Novel Automation of an Enzyme-Linked Immunosorbent Spot Assay Testing Method: Comparable Diagnostic Performance of the T-SPOT.TB Test Using Manual Density Gradient Cell Isolation versus Automated Positive Selection with the T-Cell Select Kit.

The diagnosis of latent tuberculosis (TB) infection (LTBI) is critical to improve TB treatment and control, and the T-SPOT.TB test is a commercial enzyme-linked immunosorbent spot assay used for this purpose. The objective of the study was to increase automation and extend the time between blood collection and processing for the T-SPOT.TB test from 0 to 8 h to 0 to 54 h. The previous maximum time between blood collection and processing for the T-SPOT.TB test is 32 h using T-Cell Xtend. For this, we compared the T-SPOT.TB test using manual peripheral blood mononuclear cell (PBMC) isolation by density gradient separation at 0 to 8 h (reference method, control arm) to an automated PBMC isolation method using magnetic beads (T-Cell Select kit) at 0 to 55 h postcollection. A total of 620 subjects were enrolled from 4 study sites, and blood samples were collected from each volunteer, comprising 1,850 paired samples in total. Overall agreement between both methods was 96.8% (confidence interval [CI], 95.9 to 97.6%), with 95.8% (CI, 93.5 to 97.5%) positive and 97.1% negative agreement (CI, 96.1 to 97.9%). In summary, there was a strong overall agreement between the automated and manual T-SPOT.TB test processing methods. The results suggest that the T-SPOT.TB test can be processed using automated positive selection with magnetic beads using T-Cell Select to decrease hands-on time. Also, this cell isolation method allowed for the time between blood collection and processing to range from 0 to 55 h. Additional studies in larger and diverse patient populations including immunocompromised and pediatric patients are needed.

Comparative Analytical Evaluation of Four Centralized Platforms for the Detection of Mycobacterium tuberculosis Complex and Resistance to Rifampicin and Isoniazid.

Failure to rapidly identify drug-resistant tuberculosis (TB) increases the risk of patient mismanagement, the amplification of drug resistance, and ongoing transmission. We generated comparative analytical data for four automated assays for the detection of TB and multidrug-resistant TB (MDR-TB): Abbott RealTime MTB and MTB RIF/INH (Abbott), Hain Lifescience FluoroType MTBDR (Hain), BD Max MDR-TB (BD), and Roche cobas MTB and MTB-RIF/INH (Roche). We included Xpert MTB/RIF (Xpert) and GenoType MTBDRplus as comparators for TB and drug resistance detection, respectively. We assessed analytical sensitivity for the detection of the Mycobacterium tuberculosis complex using inactivated strains (M. tuberculosis H37Rv and M. bovis) spiked into TB-negative sputa and computed the 95% limits of detection (LOD95). We assessed the accuracy of rifampicin and isoniazid resistance detection using well-characterized M. tuberculosis strains with high-confidence mutations accounting for >85% of first-line resistance mechanisms globally. For H37Rv and M. bovis, we measured LOD95 values of 3,781 and 2,926 (Xpert), 322 and 2,182 (Abbott), 826 and 4,301 (BD), 10,398 and 23,139 (Hain), and 2,416 and 2,136 (Roche) genomes/ml, respectively. Assays targeting multicopy genes or targets (Abbott, BD, and Roche) showed increased analytical sensitivity compared to Xpert. Quantification of the panel by quantitative real-time PCR prevents the determination of absolute values, and results reported here can be interpreted for comparison purposes only. All assays showed accuracy comparable to that of Genotype MTBDRplus for the detection of rifampicin and isoniazid resistance. The data from this analytical study suggest that the assays may have clinical performances similar to those of WHO-recommended molecular TB and MDR-TB assays.

"SILVAMP TB LAM" Rapid Urine Tuberculosis Test Predicts Mortality in Patients Hospitalized With Human Immunodeficiency Virus in South Africa.

