Diagnostic accuracy of Xpert MTB/RIF for extrapulmonary tuberculosis specimens: establishing a laboratory testing algorithm for South Africa.

South Africa implemented Xpert MTB/RIF as the initial diagnostic test for pulmonary tuberculosis (TB). Xpert MTB/RIF's accuracy for diagnosing extrapulmonary tuberculosis (EPTB) was investigated. EPTB specimens (n = 7,916) from hospitalized patients received over a 6-month period at a high-throughput TB referral laboratory in Johannesburg were investigated. Large-volume specimens were centrifuged, tissue biopsy specimens homogenized, and all specimens checked for growth of contaminating bacteria on blood agar. Contaminated samples received NALC-NaOH (N-acetyl-l-cysteine-sodium hydroxide) decontamination prior to liquid culture. Residual specimens (volumes > 1 ml) after inoculation of culture (n = 1,175) were tested using the Xpert MTB/RIF sputum protocol. Using culture as the reference, Xpert MTB/RIF's overall sensitivity was 59% (95% confidence interval [95% CI], 53% to 65%) and specificity was 92% (CI, 90% to 94%), with the highest sensitivities of 91% (95% CI, 78% to 97%) for pus, 80% (95% CI, 56% to 94%) for lymph node aspirates, and 51% (95% CI, 44% to 58%) for fluids (ascitic, 59%; pleural, 47%). A difference in sensitivities was noticed between specimens classified as having a thick (87% [95% CI, 76% to 94%]) versus clear (watery) (48% [95% CI, 36% to 61%]) appearance. This was unchanged with traces of blood (52% [95% CI, 44% to 60%]) or precentrifugation (57% [95% CI, 28% to 82%]) among clear specimens. Xpert MTB/RIF generated an additional 124 specimen results that were contaminated by Mycobacterial Growth Indicator Tubes (MGIT; 10.5%) and diagnosed rifampin (RIF) resistance earlier (9.6% [25/260]). Xpert MTB/RIF's performance on EPTB specimens provides very promising results and should be considered for incorporation into national TB guidelines. Xpert MTB/RIF is less affected by contaminating bacteria and reduces laboratory labor and diagnostic delay compared to traditional methods.

Performance Evaluation of Four Qualitative RT-PCR Assays for the Detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in late 2019, and its rapid spread around the globe led the World Health Organization to declare it a pandemic. Laboratory diagnostics provide important information to help control virus transmission, and molecular nucleic acid amplification tests have been recognized as the gold standard for the direct detection of viral genetic material. The main aim of this study was to independently evaluate the analytical performance of four molecular assays that were designed for the detection of SARS-CoV-2 on open testing platforms under emergency use approval, namely, the COVIWOK COVID-19 RT-PCR Meril COVID-19 One-step RT-PCR Kit, the AmoyDx Novel Coronavirus (2019-nCoV) Detection Kit, the Meril COVID-19 One-step RT-PCR Kit and the NeoPlex COVID-19 Detection Kit, as alternatives to the current standard of care (SOC) assays in-country. All of the evaluated assays showed an acceptable performance, with a specificity of 100% and a sensitivity of 93.8% to 98.4%, compared to a SOC assay, with a Cohen's kappa coefficient of ≥0.9 (95% CI). In addition, the assays detected the AccuPlex reference material at 100 copies/mL, suggesting a good limit of detection. These assays provide suitable alternatives to the SOC assays that are currently available in-country, and these alternatives are acceptable for diagnostic use in South Africa. IMPORTANCE Laboratory diagnosis plays an important role in curbing the transmission of infection and reducing harmful delays in clinical and public health responses. Alternatives to the current standard of care assays for SARS-CoV-2 are important in order to overcome the challenges that are associated with global demands and supply shortages. Four molecular assays for the detection of SARS-CoV-2 that were designed for open testing platforms were evaluated in this study under emergency use approval. These assays had acceptable performance and provide suitable alternatives to the current standard of care assays that are available in-country. Their compatibilities with existing in-country amplification platforms make these assays convenient to use for diagnostic testing, both locally and globally These assays were recommended to the South African Health Products Regulatory Authority (SAHPRA) for patient care in South Africa.

More Than a Decade of GeneXpert®Mycobacterium tuberculosis/Rifampicin (Ultra) Testing in South Africa: Laboratory Insights from Twenty-Three Million Tests.

