Accuracy of cobas MTB and MTB-RIF/INH for Detection of Mycobacterium tuberculosis and Drug Resistance.

This study evaluated the performance of cobas MTB and cobas MTB-RIF/INH for the diagnosis of tuberculosis (TB) and detection of rifampicin (RIF) and isoniazid (INH) resistance. Adults presenting with pulmonary TB symptoms were recruited in South Africa, Moldova, and India. Performance of cobas MTB was assessed using culture as reference, whereas cobas MTB-RIF/INH was assessed using phenotypic drug susceptibility testing and whole-genome sequencing as composite reference standards. Xpert MTB/RIF (Xpert) or Xpert MTB/RIF Ultra (Ultra) was used as a comparator. The overall sensitivity and specificity of cobas MTB were 95% (95% CI, 93%-96%) and 96% (95% CI, 95%-97%), respectively. Among smear-negative results, the sensitivity of cobas MTB was 75% (95% CI, 66%-83%). Among participants tested with both cobas MTB and Xpert, sensitivity was 96% (95% CI, 94%-97%) for cobas MTB and 95% (95% CI, 93%-97%) for Xpert. Among participants tested with both cobas MTB and Ultra, sensitivity was 88% (95% CI, 81%-92%) for cobas MTB and 89% (95% CI, 83%-93%) for Ultra. Sensitivity and specificity of cobas MTB-RIF/INH for RIF and INH detection were 90% (95% CI, 84%-94%) and 100% (95% CI, 99%-100%), and 89% (95% CI, 84%-93%) and 99.5% (95% CI, 98%-100%), respectively. The cobas MTB and cobas MTB-RIF/INH assays exhibited high performance in a diverse population and present a suitable option for molecular detection of TB and both RIF and INH resistance.

Storage of Mycobacterium tuberculosis culture isolates in MicrobankTM beads at a South African laboratory.

Background: Mycobacterium tuberculosis complex (MTBC) isolates are typically stored at -70 °C in cryovials containing 1 mL aliquots of a liquid medium, with or without 50% glycerol. Multiple uses of the culture stock may decrease the strain viability while increasing the risk of culture contamination. Small culture aliquots may be more practical; however, storage capacity remains challenging. MicrobankTM beads (25 beads/vial) for the long-term storage of fungal cultures is well documented, but their use for storing MTBC isolates is uninvestigated. Objective: The study aimed to determine the feasibility of using MicrobankTM beads for long-term storage of MTBC isolates at a laboratory in South Africa. Methods: In February 2020, 20 isolates in liquid culture were stored in MicrobankTM beads, following an in-house developed protocol, at -70 °C. At defined time points (16 months [15 June 2021] and 21 months [18 November 2021]), two beads were retrieved from each storage vial and assessed for viability and level of contamination. Results: Stored liquid isolates demonstrated MTBC growth within an average time-to-detection of 18 days following retrieval, even at 21 months post storage. Contaminating organisms were detected in 2 of 80 (2.5%) culture isolates. Conclusion: MicrobankTM beads will allow for the reculture of up to 25 culture isolates using a reduced culture volume compared to current storage methods. MicrobankTM beads represent a storage solution for the medium-term storage of MTBC isolates. What this study adds: This study evaluated the use of MicrobankTM beads as an alternate method for storing MTBC culture isolates at -70 °C and provided a suitable option for medium-term storage of MTBC.

Determining cost and placement decisions for moderate complexity NAATs for tuberculosis drug susceptibility testing.

