Point-of-care C-reactive protein and Xpert MTB/RIF Ultra for tuberculosis screening and diagnosis in unselected antiretroviral therapy initiators: a prospective, cross-sectional, diagnostic accuracy study.

BACKGROUND: Tuberculosis, a major cause of death in people living with HIV, remains challenging to diagnose. Diagnostic accuracy data are scarce for promising triage and confirmatory tests such as C-reactive protein (CRP), sputum and urine Xpert MTB/RIF Ultra (Xpert Ultra), and urine Determine TB LAM Ag (a lateral flow lipoarabinomannan [LF-LAM] test), without symptom selection. We evaluated novel triage and confirmatory tests in ambulatory people with HIV initiating antiretroviral therapy (ART). METHODS: 897 ART-initiators were recruited irrespective of symptoms and sputum induction offered. For triage (n=800), we evaluated point-of-care blood-based CRP testing, compared with the WHO-recommended four-symptom screen (W4SS). For sputum-based confirmatory testing (n=787), we evaluated Xpert Ultra versus Xpert MTB/RIF (Xpert). For urine-based confirmatory testing (n=732), we evaluated Xpert Ultra and LF-LAM. We used a sputum culture reference standard. FINDINGS: 463 (52%) of 897 participants were female. The areas under the receiver operator characteristic curves for CRP was 0·78 (95% CI 0·73-0·83) and for number of W4SS symptoms was 0·70 (0·64-0·75). CRP (≥10 mg/L) had similar sensitivity to W4SS (77% [95% CI 68-85; 80/104] vs 77% [68-85; 80/104]; p>0·99] but higher specificity (64% [61-68; 445/696] vs 48% [45-52; 334/696]; p<0·0001]; reducing unnecessary confirmatory testing by 138 (95% CI 117-160) per 1000 people and number-needed-to-test from 6·91 (95% CI 6·25-7·81) to 4·87 (4·41-5·51). Sputum samples with Xpert Ultra, which required induction in 49 (31%) of 158 of people (95% CI 24-39), had higher sensitivity than Xpert (71% [95% CI 61-80; 74/104] vs 56% [46-66; 58/104]; p<0·0001). Of the people with one or more confirmatory sputum or urine test results that were positive, the proportion detected by Xpert Ultra increased from 45% (26-64) to 66% (46-82) with induction. Programmatically done haemoglobin, triage test combinations, and urine tests showed comparatively worse results. INTERPRETATION: CRP is a more specific triage test than W4SS in those initiating ART. Sputum induction improves diagnostic yield. Sputum samples with Xpert Ultra is a more accurate confirmatory test than with Xpert. FUNDING: South African Medical Research Council, EDCTP2, US National Institutes of Health-National Institute of Allergy and Infectious Diseases.

Blood RNA biomarkers for tuberculosis screening in people living with HIV before antiretroviral therapy initiation: a diagnostic accuracy study.

BACKGROUND: Undiagnosed tuberculosis remains a major threat for people living with HIV. Multiple blood transcriptomic biomarkers have shown promise for tuberculosis diagnosis. We sought to evaluate their diagnostic accuracy and clinical utility for systematic pre-antiretroviral therapy (ART) tuberculosis screening. METHODS: We enrolled consecutive adults (age ≥18 years) referred to start ART at a community health centre in Cape Town, South Africa, irrespective of symptoms. Sputa were obtained (using induction if required) for two liquid cultures. Whole-blood RNA samples underwent transcriptional profiling using a custom Nanostring gene panel. We measured the diagnostic accuracy of seven candidate RNA signatures (one single gene biomarker [BATF2] and six multigene biomarkers) for the reference standard of Mycobacterium tuberculosis culture status, using area under the receiver-operating characteristic curve (AUROC) analysis, and sensitivity and specificity at prespecified thresholds (two standard scores above the mean of healthy controls; Z2). Clinical utility was assessed by calculating net benefit in decision curve analysis. We compared performance with C-reactive protein (CRP; threshold ≥5 mg/L), WHO four-symptom screen (W4SS), and the WHO target product profile for tuberculosis triage tests. FINDINGS: A total of 707 people living with HIV (407 [58%] female and 300 [42%] male) were included, with median CD4 count 306 cells per mm3 (IQR 184-486). Of 676 participants with available sputum culture results, 89 (13%) had culture-confirmed tuberculosis. The seven RNA signatures were moderately to highly correlated (Spearman rank coefficients 0·42-0·93) and discriminated tuberculosis culture positivity with similar AUROCs (0·73-0·80), but none statistically better than CRP (AUROC 0·78, 95% CI 0·72-0·83). Diagnostic accuracy was similar across CD4 count strata, but lower among participants with negative W4SS (AUROCs 0·56-0·65) compared with positive (AUROCs 0·75-0·84). The RNA biomarker with the highest AUROC point estimate was a four-gene signature (Suliman4; AUROC 0·80, 95% CI 0·75-0·86), with sensitivity 83% (95% CI 74-90) and specificity 59% (55-63) at the Z2 threshold. In decision curve analysis, Suliman4 and CRP had similar clinical utility to guide confirmatory tuberculosis testing, but both had higher net benefit than W4SS. In exploratory analyses, an approach combining CRP (≥5 mg/L) and Suliman4 (≥Z2) had sensitivity of 80% (70-87), specificity of 70% (66-74), and higher net benefit than either biomarker alone. INTERPRETATION: RNA biomarkers showed better clinical utility to guide confirmatory tuberculosis testing for people living with HIV before ART initiation than symptom-based screening, but their performance did not exceed that of CRP and fell short of WHO recommended targets. Interferon-independent approaches might be required to improve accuracy of host-response biomarkers to support tuberculosis screening before ART initiation. FUNDING: South African Medical Research Council, European and Developing Countries Clinical Trials Partnership 2, National Institutes of Health National Institute of Allergy and Infectious Diseases, The Wellcome Trust, National Institute for Health and Care Research, Royal College of Physicians London.

