GeneXpert Ultra in Urine Samples for Diagnosis of Extra-Pulmonary Tuberculosis.

Extra-pulmonary tuberculosis (EPTB) continues to be difficult to diagnose. Novel biomarkers in biological specimens offer promise. Detection of Mycobacterium tuberculosis (Mtb) DNA in urine could prove useful in diagnosis of EPTB, possibly due to disseminated disease or micro-abscesses reported in kidneys. The current study was designed to detect Mtb DNA in stored urine samples from patients with EPTB. Diagnosis of EPTB was reached using Microbiological Reference Standards (MRS) on samples from the disease site using WHO Recommended Diagnostics (WRD), [smear microscopy, liquid culture (MGIT-960)] and GX (molecular WRD, mWRD) and Comprehensive reference standards [CRS, clinical presentation, microbiological reference standards, radiology, histopathology]. GX-Ultra was performed on urine samples stored in -80oC deep freezer, retrospectively. Of 70 patients, 51 (72.9%) were classified as confirmed TB, 11 (15.7%) unconfirmed TB, and 8 (11.4%) unlikely TB. GX-Ultra in urine samples demonstrated sensitivity of 52.9% and specificity of 57.9% against MRS, and higher sensitivity of 56.5% and specificity of 100% against CRS. The sensitivity and specificity of GX-Ultra in urine was 53.6% and 75% for pus sample subset and 52.2% and 53.3% for fluid sample subset. Urine being non-invasive and easy to collect, detection of Mtb DNA using mWRD in urine samples is promising for diagnosis of EPTB.

Protocol for a cluster randomised controlled trial of secondary distribution of hepatitis C self-testing within the context of a house-to-house hepatitis C micro-elimination programme in Karachi, Pakistan.

BACKGROUND: Globally, just 21% of the estimated 58 million people living with hepatitis C virus (HCV) know their status. Thus, there is considerable need to scale-up HCV testing if the World Health Organization (WHO) 2030 hepatitis elimination goals are to be achieved. HCV self-testing may assist with this; however, there are currently no data on the real-world impact of HCV self-testing. With an estimated 5% of the general population living with HCV, Pakistan has the second highest HCV burden in the world. This study aims to evaluate the acceptability and impact of home delivery of HCV self-testing for secondary distribution in the context of a house-to-house HCV micro-elimination programme in Pakistan. METHODS: This is a parallel group, non-blinded, cluster randomised trial comparing secondary distribution of HCV self-testing with secondary distribution of information pamphlets encouraging individuals to visit a testing facility for HCV screening. The cluster allocation ratio is 1:1. Clusters will be randomised either to HCV self-testing distributed via study staff or control clusters where information on HCV will be given and the participant will be requested to attend their local hospital for HCV screening. In both clusters, only households with a member who has not yet been screened as part of the larger micro-elimination project will be included. The primary outcome is the number and proportion of participants who report completion of testing. Secondary outcomes include the number and proportion of participants who a) receive a positive result and are made aware of their status, b) are referred to and complete HCV RNA confirmatory testing, and c) start treatment. Acceptability, feasibility, attitudes towards HCV testing, and cost will also be evaluated. The target sample size is 2,000 participants. DISCUSSION: This study will provide the first ever evidence regarding secondary distribution of HCV self-testing. By comparing HCV self-testing with facility-based testing, we will assess whether HCV self-testing increases the uptake of HCV testing. The findings will inform micro-elimination programmes and determine whether HCV self-testing can enable individuals to be reached who may otherwise be missed. TRIAL REGISTRATION: This study and was registered on clinicaltrials.gov ( NCT04971538 ) 21 July 2021.

A prospective multicentre diagnostic accuracy study for the Truenat tuberculosis assays.

BACKGROUND: Bringing reliable and accurate tuberculosis (TB) diagnosis closer to patients is a key priority for global TB control. Molbio Diagnostics have developed the Truenat point-of-care molecular assays for detection of TB and rifampicin (RIF) resistance. METHODS: We conducted a prospective multicentre diagnostic accuracy study at 19 primary healthcare centres and seven reference laboratories in Peru, India, Ethiopia and Papua New Guinea to estimate the diagnostic accuracy of the point-of-care Truenat MTB, MTB Plus and MTB-RIF Dx assays for pulmonary TB using culture and phenotypic drug susceptibility testing as the reference standard, compared with Xpert MTB/RIF or Ultra. RESULTS: Of 1807 enrolled participants with TB signs/symptoms, 24% were culture-positive for Mycobacterium tuberculosis, of which 15% were RIF-resistant. In microscopy centres, the pooled sensitivity of Truenat MTB and Truenat MTB Plus was 73% (95% CI 67-78%) and 80% (95% CI 75-84%), respectively. Among smear-negative specimens, sensitivities were 36% (95% CI 27-47%) and 47% (95% CI 37-58%), respectively. Sensitivity of Truenat MTB-RIF was 84% (95% CI 62-95%). Truenat assays showed high specificity. Head-to-head comparison in the central reference laboratories suggested that the Truenat assays have similar performance to Xpert MTB/RIF. CONCLUSION: We found the performance of Molbio's Truenat MTB, MTB Plus and MTB-RIF Dx assays to be comparable to that of the Xpert MTB/RIF assay. Performing the Truenat tests in primary healthcare centres with very limited infrastructure was feasible. These data supported the development of a World Health Organization policy recommendation of the Molbio assays.

