Lara Noble

Background. Human papillomavirus (HPV) nucleic acid-based testing is the preferred method for cervical cancer screening. Leveraging point-of-care (POC) technologies can increase accessibility to screening in resource-limited settings. However, POC technologies are designed for use with fresh specimens and obtaining pre-clinical data to motivate clinical trials can be difficult. Methodology. A laboratory-based, proof-of-concept evaluation of the Pluslife HPV-16/18/45 Nucleic Acid Test Card on the Pluslife platform (Pluslife Biotech Co, Ltd., Guangzhou, Guangdong, China) was performed on swabs contrived using AccuTrak™ HPV Qualification Panel (SeraCare) reference material, or residual liquid-based cytology specimens, and stored, self-collected, vaginal swab specimens. Pluslife HPV 16/18/45 results were compared to paired BD Onclarity™ HPV (Becton Dickinson) results to provide analytical performance data (positive percentage agreement (PPA), negative percentage agreement (NPA), overall agreement (Cohen’s Kappa), and relative clinical sensitivity and specificity, all reported with 95 percent confidence intervals (95% CI)). Results. The AccuTrak™ and LBC contrived swab reproducibility results were correct for both inter- and intra-day testing (100.0% agreement). In 50 contrived clinical swabs, PPA was 100.0% (95% CI: 88.4, 100.0), NPA was 95.0% (95% CI: 75.1, 99.9), and overall agreement was 95.8% (95% CI: 87.7, 103.9). When stratified by cytology, the relative clinical sensitivity and specificity were 100.0% (100.0, 100.0) and 95.0% (95% CI: 64.4, 140.0) respectively. Amongst 24 self-collected vaginal swabs, the PPA and NPA were 92.3% (95% CI: 64.0, 99.8) and 100.0% (95% CI: 71.5, 100.0) respectively, with overall agreement of 91.7% (95% CI: 75.7, 107.6). Only one error (cartridge fill) was noted in 120 tests performed. Conclusions. This pre-clinical study described methods for using off-label biorepository specimens. The Pluslife HPV 16/18/45 assay showed similar performance to the BD On-clarity™ HPV assay on both contrived and self-collected vaginal swab specimens. These data support further clinical evaluation of the Pluslife HPV 16/18/45 assay as a solution for rapid, POC HPV screening.

Background/Objectives: Access to accurate tuberculosis (TB) diagnostics remains limited, particularly in high-burden settings. The Pluslife MTB assay is among the first molecular tests specifically designed for swab-based detection of Mycobacterium tuberculosis complex (MTBC) and offers near point-of-care use. Methods: We conducted a prospective diagnostic accuracy study in South Africa to evaluate the performance of the Pluslife assay on tongue swabs (TSs) and sputum swabs (SSs) among symptomatic and asymptomatic adults. Results were compared against liquid culture as the reference standard and Xpert MTB/RIF Ultra (Xpert Ultra) as a comparator. Operational characteristics and ease-of-use were assessed through structured observation and Likert-scale scoring by testing personnel. Results: Of the 256 participants enrolled, 92 [36%] were people with HIV (PHIV) and 217 were included in the final analysis. Culture confirmed TB in 41/217 (19%). Pluslife on SSs demonstrated a sensitivity of 85% (95% CI: 70.8–94.4), compared to 83% (95% CI: 67.9–92.8), for Xpert Ultra on sputum and detected one additional case (paired analysis, p = 1.000). Sensitivity on TSs was lower (63%, 95% CI: 46.9–77.9), particularly among PHIV. Specificity exceeded 97% across specimen types. Concordance on TSs between Pluslife and Xpert Ultra increased with higher bacterial loads. Operational evaluation showed short hands-on time and high ease-of-use scores, though limitations were noted for patient identifier recording and troubleshooting on the Pluslife MiniDock device. Conclusions: The Pluslife MTB assay on SSs demonstrated good diagnostic accuracy and favorable usability in this study. Tongue swabs remain feasible but less reliable, supporting sputum as the preferred first specimen for TB diagnosis.