Tuberculosis prevention: current strategies and future directions.

BACKGROUND: An estimated one fourth of the world's population is infected with Mycobacterium tuberculosis, and 5-10% of those infected develop tuberculosis in their lifetime. Preventing tuberculosis is one of the most underutilized but essential components of curtailing the tuberculosis epidemic. Moreover, current evidence illustrates that tuberculosis manifestations occur along a dynamic spectrum from infection to disease rather than a binary state as historically conceptualized. Elucidating determinants of transition between these states is crucial to decreasing the tuberculosis burden and reaching the END-TB Strategy goals as defined by the WHO. Vaccination, detection of infection, and provision of preventive treatment are key elements of tuberculosis prevention. OBJECTIVES: This review provides a comprehensive summary of recent evidence and state-of-the-art updates on advancements to prevent tuberculosis in various settings and high-risk populations. SOURCES: We identified relevant studies in the literature and synthesized the findings to provide an overview of the current state of tuberculosis prevention strategies and latest research developments. CONTENT: We present the current knowledge and recommendations regarding tuberculosis prevention, with a focus on M. bovis Bacille-Calmette-Guérin vaccination and novel vaccine candidates, tests for latent infection with M. tuberculosis, regimens available for tuberculosis preventive treatment and recommendations in low- and high-burden settings. IMPLICATIONS: Effective tuberculosis prevention worldwide requires a multipronged approach that addresses social determinants, and improves access to tuberculosis detection and to new short tuberculosis preventive treatment regimens. Robust collaboration and innovative research are needed to reduce the global burden of tuberculosis and develop new detection tools, vaccines, and preventive treatments that serve all populations and ages.

Blood RNA biomarkers for tuberculosis screening in people living with HIV prior to anti-retroviral therapy initiation: A diagnostic accuracy study.

Background: Undiagnosed tuberculosis (TB) remains a major threat for people living with HIV (PLHIV). Multiple blood transcriptomic biomarkers have shown promise for TB diagnosis. We sought to evaluate their diagnostic accuracy and clinical utility for systematic pre-antiretroviral therapy (ART) TB screening. Methods: We enrolled consecutive adults referred to start ART at a community health centre in Cape Town, South Africa, irrespective of symptoms. Sputa were obtained (using induction if required) for two liquid cultures. Whole-blood RNA samples underwent transcriptional profiling using a custom Nanostring gene-panel. We measured the diagnostic accuracy of seven candidate RNA biomarkers for the reference standard of Mycobacterium tuberculosis culture status, using area under the receiver-operating characteristic curve (AUROC) analysis, and sensitivity/specificity at pre-specified thresholds (two standard scores above the mean of healthy controls; Z2). Clinical utility was assessed using decision curve analysis. We compared performance to CRP (threshold ≥5mg/L), World Health Organisation (WHO) four-symptom screen (W4SS) and the WHO target product profile for TB triage tests. Results: A total of 707 PLHIV were included, with median CD4 count 306 cells/mm3. Of 676 with available sputum culture results, 89 (13%) had culture-confirmed TB. The seven RNA biomarkers were moderately to highly correlated (Spearman rank coefficients 0.42-0.93) and discriminated TB culture-positivity with similar AUROCs (0.73-0.80), but none statistically better than CRP (AUROC 0.78; 95% CI 0.72-0.83). Diagnostic accuracy was similar across CD4 count strata, but lower among W4SS-negative (AUROCs 0.56-0.65) compared to W4SS-positive participants (AUROCs 0.75-0.84). The RNA biomarker with highest AUROC point estimate was a 4-gene signature (Suliman4; AUROC 0.80; 95% CI 0.75-0.86), with sensitivity 0.83 (0.74-0.90) and specificity 0.59 (0.55-0.63) at Z2 threshold. In decision curve analysis, Suliman4 and CRP had similar clinical utility to guide confirmatory TB testing, but both had higher net benefit than W4SS. In exploratory analyses, an approach combining CRP (≥5mg/L) and Suliman4 (≥Z2) had sensitivity of 0.80 (0.70-0.87), specificity of 0.70 (0.66-0.74) and higher net benefit than either biomarker alone. Interpretation: RNA biomarkers showed better clinical utility to guide confirmatory TB testing for PLHIV prior to ART initiation than symptom-based screening, but their performance did not exceed that of CRP, and fell short of WHO recommended targets. Interferon-independent approaches may be required to improve accuracy of host-response biomarkers to support TB screening pre-ART initiation. Funding: South African MRC, EDCTP2, NIH/NIAID, Wellcome Trust, NIHR, Royal College of Physicians London.

Clinical utility of WHO-recommended screening tools and development and validation of novel clinical prediction models for pulmonary tuberculosis screening among outpatients living with HIV: an individual participant data meta-analysis.