Guidance for Studies Evaluating the Accuracy of Sputum-Based Tests to Diagnose Tuberculosis.

Tests that can replace sputum smear microscopy have been identified as a top priority diagnostic need for tuberculosis by the World Health Organization. High-quality evidence on diagnostic accuracy for tests that may meet this need is an essential requirement to inform decisions about policy and scale-up. However, test accuracy studies are often of low and inconsistent quality and poorly reported, leading to uncertainty about true test performance. Here we provide guidance for the design of diagnostic test accuracy studies of sputum smear-replacement tests. Such studies should have a cross-sectional or cohort design, enrolling either a consecutive series or a random sample of patients who require evaluation for tuberculosis. Adults with respiratory symptoms are the target population. The reference standard should at a minimum be a single, automated, liquid culture, but additional cultures, follow-up, clinical case definition, and specific measures to understand discordant results should also be included. Inclusion of smear microscopy and Xpert MTB/RIF (or MTB/RIF Ultra) as comparators is critical to allow broader comparability and generalizability of results, because disease spectrum can vary between studies and affects relative test performance. Given the complex nature of sputum (the primary specimen type used for pulmonary TB), careful design and reporting of the specimen flow is essential. Test characteristics other than accuracy (such as feasibility, implementation considerations, and data on impact on patient, population and health systems outcomes) are also important aspects.

Molecular Detection of Mycobacterium tuberculosis from Stools in Young Children by Use of a Novel Centrifugation-Free Processing Method.

The microbiological diagnosis of tuberculosis (TB) in children is challenging, as it relies on the collection of relatively invasive specimens by trained health care workers, which is not feasible in many settings. Mycobacterium tuberculosis is detectable from the stools of children using molecular methods, but processing stool specimens is resource intensive. We evaluated a novel, simple, centrifugation-free processing method for stool specimens for use on the Xpert MTB/RIF assay (Xpert), using two different stool masses: 0.6 g and a swab sample. Two hundred eighty children (median age, 15.5 months; 35 [12.5%] HIV infected) with suspected intrathoracic TB were enrolled from two sites in South Africa. Compared to a single Xpert test on respiratory specimens, the sensitivity of Xpert on stools using the 0.6-g and swab samples was 44.4% (95% confidence interval [CI], 13.7 to 78.8%) for both methods, with a specificity of >99%. The combined sensitivities of two stool tests versus the first respiratory Xpert were 70.0% (95% CI, 34.8 to 93.3) and 50.0% (95% CI, 18.7 to 81.3) for the 0.6-g and swab sample, respectively. Retesting stool specimens with nondeterminate Xpert results improved nondeterminate rates from 9.3% to 3.9% and from 8.6% to 4.3% for 0.6-g and swab samples, respectively. Overall, stool Xpert detected 14/94 (14.9%) children who initiated antituberculosis treatment, while respiratory specimens detected 23/94 (24.5%). This stool processing method is well suited for settings with low capacity for respiratory specimen collection. However, the overall sensitivity to detect confirmed and clinical TB was lower than that of respiratory specimens. More sensitive rapid molecular assays are needed to improve the utility of stools for the diagnosis of intrathoracic TB in children from resource-limited settings.

FIND Tuberculosis Strain Bank: a Resource for Researchers and Developers Working on Tests To Detect Mycobacterium tuberculosis and Related Drug Resistance.