BACKGROUND: Access to drug resistant testing for tuberculosis (TB) remains a challenge in high burden countries. Recently, the World Health Organization approved the use of several moderate complexity automated nucleic acid amplification tests (MC-NAAT) that have performance profiles suitable for placement in a range of TB laboratory tiers to improve drug susceptibility tests (DST) coverage. METHODS: We conducted cost analysis of two MC-NAATs with different testing throughput: Lower Throughput (LT, < 24 tests per run) and Higher Throughput (HT, upto 90+ tests per run) for placement in a hypothetical laboratory in a resource limited setting. We used per-test cost as the main indicator to assess 1) drivers of cost by resource types and 2) optimized levels of annual testing volumes for the respective MC-NAATs. RESULTS: The base-case per test cost of $18.52 (range: $13.79 - $40.70) for LT test and $15.37 (range: $9.61 - $37.40) for HT test. Per test cost estimates were most sensitive to the number of testing days per week, followed by equipment costs and TB-specific workloads. In general, HT NAATs were cheaper at all testing volume levels, but at lower testing volumes (less than 2,000 per year) LT tests can be cheaper if the durability of the testing system is markedly better and/or procured equipment costs are lower than that of HT NAAT. CONCLUSION: Assuming equivalent performance and infrastructural needs, placement strategies for MC-NAATs need to be prioritized by laboratory system's operational factors, testing demands, and costs.

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.

Evaluation Protocol for SARS-CoV-2 Serological Assays.

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) has been identified as the causative agent of COVID-19. Accurate detection of SARS-CoV-2 infection is not only important for management of infected individuals but also to break the chain of transmission. Although the polymerase chain reaction (PCR) is the gold standard for diagnosis of acute SARS-CoV-2 infection, there are a number of limitations of these assays, which include the inability to detect past infection and decline in sensitivity 14 days post-symptom onset. There are several serology tests developed for the detection of SARS-CoV-2 antibodies including high-throughput serology platforms and lateral flow immunoassays. These tests should be evaluated for their performance to meet local regulations acceptance criteria. To optimize the diagnostic algorithm for SARS-CoV-2, this protocol describes the evaluation of serological antibody testing using various automated serology platforms and lateral flow immunoassays. This protocol was evaluated in both serum and plasma samples. The sample preparation, procedure, and data analysis are described. The protocol can be adapted for any serological testing.

Antigen-Based Point of Care Testing (POCT) for Diagnosing SARS-CoV-2: Assessing Performance.

Currently, the most accurate way to diagnose an active SARS-CoV-2 (COVID-19) infection is through detection of viral RNA using reverse transcription polymerase chain reaction (RT-PCR) test. While RT-PCR tests are the most sensitive for identifying infection, there are significant limitations, such as global access to sufficient test kits, turnaround times (TAT) from specimen collection to test result is often greater than 24 h and the need for skilled operators in accredited laboratories requiring specialized equipment. A rapid test performed at the point of care (POC) could provide a result within an approximate time of 30 min post specimen collection, be performed by a health care worker and comprise a simple workflow, improving both turnaround time and potentially decreasing costs (e.g., transport, cold-chain, skilled laboratory staff, complex equipment). Determining the performance of SARS-CoV-2 RT-PCR tests is, however, easier to assess than antigen-based POCT, as residual clinical specimens (swabs in universal transport media [UTM]) are readily available in laboratory environments, and do not require patient informed consent. Evaluating the performance of POCT requires informed-consent driven studies, with patients required to provide a standard of care specimen as well as study evaluation specimens, which is often not acceptable as nasopharyngeal swabbing can be invasive, clinical field trials are costly and time consuming. Many institutions and regulatory bodies also require preliminary data prior to use in field settings. Therefore, we have developed a method to determine the performance of antigen based POCT that can be used by implementers in national healthcare programs, regulators and rapid test developers. The method investigates both quantitative and qualitative parameters, with the latter providing insights into the capability for implementation and national program uptake.

Performance of the Abbott SARS-CoV-2 IgG serological assay in South African 2 patients.

In late December 2019, pneumonia cases of unknown origin were reported in Wuhan, China. This virus was named SARS-CoV2 and the clinical syndrome was named coronavirus disease 19 (COVID-19). South Africa, despite strict and early lockdown has the highest infection rate in Africa. A key component of South Africa's response to SARSCoV2 was the rapid scale-up of diagnostic testing. The Abbott SARS-CoV2 assay detects IgG antibodies against the Nucleocapsid (N) protein of the SARS-CoV2 virus. This study undertook to validate and evaluate performance criteria of the Abbott assay and to establish whether this assay would show clinical utility in our population. Positive patients (n = 391) and negative controls (n = 139) were included. The Architect-i and Alinity-i systems were analyzers that were used to perform the SARS-CoV-2 IgG assay. In-house ELISA was incorporated into the study as a confirmatory serology test. A total of number of 530 participants was tested, 87% were symptomatic with infection and 13% were asymptomatic. When compared to RT-qPCR, the sensitivity of Architect and Alinity SARS-CoV2 assays was 69.5% and 64.8%, respectively. Specificity for Architect and Alinity assays was 95% and 90.3%, respectively. The Abbott assay was also compared to in house ELISA assay, with sensitivity for the Architect and Alinity assays of 94.7% and 92.5%, respectively. Specificity for Abbott Alinity assays was 91.7% higher than Abbott Architect 88.1%. Based on the current findings testing of IgG after 14 days is recommended in South Africa and supports other studies performed around the world.

