Diagnostic accuracy of Truenat MTB Plus, Truenat MTB Ultima and Xpert MTB/RIF Ultra for the diagnosis of pulmonary TB in an HIV-endemic setting.

Background: Truenat MTB Plus (MTB Plus) and MTB Ultima (Ultima) are World Health Organization-endorsed low-complexity tuberculosis (TB) tests, however, performance data are scarce. Methods: Adults (≥18 years; n=498) self-presenting with symptoms to primary care clinics in Cape Town, South Africa (19/02/2016-22/02/2023) provided sputa. We evaluated the accuracy of MTB Plus and Ultima, with Xpert MTB/RIF Ultra (Ultra) as a comparator, vs. a single culture (TB reference standard) or MTBDRplus on an isolate (rifampicin susceptibility reference standard). Results: The proportion of MTB Plus and Ultima unsuccessful results was 20% (95% confidence interval 17, 23) and 14 (11, 16), respectively, with ≥half resolving upon retesting the same eluate. In a three-way analysis, MTB Plus, Ultima and Ultra had sensitivities of 84% (78, 88), 90% (85, 93), and 92% (87, 95), and specificities of 95% (92, 97), 85% (80, 88) and 95% (92, 97) for TB. The proportion of unsuccessful results for MTB-RIF Dx done the same day as DNA extraction was 9% (3, 16; MTB Plus-positives) and 18% (10, 26; Ultima-positives) [if after day-of-extraction, these were 27% (18, 35) and 44% (35, 51)]. Same-day rifampicin susceptibility testing was often unsuccessful in samples with "very low" load [73% (58, 89) MTB Plus, 75% (65, 86) Ultima] but had 100% (40, 100) sensitivity and 99% (96, 100) specificity (for both MTB Plus- or Ultima-positive DNA). Lot variation in unsuccessful and false-positive results was observed. Conclusion: Ultima showed comparable sensitivity to Ultra but specificity, lot variation, and, like MTB-RIF Dx, unsuccessful result rates were suboptimal. Funding: European & Developing Countries Clinical Trials Partnership, and South African Medical Research Council.

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.

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 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.

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.

MicroRNA profiling and their pathways in South African individuals with prediabetes and newly diagnosed type 2 diabetes mellitus.

Early identification of individuals with elevated risk of developing diabetes mellitus, followed by the implementation of effective prevention interventions can delay the onset of the disease and related complications. In this regard, recent studies have shown that miRNAs are useful as early markers of certain disease types, including diabetes. We used high throughput sequencing to assess miRNA expression profiles from whole blood of 12 individuals with screen-detected diabetes, 12 with prediabetes and 12 with normal glucose tolerance, matched for age, blood pressure, smoking and body mass index. We identified a total of 261 (57 novel) differentially expressed miRNA profiles between the study groups. Comparison of the miRNA expression profiles between prediabetess and diabetes revealed 25 common miRNA, but highlighted some interesting differences. For instance, three miRNAs (miR-126-3p, miR-28-3p miR-486-5p) were dysregulated in prediabetes compared to screen-detected diabetes. Target gene analysis showed thousands of potential genes and KEGG pathway analysis revealed 107 significant pathways of which some are involved signal transduction, cell-cell communications, cell growth and death, immune response, endocrine system and metabolic diseases. This first detailed African study has shown both known and novel differentially expressed miRNAs in relation to glucose tolerance.