ABSTRACT
Background: Limited data are available on the diagnostic accuracy of blood RNA biomarker signatures for extrapulmonary TB (EPTB). We addressed this question among people investigated for TB lymphadenitis and TB pericarditis, in Cape Town, South Africa. Methods: We enrolled 440 consecutive adults referred to a hospital for invasive sampling for presumptive TB lymphadenitis (n=300) or presumptive TB pericarditis (n=140). Samples from the site of disease underwent culture and/or molecular testing for Mycobacterium tuberculosis complex (Mtb). Discrimination of patients with and without TB defined by microbiology or cytology reference standards was evaluated using seven previously reported blood RNA signatures by area under the receiver-operating characteristic curve (AUROC) and sensitivity/specificity at predefined thresholds, benchmarked against blood C-reactive protein (CRP) and the World Health Organization (WHO) target product profile (TPP) for a TB triage test. Decision curve analysis (DCA) was used to evaluate the clinical utility of the best performing blood RNA signature and CRP. Results: Data from 374 patients for whom results were available from at least one microbiological test from the site of disease, and blood CRP and RNA measurements, were included. Using microbiological results as the reference standard in the primary analysis (N=204 with TB), performance was similar across lymphadenitis and pericarditis patients. In the pooled analysis of both cohorts, all RNA signatures had comparable discrimination with AUROC point estimates ranging 0.77-0.82, superior to that of CRP (0.61, 95% confidence interval 0.56-0.67). The best performing signature (Roe3) achieved an AUROC of 0.82 (0.77-0.86). At a predefined threshold of 2 standard deviations (Z2) above the mean of a healthy reference control group, this signature achieved 78% (72-83%) sensitivity and 69% (62-75%) specificity. In this setting, DCA revealed that Roe3 offered greater net benefit than other approaches for services aiming to reduce the number needed to investigate with confirmatory testing to <4 to identify each case of TB. Interpretation: RNA biomarkers show better accuracy and clinical utility than CRP to trigger confirmatory TB testing in patients with TB lymphadenitis and TB pericarditis, but still fall short of the WHO TPP for TB triage tests. Funding: South African MRC, EDCTP2, NIH/NIAID, Wellcome Trust, NIHR, Royal College of Physicians London. Research in context: Evidence before this study: Blood RNA biomarker signatures and CRP measurements have emerged as potential triage tests for TB, but evidence is mostly limited to their performance in pulmonary TB. Microbiological diagnosis of extrapulmonary TB (EPTB) is made challenging by the need for invasive sampling to obtain tissue from the site of disease. This is compounded by lower sensitivity of confirmatory molecular tests for EPTB compared to their performance in pulmonary disease. We performed a systematic review of diagnostic accuracy studies of blood RNA biomarkers or CRP measurements for EPTB, which could mitigate the need for site-of-disease sampling for the diagnosis of TB. We searched PubMed up to 1 st August 2023, using the following criteria: "extrapulmonary [title/abstract] AND tuberculosis [title/abstract] AND biomarker [title/abstract]". Although extrapulmonary TB was included in several studies, none focused specifically on EPTB or included an adequate number of EPTB cases to provide precise estimates of test accuracy. Added value of this study: To the best of our knowledge, we report the first diagnostic accuracy study of blood RNA biomarkers and CRP for TB among people with EPTB syndromes. We examined the performance of seven previously identified blood RNA biomarkers as triage tests for TB lymphadenitis and TB pericarditis compared to a microbiology reference standard among people referred to hospital for invasive sampling in a high TB and HIV prevalence setting. Multiple blood RNA biomarkers showed comparable diagnostic accuracy to that previously reported for pulmonary TB in both EPTB disease cohorts, irrespective of HIV status. All seven blood RNA biomarkers showed superior diagnostic accuracy to CRP for both lymphadenitis and pericarditis, but failed to meet the combined >90% sensitivity and >70% specificity recommended for a blood-based diagnostic triage test by WHO. Nonetheless, in decision