Blood RNA biomarkers for tuberculosis screening in people living with HIV prior to anti-retroviral therapy initiation: A diagnostic accuracy study
Authors: Mann Tiffeney , Gupta Rishi K , Reeve Byron WP , Ndlangalavu Gcobisa , Chandran Aneesh , Krishna Amirtha P , Calderwood Claire J , Tshivhula Happy , Palmer Zaida , Naidoo Selisha , Mbu Desiree L , Theron Grant , Noursadeghi Mahdad 
Published: June 2023

AbstractBackgroundUndiagnosed 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.MethodsWe 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 ofMycobacterium tuberculosisculture 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.ResultsA 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.InterpretationRNA 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.FundingSouth African MRC, EDCTP2, NIH/NIAID, Wellcome Trust, NIHR, Royal College of Physicians London.Research in ContextEvidence before this studyThe World Health Organisation (WHO) commissioned a recent systematic review and individual participant data meta-analysis of tuberculosis (TB) screening strategies among ambulatory people living with HIV (PLHIV). TB is a major cause of morbidity and mortality among PLHIV, particularly among those with untreated HIV and consequent immunosuppression. Importantly, initiation of antiretroviral treatment (ART) for HIV is also associated with increased short-term risk of incident TB, attributed to immune reconstitution inflammatory syndrome, which may in turn potentiate the immunopathogenesis of TB. As a result, in high TB prevalence settings, systematic screening for TB is widely advocated for PLHIV before starting ART. In this context, universal sputum microbiological screening is not economically sustainable, and limited by practical feasibility among those who are not expectorating sputum. Patient stratification to identify those at greater risk of TB is required to target resources for microbiological testing more precisely. For this purpose, the WHO four symptom screen (W4SS) achieved an estimated 84% sensitivity and 37% specificity for pre-ART TB screening. Blood CRP ≥5mg/L offered better performance, estimated at 89% sensitivity and 54% specificity respectively, but still fell short of the WHO target product profile, aiming for ≥90% sensitivity and ≥70% specificity. Blood RNA biomarkers of TB, reflecting interferon (IFN) and tumour necrosis factor-mediated immune responses, have been gaining momentum as potential triage tests for symptomatic and pre-symptomatic TB, but their performance has not been comprehensively evaluated among PLHIV initiating ART. Untreated HIV also drives chronic IFN activity that may compromise the specificity of IFN-dependent biomarkers in this population.Added value of this studyTo our knowledge, this is the largest study to date to benchmark the performance of candidate blood RNA biomarkers for unselected and systematic pre-ART TB screening among PLHIV, against contemporary standards and aspirational performance targets. The blood RNA biomarkers showed better diagnostic accuracy and clinical utility to guide confirmatory TB testing for PLHIV than symptom-based screening with W4SS, but their performance did not exceed that of CRP, and they did not achieve WHO recommended targets. The results were comparable for microbiologically confirmed TB at enrolment to the study and for all cases starting TB treatment within six months of enrolment. Blood RNA biomarkers correlated with features of disease severity that might be attributed to either TB or HIV. Accordingly, their discrimination of TB among PLHIV was particularly limited by poor specificity. Diagnostic accuracy was significantly better among people who were symptomatic compared to those who were asymptomatic, further limiting the value of RNA biomarkers in pre-symptomatic TB. Interestingly, blood RNA biomarkers only showed moderate correlation with CRP, suggesting these two measurements provided information on different components of the host response. An exploratory analysis showed that CRP can be combined with the best performing blood RNA signature to provide better clinical utility than achieved by either test alone.Implications of all the available evidenceOur data demonstrate that blood RNA biomarkers do not perform any better than CRP as triage tests for TB among PLHIV prior to ART initiation. Since CRP is already widely available on a low cost point-of-care platform, our findings support further evaluation of the clinical and health-economic impact of CRP-based triage for pre-ART TB screening. An underlying mechanism that limits the diagnostic accuracy of RNA biomarkers for TB among PLHIV prior to ART may be upregulation of interferon signalling in untreated HIV. Since interferon activity underpins upregulated expression of TB biomarker genes, HIV-induced upregulation of interferon-stimulated genes may reduce the specificity of blood transcriptomic biomarkers for TB in this context. These findings highlight a wider need to identify interferon-independent host-response based biomarkers to support disease specific screening of PLHIV pre-ART initiation.