BACKGROUND: The World Health Organization (WHO) recommends that outpatient people living with HIV (PLHIV) undergo tuberculosis screening with the WHO four-symptom screen (W4SS) or C-reactive protein (CRP) (5 mg·L-1 cut-off) followed by confirmatory testing if screen positive. We conducted an individual participant data meta-analysis to determine the performance of WHO-recommended screening tools and two newly developed clinical prediction models (CPMs). METHODS: Following a systematic review, we identified studies that recruited adult outpatient PLHIV irrespective of tuberculosis signs and symptoms or with a positive W4SS, evaluated CRP and collected sputum for culture. We used logistic regression to develop an extended CPM (which included CRP and other predictors) and a CRP-only CPM. We used internal-external cross-validation to evaluate performance. RESULTS: We pooled data from eight cohorts (n=4315 participants). The extended CPM had excellent discrimination (C-statistic 0.81); the CRP-only CPM had similar discrimination. The C-statistics for WHO-recommended tools were lower. Both CPMs had equivalent or higher net benefit compared with the WHO-recommended tools. Compared with both CPMs, CRP (5 mg·L-1 cut-off) had equivalent net benefit across a clinically useful range of threshold probabilities, while the W4SS had a lower net benefit. The W4SS would capture 91% of tuberculosis cases and require confirmatory testing for 78% of participants. CRP (5 mg·L-1 cut-off), the extended CPM (4.2% threshold) and the CRP-only CPM (3.6% threshold) would capture similar percentages of cases but reduce confirmatory tests required by 24, 27 and 36%, respectively. CONCLUSIONS: CRP sets the standard for tuberculosis screening among outpatient PLHIV. The choice between using CRP at 5 mg·L-1 cut-off or in a CPM depends on available resources.

Point-of-care C-reactive protein and Xpert MTB/RIF Ultra for tuberculosis screening and diagnosis in unselected antiretroviral therapy initiators: a prospective diagnostic accuracy study.

Background: Tuberculosis (TB), a major cause of death in people living with HIV (PLHIV), remains challenging to diagnose. Diagnostic accuracy data are lacking for promising triage tests, such as C-reactive protein (CRP), and confirmatory tests, such as sputum and urine Xpert MTB/RIF Ultra (Ultra), and urine LAM, without prior symptom selection. Methods: 897 PLHIV initiating antiretroviral therapy were consecutively recruited in settings with high TB incidence, irrespective of symptoms. Participants were offered sputum induction, with a liquid culture reference standard. First, we evaluated point-of-care CRP testing on blood, compared to the World Health Organization (WHO)-recommended four-symptom screen (W4SS) for triage (n=800). Second, we evaluated Xpert MTB/RIF Ultra (Ultra) versus Xpert MTB/RIF (Xpert) for sputum-based confirmatory testing (n=787), with or without sputum induction. Third, we evaluated Ultra and Determine LF-LAM for urine-based confirmatory testing (n=732). Findings: CRP and number of W4SS symptoms had areas under the receiver operator characteristic curve of 0.78 (95% confidence interval 0.73, 0.83) and 0.70 (0.64, 0.75), respectively. For triage, CRP (≥10 mg/l) has similar sensitivity to W4SS [77% (68, 85) vs. 77% (68, 85); p>0.999] but higher specificity [64% (61, 68) vs. 48% (45, 52); p<0.001]; reducing unnecessary confirmatory testing by 138 per 1000 people and the number-needed-to-test from 6.91 (6.25, 7.81) to 4.87 (4.41, 5.51). Using sputum, which required induction in 31% (24, 39) of people, Ultra had higher sensitivity than Xpert [71% (61, 80) vs. 56% (46, 66); p<0.001] but lower specificity [98% (96, 100) vs. 99% (98, 100); p<0.001]. The proportion of people with ≥1 positive confirmatory result detected by Ultra increased from 45% (26, 64) to 66% (46, 82) when induction was done. Programmatically-done haemoglobin, triage test combinations, and urine tests showed comparatively worse performance. Interpretation: Among ART-initiators in a high burden setting, CRP is a more specific triage test than W4SS. Sputum induction improves yield. Sputum Ultra is a more accurate confirmatory test than Xpert. Funding: SAMRC (MRC-RFA-IFSP-01-2013), EDCTP2 (SF1401, OPTIMAL DIAGNOSIS), NIH/NIAD (U01AI152087). Research in context: Evidence before this study: Novel triage and confirmatory tests are urgently needed for TB, especially in key risk groups like PLHIV. Many TB cases do not meet World Health Organization (WHO)-recommended four-symptom screen (W4SS) criteria despite accounting for significant transmission and morbidity. W4SS also lacks specificity, which makes onward referral of triage-positive people for expensive confirmatory testing inefficient and hampers diagnostic scale-up. Alternative triage approaches like CRP have promise, but have comparatively little data in ART-initiators, especially when done without syndromic preselection and using point-of-care (POC) tools. After triage, confirmatory testing can be challenging due to sputum scarcity and paucibacillary early-stage disease. Next generation WHO-endorsed rapid molecular tests (including Xpert MTB/RIF Ultra; Ultra) are a standard-of-care for confirmatory testing. However, there are no supporting data in ART-initiators, among whom Ultra may offer large sensitivity gains over predecessors like Xpert MTB/RIF (Xpert). The added value of sputum induction to augment diagnostic sampling for confirmatory testing is also unclear. Lastly, the performance of urine tests (Ultra, Determine LF-LAM) in this population requires more data.Added value of this study: We evaluated repurposed and new tests for triage and confirmatory testing using a rigorous microbiological reference standard in a highly vulnerable high-priority patient population (ART-initiators) regardless of symptoms and ability to naturally expectorate sputum. We showed POC CRP triage is feasible, performs better than W4SS, and that combinations of different triage approaches offer no advantages over CRP alone. Sputum Ultra has superior sensitivity to Xpert; often detecting W4SS-negative TB. Furthermore, without induction, confirmatory sputum-based testing would not be possible in a third of people. Urine tests had poor performance. This study contributed unpublished data to systematic reviews and meta-analyses used by the WHO to inform global policy supporting use of CRP triage and Ultra in PLHIV.Implication of all the available evidence: POC CRP triage testing is feasible and superior to W4SS and, together with sputum induction in people who triage CRP-positive should, after appropriate cost and implementation research, be considered for roll-out in ART-initiators in high burden settings. Such people should be offered Ultra, which outperforms Xpert.