The spread of multidrug-resistant (MDR) tuberculosis (TB) and extensively drug-resistant (XDR) TB hampers global efforts in the fight against tuberculosis. To enhance the development and evaluation of diagnostic tests quickly and efficiently, well-characterized strains and samples from drug-resistant tuberculosis patients are necessary. In this project, the Foundation for Innovative New Diagnostics (FIND) has focused on the collection, characterization, and storage of such well-characterized reference materials and making them available to researchers and developers. The collection is being conducted at multiple centers in Southeast Asia, South America, Eastern Europe, and soon the sub-Saharan Africa regions. Strains are characterized for their phenotypic resistances and MICs to first-line drugs (FLDs) and second-line drugs (SLDs) using the automated MGIT 960 system following validated procedures and WHO criteria. Analysis of resistance-associated mutations is done by whole-genome sequencing (WGS) using the Illumina NextSeq system. Mycobacterial interspersed repetitive-unit-variable-number tandem-repeat analysis and WGS are used to determine strain lineages. All strains are maintained frozen at -80°C ± 10°C as distinct mother and daughter lots. All strains are extensively quality assured. The data presented here represent an analysis of the initial part of the collection. Currently, the bank contains 118 unique strains with extracted genomic DNA and matched sputum, serum, and plasma samples and will be expanded to a minimum of 1,000 unique strains over the next 3 years. Analysis of the current strains by phenotypic resistance testing shows 102 (86.4%), 10 (8.5%), and 6 (5.1%) MDR, XDR, and mono/poly resistant strains, respectively. Two of the strains are resistant to all 11 drugs that were phenotypically tested. WGS mutation analysis revealed FLD resistance-associated mutations in the rpoB, katG, inhA, embB, embA, and pncA genes; SLD resistance in the gyrA, gyrB, rrs, eis, and tlyA genes; and ethionamide resistance in the ethA genes. Most important lineages are represented in the bank, and further collections have been initiated to increase geographic and lineage diversity. The bank provides highly characterized and high-quality strains as a resource for researchers and developers in support of the development and evaluation of new diagnostics and drug resistance detection tools.

Feasibility and Operational Performance of Tuberculosis Detection by Loop-Mediated Isothermal Amplification Platform in Decentralized Settings: Results from a Multicenter Study.

Currently available nucleic acid amplification platforms for tuberculosis (TB) detection are not designed to be simple or inexpensive enough to implement in decentralized settings in countries with a high burden of disease. The loop-mediated isothermal amplification platform (LAMP) may change this. We conducted a study in adults with symptoms suggestive of TB in India, Uganda, and Peru to establish the feasibility of using TB-LAMP (Eiken Chemical Co.) in microscopy laboratories compared with using smear microscopy against a reference standard of solid and liquid cultures. Operational characteristics were evaluated as well. A total of 1,777 participants met the eligibility criteria and were included for analysis. Overall, TB-LAMP sensitivities among culture-positive samples were 97.2% (243/250; 95% confidence interval [CI], 94.3% to 98.2%) and 62.0% (88/142; 95% CI, 53.5% to 70.0%) for smear-positive and smear-negative TB, respectively, but varied widely by country and operator. Specificities ranged from 94.5% (446/472; 95% CI, 92.0% to 96.4%) to 98.0% (350/357; 95% CI, 96.0% to 99.2%) by country. A root cause analysis identified high temperatures, high humidity, and/or low reaction volumes as possible causes for false-positive results, as they may result in nonspecific amplification. The study was repeated in India with training focused on vulnerable steps and an updated protocol; 580 participants were included for analysis. Specificity in the repeat trial was 96.6% (515/533; 95% CI, 94.7% to 97.9%). To achieve acceptable performance of LAMP at the microscopy center level, significant training and infrastructure requirements are necessary.

Performance of the G4 Xpert® MTB/RIF assay for the detection of Mycobacterium tuberculosis and rifampin resistance: a retrospective case-control study of analytical and clinical samples from high- and low-tuberculosis prevalence settings.

BACKGROUND: The Xpert® MTB/RIF (Xpert) assay is a rapid PCR-based assay for the detection of Mycobacterium tuberculosis complex DNA (MTBc) and mutations associated with rifampin resistance (RIF). An updated version introduced in 2011, the G4 Xpert, included modifications to probe B and updated analytic software. METHODS: An analytical study was performed to assess Xpert detection of mutations associated with rifampin resistance in rifampin-susceptible and -resistant isolates. A clinical study was performed in which specimens from US and non-US persons suspected of tuberculosis (TB) were tested to determine Xpert performance characteristics. All specimens underwent smear microscopy, mycobacterial culture, conventional drug-susceptibility testing and Xpert testing; DNA from isolates with discordant rifampin resistance results was sequenced. RESULTS: Among 191 laboratory-prepared isolates in the analytical study, Xpert sensitivity for detection of rifampin resistance associated mutations was 97.7% and specificity was 90.8%, which increased to 99.0% after DNA sequencing analysis of the discordant samples. Of the 1,096 subjects in the four clinical studies, 49% were from the US. Overall, Xpert detected MTBc in 439 of 468 culture-positive specimens for a sensitivity of 93.8% (95% confidence interval [CI]: 91.2%-95.7%) and did not detect MTBc in 620 of 628 culture-negative specimens for a specificity of 98.7% (95% CI: 97.5%-99.4%). Sensitivity was 99.7% among smear-positive cases, and 76.1% among smear-negative cases. Non-determinate MTBc detection and false-positive RIF resistance results were low (1.2 and 0.9%, respectively). CONCLUSIONS: The updated Xpert assay retained the high sensitivity and specificity of the previous assay versions and demonstrated low rates of non-determinate and RIF resistance false positive results.