Reducing diagnostic delay is key toward decreasing tuberculosis-associated deaths in people living with human immunodeficiency virus. In tuberculosis patients with retrospective urine testing, the point-of-care Fujifilm SILVAMP TB LAM (FujiLAM) could have rapidly diagnosed tuberculosis in up to 89% who died. In FujiLAM negative patients, the probability of 12-week survival was 86-97%.

Cost analysis of rapid diagnostics for drug-resistant tuberculosis.

BACKGROUND: Growth-based drug susceptibility testing (DST) is the reference standard for diagnosing drug-resistant tuberculosis (TB), but standard time to result (TTR) is typically ≥ 3 weeks. Rapid tests can reduce that TTR to days or hours, but accuracy may be lowered. In addition to the TTR and test accuracy, the cost of a diagnostic test may affect whether it is adopted in clinical settings. We examine the cost-effectiveness of rapid diagnostics for extremely drug-resistant TB (XDR-TB) in three different high-prevalence settings. METHODS: 1128 patients with confirmed TB were enrolled at clinics in Mumbai, India; Chisinau, Moldova; and Port Elizabeth, South Africa. Patient sputum samples underwent DST for first and second line TB drugs using 2 growth-based (MGIT, MODS) and 2 molecular (Pyrosequencing [PSQ], line-probe assays [LPA]) assays. TTR was the primary measure of effectiveness. Sensitivity and specificity were also evaluated. The cost to perform each test at each site was recorded and included test-specific materials, personnel, and equipment costs. Incremental cost-effectiveness ratios were calculated in terms of $/day saved. Sensitivity analyses examine the impact of batch size, equipment, and personnel costs. RESULTS: Our prior results indicated that the LPA and PSQ returned results in a little over 1 day. Mean cost per sample without equipment or overhead was $23, $28, $33, and $41 for the MODS, MGIT, PSQ, and LPA, respectively. For diagnosing XDR-TB, MODS was the most accurate, followed by PSQ, and LPA. MODS was quicker and less costly than MGIT. PSQ and LPA were considerably faster but cost more than MODS. Batch size and personnel costs were the main drivers of cost variation. CONCLUSIONS: Multiple factors must be weighed when selecting a test for diagnosis of XDR-TB. Rapid tests can greatly improve the time required to diagnose drug-resistant TB, potentially improving treatment success, and preventing the spread of XDR-TB. Faster time to result must be weighed against the potential for reduced accuracy, and increased costs. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02170441 .

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).

Sequential and parallel testing for microbiological confirmation of tuberculosis disease in children in five low-income and middle-income countries: a secondary analysis of the RaPaed-TB study.