Detection of isoniazid, fluoroquinolone, ethionamide, amikacin, kanamycin, and capreomycin resistance by the Xpert MTB/XDR assay: a cross-sectional multicentre diagnostic accuracy study.

BACKGROUND: The WHO End TB Strategy requires drug susceptibility testing and treatment of all people with tuberculosis, but second-line diagnostic testing with line-probe assays needs to be done in experienced laboratories with advanced infrastructure. Fewer than half of people with drug-resistant tuberculosis receive appropriate treatment. We assessed the diagnostic accuracy of the rapid Xpert MTB/XDR automated molecular assay (Cepheid, Sunnyvale, CA, USA) to overcome these limitations. METHODS: We did a prospective study involving individuals presenting with pulmonary tuberculosis symptoms and at least one risk factor for drug resistance in four sites in India (New Delhi and Mumbai), Moldova, and South Africa between July 31, 2019, and March 21, 2020. The Xpert MTB/XDR assay was used as a reflex test to detect resistance to isoniazid, fluoroquinolones, ethionamide, amikacin, kanamycin, and capreomycin in adults with positive results for Mycobacterium tuberculosis complex on Xpert MTB/RIF or Ultra (Cepheid). Diagnostic performance was assessed against a composite reference standard of phenotypic drug-susceptibility testing and whole-genome sequencing. This study is registered with ClinicalTrials.gov, number NCT03728725. FINDINGS: Of 710 participants, 611 (86%) had results from both Xpert MTB/XDR and the reference standard for any drug and were included in analysis. Sensitivity for Xpert MTB/XDR detection of resistance was 94% (460 of 488, 95% CI 92-96) for isoniazid, 94% (222 of 235, 90-96%) for fluoroquinolones, 54% (178 of 328, 50-61) for ethionamide, 73% (60 of 82, 62-81) for amikacin, 86% (181 of 210, 81-91) for kanamycin, and 61% (53 of 87, 49-70) for capreomycin. Specificity was 98-100% for all drugs. Performance was equivalent to that of line-probe assays. The non-determinate rate of Xpert MTB/XDR (ie, invalid M tuberculosis complex detection) was 2·96%. INTERPRETATION: The Xpert MTB/XDR assay showed high diagnostic accuracy and met WHO's minimum target product profile criteria for a next-generation drug susceptibility test. The assay has the potential to diagnose drug-resistant tuberculosis rapidly and accurately and enable optimum treatment. FUNDING: German Federal Ministry of Education and Research through KfW, Dutch Ministry of Foreign Affairs, and Australian Department of Foreign Affairs and Trade.

Operational characteristics of 30 lateral flow immunoassays used to identify COVID-19 immune response.

Serology or antibody tests for COVID-19 are designed to detect antibodies (mainly Immunoglobulin M (IgM) and Immunoglobulin G (IgG) produced in response to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS CoV-2) infection. In this study, 30 lateral flow immunoassays were tested using serum or plasma from patients with confirmed SARS CoV-2 infection. Negative serological controls were accessed from a well-characterised bank of sera which were stored prior to February 2020. Operational characteristics and ease of use of the assays are reported. 4/30 (13%) of kits (Zheihang Orient Gene COVID-19 IgG/IgM, Genrui Novel Coronavirus (2019-nCoV) IgG/IgM, Biosynex COVID-19 BSS IgG/IgM, Boson Biotech 2019-nCoV IgG/IgM) were recommended for SAHPRA approval based on kit sensitivity. Of these, only the Orientgene was recommended by SAHPRA in August 2020 for use within the approved national testing algorithm while the remaining three received limited authorization for evaluation. All kits evaluated work on the same basic principle of immunochromatography with minor differences noted in the shape and colour of cartridges, the amount of specimen volume required and the test duration. Performance of the lateral flow tests were similar to sensitivities and specificities reported in other studies. The cassettes of the majority of kits evaluated (90%) detected both IgG and IgM. Only 23% of kits evaluated contained all consumables required for point-of-care testing. The study highlights the need for thorough investigation of kits prior to implementation.