Diagnostic accuracy of a three-gene Mycobacterium tuberculosis host response cartridge using fingerstick blood for childhood tuberculosis: a multicentre prospective study in low-income and middle-income countries.

BACKGROUND: Childhood tuberculosis remains a major cause of morbidity and mortality in part due to missed diagnosis. Diagnostic methods with enhanced sensitivity using easy-to-obtain specimens are needed. We aimed to assess the diagnostic accuracy of the Cepheid Mycobacterium tuberculosis Host Response prototype cartridge (MTB-HR), a candidate test measuring a three-gene transcriptomic signature from fingerstick blood, in children with presumptive tuberculosis disease. METHODS: RaPaed-TB was a prospective diagnostic accuracy study conducted at four sites in African countries (Malawi, Mozambique, South Africa, and Tanzania) and one site in India. Children younger than 15 years with presumptive pulmonary or extrapulmonary tuberculosis were enrolled between Jan 21, 2019, and June 30, 2021. MTB-HR was performed at baseline and at 1 month in all children and was repeated at 3 months and 6 months in children on tuberculosis treatment. Accuracy was compared with tuberculosis status based on standardised microbiological, radiological, and clinical data. FINDINGS: 5313 potentially eligible children were screened, of whom 975 were eligible. 784 children had MTB-HR test results, of whom 639 had a diagnostic classification and were included in the analysis. MTB-HR differentiated children with culture-confirmed tuberculosis from those with unlikely tuberculosis with a sensitivity of 59·8% (95% CI 50·8-68·4). Using any microbiological confirmation (culture, Xpert MTB/RIF Ultra, or both), sensitivity was 41·6% (34·7-48·7), and using a composite clinical reference standard, sensitivity was 29·6% (25·4-34·2). Specificity for all three reference standards was 90·3% (95% CI 85·5-94·0). Performance was similar in different age groups and by malnutrition status. Among children living with HIV, accuracy against the strict reference standard tended to be lower (sensitivity 50·0%, 15·7-84·3) compared with those without HIV (61·0%, 51·6-69·9), although the difference did not reach statistical significance. Combining baseline MTB-HR result with one Ultra result identified 71·2% of children with microbiologically confirmed tuberculosis. INTERPRETATION: MTB-HR showed promising diagnostic accuracy for culture-confirmed tuberculosis in this large, geographically diverse, paediatric cohort and hard-to-diagnose subgroups. FUNDING: European and Developing Countries Clinical Trials Partnership, UK Medical Research Council, Swedish International Development Cooperation Agency, Bundesministerium für Bildung und Forschung; German Center for Infection Research (DZIF).

Xpert MTB/RIF Ultra versus mycobacterial growth indicator tube liquid culture for detection of Mycobacterium tuberculosis in symptomatic adults: a diagnostic accuracy study.

BACKGROUND: Xpert MTB/RIF Ultra (Ultra) is an automated molecular test for the detection of Mycobacterium tuberculosis in sputum. We compared the sensitivity of Ultra to that of mycobacterial growth indicator tube (MGIT) liquid culture, considered the most sensitive assay in routine clinical use. METHODS: In this prospective, multicentre, cross-sectional diagnostic accuracy study, we used a non-inferiority design to assess whether the sensitivity of a single Ultra test was non-inferior to that of a single liquid culture for detection of M tuberculosis in sputum. We enrolled adults (age ≥18 years) with pulmonary tuberculosis symptoms in 11 countries and each adult provided three sputum specimens with a minimum volume of 2 mL over 2 days. Ultra was done directly on sputum 1, and Ultra and MGIT liquid culture were done on resuspended pellet from sputum 2. Results of MGIT and solid media cultures done on sputum 3 were considered the reference standard. The pre-defined non-inferiority margin was 5·0%. FINDINGS: Between Feb 18, 2016, and Dec 4, 2019, we enrolled 2906 participants. 2600 (89%) participants were analysed, including 639 (25%) of 2600 who were positive for tuberculosis by the reference standard. Of the 2357 included in the non-inferiority analysis, 877 (37%) were HIV-positive and 984 (42%) were female. Sensitivity of Ultra performed directly on sputum 1 was non-inferior to that of sputum 2 MGIT culture (MGIT 91·1% vs Ultra 91·9%; difference -0·8 percentage points; 95% CI -2·8 to 1·1). Sensitivity of Ultra performed on sputum 2 pellet was also non-inferior to that of sputum 2 MGIT (MGIT 91·1% vs Ultra 91·9%; difference -0·8 percentage points; -2·7 to 1·0). INTERPRETATION: For the detection of M tuberculosis in sputum from adults with respiratory symptoms, there was no difference in sensitivity of a single Ultra test to that of a single MGIT culture. Highly sensitive, rapid molecular approaches for M tuberculosis detection, combined with advances in genotypic methods for drug resistance detection, have potential to replace culture. FUNDING: US National Institute of Allergy and Infectious Diseases.