Predictive performance of interferon-gamma release assays and the tuberculin skin test for incident tuberculosis: an individual participant data meta-analysis.

Background: Evidence on the comparative performance of purified protein derivative tuberculin skin tests (TST) and interferon-gamma release assays (IGRA) for predicting incident active tuberculosis (TB) remains conflicting. We conducted an individual participant data meta-analysis to directly compare the predictive performance for incident TB disease between TST and IGRA to inform policy. Methods: We searched Medline and Embase from 1 January 2002 to 4 September 2020, and studies that were included in previous systematic reviews. We included prospective longitudinal studies in which participants received both TST and IGRA and estimated performance as hazard ratios (HR) for the development of all diagnoses of TB in participants with dichotomised positive test results compared to negative results, using different thresholds of positivity for TST. Secondary analyses included an evaluation of the impact of background TB incidence. We also estimated the sensitivity and specificity for predicting TB. We explored heterogeneity through pre-defined sub-group analyses (e.g. country-level TB incidence). Publication bias was assessed using funnel plots and Egger's test. This review is registered with PROSPERO, CRD42020205667. Findings: We obtained data from 13 studies out of 40 that were considered eligible (N = 32,034 participants: 36% from countries with TB incidence rate ≥100 per 100,000 population). All reported data on TST and QuantiFERON Gold in-Tube (QFT-GIT). The point estimate for the TST was highest with higher cut-offs for positivity and particularly when stratified by bacillus Calmette-Guérin vaccine (BCG) status (15 mm if BCG vaccinated and 5 mm if not [TST5/15 mm]) at 2.88 (95% CI 1.69-4.90). The pooled HR for QFT-GIT was higher than for TST at 4.15 (95% CI 1.97-8.75). The difference was large in countries with TB incidence rate <100 per 100,000 population (HR 10.38, 95% CI 4.17-25.87 for QFT-GIT VS. HR 5.36, 95% CI 3.82-7.51 for TST5/15 mm) but much of this difference was driven by a single study (HR 5.13, 95% CI 3.58-7.35 for TST5/15 mm VS. 7.18, 95% CI 4.48-11.51 for QFT-GIT, when excluding the study, in which all 19 TB cases had positive QFT-GIT results). The comparative performance was similar in the higher burden countries (HR 1.61, 95% CI 1.23-2.10 for QFT-GIT VS. HR 1.72, 95% CI 0.98-3.01 for TST5/15 mm). The predictive performance of both tests was higher in countries with TB incidence rate <100 per 100,000 population. In the lower TB incidence countries, the specificity of TST (76% for TST5/15 mm) and QFT-GIT (74%) for predicting active TB approached the minimum World Health Organization target (≥75%), but the sensitivity was below the target of ≥75% (63% for TST5/15 mm and 65% for QFT-GIT). The absolute differences in positive and negative predictive values between TST15 mm and QFT-GIT were small (positive predictive values 2.74% VS. 2.46%; negative predictive values 99.42% VS. 99.52% in low-incidence countries). Egger's test did not show evidence of publication bias (0.74 for TST15 mm and p = 0.68 for QFT-GIT). Interpretation: IGRA appears to have higher predictive performance than the TST in low TB incidence countries, but the difference was driven by a single study. Any advantage in clinical performance may be small, given the numerically similar positive and negative predictive values. Both IGRA and TST had lower performance in countries with high TB incidence. Test choice should be contextual and made considering operational and likely clinical impact of test results. Funding: YH, IA, and MXR were supported by the National Institute for Health and Care Research (NIHR), United Kingdom (RP-PG-0217-20009). MQ was supported by the Medical Research Council [MC_UU_00004/07].

Clinical utility of C-reactive protein-based triage for presumptive pulmonary tuberculosis in South African adults.