BACKGROUND: Despite causing high mortality worldwide, paediatric tuberculosis is often undiagnosed. We aimed to investigate optimal testing strategies for microbiological confirmation of tuberculosis in children younger than 15 years, including the yield in high-risk subgroups (eg, children younger than 5 years, with HIV, or with severe acute malnutrition [SAM]). METHODS: For this secondary analysis, we used data from RaPaed-TB, a multicentre diagnostic accuracy study evaluating novel diagnostic assays and testing approaches for tuberculosis in children recruited from five health-care centres in Malawi, Mozambique, South Africa, Tanzania, and India conducted between Jan 21, 2019, and June 30, 2021. Children were included if they were younger than 15 years and had signs or symptoms of pulmonary or extrapulmonary tuberculosis; they were excluded if they weighed less than 2 kg, had received three or more doses of anti-tuberculosis medication at time of enrolment, were in a condition deemed critical by the local investigator, or if they did not have at least one valid microbiological result. We collected tuberculosis-reference specimens via spontaneous sputum, induced sputum, gastric aspirate, and nasopharyngeal aspirates. Microbiological tests were Xpert MTB/RIF Ultra (hereafter referred to as Ultra), liquid culture, and Löwenstein-Jensen solid culture, which were followed by confirmatory testing for positive cultures. The main outcome of this secondary analysis was categorising children as having confirmed tuberculosis if culture or Ultra positive on any sample, unconfirmed tuberculosis if clinically diagnosed, and unlikely tuberculosis if neither of these applied. FINDINGS: Of 5313 children screened, 975 were enrolled, of whom 965 (99%) had at least one valid microbiological result. 444 (46%) of 965 had unlikely tuberculosis, 282 (29%) had unconfirmed tuberculosis, and 239 (25%) had confirmed tuberculosis. Median age was 5·0 years (IQR 1·8-9·0); 467 (48%) of 965 children were female and 498 (52%) were male. 155 (16%) of 965 children had HIV and 110 (11%) children had SAM. 196 (82%) of 239 children with microbiological detection tested positive on Ultra. 110 (46%) of 239 were confirmed by both Ultra and culture, 86 (36%) by Ultra alone, and 43 (18%) by culture alone. 'Trace' was the most common semiquantitative result (93 [40%] of 234). 481 (50%) of 965 children had only one specimen type collected, 99 (21%) of whom had M tuberculosis detected. 484 (50%) of 965 children had multiple specimens collected, 141 (29%) of whom were positive on at least one specimen type. Of the 102 children younger than 5 years with M tuberculosis detected, 80 (78%) tested positive on sputum. 64 (80%) of 80 children who tested positive on sputum were positive on sputum alone; 61 (95%) of 64 were positive on induced sputum, two (3%) of 64 were positive on spontaneous sputum, and one (2%) was positive on both. INTERPRETATION: High rates of microbiological confirmation of tuberculosis in children can be achieved via parallel sampling and concurrent testing procedures. Sample types and choice of test to be used sequentially should be considered when applying to groups such as children younger than 5 years, living with HIV, or with SAM. FUNDING: European and Developing Countries Clinical Trials Partnership programme, supported by the EU, the UK Medical Research Council, Swedish International Development Cooperation Agency, Bundesministerium für Bildung und Forschung, the German Center for Infection Research, and Beckman Coulter.

Emergence and spread of extensively and totally drug-resistant tuberculosis, South Africa.

Factors driving the increase in drug-resistant tuberculosis (TB) in the Eastern Cape Province, South Africa, are not understood. A convenience sample of 309 drug-susceptible and 342 multidrug-resistant (MDR) TB isolates, collected July 2008-July 2009, were characterized by spoligotyping, DNA fingerprinting, insertion site mapping, and targeted DNA sequencing. Analysis of molecular-based data showed diverse genetic backgrounds among drug-sensitive and MDR TB sensu stricto isolates in contrast to restricted genetic backgrounds among pre-extensively drug-resistant (pre-XDR) TB and XDR TB isolates. Second-line drug resistance was significantly associated with the atypical Beijing genotype. DNA fingerprinting and sequencing demonstrated that the pre-XDR and XDR atypical Beijing isolates evolved from a common progenitor; 85% and 92%, respectively, were clustered, indicating transmission. Ninety-three percent of atypical XDR Beijing isolates had mutations that confer resistance to 10 anti-TB drugs, and some isolates also were resistant to para-aminosalicylic acid. These findings suggest the emergence of totally drug-resistant TB.

Feasibility, Ease-of-Use, and Operational Characteristics of World Health Organization-Recommended Moderate-Complexity Automated Nucleic Acid Amplification Tests for the Detection of Tuberculosis and Resistance to Rifampicin and Isoniazid.