Rapid molecular detection of tuberculosis and rifampin resistance.

BACKGROUND: Global control of tuberculosis is hampered by slow, insensitive diagnostic methods, particularly for the detection of drug-resistant forms and in patients with human immunodeficiency virus infection. Early detection is essential to reduce the death rate and interrupt transmission, but the complexity and infrastructure needs of sensitive methods limit their accessibility and effect. METHODS: We assessed the performance of Xpert MTB/RIF, an automated molecular test for Mycobacterium tuberculosis (MTB) and resistance to rifampin (RIF), with fully integrated sample processing in 1730 patients with suspected drug-sensitive or multidrug-resistant pulmonary tuberculosis. Eligible patients in Peru, Azerbaijan, South Africa, and India provided three sputum specimens each. Two specimens were processed with N-acetyl-L-cysteine and sodium hydroxide before microscopy, solid and liquid culture, and the MTB/RIF test, and one specimen was used for direct testing with microscopy and the MTB/RIF test. RESULTS: Among culture-positive patients, a single, direct MTB/RIF test identified 551 of 561 patients with smear-positive tuberculosis (98.2%) and 124 of 171 with smear-negative tuberculosis (72.5%). The test was specific in 604 of 609 patients without tuberculosis (99.2%). Among patients with smear-negative, culture-positive tuberculosis, the addition of a second MTB/RIF test increased sensitivity by 12.6 percentage points and a third by 5.1 percentage points, to a total of 90.2%. As compared with phenotypic drug-susceptibility testing, MTB/RIF testing correctly identified 200 of 205 patients (97.6%) with rifampin-resistant bacteria and 504 of 514 (98.1%) with rifampin-sensitive bacteria. Sequencing resolved all but two cases in favor of the MTB/RIF assay. CONCLUSIONS: The MTB/RIF test provided sensitive detection of tuberculosis and rifampin resistance directly from untreated sputum in less than 2 hours with minimal hands-on time. (Funded by the Foundation for Innovative New Diagnostics.)

Rapid and Accurate Diagnosis of Pediatric Tuberculosis Disease: A Diagnostic Accuracy Study for Pediatric Tuberculosis.

INTRODUCTION: An estimated 1.2 million children develop tuberculosis (TB) every year with 240,000 dying because of missed diagnosis. Existing tools suffer from lack of accuracy and are often unavailable. Here, we describe the scientific and clinical methodology applied in RaPaed-TB, a diagnostic accuracy study. METHODS: This prospective diagnostic accuracy study evaluating several candidate tests for TB was set out to recruit 1000 children <15 years with presumptive TB in 5 countries (Malawi, Mozambique, South Africa, Tanzania, India). Assessments at baseline included documentation of TB signs and symptoms, TB history, radiography, tuberculin skin test, HIV testing and spirometry. Respiratory samples for reference standard testing (culture, Xpert Ultra) included sputum (induced/spontaneous) or gastric aspirate, and nasopharyngeal aspirate (if <5 years). For novel tests, blood, urine and stool were collected. All participants were followed up at months 1 and 3, and month 6 if on TB treatment or unwell. The primary endpoint followed NIH-consensus statements on categorization of TB disease status for each participant. The study was approved by the sponsor's and all relevant local ethics committees. DISCUSSION: As a diagnostic accuracy study for a disease with an imperfect reference standard, Rapid and Accurate Diagnosis of Pediatric Tuberculosis Disease (RaPaed-TB) was designed following a rigorous and complex methodology. This allows for the determination of diagnostic accuracy of novel assays and combination of testing strategies for optimal care for children, including high-risk groups (ie, very young, malnourished, children living with HIV). Being one of the largest of its kind, RaPaed-TB will inform the development of improved diagnostic approaches to increase case detection in pediatric TB.

Feasibility, diagnostic accuracy, and effectiveness of decentralised use of the Xpert MTB/RIF test for diagnosis of tuberculosis and multidrug resistance: a multicentre implementation study.

