Evaluation of the Xpert MTB Host Response assay for the triage of patients with presumed pulmonary tuberculosis: a prospective diagnostic accuracy study in Viet Nam, India, the Philippines, Uganda, and South Africa.

BACKGROUND: Non-sputum-based triage tests for tuberculosis are a priority for ending tuberculosis. We aimed to evaluate the diagnostic accuracy of the late-prototype Xpert MTB Host Response (Xpert HR) blood-based assay. METHODS: We conducted a prospective diagnostic accuracy study among outpatients with presumed tuberculosis in outpatient clinics in Viet Nam, India, the Philippines, Uganda, and South Africa. Eligible participants were aged 18 years or older and reported cough lasting at least 2 weeks. We excluded those receiving tuberculosis treatment in the preceding 12 months and those who were unwilling to consent. Xpert HR was performed on capillary or venous blood. Reference standard testing included sputum Xpert MTB/RIF Ultra and mycobacterial culture. We performed receiver operating characteristic (ROC) analysis to identify the optimal cutoff value for the Xpert HR to achieve the target sensitivity of 90% or more while maximising specificity, then calculated diagnostic accuracy using this cutoff value. This study was prospectively registered with ClinicalTrials.gov, NCT04923958. FINDINGS: Between July 13, 2021, and Aug 15, 2022, 2046 adults with at least 2 weeks of cough were identified, of whom 1499 adults (686 [45·8%] females and 813 [54·2%] males) had valid Xpert HR and reference standard results. 329 (21·9%) had microbiologically confirmed tuberculosis. Xpert HR had an area under the ROC curve of 0·89 (95% CI 0·86-0·91). The optimal cutoff value was less than or equal to -1·25, giving a sensitivity of 90·3% (95% CI 86·5-93·3; 297 of 329) and a specificity of 62·6% (95% CI 59·7-65·3; 732 of 1170). Sensitivity was similar across countries, by sex, and by subgroups, although specificity was lower in people living with HIV (45·1%, 95% CI 37·8-52·6) than in those not living with HIV (65·9%, 62·8-68·8; difference of 20·8%, 95% CI 13·0-28·6; p<0·0001). Xpert HR had high negative predictive value (95·8%, 95% CI 94·1-97·1), but positive predictive value was only 40·1% (95% CI 36·8-44·1). Using the Xpert HR as a triage test would have reduced confirmatory sputum testing by 57·3% (95% CI 54·2-60·4). INTERPRETATION: Xpert HR did not meet WHO minimum specificity targets for a non-sputum-based triage test for pulmonary tuberculosis. Despite promise as a rule-out test that could reduce confirmatory sputum testing, further cost-effectiveness modelling and data on acceptability and usability are needed to inform policy recommendations. FUNDING: National Institute of Allergy and Infectious Diseases of the US National Institutes of Health. TRANSLATIONS: For the Vietnamese and Tagalog translations of the abstract see Supplementary Materials section.

The regulatory TnaC nascent peptide preferentially inhibits release factor 2-mediated hydrolysis of peptidyl-tRNA.

The tnaC regulatory gene from the tna operon of Escherichia coli controls the transcription of its own operon through an attenuation mechanism relying on the accumulation of arrested ribosomes during inhibition of its own translation termination. This free l-Trp-dependent mechanism of inhibition of translation termination remains unclear. Here, we analyzed the inhibitory effects of l-Trp on the function of two known E. coli translation termination factors, RF1 and RF2. Using a series of reporter genes, we found that the in vivo l-Trp sensitivity of tnaC gene expression is influenced by the identity of its stop codon, with the UGA stop codon producing higher expression efficiency of the tnaA-lacZ gene construct than the UAG stop codon. In vitro TnaC-peptidyl-tRNA accumulation and toe-printing assays confirmed that in the presence of l-Trp, the UGA stop codon generates higher accumulation of both TnaC-peptidyl-tRNA and arrested ribosomes than does the UAG stop codon. RF-mediated hydrolysis assays corroborated that l-Trp blocks RF2 function more than that of RF1. Mutational analyses disclosed that amino acids substitutions at the 246 and 256 residue positions surrounding the RF2-GGQ functional motif reduce l-Trp-dependent expression of the tnaC(UGA) tnaA-lacZ construct and the ability of l-Trp to inhibit RF2-mediated cleavage of the TnaC-peptidyl-tRNA. Altogether, our results indicate that l-Trp preferentially blocks RF2 activity during translation termination of the tnaC gene. This inhibition depends on the identities of amino acid residues surrounding the RF2-GGQ functional motif.