD) and 8 (80%) case-control (353 TB cases; 606 controls) design. All cohorts (10/10) were recruited in Sub-Saharan Africa and 9/10 (90%) had a high risk of bias. Ten signatures (10/31; 32%) met minimum WHO Target Product Profile (TPP) criteria for TB triage tests. Only one study (1/12; 8%) evaluated prognostic performance of a transcriptomic signature for progression to TB in PLHIV, which did not meet the minimum WHO prognostic TPP. Conclusions Generalisability of reported findings is limited by few studies enrolling PLHIV, limited geographical diversity, and predominantly case-control design, which also introduces spectrum bias. New prospective cohort studies are needed that include PLHIV and are conducted in diverse settings. Further research exploring the effect of HIV clinical, virological, and immunological factors on diagnostic performance is necessary for development and implementation of TB transcriptomic signatures in PLHIV.

Tongue swab testing on two automated tuberculosis diagnostic platforms, Cepheid Xpert® MTB/RIF Ultra and Molbio Truenat® MTB Ultima.

Tongue dorsum swabbing is a potential alternative to sputum collection for tuberculosis (TB) testing. Previous studies showed that Cepheid Xpert® MTB/RIF Ultra (Xpert Ultra) can detect Mycobacterium tuberculosis (MTB) DNA in tongue swabs stored in buffer, with 72% sensitivity and 100% specificity relative to a sputum microbiological reference standard (sputum MRS). The present study evaluated a more convenient sample collection protocol (dry swab storage), combined with streamlined sample processing protocols, for side-by-side analysis using two commercial TB diagnostic tests: Xpert Ultra and Molbio Truenat® MTB Ultima (MTB Ultima). Copan FLOQSwabs were self-collected, or collected by study workers, from 321 participants in Western Cape, South Africa. All participants had symptoms suggestive of TB, and 245 of them had sputum MRS-confirmed TB (by sputum culture and/or Xpert Ultra). One tongue swab per participant was tested on Xpert Ultra and another tongue swab was tested with MTB Ultima. Xpert Ultra was 75.4% sensitive and 100% specific, and MTB Ultima was 71.6% sensitive and 96.9% specific, relative to sputum MRS. When sample lysates that were false-negative by MTB Ultima were frozen, thawed, and re-tested, MTB Ultima sensitivity rose to 79.1%. Both tests were more sensitive with swabs from participants with higher sputum Xpert semi-quantitative results. The protocol for Xpert Ultra enabled fast and easy testing of dry-stored swabs with no loss of accuracy relative to previous methods. MTB Ultima testing of dry-stored swabs exhibited comparable performance to Xpert Ultra. These results further support tongue swabs as easy-to-collect samples for high-throughput TB testing.

Dose-finding study of the novel tuberculosis vaccine, MVA85A, in healthy BCG-vaccinated infants.

BACKGROUND: BCG, the only licensed tuberculosis vaccine, affords poor protection against lung tuberculosis in infants and children. A new tuberculosis vaccine, which may enhance the BCG-induced immune response, is urgently needed. We assessed the safety of and characterized the T cell response induced by 3 doses of the candidate vaccine, MVA85A, in BCG-vaccinated infants from a setting where tuberculosis is endemic. METHODS: Infants aged 5-12 months were vaccinated intradermally with either 2.5 × 10(7), 5 × 10(7), or 10 × 10(7) plaque-forming units of MVA85A, or placebo. Adverse events were documented, and T-cell responses were assessed by interferon γ (IFN-γ) enzyme-linked immunospot assay and intracellular cytokine staining. RESULTS: The 3 MVA85A doses were well tolerated, and no vaccine-related serious adverse events were recorded. MVA85A induced potent, durable T-cell responses, which exceeded prevaccination responses up to 168 d after vaccination. No dose-related differences in response magnitude were observed. Multiple CD4 T cell subsets were induced; polyfunctional CD4 T cells co-expressing T-helper cell 1 cytokines with or without granulocyte-macrophage colony-stimulating factor predominated. IFN-γ-expressing CD8 T cells, which peaked later than CD4 T cells, were also detectable. CONCLUSIONS: MVA85A was safe and induced robust, polyfunctional, durable CD4 and CD8 T-cell responses in infants. These data support efficacy evaluation of MVA85A to prevent tuberculosis in infancy. Clinical Trials Registration. NCT00679159.