Four moderate-complexity automated nucleic acid amplification tests for the diagnosis of tuberculosis are reported as having laboratory analytical and clinical performance similar to that of the Cepheid Xpert MTB/RIF assay. These assays are the Abbott RealTime MTB and RealTime MTB RIF/INH Resistance, Becton Dickinson MAX MDR-TB, the Hain Lifescience/Bruker FluoroType MTBDR, and the Roche cobas MTB and MTB RIF/INH assays. The study compared feasibility, ease of use, and operational characteristics of these assays/platforms. Manufacturer input was obtained for technical characteristics. Laboratory operators were requested to complete a questionnaire on the assays' ease of use. A time-in-motion analysis was also undertaken for each platform. For ease-of-use and operational requirements, the BD MAX MDR-TB assay achieved the highest scores (86% and 90%) based on information provided by the user and manufacturer, respectively, followed by the cobas MTB and MTB-RIF/INH assay (68% and 86%), the FluoroType MTBDR assay (67% and 80%), and the Abbott RT-MTB and RT MTB RIF/INH assays (64% and 76%). The time-in-motion analysis revealed that for 94 specimens, the RealTime MTB assay required the longest processing time, followed by the cobas MTB assay and the FluoroType MTBDR assay. The BD MAX MDR-TB assay required 4.6 hours for 22 specimens. These diagnostic assays exhibited different strengths and weaknesses that should be taken into account, in addition to affordability, when considering placement of a new platform.

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.

Population structure of multi- and extensively drug-resistant Mycobacterium tuberculosis strains in South Africa.

Genotyping of multidrug-resistant (MDR) Mycobacterium tuberculosis strains isolated from tuberculosis (TB) patients in four South African provinces (Western Cape, Eastern Cape, KwaZulu-Natal, and Gauteng) revealed a distinct population structure of the MDR strains in all four regions, despite the evidence of substantial human migration between these settings. In all analyzed provinces, a negative correlation between strain diversity and an increasing level of drug resistance (from MDR-TB to extensively drug-resistant TB [XDR-TB]) was observed. Strains predominating in XDR-TB in the Western and Eastern Cape and KwaZulu-Natal Provinces were strongly associated with harboring an inhA promoter mutation, potentially suggesting a role of these mutations in XDR-TB development in South Africa. Approximately 50% of XDR-TB cases detected in the Western Cape were due to strains probably originating from the Eastern Cape. This situation may illustrate how failure of efficient health care delivery in one setting can burden health clinics in other areas.

Implementation of a customised antimicrobial resistance laboratory scorecard in Cameroon, Ethiopia and Kenya.

Background: In low-resource settings, antimicrobial resistance (AMR) is detected by traditional culture-based methods and ensuring the quality of such services is a challenge. The AMR Scorecard provides laboratories with a technical assessment tool for strengthening the quality of bacterial culture, identification, and antimicrobial testing procedures. Objective: To evaluate the performance of the AMR Scorecard in 11 pilot laboratory evaluations in three countries also assessed with the Stepwise Laboratory Quality Improvement Process Towards Accreditation (SLIPTA) checklist. Methods: Pilot laboratory evaluations were conducted in Cameroon, Ethiopia and Kenya between February 2019 and March 2019. Assessors with previous SLIPTA and microbiology experience were trained. Assessors performed the laboratory assessments using the SLIPTA and AMR Scorecard tools. Results: Weaknesses in technical procedures and the quality management systems were identified in all areas and all laboratories. Safety had the highest mean performance score (SLIPTA: 68%; AMR Scorecard: 73%) while management review had the lowest (SLIPTA: 32%; AMR Scorecard: 8%) across all laboratories. The AMR Scorecard scores were generally consistent with SLIPTA scores. The AMR Scorecard identified technical weaknesses in AMR testing, and SLIPTA identified weaknesses in the quality management systems in the laboratories. Conclusion: Since the AMR Scorecard identified important gaps in AMR testing not detected by SLIPTA, it is recommended that microbiology laboratories use SLIPTA and the AMR Scorecard in parallel when preparing for accreditation. Expanding the use of the AMR Scorecard is a priority to address the need for quality clinical microbiology laboratory services in support of optimal patient care and AMR surveillance.

Situating trade-offs: Stakeholder perspectives on overtreatment versus missed diagnosis in transition to Xpert MTB/RIF Ultra in Kenya and Swaziland.