BACKGROUND: Identification of an accurate, low-cost triage test for pulmonary TB among people presenting to healthcare facilities is an urgent global research priority. We assessed the diagnostic accuracy and clinical utility of C-reactive protein (CRP) for TB triage among symptomatic adult outpatients, irrespective of HIV status. METHODS: We prospectively enrolled adults reporting at least one (for people with HIV) or two (for people without HIV) symptoms of cough, fever, night sweats, or weight loss at two TB clinics in Cape Town, South Africa. Participants provided sputum for culture and Xpert MTB/RIF Ultra. We evaluated the diagnostic accuracy of CRP (measured using a laboratory-based assay) against a TB-culture reference standard as the area under the receiver operating characteristic curve (AUROC), and sensitivity and specificity at pre-specified thresholds. We assessed clinical utility using decision curve analysis and benchmarked against WHO recommendations. RESULTS: Of 932 included individuals, 255 (27%) had culture-confirmed pulmonary TB and 389 (42%) were living with HIV. CRP demonstrated an AUROC of 0·80 (95% confidence interval 0·77-0·83), with sensitivity 93% (89-95%) and specificity 54% (50-58%) using a primary cut-off of ≥10 mg/L. Performance was similar among people with HIV to those without. In decision curve analysis, CRP-based triage offered greater clinical utility than confirmatory testing for all up to a number willing to test threshold of 20 confirmatory tests per true positive pulmonary TB case diagnosed (threshold probability 5%). If it is possible to perform more confirmatory tests than this, a 'confirmatory test for all' strategy performed better. CONCLUSIONS: CRP achieved the WHO-defined sensitivity, but not specificity, targets for a triage test for pulmonary TB and showed evidence of clinical utility among symptomatic outpatients, irrespective of HIV status. FUNDING: South African Medical Research Council, EDCTP2, Royal Society Newton Advanced Fellowship, Wellcome Trust, National Institute of Health Research, Royal College of Physicians.

Toward a more generalizable blood RNA signature for bacterial and viral infections.

Host-response profiles can discriminate different infections. A new 8-gene blood RNA signature to discriminate bacterial and viral infections extends our focus hitherto on the case mix from the US and Europe to include that of low- and middle-income countries.1 Challenges remain.

Clinical prediction models for mortality in patients with covid-19: external validation and individual participant data meta-analysis.

OBJECTIVE: To externally validate various prognostic models and scoring rules for predicting short term mortality in patients admitted to hospital for covid-19. DESIGN: Two stage individual participant data meta-analysis. SETTING: Secondary and tertiary care. PARTICIPANTS: 46 914 patients across 18 countries, admitted to a hospital with polymerase chain reaction confirmed covid-19 from November 2019 to April 2021. DATA SOURCES: Multiple (clustered) cohorts in Brazil, Belgium, China, Czech Republic, Egypt, France, Iran, Israel, Italy, Mexico, Netherlands, Portugal, Russia, Saudi Arabia, Spain, Sweden, United Kingdom, and United States previously identified by a living systematic review of covid-19 prediction models published in The BMJ, and through PROSPERO, reference checking, and expert knowledge. MODEL SELECTION AND ELIGIBILITY CRITERIA: Prognostic models identified by the living systematic review and through contacting experts. A priori models were excluded that had a high risk of bias in the participant domain of PROBAST (prediction model study risk of bias assessment tool) or for which the applicability was deemed poor. METHODS: Eight prognostic models with diverse predictors were identified and validated. A two stage individual participant data meta-analysis was performed of the estimated model concordance (C) statistic, calibration slope, calibration-in-the-large, and observed to expected ratio (O:E) across the included clusters. MAIN OUTCOME MEASURES: 30 day mortality or in-hospital mortality. RESULTS: Datasets included 27 clusters from 18 different countries and contained data on 46 914patients. The pooled estimates ranged from 0.67 to 0.80 (C statistic), 0.22 to 1.22 (calibration slope), and 0.18 to 2.59 (O:E ratio) and were prone to substantial between study heterogeneity. The 4C Mortality Score by Knight et al (pooled C statistic 0.80, 95% confidence interval 0.75 to 0.84, 95% prediction interval 0.72 to 0.86) and clinical model by Wang et al (0.77, 0.73 to 0.80, 0.63 to 0.87) had the highest discriminative ability. On average, 29% fewer deaths were observed than predicted by the 4C Mortality Score (pooled O:E 0.71, 95% confidence interval 0.45 to 1.11, 95% prediction interval 0.21 to 2.39), 35% fewer than predicted by the Wang clinical model (0.65, 0.52 to 0.82, 0.23 to 1.89), and 4% fewer than predicted by Xie et al's model (0.96, 0.59 to 1.55, 0.21 to 4.28). CONCLUSION: The prognostic value of the included models varied greatly between the data sources. Although the Knight 4C Mortality Score and Wang clinical model appeared most promising, recalibration (intercept and slope updates) is needed before implementation in routine care.

Rapid synchronous type 1 IFN and virus-specific T cell responses characterize first wave non-severe SARS-CoV-2 infections.

Effective control of SARS-CoV-2 infection on primary exposure may reveal correlates of protective immunity to future variants, but we lack insights into immune responses before or at the time virus is first detected. We use blood transcriptomics, multiparameter flow cytometry, and T cell receptor (TCR) sequencing spanning the time of incident non-severe infection in unvaccinated virus-naive individuals to identify rapid type 1 interferon (IFN) responses common to other acute respiratory viruses and cell proliferation responses that discriminate SARS-CoV-2 from other viruses. These peak by the time the virus is first detected and sometimes precede virus detection. Cell proliferation is most evident in CD8 T cells and associated with specific expansion of SARS-CoV-2-reactive TCRs, in contrast to virus-specific antibodies, which lag by 1-2 weeks. Our data support a protective role for early type 1 IFN and CD8 T cell responses, with implications for development of universal T cell vaccines.

Prospective validation of the 4C prognostic models for adults hospitalised with COVID-19 using the ISARIC WHO Clinical Characterisation Protocol.