This study seeks to describe the rollout and current state of South Africa's GeneXpert molecular diagnostic program for tuberculosis (TB). Xpert MTB/RIF was introduced in 2011 with a subsequent expansion to include extra-pulmonary and paediatric testing, followed by Xpert MTB/RIF Ultra in 2017. Through a centralised laboratory information system and the use of a standardised platform for more than a decade, over 23 million tests were analysed, describing the numbers tested, Mycobacterium tuberculosis complex detection, rifampin resistance, and the unsuccessful test rates. The stratification by province, specimen type, age, and sex identified significant heterogeneity across the program and highlighted testing gaps for men, low detection yield for paediatric pulmonary TB, and the effects of inadequate specimen quality on the detection rate. The insights gained from these data can aid in the monitoring of interventions in support of the national TB program beyond laboratory operational aspects.

How South Africa Used National Cycle Threshold (Ct) Values to Continuously Monitor SARS-CoV-2 Laboratory Test Quality.

The high demand for SARS-CoV-2 tests but limited supply to South African laboratories early in the COVID-19 pandemic resulted in a heterogenous diagnostic footprint of open and closed molecular testing platforms being implemented. Ongoing monitoring of the performance of these multiple and varied systems required novel approaches, especially during the circulation of variants. The National Health Laboratory Service centrally collected cycle threshold (Ct) values from 1,497,669 test results reported from 6 commonly used PCR assays in 36 months, and visually monitored changes in their median Ct within a 28-day centered moving average for each assays' gene targets. This continuous quality monitoring rapidly identified delayed hybridization of RdRp in the Allplex™ SARS-CoV-2 assay due to the Delta (B.1.617.2) variant; S-gene target failure in the TaqPath™ COVID-19 assay due to B.1.1.7 (Alpha) and the B.1.1.529 (Omicron); and recently E-gene delayed hybridization in the Xpert® Xpress SARS-CoV-2 due to XBB.1.5. This near "real-time" monitoring helped inform the need for sequencing and the importance of multiplex molecular nucleic acid amplification technology designs used in diagnostics for patient care. This continuous quality monitoring approach at the granularity of Ct values should be included in ongoing surveillance and with application to other disease use cases that rely on molecular diagnostics.

Integrating Molecular Diagnostics and GIS Mapping: A Multidisciplinary Approach to Understanding Tuberculosis Disease Dynamics in South Africa Using Xpert MTB/RIF.

An investigation was carried out to examine the use of national Xpert MTB/RIF data (2013-2017) and GIS technology for MTB/RIF surveillance in South Africa. The aim was to exhibit the potential of using molecular diagnostics for TB surveillance across the country. The variables analysed include Mycobacterium tuberculosis (Mtb) positivity, the mycobacterial proportion of rifampicin-resistant Mtb (RIF), and probe frequency. The summary statistics of these variables were generated and aggregated at the facility and municipal level. The spatial distribution patterns of the indicators across municipalities were determined using the Moran's I and Getis Ord (Gi) statistics. A case-control study was conducted to investigate factors associated with a high mycobacterial load. Logistic regression was used to analyse this study's results. There was striking spatial heterogeneity in the distribution of Mtb and RIF across South Africa. The median patient age, urban setting classification, and number of health care workers were found to be associated with the mycobacterial load. This study illustrates the potential of using data generated from molecular diagnostics in combination with GIS technology for Mtb surveillance in South Africa. Spatially targeted interventions can be implemented in areas where high-burden Mtb persists.

Painless Capillary Blood Collection: A Rapid Evaluation of the Onflow Device.

Blood-based diagnostics are critical for many medical decisions, but mostly rely on venepuncture, which can be inconvenient and painful. The Onflow Serum Gel (Loop Medical SA, Vaud, Lausanne, Switzerland) is a novel blood collection device that utilises needle-free technology to collect capillary blood. In this pilot study, 100 healthy participants were enrolled and provided two Onflow collected specimens and one venous blood specimen. Five chemistry analytes (AST, ALT, LDH, potassium, creatinine) and haemolysis were measured per specimen, and laboratory analyte results were compared. Onflow was found to be more acceptable than venepuncture with lower pain ratings, and 96.5% of participants would use the Onflow method again. All phlebotomists (100%) found Onflow intuitive and user-friendly, with ~1 mL of Onflow blood successfully collected from 99% of participants in <12 min (mean: 6 min, 40 s) and 91% collected on the first attempt. ALT and AST analytes showed no difference in performance, while creatinine generated a negative bias (-5.6 µmol/L), and increased variability was noted with potassium (3.6%CV) and LDH (6.7%CV), although none were clinically relevant. These differences may be due to 35% of Onflow collected specimens having "mild" haemolysis. Onflow is a promising alternative blood collection device that should now be evaluated in participants with expected abnormal chemistries and as an option for self-collection.

Rapid Evaluation of the Xpert® Xpress CoV-2 plus and Xpert® Xpress CoV-2/Flu/RSV plus Tests.