Clinical predictors of pulmonary tuberculosis among South African adults with HIV.

Background: Tuberculosis (TB) clinical prediction rules rely on presence of symptoms, however many undiagnosed cases in the community are asymptomatic. This study aimed to explore the utility of clinical factors in predicting TB among people with HIV not seeking care. Methods: Baseline data were analysed from an observational cohort of ambulant adults with HIV in South Africa. Participants were tested for Mycobacterium tuberculosis (Mtb) sensitisation (interferon-γ release assay, IGRA) and microbiologically-confirmed prevalent pulmonary TB disease at baseline, and actively surveilled for incident TB through 15 months. Multivariable LASSO regression with post-selection inference was used to test associations with Mtb sensitisation and TB disease. Findings: Between March 22, 2017, and May 15, 2018, 861 participants were enrolled; Among 851 participants included in the analysis, 94·5% were asymptomatic and 45·9% sensitised to Mtb. TB prevalence was 2·0% at baseline and incidence 2·3/100 person-years through 15 months follow-up. Study site was associated with baseline Mtb sensitisation (p < 0·001), prevalent (p < 0·001), and incident TB disease (p = 0·037). Independent of site, higher CD4 counts (per 50 cells/mm3, aOR 1·48, 95%CI 1·12-1·77, p = 0·006) were associated with increased IGRA positivity, and participants without TB disease (aOR 0·80, 95%CI 0·69-0·94, p = 0·006) had reduced IGRA positivity; no variables were independently associated with prevalent TB. Mixed ancestry (aHR 1·49, 95%CI 1·30->1000, p = 0·005) and antiretroviral initiation (aHR 1·48, 95%CI 1·01-929·93, p = 0·023) were independently associated with incident TB. Models incorporating clinical features alone performed poorly in diagnosing prevalent (AUC 0·65, 95%CI 0·44-0·85) or predicting progression to incident (0·67, 0·46-0·88) TB. Interpretation: CD4 count and antiretroviral initiation, proxies for immune status and HIV stage, were associated with Mtb sensitisation and TB disease. Inadequate performance of clinical prediction models may reflect predominantly subclinical disease diagnosed in this setting and unmeasured local site factors affecting transmission and progression. Funding: The CORTIS-HR study was funded by the Bill & Melinda Gates Foundation (OPP1151915) and by the Strategic Health Innovation Partnerships Unit of the South African Medical Research Council with funds received from the South African Department of Science and Technology. The regulatory sponsor was the University of Cape Town.

Evaluation of a transcriptomic signature of tuberculosis risk in combination with an interferon gamma release assay: A diagnostic test accuracy study.

Background: We evaluated the diagnostic and prognostic performance of a transcriptomic signature of tuberculosis (TB) risk (RISK11) and QuantiFERON-TB Gold-plus (QFTPlus) as combination biomarkers of TB risk. Methods: Healthy South Africans who were HIV-negative aged 18-60 years with baseline RISK11 and QFTPlus results were evaluated in a prospective cohort study conducted between Sept 20, 2016 and Dec 20, 2019. Prevalence and incidence-rate ratios were used to evaluate risk of TB. Positive (LR+) and negative (LR-) likelihood ratios were used to compare individual tests versus Both-Positive (RISK11+/QFTPlus+) and Either-Positive (RISK11+ or QFTPlus+) combinations. Findings: Among 2912 participants, prevalent TB in RISK11+/QFTPlus+ participants was 13·3-fold (95% CI 4·2-42·7) higher than RISK11-/QFTPlus-; 2·4-fold (95% CI 1·2-4·8) higher than RISK11+/QFTPlus-; and 4·5-fold (95% CI 2·5-8·0) higher than RISK11-/QFTPlus+ participants. Risk of incident TB in RISK11+/QFTPlus+ participants was 8·3-fold (95% CI 2·5-27·0) higher than RISK11-/QFTPlus-; 2·5-fold (95% CI 1·0-6·6) higher than RISK11+/QFTPlus-; and 2·1-fold (95% CI 1·2-3·4) higher than RISK11-/QFTPlus+ participants, respectively. Compared to QFTPlus, the Both-Positive test combination increased diagnostic LR+ from 1·3 (95% CI 1·2-1·5) to 4·7 (95% CI 3·2-7·0), and prognostic LR+ from 1·4 (95% CI 1·2-1·5) to 2·8 (95% CI 1·5-5·1), but did not improve upon RISK11 alone. Compared with RISK11, the Either-Positive test combination decreased diagnostic LR- from 0·7 (95% CI 0·6-0·9) to 0·3 (95% CI 0·2-0·6), and prognostic LR- from 0·9 (95% CI 0·8-1·0) to 0·3 (0·1-0·7), but did not improve upon QFTPlus alone. Interpretation: Combining two tests such as RISK11 and QFTPlus, with discordant individual performance characteristics does not improve overall discriminatory performance, relative to the individual tests. Funding: Bill and Melinda Gates Foundation, South African Medical Research Council.