PURPOSE: To prospectively validate two risk scores to predict mortality (4C Mortality) and in-hospital deterioration (4C Deterioration) among adults hospitalised with COVID-19. METHODS: Prospective observational cohort study of adults (age ≥18 years) with confirmed or highly suspected COVID-19 recruited into the International Severe Acute Respiratory and emerging Infections Consortium (ISARIC) WHO Clinical Characterisation Protocol UK (CCP-UK) study in 306 hospitals across England, Scotland and Wales. Patients were recruited between 27 August 2020 and 17 February 2021, with at least 4 weeks follow-up before final data extraction. The main outcome measures were discrimination and calibration of models for in-hospital deterioration (defined as any requirement of ventilatory support or critical care, or death) and mortality, incorporating predefined subgroups. RESULTS: 76 588 participants were included, of whom 27 352 (37.4%) deteriorated and 12 581 (17.4%) died. Both the 4C Mortality (0.78 (0.77 to 0.78)) and 4C Deterioration scores (pooled C-statistic 0.76 (95% CI 0.75 to 0.77)) demonstrated consistent discrimination across all nine National Health Service regions, with similar performance metrics to the original validation cohorts. Calibration remained stable (4C Mortality: pooled slope 1.09, pooled calibration-in-the-large 0.12; 4C Deterioration: 1.00, -0.04), with no need for temporal recalibration during the second UK pandemic wave of hospital admissions. CONCLUSION: Both 4C risk stratification models demonstrate consistent performance to predict clinical deterioration and mortality in a large prospective second wave validation cohort of UK patients. Despite recent advances in the treatment and management of adults hospitalised with COVID-19, both scores can continue to inform clinical decision making. TRIAL REGISTRATION NUMBER: ISRCTN66726260.

The diagnostic performance of novel skin-based in-vivo tests for tuberculosis infection compared with purified protein derivative tuberculin skin tests and blood-based in vitro interferon-γ release assays: a systematic review and meta-analysis.

BACKGROUND: Novel skin-based tests for tuberculosis infection might present suitable alternatives to current tests; however, diagnostic performance of new tests compared with the purified protein derivative-tuberculin skin test (TST) or interferon-γ release assays (IGRA) needs systematic assessment. METHODS: In this systematic review and meta-analysis, we searched English (Medline OVID), Chinese (Chinese Biomedical Literature Database and the China National Knowledge Infrastructure), and Russian (e-library) databases from the inception of each database to May 15, 2019, (with updated search of the Russian and English databases on Oct, 20 2020) using terms "ESAT6" OR "CFP10" AND "skin test" AND "Tuberculosis" OR "C-Tb" OR "Diaskintest". We included studies reporting on the performance of index tests alone or compared with a comparator. Inclusion criteria varied according to review objectives and performance outcome, but reporting of test cut-offs for positivity applied to study population was required from all studies. We used a hierarchy of reference standards for tuberculosis infection consistent with the 2020 WHO framework to evaluate diagnostic performance. Two authors independently reviewed the titles and abstracts for English and Chinese (LF and MK) and Russian studies (MK and VN). Study quality was assessed with QUADAS-2. Pooled random-effects estimates are presented when appropriate for total agreement proportion, sensitivity in microbiologically confirmed tuberculosis and specificity in cohorts with low risk of tuberculosis infection. This study is registered with PROSPERO, CRD42019135572. FINDINGS: We identified 1466 original articles, of which 37 (2·5%) studies, including 10 915 individuals (7111 Diaskintest, 2744 C-Tb, 887 EC, 173 DPPD), were included in the qualitative analysis (29 [78%] studies of Diaskintest, five [15%] studies of C-Tb, two [5%] studies of EC-skintest, and one [3%] study of DPPD). 22 (1·5%) studies including 5810 individuals (3143 Diaskintest, 2129 C-Tb, 538 EC-skintest) were included in the quantitative analysis: 15 (68%) of Diaskintest, five (23%) of C-Tb, and two (9%) of EC-skintest. Tested sub-populations included individuals with HIV, children (0-18 years), and individuals exposed to tuberculosis. Studies were heterogeneous with moderate to high risk of bias. Nine head-to-head studies of index test versus TST and IGRA permitted direct comparisons and pooling. In a mixed cohort of people with and without tuberculosis, Diaskintest pooled agreement with IGRA was 87·16% (95% CI 79·47-92·24) and 55·45% (46·08-64·45) with TST-5 mm cut-off (TST5 mm). Diaskintest sensitivity was 91·18% (95% CI 81·72-95·98) compared with 88·24% (78·20-94·01) for TST5 mm, 89·66 (78·83-95·28) for IGRA QuantiFERON, and 90·91% (79·95-96·16) for TSPOT.TB. C-Tb agreement with IGRA in individuals with active tuberculosis was 79·80% (95% CI 76·10-83·07) compared with 78·92% (74·65-82·63) for TST5 mm/15 mm cut-off (TST5 mm/15 mm). TST5/15mm reflects threshold in cohorts that applied stratified cutoffs: 5 mm for HIV-infected, immunocompromised, or BCG-naive individuals, and 15mm for BCG-vaccinated immunocompetent individuals. C-Tb sensitivity was 74·52% (95% CI 70·39-78·25) compared with a sensitivity of 78·18% (67·75-85·94) for TST5 mm/15 mm, and 71·67% (63·44-78·68) for IGRA. Specificity was 97·85% (95% CI 93·96-99·25) for C-Tb versus 93·31% (90·22-95·48) for TST 15 mm cut-off and 99·15% (79·66-99·97) for IGRA. EC-skintest sensitivity was 86·06% (95% CI 82·39-89·07). INTERPRETATION: Novel skin-based tests for tuberculosis infection appear to perform similarly to IGRA or TST; however, study quality varied. Evaluation of test performance, patient-important outcomes, and diagnostic use in current clinical algorithms will inform implementation in key populations. FUNDING: StopTB (New Diagnostics Working Group) and FIND. TRANSLATIONS: For the Chinese and Russian translations of the abstract see Supplementary Materials section.