The Xpert® Xpress SARS-CoV-2 and Xpert® Xpress SARS-CoV-2/Flu/RSV tests were rapidly developed and widely used during the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. In response to emerging genetic variability, a new SARS-CoV-2 target (RNA-dependent RNA-polymerase) has been added to both tests: Xpert® Xpress CoV-2 plus and Xpert® Xpress CoV-2/Flu/RSV plus test. A rapid evaluation of both tests was performed in South Africa, using residual respiratory specimens. Residual respiratory specimens (n = 125) were used to evaluate the Xpert® Xpress CoV-2 plus test and included 50 genotyped specimens. The Xpert® Xpress CoV-2/Flu/RSV plus test was assessed using 45 genotyped SARS-CoV-2 specimens, 10 influenza A, 10 influenza B and 20 respiratory syncytial virus specimens. Results were compared to in-country standard-of-care tests. Genotyped specimens tested the performance of the test under pressure from circulating SARS-CoV-2 variants of concern. Reference material was included to assess the test limits and linearity. The Xpert® Xpress CoV-2 plus test performance compared to reference results across residual respiratory specimens was good (positive percentage agreement (PPA) = 95.2%, negative percentage agreement (NPA) = 95.0%) The Xpert® Xpress CoV-2/Flu/RSV plus test showed good performance across all residual respiratory specimens (PPA = 100%, NPA = 98.3%). All genotyped variants of concern were detected by both tests. The Xpert® Xpress CoV-2 plus and Xpert® Xpress CoV-2/Flu/RSV plus tests can be used to diagnose SARS-CoV-2, and to diagnose and differentiate SARS-CoV-2, influenza A, influenza B and respiratory syncytial virus, respectively. The NPA was lower than the recommended 99%, but was influenced by the low number of negative specimens tested. The variants of concern assessed did not affect test performance. It is recommended that sites perform their own assessments compared to in-country standard-of-care tests.

Monitored Implementation of COVID-19 Rapid Antigen Screening at Taxi Ranks in Johannesburg, South Africa.

Digital tools can support community-based decentralized testing initiatives to broaden access to COVID-19 diagnosis, especially in high-transmission settings. This operational study investigated the use of antigen-detecting rapid diagnostic tests (Ag-RDTs) for COVID-19 combined with an end-to-end digital health solution, in three taxi ranks in Johannesburg, South Africa. Members of the public were eligible if they were aged ≥18 years, could read, and had a cellphone. Over 15,000 participants, enrolled between June and September 2021, were screened for COVID-19 risk factors. A digital risk questionnaire identified 2061 (13%) participants as moderate risk and 2987 (19%) as high risk, based on symptoms and/or recent exposure to a known case. Of this group referred for testing, 3997 (79%) received Ag-RDTs, with positivity rates of 5.1% in the "high-risk" group and 0.8% in the "moderate-risk" group. A subset of 569 randomly selected participants received additional PCR testing. Sensitivity of the Ag-RDT in this setting was 40% (95% CI: 30.3%, 50.3%); most false negatives had high cycle threshold values (>25), hence low viral loads. Over 80% of participants who tested positive completed a 2-week phone-based follow-up questionnaire. Overall, the digital tool combined with Ag-RDTs enhanced community-based decentralized COVID-19 testing service delivery, reporting and follow-up.

The Development of a Standardized Quality Assessment Material to Support Xpert® HIV-1 Viral Load Testing for ART Monitoring in South Africa.

The tiered laboratory framework for human immunodeficiency virus (HIV) viral load monitoring accommodates a range of HIV viral load testing platforms, with quality assessment critical to ensure quality patient testing. HIV plasma viral load testing is challenged by the instability of viral RNA. An approach using an RNA stabilizing buffer is described for the Xpert® HIV-1 Viral Load (Cepheid) assay and was tested in remote laboratories in South Africa. Plasma panels with known HIV viral titres were prepared in PrimeStore molecular transport medium for per-module verification and per-instrument external quality assessment. The panels were transported at ambient temperatures to 13 testing laboratories during 2017 and 2018, tested according to standard procedures and uploaded to a web portal for analysis. A total of 275 quality assessment specimens (57 verification panels and two EQA cycles) were tested. All participating laboratories met study verification criteria (n = 171 specimens) with an overall concordance correlation coefficient (ρc) of 0.997 (95% confidence interval (CI): 0.996 to 0.998) and a mean bias of -0.019 log copies per milliliter (cp/mL) (95% CI: -0.044 to 0.063). The overall EQA ρc (n = 104 specimens) was 0.999 (95% CI: 0.998 to 0.999), with a mean bias of 0.03 log cp/mL (95% CI: 0.02 to 0.05). These panels are suitable for use in quality monitoring of Xpert® HIV-1 VL and are applicable to laboratories in remote settings.