Performance of the EUROIMMUN Anti-SARS-CoV-2 ELISA Assay for detection of IgA and IgG antibodies in South Africa.

Severe Acute Respiratory Syndrome-Coronavirus 2 (SARS-CoV-2) has been identified as the causative agent for causing the clinical syndrome of COVID -19. Accurate detection of SARS-CoV-2 infection is not only important for management of infected individuals but also to break the chain of transmission. South Africa is the current epicenter of SARS-CoV-2 infection in Africa. To optimize the diagnostic algorithm for SARS-CoV-2 in the South African setting, the study aims to evaluate the diagnostic performance of the EUROIMMUN Anti-SARS-CoV-2 assays. This study reported the performance of EUROIMMUN enzyme-linked immunosorbent assay (ELISA) for semi-quantitative detection of IgA and IgG antibodies in serum and plasma samples targeting the recombinant S1 domain of the SARS-CoV-2 spike protein as antigen. Samples were collected from 391 individuals who had tested positive for SARS-CoV-2 and 139 SARS CoV-2 negative controls. Samples were stratified by number of days' post-PCR diagnosis and symptoms. The sensitivity of EUROIMMUN IgG was 64.1% (95% CI: 59.1-69.0%) and 74.3% (95% CI: 69.6-78.6%) for IgA and the specificity was lower for IgA [84.2% (95% CI: 77-89.2%)] than IgG [95.2% (95% CI: 90.8-98.4%)]. The EUROIMMUN Anti-SARS-CoV-2 ELISA Assay sensitivity was higher for IgA but low for IgG and improved for both assays in symptomatic individuals and at later timepoints post PCR diagnosis.

SARS-CoV-2 Antigens Expressed in Plants Detect Antibody Responses in COVID-19 Patients.

Background: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has swept the world and poses a significant global threat to lives and livelihoods, with 115 million confirmed cases and at least 2.5 million deaths from Coronavirus disease 2019 (COVID-19) in the first year of the pandemic. Developing tools to measure seroprevalence and understand protective immunity to SARS-CoV-2 is a priority. We aimed to develop a serological assay using plant-derived recombinant viral proteins, which represent important tools in less-resourced settings. Methods: We established an indirect ELISA using the S1 and receptor-binding domain (RBD) portions of the spike protein from SARS-CoV-2, expressed in Nicotiana benthamiana. We measured antibody responses in sera from South African patients (n = 77) who had tested positive by PCR for SARS-CoV-2. Samples were taken a median of 6 weeks after the diagnosis, and the majority of participants had mild and moderate COVID-19 disease. In addition, we tested the reactivity of pre-pandemic plasma (n = 58) and compared the performance of our in-house ELISA with a commercial assay. We also determined whether our assay could detect SARS-CoV-2-specific IgG and IgA in saliva. Results: We demonstrate that SARS-CoV-2-specific immunoglobulins are readily detectable using recombinant plant-derived viral proteins, in patients who tested positive for SARS-CoV-2 by PCR. Reactivity to S1 and RBD was detected in 51 (66%) and 48 (62%) of participants, respectively. Notably, we detected 100% of samples identified as having S1-specific antibodies by a validated, high sensitivity commercial ELISA, and optical density (OD) values were strongly and significantly correlated between the two assays. For the pre-pandemic plasma, 1/58 (1.7%) of samples were positive, indicating a high specificity for SARS-CoV-2 in our ELISA. SARS-CoV-2-specific IgG correlated significantly with IgA and IgM responses. Endpoint titers of S1- and RBD-specific immunoglobulins ranged from 1:50 to 1:3,200. S1-specific IgG and IgA were found in saliva samples from convalescent volunteers. Conclusion: We demonstrate that recombinant SARS-CoV-2 proteins produced in plants enable robust detection of SARS-CoV-2 humoral responses. This assay can be used for seroepidemiological studies and to measure the strength and durability of antibody responses to SARS-CoV-2 in infected patients in our setting.