BACKGROUND: The Xpert MTB/RIF test (Cepheid, Sunnyvale, CA, USA) can detect tuberculosis and its multidrug-resistant form with very high sensitivity and specificity in controlled studies, but no performance data exist from district and subdistrict health facilities in tuberculosis-endemic countries. We aimed to assess operational feasibility, accuracy, and effectiveness of implementation in such settings. METHODS: We assessed adults (≥18 years) with suspected tuberculosis or multidrug-resistant tuberculosis consecutively presenting with cough lasting at least 2 weeks to urban health centres in South Africa, Peru, and India, drug-resistance screening facilities in Azerbaijan and the Philippines, and an emergency room in Uganda. Patients were excluded from the main analyses if their second sputum sample was collected more than 1 week after the first sample, or if no valid reference standard or MTB/RIF test was available. We compared one-off direct MTB/RIF testing in nine microscopy laboratories adjacent to study sites with 2-3 sputum smears and 1-3 cultures, dependent on site, and drug-susceptibility testing. We assessed indicators of robustness including indeterminate rate and between-site performance, and compared time to detection, reporting, and treatment, and patient dropouts for the techniques used. FINDINGS: We enrolled 6648 participants between Aug 11, 2009, and June 26, 2010. One-off MTB/RIF testing detected 933 (90·3%) of 1033 culture-confirmed cases of tuberculosis, compared with 699 (67·1%) of 1041 for microscopy. MTB/RIF test sensitivity was 76·9% in smear-negative, culture-positive patients (296 of 385 samples), and 99·0% specific (2846 of 2876 non-tuberculosis samples). MTB/RIF test sensitivity for rifampicin resistance was 94·4% (236 of 250) and specificity was 98·3% (796 of 810). Unlike microscopy, MTB/RIF test sensitivity was not significantly lower in patients with HIV co-infection. Median time to detection of tuberculosis for the MTB/RIF test was 0 days (IQR 0-1), compared with 1 day (0-1) for microscopy, 30 days (23-43) for solid culture, and 16 days (13-21) for liquid culture. Median time to detection of resistance was 20 days (10-26) for line-probe assay and 106 days (30-124) for conventional drug-susceptibility testing. Use of the MTB/RIF test reduced median time to treatment for smear-negative tuberculosis from 56 days (39-81) to 5 days (2-8). The indeterminate rate of MTB/RIF testing was 2·4% (126 of 5321 samples) compared with 4·6% (441 of 9690) for cultures. INTERPRETATION: The MTB/RIF test can effectively be used in low-resource settings to simplify patients' access to early and accurate diagnosis, thereby potentially decreasing morbidity associated with diagnostic delay, dropout and mistreatment. FUNDING: Foundation for Innovative New Diagnostics, Bill & Melinda Gates Foundation, European and Developing Countries Clinical Trials Partnership (TA2007.40200.009), Wellcome Trust (085251/B/08/Z), and UK Department for International Development.

A multisite assessment of the quantitative capabilities of the Xpert MTB/RIF assay.

RATIONALE: The Xpert MTB/RIF is an automated molecular test for Mycobacterium tuberculosis that estimates bacterial burden by measuring the threshold-cycle (Ct) of its M. tuberculosis-specific real-time polymerase chain reaction. Bacterial burden is an important biomarker for disease severity, infection control risk, and response to therapy. OBJECTIVES: Evaluate bacterial load quantitation by Xpert MTB/RIF compared with conventional quantitative methods. METHODS: Xpert MTB/RIF results were compared with smear-microscopy, semiquantiative solid culture, and time-to-detection in liquid culture for 741 patients and 2,008 samples tested in a multisite clinical trial. An internal control real-time polymerase chain reaction was evaluated for its ability to identify inaccurate quantitative Xpert MTB/RIF results. MEASUREMENTS AND MAIN RESULTS: Assays with an internal control Ct greater than 34 were likely to be inaccurately quantitated; this represented 15% of M. tuberculosis-positive tests. Excluding these, decreasing M. tuberculosis Ct was associated with increasing smear microscopy grade for smears of concentrated sputum pellets (r(s) = -0.77) and directly from sputum (r(s) =-0.71). A Ct cutoff of approximately 27.7 best predicted smear-positive status. The association between M. tuberculosis Ct and time-to-detection in liquid culture (r(s) = 0.68) and semiquantitative colony counts (r(s) = -0.56) was weaker than smear. Tests of paired same-patient sputum showed that high viscosity sputum samples contained ×32 more M. tuberculosis than nonviscous samples. Comparisons between the grade of the acid-fast bacilli smear and Xpert MTB/RIF quantitative data across study sites enabled us to identify a site outlier in microscopy. CONCLUSIONS: Xpert MTB/RIF quantitation offers a new, standardized approach to measuring bacterial burden in the sputum of patients with tuberculosis.

Pediatric Tuberculosis Diagnostics: Present and Future.

The current diagnostic abilities for the detection of pediatric tuberculosis are suboptimal. Multiple factors contribute to the under-diagnosis of intrathoracic tuberculosis in children, namely the absence of pathognomonic features of the disease, low bacillary loads in respiratory specimens, challenges in sample collection, and inadequate access to diagnostic tools in high-burden settings. Nonetheless, the 2020s have witnessed encouraging progress in the area of novel diagnostics. Recent WHO-endorsed rapid molecular assays hold promise for use in service decentralization strategies, and new policy recommendations include stools as an alternative, child-friendly specimen for testing with the GeneXpert assay. The pipeline of promising assays in mid/late-stage development is expanding, and novel pediatric candidate biomarkers based on the host immune response are being identified for use in diagnostic and triage tests. For a new test to meet the pediatric target product profiles prioritized by the WHO, it is key that the peculiarities and needs of the hard-to-reach pediatric population are considered in the early planning phases of discovery, validation, and implementation studies.