The effect of host factors on discriminatory performance of a transcriptomic signature of tuberculosis risk.

BACKGROUND: We aimed to understand host factors that affect discriminatory performance of a transcriptomic signature of tuberculosis risk (RISK11). METHODS: HIV-negative adults aged 18-60 years were evaluated in a prospective study of RISK11 and surveilled for tuberculosis through 15 months. Generalised linear models and receiver-operating characteristic (ROC) regression were used to estimate effect of host factors on RISK11 score (%marginal effect) and on discriminatory performance for tuberculosis disease (area under the curve, AUC), respectively. FINDINGS: Among 2923 participants including 74 prevalent and 56 incident tuberculosis cases, percentage marginal effects on RISK11 score were increased among those with prevalent tuberculosis (+18·90%, 95%CI 12·66-25·13), night sweats (+14·65%, 95%CI 5·39-23·91), incident tuberculosis (+7·29%, 95%CI 1·46-13·11), flu-like symptoms (+5·13%, 95%CI 1·58-8·68), and smoking history (+2·41%, 95%CI 0·89-3·93) than those without; and reduced in males (-6·68%, 95%CI -8·31- -5·04) and with every unit increase in BMI (-0·13%, 95%CI -0·25- -0·01). Adjustment for host factors affecting controls did not change RISK11 discriminatory performance. Cough was associated with 72·55% higher RISK11 score in prevalent tuberculosis cases. Stratification by cough improved diagnostic performance from AUC = 0·74 (95%CI 0·67-0·82) overall, to 0·97 (95%CI 0·90-1·00, p < 0·001) in cough-positive participants. Combining host factors with RISK11 improved prognostic performance, compared to RISK11 alone, (AUC = 0·76, 95%CI 0·69-0·83 versus 0·56, 95%CI 0·46-0·68, p < 0·001) over a 15-month predictive horizon. INTERPRETATION: Several host factors affected RISK11 score, but only adjustment for cough affected diagnostic performance. Combining host factors with RISK11 should be considered to improve prognostic performance. FUNDING: Bill and Melinda Gates Foundation, South African Medical Research Council.

Inflammation and immune activation are associated with risk of Mycobacterium tuberculosis infection in BCG-vaccinated infants.

Tuberculosis vaccine development is hindered by the lack of validated immune correlates of protection. Exploring immune correlates of risk of disease and/or infection in prospective samples can inform this field. We investigate whether previously identified immune correlates of risk of TB disease also associate with increased risk of M.tb infection in BCG-vaccinated South African infants, who became infected with M.tb during 2-3 years of follow-up. M.tb infection is defined by conversion to positive reactivity in the QuantiFERON test. We demonstrate that inflammation and immune activation are associated with risk of M.tb infection. Ag85A-specific IgG is elevated in infants that were subsequently infected with M.tb, and this is coupled with upregulated gene expression of immunoglobulin-associated genes and type-I interferon. Plasma levels of IFN-[Formula: see text]2, TNF-[Formula: see text], CXCL10 (IP-10) and complement C2 are also higher in infants that were subsequently infected with M.tb.

Prospective multicentre head-to-head validation of host blood transcriptomic biomarkers for pulmonary tuberculosis by real-time PCR.