Implementing new diagnostics in public health programs can involve difficult trade-off decisions between individual patient benefits and public health considerations. Such decision-making processes are often not documented and may not include engagement of affected communities. This paper examines the perspectives of stakeholders on the trade-off between over-treatment and missed diagnosis captured during decision-making workshops on the transition from use of Xpert MTB/RIF to diagnose tuberculosis to Xpert MTB/RIF Ultra in Kenya and Swaziland. Xpert MTB/RIF Ultra has an overall increase in sensitivity but a decrease in specificity when compared to its predecessor. We conducted a qualitative study using four focus group discussions with a total of 47 participants and non-participant observation. The analysis reveals how participants deemed Xpert MTB/RIF Ultra's reduced specificity vis-à-vis its increased sensitivity to be an acceptable trade-off. The way participants assessed this trade-off was shaped by their experiences with the general uncertainty of all diagnostic tests, alternative testing options, historical evolution of diagnostic practices, epidemiological factors and resource constraints. In assessing the trade-off community and individual benefit and harm was frequently discussed together. Qualitative research on stakeholder engagement activities for diagnostic development and implementation can identify everyday experiences and situate assessments and perspectives of key stakeholders and as such aid in decision-making, improving implementation as well as patient outcomes. Further research is needed on the intended and unintended consequences of such engagement activities, how findings are being incorporated by decision-makers, and the impact on programmatic implementation.

Performance of FASTPlaqueTB and a modified protocol in a high HIV prevalence community in South Africa.

Modifications in the FASTPlaqueTB test protocol have resulted in an increase in the analytical limits of detection. This study investigated whether the performance of a modified prototype was able to increase the detection of smear-negative, culture-positive sputum samples as compared to the first generation FASTPlaqueTB test. Modifications to the FASTPlaqueTB did result in increased detection of smear-negative samples, but this was associated with a decrease in the specificity of the test. Before the FASTPlaqueTB can be considered as a viable replacement for smear microscopy and culture for the identification of tuberculosis, further work is required to resolve the performance issues identified in this study.

Novel lipoarabinomannan point-of-care tuberculosis test for people with HIV: a diagnostic accuracy study.

BACKGROUND: Most tuberculosis-related deaths in people with HIV could be prevented with earlier diagnosis and treatment. The only commercially available tuberculosis point-of-care test (Alere Determine TB LAM Ag [AlereLAM]) has suboptimal sensitivity, which restricts its use in clinical practice. The novel Fujifilm SILVAMP TB LAM (FujiLAM) assay has been developed to improve the sensitivity of AlereLAM. We assessed the diagnostic accuracy of the FujiLAM assay for the detection of tuberculosis in hospital inpatients with HIV compared with the AlereLAM assay. METHODS: For this diagnostic accuracy study, we assessed biobanked urine samples obtained from the FIND Specimen Bank and the University of Cape Town Biobank, which had been collected from hospital inpatients (aged ≥18 years) with HIV during three independent prospective cohort studies done at two South African hospitals. Urine samples were tested using FujiLAM and AlereLAM assays. The conduct and reporting of each test was done blind to other test results. The primary objective was to assess the diagnostic accuracy of FujiLAM compared with AlereLAM, against microbiological and composite reference standards (including clinical diagnoses). FINDINGS: Between April 18, 2018, and May 3, 2018, urine samples from 968 hospital inpatients with HIV were evaluated. The prevalence of microbiologically-confirmed tuberculosis was 62% and the median CD4 count was 86 cells per µL. Using the microbiological reference standard, the estimated sensitivity of FujiLAM was 70·4% (95% CI 53·0 to 83·1) compared with 42·3% (31·7 to 51·8) for AlereLAM (difference 28·1%) and the estimated specificity of FujiLAM was 90·8% (86·0 to 94·4) and 95·0% (87·7-98·8) for AlereLAM (difference -4·2%). Against the composite reference standard, the specificity of both assays was higher (95·7% [92·0 to 98·0] for FujiLAM vs 98·2% [95·7 to 99·6] for AlereLAM; difference -2·5%), but the sensitivity of both assays was lower (64·9% [50·1 to 76·7] for FujiLAM vs 38·2% [28·1 to 47·3] for AlereLAM; difference 26·7%). INTERPRETATION: In comparison to AlereLAM, FujiLAM offers superior diagnostic sensitivity, while maintaining specificity, and could transform rapid point-of-care tuberculosis diagnosis for hospital inpatients with HIV. The applicability of FujiLAM for settings of intended use requires prospective assessment. FUNDING: Global Health Innovative Technology Fund, UK Department for International Development, Dutch Ministry of Foreign Affairs, Bill & Melinda Gates Foundation, German Federal Ministry of Education and Research, Australian Department of Foreign Affairs and Trade, Wellcome Trust, Department of Science and Technology and National Research Foundation of South Africa, and South African Medical Research Council.