Background: The International Normalized Ratio (INR) monitors anticoagulant treatment but relies on laboratory-based services. This could limit access to rapid monitoring and increase the diagnostic delay, both of which may be addressed by point-of-care testing (POCT). This study investigated the LumiraDx POC platform for INR monitoring. Methods: INR was measured on recalcified residual venous (n = 94) specimens from Chris Hani Baragwanath Hospital and capillary blood specimens (n = 254) from consenting enrolled participants at Charlotte Maxeke Johannesburg Academic Hospital Anticoagulation clinic, Johannesburg, South Africa. Standard-of-care (SOC) INR was measured on sodium-citrated venous blood using the Sysmex-CS2500 platform (Siemens Healthcare) and Neoplastin-R (Roche Diagnostics and Diagnostica Stago, Paris, France) within 2 h post-venipuncture. Within run, precision was measured using 2 LumiraDx control levels. The statistical agreement of paired INR measurements was also stratified by dosing decision. Results: The precision was within the manufacturer’s claim for controls (level 1%CV: 3.63, level 2%CV: 2.24). Accuracy analysis showed a moderate overall agreement compared to the SOC INR results with a correlation coefficient of 0.94 (95% Cl, (0.9267 to 0.9497)). The overall precision (ρ > 0.9) and accuracy (C_b = 0.9842) were good with an absolute bias of 0.07. The 95% confidence intervals for the slope and intercept did not include 1.00 and 0.00, respectively; however, the total calculated error was within the minimal acceptable limits. Conclusion: The LumiraDx INR Test showed a good performance compared to laboratory-based testing and provided opportunity for rapid and patient-centric care. Owing to an increasing positive bias for INR > 3.5, confirmation with laboratory INR measurements may be required.

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.

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.

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, ten influenza A, ten Influenza B and twenty respiratory syncytial virus specimens. Results were com-pared 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 per-centage 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.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is frequently diagnosed through detection of viral RNA using nucleic acid amplification testing (NAAT) assays that are usually used in centralized settings. Following the publication of the SARS-CoV-2 genetic sequence, multiple diagnostic assays were launched in 2020. These assays require evaluation beyond manufacturer self-reported performance to determine whether they are suitable for use, meet country acceptance criteria, and are compatible with existing in-country platforms. In order to meet the demand for testing services, rapid yet robust assay performance evaluations are required. In our setting, these evaluation protocols required the use of residual patient specimens and reference materials, as typical clinical trials are time-consuming and limited by cost and the cyclical nature of SARS-CoV-2 infection. This protocol is designed to assist in the rapid and robust evaluation of nucleic acid-based assays for the detection of SARS-CoV-2 using limited specimens, reference materials, and test kits. While it is specific for RNA-based assays, it can be adapted for fully automated analyses. The preparation and processing of evaluation panels is described, followed by methods for analytical precision analysis and data visualization. Assay robustness and scalability are briefly discussed as these can be critical for implementation. This protocol is designed to be flexible and alternative options are provided throughout the text where possible. Key words SARS-CoV-2 molecular diagnostics Nucleic acid amplification testing (NAAT) Real-time polymerase chain reaction (PCR) Analytical performance Implementation readiness

The African Journal of Laboratory Medicine focuses on the role of the laboratory and its professionals in the clinical and public healthcare sectors, and is specifically based on an African frame of reference.

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 (ρ_c) 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 ρ_c (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.

The tiered laboratory framework for 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. EDTA-plasma panels with known HIV viral titres was prepared in PrimeStore molecular transport medium for per-module verification and per-instrument external quality assessment. The panels were transported at ambient temperature to 13 testing laboratories during 2017-2018, 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 participants passed verification (n=171 specimens) with an overall concordance correlation (ρc) of 0.997 (95%confidence interval [CI]:0.996,0.998) and a mean log bias of -0.019log cp/mL (95%CI:-0.044,0.063). The overall EQA ρc (n=104 specimens) was 0.999 (95%CI:0.998,0.999), with a mean log bias of 0.03 log cp/mL 95%(CI:0.02,0.05). The panels are suitable for use in quality monitoring of Xpert HIV-1 VL and are applicable to laboratories in remote settings.