Diagnostic Advances in Childhood Tuberculosis-Improving Specimen Collection and Yield of Microbiological Diagnosis for Intrathoracic Tuberculosis.

There is no microbiological gold standard for childhood tuberculosis (TB) diagnosis. The paucibacillary nature of the disease, challenges in sample collection in young children, and the limitations of currently available microbiological tests restrict microbiological confirmation of intrathoracic TB to the minority of children. Recent WHO guidelines recommend the use of novel rapid molecular assays as initial diagnostic tests for TB and endorse alternative sample collection methods for children. However, the uptake of these tools in high-endemic settings remains low. In this review, we appraise historic and new microbiological tests and sample collection techniques that can be used for the diagnosis of intrathoracic TB in children. We explore challenges and possible ways to improve diagnostic yield despite limitations, and identify research gaps to address in order to improve the microbiological diagnosis of intrathoracic TB in children.

A quasi-randomised controlled trial of online distribution of home-based hepatitis C self-testing for key populations in Malaysia: a study protocol.

BACKGROUND: Malaysia has an estimated hepatitis C virus (HCV) prevalence of 1.9% among its adult population and a history of providing HCV treatment in the public sector. In 2019, Malaysia launched a 5-year national strategic plan for viral hepatitis control and has been expanding HCV testing and treatment to the primary care and community levels, while actively engaging key populations in services for hepatitis care. The Ministry of Health (MoH) is seeking to specifically understand how to better target HCV services at men who have sex with men (MSM); HCV self-testing could increase the uptake of HCV testing among this group. METHODS: We aim to integrate HCV antibody self-testing into an existing online platform used for HIV self-testing, to evaluate the acceptability and impact of an online HCV self-testing programme in Malaysia. This is a non-blinded parallel group quasi-randomised superiority study comparing HCV self-testing via an online distribution model with the standard care, which involves attending a clinic for facility-based HCV antibody testing (control, 2:1). Participants will be randomised to either the HCV self-testing via online distribution arm, in which either an oral fluid- or blood-based HCV self-test kit will be mailed to them, or the control arm, where they will be provided with information about the nearest centre with HCV testing. The primary outcome is the number and proportion of participants who report completion of testing. Secondary outcomes include the number and proportion of participants who (a) receive a positive result and are made aware of their status, (b) are referred to and complete HCV RNA confirmatory testing, and (c) start treatment. Acceptability, feasibility, attitudes around HCV testing, and cost will also be evaluated. The target sample size is 750 participants. DISCUSSION: This study is one of the first in the world to explore the real-world impact of HCV self-testing on key populations using online platforms and compare this with standard HCV testing services. The outcomes of this study will provide critical evidence about testing uptake, linkage to care, acceptability, and any social harms that may emerge due to HCV self-testing. TRIAL REGISTRATION: ClinicalTrials.gov NCT04982718.

Home-based hepatitis C self-testing in people who inject drugs and men who have sex with men in Georgia: a protocol for a randomised controlled trial.

INTRODUCTION: Globally, it is estimated that more than three-quarters of people with chronic hepatitis C virus (HCV) are unaware of their HCV status. HCV self-testing (HCVST) may improve access and uptake of HCV testing particularly among key populations such as people who inject drugs (PWID) and men who have sex with men (MSM) where HCV prevalence and incidence are high and barriers to accessing health services due to stigma and discrimination are common. METHODS AND ANALYSIS: This randomised controlled trial compares an online programme offering oral fluid-based HCVST delivered to the home with referral to standard-of-care HCV testing at HCV testing sites. Eligible participants are adults self-identifying as either MSM or PWID who live in Tbilisi or Batumi, Georgia, and whose current HCV status is unknown. Participants will be recruited through an online platform and randomised to one of three arms for MSM (courier delivery, peer delivery and standard-of-care HCV testing (control)) and two for PWID (peer delivery and standard-of-care HCV testing (control)). Participants in the postal delivery group will receive an HCVST kit delivered by an anonymised courier. Participants in the peer delivery groups will schedule delivery of the HCVST by a peer. Control groups will receive information on how to access standard-of-care testing at a testing site. The primary outcome is the number and proportion of participants who report completion of testing. Secondary outcomes include the number and proportion of participants who (a) receive a positive result and are made aware of their status, (b) are referred to and complete HCV RNA confirmatory testing, and (c) start treatment. Acceptability, feasibility, and attitudes around HCV testing and cost will also be evaluated. The target sample size is 1250 participants (250 per arm). ETHICS AND DISSEMINATION: Ethical approval has been obtained from the National Centers for Disease Control and Public Health Georgia Institutional Review Board (IRB) (IRB# 2021-049). Study results will be disseminated by presentations at conferences and via peer-reviewed journals. Protocol version 1.1; 14 July 2021. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov Registry (NCT04961723).

