Lucia Hans

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.

Rapid antigen tests targeting SARS-CoV-2 nucleocapsid protein were essential for decentralised testing during the COVID-19 pandemic. Independent performance evaluations are essential to support regulatory approval and inform clinical implementation, particularly in resource-limited settings. This study presents a retrospective analytical and operational evaluation of two instrument-based fluorescent immunoassays (FIAs): the PCL COVID-19 Ag Rapid FIA and LumiraDx SARS-CoV-2 Ag Test. Analytical sensitivity was determined using recombinant nucleocapsid protein and viral cultures. Clinical performance was assessed using residual clinical specimens (n = 110) with RT-PCR as a reference, stratified by cycle threshold (Ct). Operational characteristics were assessed using a structured Likert framework. Overall sensitivity was 63% (51–73) for PCL and 95% (88–99) for LumiraDx. For Ct ≤ 25, sensitivity increased to 93% and 100%. Specificity was ≥97% for both. LumiraDx maintained sensitivity (83–94%) at Ct 25–30, whereas PCL did not detect any positives in this range. The limit of detection was 39 pM (PCL) and 0.6 pM (LumiraDx). Operational usability was high for both (90% PCL, 87% LumiraDx). LumiraDx showed higher analytical sensitivity across a broader viral load range, supporting primary diagnostic use, whereas PCL was limited to high viral loads. This evaluation provides a reproducible framework for rapid diagnostic assessment during emerging outbreaks.

Rapid antigen tests targeting SARS-CoV-2 nucleocapsid protein were essential for decentralised testing during the COVID-19 pandemic. Independent performance evaluations are critical to guide regulatory approval and implementation, particularly in resource-limited settings. This study presents a retrospective analytical and operational evaluation of two instrument-based fluorescent immunoassays (FIA): the PCL COVID-19 Ag Rapid FIA (PCL Inc., South Korea) and LumiraDx SARS-CoV-2 Ag Test (LumiraDx Ltd., UK). Analytical sensitivity was determined using recombinant nucleocapsid protein and viral cultures. Clinical performance was assessed using residual clinical specimens (n=110) with RT-PCR as reference, stratified by cycle threshold (Ct). Operational characteristics were assessed using a structured Likert framework. Overall sensitivity was 63% (51-73) for PCL and 95% (88-99) for LumiraDx. For Ct≤25, sensitivity increased to 93% and 100%. Specificity was ≥97% for both. LumiraDx maintained sensitivity (83-94%) at Ct 25-30, whereas PCL detected no positives in this range. Limit of detection was 0.039 nM (PCL) and 0.6 pM (LumiraDx). Operational usability was high for both (90% PCL, 87% LumiraDx). LumiraDx showed higher analytical sensitivity across a broader viral load range, supporting primary diagnostic use, whereas PCL was limited to high viral loads. This provides a reproducible framework for rapid diagnostic assessment during emerging outbreaks.

Background: The hepatitis C virus (HCV) testing algorithm used in South African laboratories involves two tests; a serology test to detect anti-HCV antibodies and a confirmatory test to detect the presence of HCV ribonucleic acid (RNA). These tests usually require consecutive blood samples taken at more than one hospital visit, leaving patients with inconclusive diagnosis for weeks. Objectives: This study aimed to describe the prevalence and proportion of HCV seropositive cases where diagnostic opportunities were missed among patients who attended public healthcare facilities in Johannesburg, South Africa. Method: This descriptive study was conducted as a retrospective analysis of laboratory data from the Department of Medical Virology, National Health Laboratory Service, Johannesburg, South Africa. Patients for whom HCV diagnosis were requested from 2017 to 2021 were included in the study. Results: Over the five-year period, a total of 212 670 patients were tested for HCV antibodies and 8575 (4.04%) tested positive. The HCV seropositivity was recorded for 74.8% (6421/8575) of males compared to females (23.6%; 2030/8575; p < 0.001). Only 25% (2183/8575) of HCV seropositive patients had a linked HCV RNA quantitative PCR test result. Conclusion: Despite an overall HCV seroprevalence of 4% in this study, only one quarter received a definitive diagnosis. This highlights that most patients are lost to follow-up because of the two-step diagnostic process, which requires them to return to the health facility for confirmatory tests. Contribution: The study highlights the need to adopt HCV reflex testing as the standard in South Africa to meet the 2030 elimination targets.