Discovery and validation of a three-gene signature to distinguish COVID-19 and other viral infections in emergency infectious disease presentations: a case-control and observational cohort study.

BACKGROUND: Emergency admissions for infection often lack initial diagnostic certainty. COVID-19 has highlighted a need for novel diagnostic approaches to indicate likelihood of viral infection in a pandemic setting. We aimed to derive and validate a blood transcriptional signature to detect viral infections, including COVID-19, among adults with suspected infection who presented to the emergency department. METHODS: Individuals (aged ≥18 years) presenting with suspected infection to an emergency department at a major teaching hospital in the UK were prospectively recruited as part of the Bioresource in Adult Infectious Diseases (BioAID) discovery cohort. Whole-blood RNA sequencing was done on samples from participants with subsequently confirmed viral, bacterial, or no infection diagnoses. Differentially expressed host genes that met additional filtering criteria were subjected to feature selection to derive the most parsimonious discriminating signature. We validated the signature via RT-qPCR in a prospective validation cohort of participants who presented to an emergency department with undifferentiated fever, and a second case-control validation cohort of emergency department participants with PCR-positive COVID-19 or bacterial infection. We assessed signature performance by calculating the area under receiver operating characteristic curves (AUROCs), sensitivities, and specificities. FINDINGS: A three-gene transcript signature, comprising HERC6, IGF1R, and NAGK, was derived from the discovery cohort of 56 participants with bacterial infections and 27 with viral infections. In the validation cohort of 200 participants, the signature differentiated bacterial from viral infections with an AUROC of 0·976 (95% CI 0·919-1·000), sensitivity of 97·3% (85·8-99·9), and specificity of 100% (63·1-100). The AUROC for C-reactive protein (CRP) was 0·833 (0·694-0·944) and for leukocyte count was 0·938 (0·840-0·986). The signature achieved higher net benefit in decision curve analysis than either CRP or leukocyte count for discriminating viral infections from all other infections. In the second validation analysis, which included SARS-CoV-2-positive participants, the signature discriminated 35 bacterial infections from 34 SARS-CoV-2-positive COVID-19 infections with AUROC of 0·953 (0·893-0·992), sensitivity 88·6%, and specificity of 94·1%. INTERPRETATION: This novel three-gene signature discriminates viral infections, including COVID-19, from other emergency infection presentations in adults, outperforming both leukocyte count and CRP, thus potentially providing substantial clinical utility in managing acute presentations with infection. FUNDING: National Institute for Health Research, Medical Research Council, Wellcome Trust, and EU-FP7.

Blood transcriptional biomarkers of acute viral infection for detection of pre-symptomatic SARS-CoV-2 infection: a nested, case-control diagnostic accuracy study.

BACKGROUND: We hypothesised that host-response biomarkers of viral infections might contribute to early identification of individuals infected with SARS-CoV-2, which is critical to breaking the chains of transmission. We aimed to evaluate the diagnostic accuracy of existing candidate whole-blood transcriptomic signatures for viral infection to predict positivity of nasopharyngeal SARS-CoV-2 PCR testing. METHODS: We did a nested case-control diagnostic accuracy study among a prospective cohort of health-care workers (aged ≥18 years) at St Bartholomew's Hospital (London, UK) undergoing weekly blood and nasopharyngeal swab sampling for whole-blood RNA sequencing and SARS-CoV-2 PCR testing, when fit to attend work. We identified candidate blood transcriptomic signatures for viral infection through a systematic literature search. We searched MEDLINE for articles published between database inception and Oct 12, 2020, using comprehensive MeSH and keyword terms for "viral infection", "transcriptome", "biomarker", and "blood". We reconstructed signature scores in blood RNA sequencing data and evaluated their diagnostic accuracy for contemporaneous SARS-CoV-2 infection, compared with the gold standard of SARS-CoV-2 PCR testing, by quantifying the area under the receiver operating characteristic curve (AUROC), sensitivities, and specificities at a standardised Z score of at least 2 based on the distribution of signature scores in test-negative controls. We used pairwise DeLong tests compared with the most discriminating signature to identify the subset of best performing biomarkers. We evaluated associations between signature expression, viral load (using PCR cycle thresholds), and symptom status visually and using Spearman rank correlation. The primary outcome was the AUROC for discriminating between samples from participants who tested negative throughout the study (test-negative controls) and samples from participants with PCR-confirmed SARS-CoV-2 infection (test-positive participants) during their first week of PCR positivity. FINDINGS: We identified 20 candidate blood transcriptomic signatures of viral infection from 18 studies and evaluated their accuracy among 169 blood RNA samples from 96 participants over 24 weeks. Participants were recruited between March 23 and March 31, 2020. 114 samples were from 41 participants with SARS-CoV-2 infection, and 55 samples were from 55 test-negative controls. The median age of participants was 36 years (IQR 27-47) and 69 (72%) of 96 were women. Signatures had little overlap of component genes, but were mostly correlated as components of type I interferon responses. A single blood transcript for IFI27 provided the highest accuracy for discriminating between test-negative controls and test-positive individuals at the time of their first positive SARS-CoV-2 PCR result, with AUROC of 0·95 (95% CI 0·91-0·99), sensitivity 0·84 (0·70-0·93), and specificity 0·95 (0·85-0·98) at a predefined threshold (Z score >2). The transcript performed equally well in individuals with and without symptoms. Three other candidate signatures (including two to 48 transcripts) had statistically equivalent discrimination to IFI27 (AUROCs 0·91-0·95). INTERPRETATION: Our findings support further urgent evaluation and development of blood IFI27 transcripts as a biomarker for early phase SARS-CoV-2 infection for screening individuals at high risk of infection, such as contacts of index cases, to facilitate early case isolation and early use of antiviral treatments as they emerge. FUNDING: Barts Charity, Wellcome Trust, and National Institute of Health Research.