Xpert MTB/RIF: a new pillar in diagnosis of extrapulmonary tuberculosis?

Approximately 10 to 15% of tuberculosis (TB) cases in India are estimated to have extrapulmonary disease, and due to a lack of diagnostic means, they often remain untreated. The early detection of Mycobacterium tuberculosis and multidrug resistance is a priority in TB diagnosis to improve the successful treatment rate of TB and reduce transmission. The Xpert MTB/RIF (Xpert) test, recently endorsed by the World Health Organization for the detection of pulmonary TB, was evaluated to test its utility in 547 patients with suspected extrapulmonary tuberculosis. Five hundred forty-seven extrapulmonary specimens were split and processed simultaneously for both culture (solid and liquid) and Xpert testing. For culture, the sensitivity was low, 53% (150/283 specimens). Xpert sensitivity and specificity results were assessed in comparison to a composite reference standard made up of smear and culture results and clinical, radiological, and histological findings. The sensitivity of the Xpert assay was 81% (228/283 specimens) (64% [89/138] for smear-negative cases and 96% [139/145] for smear-positive cases), with a specificity of 99.6%. The sensitivity was found to be high for the majority of specimen types (63 to 100%) except for cerebrospinal fluid, the sensitivity of which was 29% (2/7 specimens). The Xpert test correctly identified 98% of phenotypic rifampin (RIF)-resistant cases and 94% of phenotypic RIF-susceptible cases. Sequencing of the 6 discrepant samples resolved 3 of them, resulting in an increased specificity of 98%. In conclusion, the results of this study suggest that the Xpert test also shows good potential for the diagnosis of extrapulmonary TB and that its ease of use makes it applicable for countries where TB is endemic.

Retrospective Diagnostic Accuracy Study of Abbott RealTime MTB against Xpert MTB/RIF Ultra and Xpert MTB/RIF for the Diagnosis of Pulmonary Tuberculosis and Susceptibility to Rifampin and Isoniazid Treatment.

High-throughput centralized testing for tuberculosis (TB) and drug resistance is important, but comparative data are limited. In this retrospective cross-sectional study, participants were recruited from Johannesburg, South Africa, and Tbilisi, Georgia. The index tests, Abbott RealTime MTB (RT-MTB) and RealTime MTB RIF/INH (RT-MTB RIF/INH), were performed on specimens stored frozen for an extended period of time (beyond manufacturer-validated specifications) and compared to paired Xpert MTB/RIF Ultra (Xpert Ultra) and Xpert MTB/RIF (Xpert) results obtained with fresh specimens. The detection reference standard was the Mycobacterium tuberculosis complex culture, and for resistance detection, it was phenotypic drug susceptibility testing. The median age of 474 participants was 39 (interquartile range [IQR], 31 to 51) years. On decontaminated sputum, Xpert Ultra had a sensitivity of 91%, compared to 77% for RT-MTB, with a difference of +14% (95% confidence interval [CI], +9.2 to +21%; 18/127). On raw sputum, Xpert Ultra exhibited a sensitivity of 89% and Xpert one of 88%, compared to 80% for RT-MTB, exhibiting differences of +10% (95% CI, +3.3 to +18%; 9/93) and +8.6% (95% CI, +2.4 to +17%; 8/93), respectively. Specificity was ≥98% for all tests. All three tests showed high sensitivity and specificity for detection of rifampin resistance. Abbott assays may have lower sensitivity than Xpert and Xpert Ultra for TB detection but similar performance for detection of resistance. The differences in TB detection may be attributable to differences in testing of frozen (Abbott) versus fresh (Xpert) samples. Studies in compliance with manufacturer's instructions are required to compare performance. IMPORTANCE In 2019, 10 million people fell ill with tuberculosis (TB), of whom 1.4 million died. There are few comparative studies of diagnostic assays, particularly those aiming to be used in high-throughput laboratories. One such assay is the Abbott RealTime MTB (RT-MTB) and RealTime MTB RIF/INH (RT-MTB RIF/INH), which uses the m2000 platform already in use in many settings for HIV load testing and allows the diagnosis of TB and resistance to two first-line drugs, rifampin and isoniazid. Our study compared the RT-MTB and RT-MTB RIF/INH to the WHO-recommended Xpert MTB/RIF Ultra and Xpert MTB/RIF. The study is the largest comparative study to date and was performed independent of the manufacturer. The study results suggest that the Abbott RealTime MTB may have a lower sensitivity, but the study may have placed the Abbott test at a disadvantage by using frozen samples and comparing the results to those for fresh samples for the Xpert.