Background: Dolutegravir- has superior viral suppression compared to efavirenz-based antiretroviral therapy (ART). However, there are limited programmatic data on suppression in rural areas of South Africa.Objectives: We aimed to compare 6- and 12-month viral suppression of dolutegravir and efavirenz regimens and determine factors available in TIER.Net (the national electronic database for HIV and tuberculosis care) associated with suppression.Method: We conducted a retrospective cohort study using Mopani District programme data from TIER.Net. Clients aged ≥ 15 years initiated on tenofovir-lamivudine-dolutegravir (TLD) or tenofovir-emtricitabine-efavirenz (TEE) between 01 October 2021 and 31 March 2023, with ≥ 150 days in care, were included. We analysed 6- and 12-month suppression proportions and factors associated with suppression using logistic regression.Results: A total of 472 clients on TEE and 944 on TLD were included. Six-month viral loads were available for 47.7% (225/472) of TEE and 57.4% (542/944) of TLD clients. Six-month suppression ( 50 copies/mL) was 65.5% (355/542) for TLD and 53.8% (121/225) for TEE (P = 0.002). TLD was associated with increased odds of suppression at 6 months (adjusted odds ratio [aOR] 1.6; 95% CI: 1.1–2.2). At 12 months, viral loads were available for 60.7% (573/944) of TLD and 56.1% (265/472) of TEE clients. Twelve-month suppression ( 50 copies/mL) was 70.0% (401/573) for TLD and 68.3% (181/265) for TEE with no statistically significant differences between TEE and TLD clients. Low-level viraemia (50 copies/mL – 999 copies/mL) at 12 months was 25.0% for TLD and 20.8% for TEE.Conclusion: TLD showed improved suppression compared to TEE at 6 but not 12 months. The high proportion of clients with low-level viraemia is concerning. All clients, regardless of regimen, need evaluation for adherence support.

Background: Following a major service disruption across the National Health Laboratory Service, backlogged HIV viral load (VL) specimens from Limpopo province were rerouted to Groote Schuur Hospital (GSH) laboratory in the Western Cape province. During this time, an increase in low-level viraemia (LLV; 50 copies/mL – 1000 copies/mL) was observed at the GSH laboratory, raising concerns about possible pre-analytical and analytical issues, including compromised specimen quality and possible contamination. Objective: To determine whether the observed increased LLV was due to analytical errors (e.g. contamination), pre-analytical factors such as prolonged turnaround times (TAT), or underlying epidemiological differences. Methods: HIV VL data from 2023–2024 for Limpopo and the Western Cape were analysed using Python. Viral load results were grouped into predefined categories and compared. Turnaround times were plotted and Thembisa model estimates were used to assess HIV prevalence and antiretroviral therapy (ART) coverage. Longitudinal patient-level analysis evaluated VL trends as a proxy for adherence. In addition, quality control data were evaluated at testing sites. Results: Limpopo specimens showed higher LLV (20%) and lower viral suppression (< 50 copies/mL) at 70%, compared to Western Cape (13% LLV, 81% suppression), where follow-up and suppression outcomes were also higher. No clear evidence indicated that extended TAT or potential instrument contamination affected VL results significantly. Conclusion: The increase in LLV at GSH was linked primarily to processing specimens from Limpopo, highlighting regional differences in HIV VL result distributions. These differences probably reflect variations in ART access and adherence, rather than laboratory-related issues such as delayed TAT, sample quality, or contamination. What this study adds: This study shows how province-specific HIV result patterns correlate with ART adherence, using a Python script to assess serial VLs and follow-up suppression. It demonstrates the value of routine data analysis in monitoring high-throughput HIV VL tests, which are often auto-released without pathologist oversight.

Background: Specimen losses across the pathology value chain (PVC) result in missed diagnostic opportunities. It is difficult to fully assess these due to the current paper-based systems, with tracking of specimens only possible on the laboratory information system (LIS). Aim: This study aimed to assess specimen losses using the paper-based register. Setting: Randomly selected Primary health care (PHC) facilities, City of Johannesburg, South Africa. Methods: The retrospective descriptive study design was used to scan 1,000 barcodes from facilities in sub-districts A to G. Data was limited to barcodes from the request form and excluded surveillance testing. Matching data from the laboratory repository was extracted. PVC losses were assessed by determining the percentage of scanned barcodes that had a registered, tested, reviewed and/or rejected date. The analysis was stratified according to sub-district, health facility type and test code. Results: The dataset analysed included 33 867 barcodes with 121 697 test codes, equating to 3.59 tests per barcode. Matching registered, tested and reviewed dates were detected for 33 107 (97.76%) barcodes. In total, a rejection for one or more test codes was detected for 1,961 barcodes (5.79%). At the sub-district level, between 95.95% (D) and 98.90% (E) of barcodes were reviewed. The rejection rate ranged from 3.27% (F) to 10.93% (D). For community health centres and clinics, 97.37% and 97.97% of the barcodes had a matching reviewed date. Conclusion: PVC losses reported were 4.05%, excluding rejections (5.79%), with slightly higher levels noted at the sub-district level. Contribution: The continuous audit of PVC losses is recommended.