Improved contamination control for a rapid phage-based rifampicin resistance test for Mycobacterium tuberculosis.

A prospective study was conducted of the rapid FASTPlaque-Response test for determination of rifampicin resistance in Mycobacterium tuberculosis with and without the addition of an antimicrobial supplement containing nystatin, oxacillin and aztreonam (NOA) to control specimen-related contamination. A total of 631 smear-positive sputum specimens was tested. The age of specimens ranged from 0 to 21 days. The NOA antimicrobial was effective at controlling contamination, with 4.1 % of specimens contaminated when the NOA antimicrobial supplement was used compared with 13.9 % contamination without NOA. Overall levels of interpretability of the test with NOA were 87.8 % with specimens of < or =3 days and 79.0 % for all specimens. This compared with 70.1 and 73.8 % readable results, respectively, from conventional culture-based drug susceptibility testing (DST). Sensitivity, specificity and overall accuracy of the FASTPlaque-Response test for rifampicin resistance were 98.1, 96.3 and 96.6 %, respectively, for all specimens with NOA, and 93.2, 96.3 and 95.9 % without NOA, when compared with resolved conventional DST results. Inclusion of the NOA supplement reduced contamination, increased the number of interpretable results and did not adversely affect the performance of the FASTPlaque-Response test. Thus, the use of NOA improves the robustness of the test, facilitating its wider implementation.

Developing a customised approach for strengthening tuberculosis laboratory quality management systems toward accreditation.

BACKGROUND: Quality-assured tuberculosis laboratory services are critical to achieve global and national goals for tuberculosis prevention and care. Implementation of a quality management system (QMS) in laboratories leads to improved quality of diagnostic tests and better patient care. The Strengthening Laboratory Management Toward Accreditation (SLMTA) programme has led to measurable improvements in the QMS of clinical laboratories. However, progress in tuberculosis laboratories has been slower, which may be attributed to the need for a structured tuberculosis-specific approach to implementing QMS. We describe the development and early implementation of the Strengthening Tuberculosis Laboratory Management Toward Accreditation (TB SLMTA) programme. DEVELOPMENT: The TB SLMTA curriculum was developed by customizing the SLMTA curriculum to include specific tools, job aids and supplementary materials specific to the tuberculosis laboratory. The TB SLMTA Harmonized Checklist was developed from the World Health Organisation Regional Office for Africa Stepwise Laboratory Quality Improvement Process Towards Accreditation checklist, and incorporated tuberculosis-specific requirements from the Global Laboratory Initiative Stepwise Process Towards Tuberculosis Laboratory Accreditation online tool. IMPLEMENTATION: Four regional training-of-trainers workshops have been conducted since 2013. The TB SLMTA programme has been rolled out in 37 tuberculosis laboratories in 10 countries using the Workshop approach in 32 laboratories in five countries and the Facility-based approach in five tuberculosis laboratories in five countries. CONCLUSION: Lessons learnt from early implementation of TB SLMTA suggest that a structured training and mentoring programme can build a foundation towards further quality improvement in tuberculosis laboratories. Structured mentoring, and institutionalisation of QMS into country programmes, is needed to support tuberculosis laboratories to achieve accreditation.