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.

The Performance of the Abbott Real Time MTB RIF/INH Compared to the MTBDRplus V2 for the Identification of MDR-TB Among Isolates.

BACKGROUND: The GenoType MTBDRplus V2 line-probe assay (LPA) is routinely used in clinical patient management to characterise the susceptibility of Mycobacterium tuberculosis complex to rifampicin (Rif) and isoniazid (INH) directly from sputum and cultured isolates. The laboratory workflow requires skill and three separate areas to minimize contamination and banding pattern interpretation requires experienced laboratory personnel. We explored the use of the RT MTB RIF/INH assay performed on the Abbott m2000 platform as an alternative laboratory platform. METHODS: Isolates (n=93) consisting of fully susceptible, Rif- or INH-mono-resistant and multi-drug resistant (MDR) strains were tested on both MTBDRplus v2 and RT MTB RIF/INH assays. Both assays target the rpoB, katG and inhA genes for resistance-detection mutations. Concordance was assessed using percent agreement and the kappa statistic. Those specimens with discordant results were further assessed using Sanger sequencing. RESULTS: A total of 89% (83/93) of cultured isolates generated successful results on the RT MTB/RIF-INH assay and MTBDRplus assays. Of the 10 discordant results, where sequencing was used as the reference method, the RT MTB RIF/INH assays misclassified six resistance isolates, while the LPA misclassified seven. DISCUSSION: Overall, the RT MTB RIF/INH demonstrated good agreement with the LPA, and a better correlation with sequencing on discrepant isolates specifically with mutations occurring in codon 511 of the rpoB gene. The RT MTB RIF/INH therefore can be used to complement existing laboratory algorithms determining Rif and INH resistance profiles, with less emphasis on manual laboratory processing.

Performance of the Roche cobas MTB Assay for the Molecular Diagnosis of Pulmonary Tuberculosis in a High HIV Burden Setting.

Increased access to and improved sensitivities of methods for diagnosing Mycobacterium tuberculosis infection and detecting rifampicin and isoniazid resistance are needed. Herein, the performance of the new cobas MTB assay for use on cobas 6800/8800 Systems (Roche) was assessed and compared with two other commercial assays: RealTime MTB (Abbott), and Xpert MTB/RIF (Cepheid). Molecular PCR-based assays were conducted on sputum specimens from individuals with presumptive and confirmed tuberculosis (n = 294) from two clinical facilities in South Africa between December 2016 and October 2017. Liquid mycobacterial culture was the reference. Test sensitivities were 94.7% (95% CI, 88%-98%), 92.6% (95% CI, 85%-97%), and 91.6% (95% CI, 84%-96%) for cobas MTB, RealTime MTB, and Xpert MTB/RIF assays, respectively. cobas MTB sensitivity was unaffected by HIV coinfection (95.7%; 95% CI, 88%-99%; n = 176) and sediment testing (94.7%; 95% CI, 88%-98%). Sensitivities were 81.8% (95% CI, 60%-95%), 72.7% (95% CI, 50%-89%), and 72.7% (95% CI, 50%-89%) among smear-negative, culture-positive individuals (n = 221) for cobas MTB, RealTime MTB, and Xpert MTB/RIF assays, respectively. cobas MTB specificity was 95.7% (95% CI, 89%-99%) and 99% (95% CI, 94%-100%) among HIV coinfected and uninfected individuals, respectively. The cobas 6800/8800 system is already implemented in South Africa for high-throughput HIV viral load testing, making it suitable for integrated HIV/tuberculosis diagnostics.

Performance of Xpert MTB/RIF, Xpert Ultra, and Abbott RealTime MTB for Diagnosis of Pulmonary Tuberculosis in a High-HIV-Burden Setting.