curve analysis, an approach of using the best performing blood RNA biomarker to trigger confirmatory microbiological testing showed superior clinical utility in clinical services seeking to reduce the number needed to test (using invasive confirmatory testing) to less than 4 for each EPTB case detected. If acceptable to undertake invasive testing in more than 4 people for each true case detected, then a test-all approach will provide greater net benefit in this TB/HIV hyperendemic setting.Implications of all the available evidence: Blood RNA biomarkers show some potential as diagnostic triage tests for TB lymphadenitis and TB pericarditis, but do not provide the level of accuracy for blood-based triage tests recommended by WHO for community-based tests. CRP has inferior diagnostic accuracy to blood RNA biomarkers and cannot be recommended for diagnostic triage among people with EPTB syndromes referred for invasive sampling.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
Subject(s)
HIV Infections , Mycobacterium tuberculosis , Tuberculosis, Pulmonary , Adult , Humans , C-Reactive Protein , South Africa/epidemiology , Triage , Tuberculosis, Pulmonary/diagnosis , Sensitivity and Specificity , Sputum , HIV Infections/complicationsABSTRACT
BACKGROUND: Blood transcriptional signatures are candidates for non-sputum triage or confirmatory tests of tuberculosis. Prospective head-to-head comparisons of their diagnostic accuracy in real-world settings are necessary to assess their clinical use. We aimed to compare the diagnostic accuracy of candidate transcriptional signatures identified by systematic review, in a setting with a high burden of tuberculosis and HIV. METHODS: We did a prospective observational study nested within a diagnostic accuracy study of sputum Xpert MTB/RIF (Xpert) and Xpert MTB/RIF Ultra (Ultra) tests for pulmonary tuberculosis. We recruited consecutive symptomatic adults aged 18 years or older self-presenting to a tuberculosis clinic in Cape Town, South Africa. Participants provided blood for RNA sequencing, and sputum samples for liquid culture and molecular testing using Xpert and Ultra. We assessed the diagnostic accuracy of candidate blood transcriptional signatures for active tuberculosis (including those intended to distinguish active tuberculosis from other diseases) identified by systematic review, compared with culture or Xpert MTB/RIF positivity as the standard reference. In our primary analysis, patients with tuberculosis were defined as those with either a positive liquid culture or Xpert result. Patients with missing blood RNA or sputum results were excluded. Our primary objective was to benchmark the diagnostic accuracy of candidate transcriptional signatures against the WHO target product profile (TPP) for a tuberculosis triage test. FINDINGS: Between Feb 12, 2016, and July 18, 2017, we obtained paired sputum and RNA sequencing data from 181 participants, 54 (30%) of whom had confirmed pulmonary tuberculosis. Of 27 eligible signatures identified by systematic review, four achieved the highest diagnostic accuracy with similar area under the receiver operating characteristic curves (Sweeney3: 90·6% [95% CI 85·6-95·6]; Kaforou25: 86·9% [80·9-92·9]; Roe3: 86·9% [80·3-93·5]; and BATF2: 86·8% [80·6-93·1]), independent of age, sex, HIV status, previous tuberculosis, or sputum smear result. At test thresholds that gave 70% specificity (the minimum WHO TPP specificity for a triage test), these four signatures achieved sensitivities between 83·3% (95% CI 71·3-91·0) and 90·7% (80·1-96·0). No signature met the optimum criteria, of 95% sensitivity and 80% specificity proposed by WHO for a triage test, or the minimum criteria (of 65% sensitivity and 98% specificity) for a confirmatory test, but all four correctly identified Ultra-positive, culture-negative patients. INTERPRETATION: Selected blood transcriptional signatures met the minimum WHO benchmarks for a tuberculosis triage test but not for a confirmatory test. Further development of the signatures is warranted to investigate their possible effects on clinical and health economic outcomes as part of a triage strategy, or when used as add-on confirmatory test in conjunction with the highly sensitive Ultra test for Mycobacterium tuberculosis DNA. FUNDING: Royal Society Newton Advanced Fellowship, Wellcome Trust, National Institute of Health Research, and UK Medical Research Council.