Performance Comparison of Three Rapid Tests for the Diagnosis of Drug-Resistant Tuberculosis.

BACKGROUND: The aim of this study was to compare the performance of several recently developed assays for the detection of multi- and extensively drug-resistant tuberculosis (M/XDR-TB) in a large, multinational field trial. METHODS: Samples from 1,128 M/XDR-TB suspects were examined by Line Probe Assay (LPA), Pyrosequencing (PSQ), and Microscopic Observation of Drug Susceptibility (MODS) and compared to the BACTEC MGIT960 reference standard to detect M/XDR-TB directly from patient sputum samples collected at TB clinics in India, Moldova, and South Africa. RESULTS: Specificity for all three assays was excellent: 97-100% for isoniazid (INH), rifampin (RIF), moxifloxacin (MOX) and ofloxacin (OFX) and 99-100% for amikacin (AMK), capreomycin (CAP) and kanamycin (KAN) resistance. Sensitivities were lower, but still very good: 94-100% for INH, RIF, MOX and OFX, and 84-90% for AMK and CAP, but only 48-62% for KAN. In terms of agreement, statistically significant differences were only found for detection of RIF (MODS outperformed PSQ) and KAN (MODS outperformed LPA and PSQ) resistance. Mean time-to-result was 1.1 days for LPA and PSQ, 14.3 days for MODS, and 24.7 days for MGIT. CONCLUSIONS: All three rapid assays evaluated provide clinicians with timely detection of resistance to the drugs tested; with molecular results available one day following laboratory receipt of samples. In particular, the very high specificity seen for detection of drug resistance means that clinicians can use the results of these rapid tests to avoid the use of toxic drugs to which the infecting organism is resistant and develop treatment regiments that have a higher likelihood of yielding a successful outcome.

The Global Consortium for Drug-resistant Tuberculosis Diagnostics (GCDD): design of a multi-site, head-to-head study of three rapid tests to detect extensively drug-resistant tuberculosis.

BACKGROUND: Drug-resistant tuberculosis (DR-TB) remains a threat to global public health, owing to the complexity and delay of diagnosis and treatment. The Global Consortium for Drug-resistant Tuberculosis Diagnostics (GCDD) was formed to develop and evaluate assays designed to rapidly detect DR-TB, so that appropriate treatment might begin more quickly. This paper describes the methodology employed in a prospective cohort study for head-to-head assessment of three different rapid diagnostic tools. METHODS: Subjects at risk of DR-TB were enrolled from three countries. Data were gathered from a combination of patient interviews, chart reviews, and laboratory testing from each site's reference laboratory. The primary outcome of interest was reduction in time from specimen arrival in the laboratory to results of rapid drug susceptibility tests, as compared with current standard mycobacterial growth indicator tube (MGIT) drug susceptibility tests. RESULTS: Successful implementation of the trial in diverse multinational populations is explained, in addition to challenges encountered and recommendations for future studies with similar aims or populations. CONCLUSIONS: The GCDD study was a head-to-head study of multiple rapid diagnostic assays aimed at improving accuracy and precision of diagnostics and reducing overall time to detection of DR-TB. By conducting a large prospective study, which captured epidemiological, clinical, and biological data, we have produced a high-quality unique dataset, which will be beneficial for analyzing study aims as well as answering future DR-TB research questions. Reduction in detection time for XDR-TB would be a major public health success as it would allow for improved treatment and more successful patient outcomes. Executing successful trials is critical in assessment of these reductions in highly variable populations. TRIAL REGISTRATION: ClinicalTrials.gov NCT02170441.