Stephanie Minnies

Background: Oral samples show promise for tuberculosis (TB) diagnosis, but data from different sample types and sputum-scarce individuals remain limited. Methods: We evaluated Xpert MTB/RIF Ultra (Ultra) in symptomatic clinic attendees (cohort A, n = 891) and people initiating antiretroviral therapy without symptom screening (cohort B, n = 258). In cohort A, we collected oral washes (OWs) and, separately, tongue swabs (flocked or foam with heat). In cohort B, we collected OWs, 3 flocked tongue swabs (1 heated, 2 pooled), and, separately, buccal swabs and periodontal brushes. Sputum induction was offered, and different culture methods were applied to a subset of cohort B tongue swabs. Results: In cohort A, Ultra sensitivity was 80% (95% confidence interval [CI], 56%–94%) for OWs, 59% (95% CI, 53%–65%) for flocked swabs, and 65% (95% CI, 58%–72%) for foam swabs, with high specificity. Foam swabs detected more people with Ultra sputum semi-quantitation categories of low or less than flocked swabs (53% [95% CI, 41%–64%] vs 37% [95% CI, 29%–46%]). In cohort B, OWs and single heated swabs had sensitivities of 71% (95% CI, 42%–92%) and 64% (95% CI, 35%–87%), respectively. Pooled tongue swabs, buccal swabs, and brushes had lower sensitivity. MGIT960 showed the highest sensitivity (64% [95% CI, 35%–87%]) among culture methods. Oral sampling identified TB in sputum-scarce people: 25% (7/28) positive by flocked or foam swabs (cohort A); 18% (10/56) were OW and 23% (13/56) single swab positive (cohort B). In cohort B, this could double Ultra positivity if induction were unavailable. Conclusions: Ultra on OWs or foam swabs offers higher sensitivity than other oral methods and effectively detects TB in sputum-scarce individuals.

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 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.

Background: Lymphadenitis is the most common extrapulmonary tuberculosis (EPTB) manifestation and a major cause of death. The microbiome is important to human health but uninvestigated in EPTB. We profiled the site-of-disease lymph node microbiome in tuberculosis lymphadenitis (TBL). Methods: Fine needle aspiration biopsies (FNABs) were collected from 159 pre-treatment presumptive TBL patients in Cape Town, South Africa. 16S Illumina MiSeq rRNA gene sequencing was done. Results: We analysed 89 definite TBLs (dTBLs) and 61 non-TBLs (nTBLs), which had similar α-but different β-diversities (p=0.001). Clustering identified five lymphotypes prior to TB status stratification: Mycobacterium-, Prevotella - and Streptococcus -dominant lymphotypes were more frequent in dTBLs whereas a Corynebacterium-dominant lymphotype and a fifth lymphotype (no dominant taxon) were more frequent in nTBLs. When restricted to dTBLs, clustering identified a Mycobacterium-dominant lymphotype with low α-diversity and other non- Mycobacterium -dominated lymphotypes (termed Prevotella-Corynebacterium and Prevotella-Streptococcus ). The Mycobacterium dTBL lymphotype was associated with HIV-positivity and clinical features characteristic of severe lymphadenitis (e.g., node size). dTBL microbial communities were enriched with potentially proinflammatory microbial short chain fatty acid metabolic pathways (propanoate, butanoate) vs. those in nTBLs. 11% (7/61) of nTBLs had Mycobacterium reads. Conclusions: TBL at the site-of-disease is not microbially homogenous and distinct microbial community clusters exist that are associated with different immunomodulatory potentials and clinical characteristics. Non- Mycobacterium -dominated dTBL lymphotypes, which contain taxa potentially targeted by TB treatment, represent less severe potentially earlier stage disease. These investigations lay foundations for studying the microbiome’s role in lymphatic TB and the long-term clinical significance of lymphotypes requires prospective evaluation.

Background Tuberculosis lymphadenitis (TBL) is the most common extrapulmonary TB (EPTB) manifestation. Xpert MTB/RIF Ultra (Ultra) is a World Health Organization-endorsed diagnostic test, but performance data for TBL, including on non-invasive specimens, are limited. Methods Fine needle aspiration biopsies (FNABs) from outpatients (≥18 years) with presumptive TBL (n=135) underwent: 1) routine Xpert (later Ultra once programmatically available), 2) a MGIT960 culture (if Xpert- or Ultra-negative, or rifampicin-resistant), and 3) study Ultra. Concentrated paired urine underwent Ultra. Primary analyses used a microbiological reference standard (MRS). Results In a head-to-head comparison (n=92) of FNAB study Ultra and Xpert, Ultra had increased sensitivity [91% (95% confidence interval 79, 98) vs. 72% (57, 84); p=0.016] and decreased specificity [76% (61, 87) vs. 93% (82, 99); p=0.020], and detected patients not on treatment. HIV nor alternative reference standards affected sensitivity and specificity. In patients with both routine and study Ultras, the latter detected more cases [+20% (0, 42); p=0.034] and, further indicative of potential laboratory-based room-for-improvement, false-negative study Ultras had more PCR inhibition than true-positives. Study Ultra “false-positives” had less mycobacterial DNA than “true-positives” [trace-positive proportions 59% (13/22) vs. 12% (5/51); pConclusions Ultra on FNABs is highly sensitive and detects more TBL than Xpert. Patients with FNAB Ultra-positive “trace” results, most of whom will be culture-negative, may require additional clinical investigation. Urine Ultra could reduce the number of patients needing invasive sampling.

Strengthening the detection of second-line drug-resistance is a key tuberculosis (TB) control priority. The performance of MTBDR plus , a multidrug-resistant (MDR)-TB assay is reduced when suboptimal ramp rates are used. We investigated ramp rate’s effect on MTBDR sl ; the most widely-used molecular second-line drug-resistant TB assay. We tested 52 smear-negative Xpert MTB/RIF Ultra-positive sputa and a Mycobacterium tuberculosis ( Mtb ) dilution series at manufacturer-recommended (2.2°C/s) or most common suboptimal ramp rate (4.0°C/s; identified via an earlier survey). Mtb- complex DNA (TUB-band)-positivity, indeterminate rates, fluoroquinolone- and second-line injectable-resistance accuracy, banding differences and, separately, inter-reader variability were assessed. 39% of re-surveyed laboratories (5/13) did not use the manufacturer-recommended MTBDR sl ramp rate. On sputum, this ramp rate improved indeterminates vs. 4.0°C/s (0/52 vs. 7/51; p=0.006), false drug-resistance calls (0/104 vs. 6/102; p=0.013), and incorrect banding calls (0/1300 vs. 55/1275; psl performance. Ramp rate correction will improve second-line drug-resistant TB diagnoses. Laboratories must ensure the optimal manufacturer-recommended ramp rate is used.