Background: Sensitive point-of-care screening tests are urgently needed to identify individuals at highest risk of tuberculosis. We prospectively tested performance of host-blood transcriptomic tuberculosis signatures. Methods: Adults without suspicion of tuberculosis were recruited from five endemic South African communities. Eight parsimonious host-blood transcriptomic tuberculosis signatures were measured by microfluidic RT-qPCR at enrolment. Upper respiratory swab specimens were tested with a multiplex bacterial-viral RT-qPCR panel in a subset of participants. Diagnostic and prognostic performance for microbiologically confirmed prevalent and incident pulmonary tuberculosis was tested in all participants at baseline and during active surveillance through 15 months follow-up, respectively. Results: Among 20,207 HIV-uninfected and 963 HIV-infected adults screened; 2923 and 861 were enroled. There were 61 HIV-uninfected (weighted prevalence 1.1%) and 10 HIV-infected (prevalence 1.2%) tuberculosis cases at baseline. Parsimonious signature diagnostic performance was superior among symptomatic (AUCs 0.85-0.98) as compared to asymptomatic (AUCs 0.61-0.78) HIV-uninfected participants. Thereafter, 24 HIV-uninfected and 9 HIV-infected participants progressed to incident tuberculosis (1.1 and 1.0 per 100 person-years, respectively). Among HIV-uninfected individuals, prognostic performance for incident tuberculosis occurring within 6-12 months was higher relative to 15 months. 1000 HIV-uninfected participants were tested for respiratory microorganisms and 413 HIV-infected for HIV plasma viral load; 7/8 signature scores were higher (p < 0.05) in participants with viral respiratory infections or detectable HIV viraemia than those without. Conclusions: Several parsimonious tuberculosis transcriptomic signatures met triage test targets among symptomatic participants, and incipient test targets within 6 months. However, the signatures were upregulated with viral infection and offered poor specificity for diagnosing sub-clinical tuberculosis.

Tuberculosis alters immune-metabolic pathways resulting in perturbed IL-1 responses.

Tuberculosis (TB) remains a major public health problem and we lack a comprehensive understanding of how Mycobacterium tuberculosis (M. tb) infection impacts host immune responses. We compared the induced immune response to TB antigen, BCG and IL-1ß stimulation between latently M. tb infected individuals (LTBI) and active TB patients. This revealed distinct responses between TB/LTBI at transcriptomic, proteomic and metabolomic levels. At baseline, we identified a novel immune-metabolic association between pregnane steroids, the PPARγ pathway and elevated plasma IL-1ra in TB. We observed dysregulated IL-1 responses after BCG stimulation in TB patients, with elevated IL-1ra responses being explained by upstream TNF differences. Additionally, distinct secretion of IL-1α/IL-1ß in LTBI/TB after BCG stimulation was associated with downstream differences in granzyme mediated cleavage. Finally, IL-1ß driven signalling was dramatically perturbed in TB disease but was completely restored after successful treatment. This study improves our knowledge of how immune responses are altered during TB disease, and may support the design of improved preventive and therapeutic tools, including host-directed strategies.

Host blood transcriptomic biomarkers of tuberculosis disease in people living with HIV: a systematic review protocol.

INTRODUCTION: Current tuberculosis triage and predictive tools offer poor accuracy and are ineffective for detecting asymptomatic disease in people living with HIV (PLHIV). Host tuberculosis transcriptomic biomarkers hold promise for diagnosing prevalent and predicting progression to incident tuberculosis and guiding further investigation, preventive therapy and follow-up. We aim to conduct a systematic review of performance of transcriptomic signatures of tuberculosis in PLHIV. METHODS AND ANALYSIS: We will search MEDLINE (PubMed), WOS Core Collection, Biological Abstracts, and SciELO Citation Index (Web of Science), Africa-Wide Information and General Science Abstracts (EBSCOhost), Scopus, and Cochrane Central Register of Controlled Trials databases for articles published in English between 1990 and 2020. Case-control, cross-sectional, cohort and randomised controlled studies evaluating performance of diagnostic and prognostic host-response transcriptomic signatures in PLHIV of all ages and settings will be included. Eligible studies will include PLHIV in signature test or validation cohorts, and use microbiological, clinical, or composite reference standards for pulmonary or extrapulmonary tuberculosis diagnosis. Study quality will be evaluated using the 'Quality Assessment of Diagnostic Accuracy Studies-2' tool and cumulative review evidence assessed using the 'Grading of Recommendations Assessment, Development and Evaluation' approach. Study selection, quality appraisal and data extraction will be performed independently by two reviewers. Study, cohort and signature characteristics of included studies will be tabulated, and a narrative synthesis of findings presented. Primary outcomes of interest, biomarker sensitivity and specificity with estimate precision, will be summarised in forest plots. Expected heterogeneity in signature characteristics, study settings, and study designs precludes meta-analysis and pooling of results. Review reporting will follow the Preferred Reporting Items for Systematic Reviews and Meta-Analyses of Diagnostic Test Accuracy Studies guidelines. ETHICS AND DISSEMINATION: Formal ethics approval is not required as primary human participant data will not be collected. Results will be disseminated through peer-reviewed publication and conference presentation. PROSPERO REGISTRATION NUMBER: CRD42021224155.