Introduction: To ensure there is adequate investment into diagnostics, an understanding of the magnitude of impact and return on investment is necessary. We, therefore, sought to understand the health and economic impacts of the molecular diagnostic programme in South Africa, to deepen the understanding of the broad value of diagnostics and guide future healthcare investments. Methods: We calculated the 10-year (where data were available) total cost and disability-adjusted life-years (DALYs) averted associated with molecular testing for tuberculosis diagnosis (2013–2022), HIV viral load monitoring (2013–2022), early infant diagnosis of HIV infection (2013–2022) and SARS-CoV-2 testing (2020–2022), based on the actual number of molecular tests conducted in South Africa for the respective time periods. We then calculated the economic value associated with those health gains and subsequent return on investment. Results: Since the inception of the molecular diagnostics programme in South Africa, approximately 4.3 million DALYs (uncertainty range (UR): 2.8–5.8 million) have been averted as a direct consequence of this programme. This has generated an estimated US$28.3 billion in economic value due to these health gains (UR$18.4–UR$38.7 billion). The return on investment varied by specific diagnostic test (20.3 (UR 15.2–25.4) for tuberculosis, 7.7 (UR 1.6–13.9) for HIV viral load testing, 63.0 (UR 63.0–65.5) for early infant diagnosis of HIV and 2.5 (UR 0.7–4.6) for SARS-CoV-2), for an average of 13.9 (UR 9.0–18.9) for the entire molecular diagnostics programme or US$13.9 of value for each UR$1 invested. Conclusions: The molecular diagnostics programme in South Africa generated a significant amount of health gains and economic value associated with these health gains. The return on investment rivals other high-impact public health interventions such as childhood vaccination. The molecular diagnostics programme in South Africa is highly impactful and will continue to be an excellent investment in South African public health expenditure.

Background: Understanding factors that impact HIV viral load (VL) accuracy in resource-limited settings is key to quality improvement. Objective: We evaluated whether testing delay and specimen storage between 25 °C and 30 °C before testing affected results. Methods: Between November 2019 and June 2023, 249 individuals on antiretroviral therapy, or with newly diagnosed HIV, were recruited from clinics in Cape Town and Gqeberha, South Africa, and three plasma preparation tubes were collected. One tube was tested within 24 h, while the others were stored uncentrifuged at ambient temperatures before testing. Centrifugation and testing of matched samples were performed on Day 4 and Day 7 after collection. Results: Time delay and ambient storage had minimal impact in specimens with a Day 1 VL of > 100 copies/mL. When grouped by Day 1 VL range, 96% – 100% of specimens at Day 4 and 93% – 100% at Day 7 had VLs within 0.5 log copies/mL of the first result. The greatest variability at Days 4 and 7 was observed when the Day 1 VL was < 100 copies/mL. However, there was no trend of increasing difference over time. Of Day 1 specimens with undetectable VL, or VL < 50 copies/mL, 80% had concordant results at Day 4 and 78% at Day 7. Conclusion: These results show that VL is stable in plasma preparation tubes for 7 days when stored at room temperature. There is significant variability in specimens with low VL, but variability is not affected by testing delay. What this study adds: Ideal HIV VL testing conditions are frequently unachievable in resource-limited settings. Data are needed on whether this impacts on the validity of test results. Our results provide reassurance that storage at ambient temperature for up to 7 days before testing does not substantially affect the VL result.

Background: Coronavirus disease (COVID-19) severely disrupted routine health care globally. This study assessed the impact of successive COVID-19 waves on HIV viral load (VL) suppression in South Africa, using the national public sector laboratory database. Guidelines recommend VL monitoring at 6 months after treatment initiation, annually once if suppressed, or more frequently if unsuppressed. Methods: Specimen-level VL data were extracted for the period January 2019–December 2021. We assessed the national percentage of samples with a VL <50 (virological suppression), 50–999 (low-level viremia), and ≥1000 (viremia) copies/mL. Data were analyzed by calendar year and month. Data for 2019 (pre–COVID-19) were compared with the 2020 and 2021 calendar years (lockdowns imposed). The national number of COVID-19 cases was reported to indicate the wave periods as follows: 1 (ancestral)-June–August 2020; 2 (Beta)-December 2020–January 2021; 3 (Delta)-June–August 2021, and 4 (Omicron)-December 2021. Results: Data are reported for 17,460,264 samples, with 5,608,733, 5,840,056, and 6,011,475 tests performed in 2019, 2020, and 2021 respectively. Overall, a VL of <50, 50–999, and ≥1000 copies/mL were reported for 69.4%, 17.3%, and 13.4% of samples, respectively. A VL <50 copies/mL was reported for 67.7%, 70.3%, and 70.0% of patients in 2019, 2020, and 2021, respectively. For the 2020 and 2021 calendar years, the monthly percentage of patients with a VL <50 copies/mL ranged between 64.6% and 72.7%. Conclusion: Our findings indicate that COVID-19 has not had a substantial impact on the percentage of samples with virological suppression at the national level.