More sensitive tests are needed for the diagnosis of smear-negative and HIV-associated tuberculosis. This study compares the sensitivities and specificities of three molecular tests, namely, the Xpert MTB/RIF test, the Xpert Ultra (Ultra), and RealTime MTB (RT-MTB), in a high HIV prevalence setting. Symptomatic adults were recruited from three outpatient sites, and each provided 4 sputum specimens. The diagnostic performance of Xpert MTB/RIF, Ultra, and RT-MTB was evaluated, with culture as a reference standard. HIV infection occurred in 62% of patients, with a median CD4 count of 220 cells/µl. The Ultra test had the highest sensitivity of 89.3% (95% confidence interval [CI], 78.1 to 96) compared to those of the Xpert MTB/RIF at 82.1% (95% CI, 69.6 to 91.1; P = 0.12) and RT-MTB at 78.6% (95% CI, 65.6 to 88.4; P = 0.68). The specificity was highest with the Xpert MTB/RIF at 100% (95% CI, 98 to 100), followed by RealTime MTB at 96.7% (95% CI, 92.9 to 98.8; P = 0.03) and the Ultra at 95.6% (95% CI, 91.5 to 98.1; P = 0.08). In patients with smear-negative disease, the Ultra was more sensitive than the Xpert MTB/RIF (64.7% [95% CI, 38.3 to 85.8] versus 41.2% [95% CI, 18.4 to 67.1], respectively; P = 0.12), and RT-MTB performed equally to Xpert MTB/RIF. In a comparison of the Ultra and RT-MTB on the same sputum specimen pellets, the Ultra was more sensitive than RT-MTB in the overall cohort (88.9% [95% CI, 77.4 to 95.8] versus 77.8% [95% CI, 64.4 to 88], respectively; P = 0.03) and among people with HIV (87.5% [95% CI, 71 to 96.5] versus 68.6% [95% CI, 50 to 83.9], respectively; P = 0.03). Although these results did not reach statistical significance, they suggest that the Ultra is more sensitive than the Xpert MTB/RIF and RT-MTB, most prominently in smear-negative disease. This was accompanied by a loss of specificity.

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.

Performance of the Abbott RealTime MTB and MTB RIF/INH Assays in a Setting of High Tuberculosis and HIV Coinfection in South Africa.

South Africa is a country with a high incidence of tuberculosis (TB), complicated by coinfection with human immunodeficiency virus (HIV). The Xpert MTB/RIF (Cepheid, Sunnyvale, CA, USA) is used in South Africa as the test for the initial diagnosis of TB, and other molecular platforms such as the m2000 (Abbott Molecular, Des Plaines, IL, USA) are widely used for molecular monitoring of HIV load. The latter platform is now also equipped with the RealTime (RT) MTB and RealTime MTB RIF/INH assays for TB and first-line drug resistance screening but has not been evaluated in settings of HIV and TB coinfection. A prospective clinical validation study was conducted at a community health center in Johannesburg, South Africa, and consenting individuals with presumptive pulmonary TB were enrolled. The performance of the Abbott assays was compared with those of the Xpert MTB/RIF, liquid culture, drug susceptibility testing, and clinical case definitions. A statistical analysis was performed on 206 individuals (73% were HIV positive). The sensitivity and specificity of the RT MTB were 82.5% (confidence interval [CI], 67.2 to 92.7) and 93.1% (CI, 86.2 to 97.2) on raw sputum and 77.5% (CI, 61.5 to 89.2) and 95.1% (CI, 88.9 to 98.4) on concentrated sputum, respectively, compared with those from liquid culture. The RT MTB correctly identified 17/35 more smear-negative culture-positive specimens than the Xpert MTB/RIF. Both the RT MTB and the Xpert MTB/RIF displayed sensitivities >70% and specificities >90% in HIV-positive individuals. The available drug resistance results concurred with MTBDRplus and drug susceptibility profiles. The RT MTB assay has similar diagnostic performance to the Xpert MTB/RIF and is suited to testing presumptive TB patients coinfected with HIV. The existing laboratory information system connectivity, training, and technical support make this a viable polyvalent option to scale up TB alongside HIV laboratory testing services in South Africa.