Biomarker-guided tuberculosis preventive therapy (CORTIS): a randomised controlled trial.

BACKGROUND: Targeted preventive therapy for individuals at highest risk of incident tuberculosis might impact the epidemic by interrupting transmission. We tested performance of a transcriptomic signature of tuberculosis (RISK11) and efficacy of signature-guided preventive therapy in parallel, using a hybrid three-group study design. METHODS: Adult volunteers aged 18-59 years were recruited at five geographically distinct communities in South Africa. Whole blood was sampled for RISK11 by quantitative RT-PCR assay from eligible volunteers without HIV, recent previous tuberculosis (ie, <3 years before screening), or comorbidities at screening. RISK11-positive participants were block randomised (1:2; block size 15) to once-weekly, directly-observed, open-label isoniazid and rifapentine for 12 weeks (ie, RISK11 positive and 3HP positive), or no treatment (ie, RISK11 positive and 3HP negative). A subset of eligible RISK11-negative volunteers were randomly assigned to no treatment (ie, RISK11 negative and 3HP negative). Diagnostic discrimination of prevalent tuberculosis was tested in all participants at baseline. Thereafter, prognostic discrimination of incident tuberculosis was tested in the untreated RISK11-positive versus RISK11-negative groups, and treatment efficacy in the 3HP-treated versus untreated RISK11-positive groups, during active surveillance through 15 months. The primary endpoint was microbiologically confirmed pulmonary tuberculosis. The primary outcome measures were risk ratio [RR] for tuberculosis of RISK11-positive to RISK11-negative participants, and treatment efficacy. This trial is registered with ClinicalTrials.gov, NCT02735590. FINDINGS: 20 207 volunteers were screened, and 2923 participants were enrolled, including RISK11-positive participants randomly assigned to 3HP (n=375) or no 3HP (n=764), and 1784 RISK11-negative participants. Cumulative probability of prevalent or incident tuberculosis disease was 0·066 (95% CI 0·049 to 0·084) in RISK11-positive (3HP negative) participants and 0·018 (0·011 to 0·025) in RISK11-negative participants (RR 3·69, 95% CI 2·25-6·05) over 15 months. Tuberculosis prevalence was 47 (4·1%) of 1139 versus 14 (0·78%) of 1984 in RISK11-positive compared with RISK11-negative participants, respectively (diagnostic RR 5·13, 95% CI 2·93 to 9·43). Tuberculosis incidence over 15 months was 2·09 (95% CI 0·97 to 3·19) vs 0·80 (0·30 to 1·30) per 100 person years in RISK11-positive (3HP-negative) participants compared with RISK11-negative participants (cumulative incidence ratio 2·6, 95% CI 1·2 to 5·9). Serious adverse events related to 3HP included one hospitalisation for seizures (unintentional isoniazid overdose) and one death of unknown cause (possibly temporally related). Tuberculosis incidence over 15 months was 1·94 (95% CI 0·35 to 3·50) versus 2·09 (95% CI 0·97 to 3·19) per 100 person-years in 3HP-treated RISK11-positive participants compared with untreated RISK11-positive participants (efficacy 7·0%, 95% CI -145 to 65). INTERPRETATION: The RISK11 signature discriminated between individuals with prevalent tuberculosis, or progression to incident tuberculosis, and individuals who remained healthy, but provision of 3HP to signature-positive individuals after exclusion of baseline disease did not reduce progression to tuberculosis over 15 months. FUNDING: Bill and Melinda Gates Foundation, South African Medical Research Council.