Time series analysis and mechanistic modelling of heterogeneity and sero-reversion in antibody responses to mild SARS­CoV-2 infection.

BACKGROUND: SARS-CoV-2 serology is used to identify prior infection at individual and at population level. Extended longitudinal studies with multi-timepoint sampling to evaluate dynamic changes in antibody levels are required to identify the time horizon in which these applications of serology are valid, and to explore the longevity of protective humoral immunity. METHODS: Healthcare workers were recruited to a prospective cohort study from the first SARS-CoV-2 epidemic peak in London, undergoing weekly symptom screen, viral PCR and blood sampling over 16-21 weeks. Serological analysis (n =12,990) was performed using semi-quantitative Euroimmun IgG to viral spike S1 domain and Roche total antibody to viral nucleocapsid protein (NP) assays. Comparisons were made to pseudovirus neutralizing antibody measurements. FINDINGS: A total of 157/729 (21.5%) participants developed positive SARS-CoV-2 serology by one or other assay, of whom 31.0% were asymptomatic and there were no deaths. Peak Euroimmun anti-S1 and Roche anti-NP measurements correlated (r = 0.57, p<0.0001) but only anti-S1 measurements correlated with near-contemporary pseudovirus neutralising antibody titres (measured at 16-18 weeks, r = 0.57, p<0.0001). By 21 weeks' follow-up, 31/143 (21.7%) anti-S1 and 6/150 (4.0%) anti-NP measurements reverted to negative. Mathematical modelling revealed faster clearance of anti-S1 compared to anti-NP (median half-life of 2.5 weeks versus 4.0 weeks), earlier transition to lower levels of antibody production (median of 8 versus 13 weeks), and greater reductions in relative antibody production rate after the transition (median of 35% versus 50%). INTERPRETATION: Mild SARS-CoV-2 infection is associated with heterogeneous serological responses in Euroimmun anti-S1 and Roche anti-NP assays. Anti-S1 responses showed faster rates of clearance, more rapid transition from high to low level production rate and greater reduction in production rate after this transition. In mild infection, anti-S1 serology alone may underestimate incident infections. The mechanisms that underpin faster clearance and lower rates of sustained anti-S1 production may impact on the longevity of humoral immunity. FUNDING: Charitable donations via Barts Charity, Wellcome Trust, NIHR.

Evaluation and improvement of the National Early Warning Score (NEWS2) for COVID-19: a multi-hospital study.

BACKGROUND: The National Early Warning Score (NEWS2) is currently recommended in the UK for the risk stratification of COVID-19 patients, but little is known about its ability to detect severe cases. We aimed to evaluate NEWS2 for the prediction of severe COVID-19 outcome and identify and validate a set of blood and physiological parameters routinely collected at hospital admission to improve upon the use of NEWS2 alone for medium-term risk stratification. METHODS: Training cohorts comprised 1276 patients admitted to King's College Hospital National Health Service (NHS) Foundation Trust with COVID-19 disease from 1 March to 30 April 2020. External validation cohorts included 6237 patients from five UK NHS Trusts (Guy's and St Thomas' Hospitals, University Hospitals Southampton, University Hospitals Bristol and Weston NHS Foundation Trust, University College London Hospitals, University Hospitals Birmingham), one hospital in Norway (Oslo University Hospital), and two hospitals in Wuhan, China (Wuhan Sixth Hospital and Taikang Tongji Hospital). The outcome was severe COVID-19 disease (transfer to intensive care unit (ICU) or death) at 14 days after hospital admission. Age, physiological measures, blood biomarkers, sex, ethnicity, and comorbidities (hypertension, diabetes, cardiovascular, respiratory and kidney diseases) measured at hospital admission were considered in the models. RESULTS: A baseline model of 'NEWS2 + age' had poor-to-moderate discrimination for severe COVID-19 infection at 14 days (area under receiver operating characteristic curve (AUC) in training cohort = 0.700, 95% confidence interval (CI) 0.680, 0.722; Brier score = 0.192, 95% CI 0.186, 0.197). A supplemented model adding eight routinely collected blood and physiological parameters (supplemental oxygen flow rate, urea, age, oxygen saturation, C-reactive protein, estimated glomerular filtration rate, neutrophil count, neutrophil/lymphocyte ratio) improved discrimination (AUC = 0.735; 95% CI 0.715, 0.757), and these improvements were replicated across seven UK and non-UK sites. However, there was evidence of miscalibration with the model tending to underestimate risks in most sites. CONCLUSIONS: NEWS2 score had poor-to-moderate discrimination for medium-term COVID-19 outcome which raises questions about its use as a screening tool at hospital admission. Risk stratification was improved by including readily available blood and physiological parameters measured at hospital admission, but there was evidence of miscalibration in external sites. This highlights the need for a better understanding of the use of early warning scores for COVID.