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.

Multicentre verification of haematology laboratory blood collection tubes during a global blood collection tube shortage.

INTRODUCTION: Verification of blood collection tubes is essential for clinical laboratories. The aim of this study was to assess performance of candidate tubes from four alternative suppliers for routine diagnostic haematology testing during an impending global shortage of blood collection tubes. METHODS: A multicentre verification study was performed in Cape Town, South Africa. Blood from 300 healthy volunteers was collected into K2 EDTA and sodium citrate tubes of BD Vacutainer® comparator tubes and one of four candidate tubes (Vacucare, Vacuette®, V-TUBE™ and Vacutest®). A technical verification was performed, which included tube physical properties and safety. Routine haematology testing was performed for clinical verification. RESULTS: Vacucare tubes did not have a fill-line indicator, Vacuette® tubes had external blood contamination on the caps post-venesection and Vacutest® tubes had hard rubber stoppers. K2 EDTA tubes of Vacuette®, Vacucare and Vacutest® performed similarly to the comparator. Unacceptable constant bias was seen for PT in Vacucare (95% CI -2.38 to -0.10), Vacutest® (95% CI -1.91 to -0.49) and Vacuette® (95% CI 0.10-1.84) tubes and for aPTT in Vacuette® (95% CI 0.22-2.00) and V-TUBE™ (95% CI -2.88 to -0.44). Unacceptable %bias was seen for aPTT in Vacucare (95% CI 2.78-4.59) and Vacutest® tubes (95% CI 2.53-3.82; desirable ±2.30), and in V-TUBE™ for mean cell volume (95% CI 1.15-1.47, desirable ±0.95%) and mean cell haemoglobin concentration (95% CI -1.65 to -0.93, desirable ±0.43%). CONCLUSION: Blood collection tubes introduce variability to routine haematology results. We recommend that laboratories use one brand of tube. Verification of new candidate tubes should be performed to ensure consistency and reliable reporting of results.

Evaluating systematic targeted universal testing for tuberculosis in primary care clinics of South Africa: A cluster-randomized trial (The TUTT Trial).

BACKGROUND: The World Health Organization (WHO) recommends systematic symptom screening for tuberculosis (TB). However, TB prevalence surveys suggest that this strategy does not identify millions of TB patients, globally. Undiagnosed or delayed diagnosis of TB contribute to TB transmission and exacerbate morbidity and mortality. We conducted a cluster-randomized trial of large urban and rural primary healthcare clinics in 3 provinces of South Africa to evaluate whether a novel intervention of targeted universal testing for TB (TUTT) in high-risk groups diagnosed more patients with TB per month compared to current standard of care (SoC) symptom-directed TB testing. METHODS AND FINDINGS: Sixty-two clinics were randomized; with initiation of the intervention clinics over 6 months from March 2019. The study was prematurely stopped in March 2020 due to clinics restricting access to patients, and then a week later due to the Coronavirus Disease 2019 (COVID-19) national lockdown; by then, we had accrued a similar number of TB diagnoses to that of the power estimates and permanently stopped the trial. In intervention clinics, attendees living with HIV, those self-reporting a recent close contact with TB, or a prior episode of TB were all offered a sputum test for TB, irrespective of whether they reported symptoms of TB. We analyzed data abstracted from the national public sector laboratory database using Poisson regression models and compared the mean number of TB patients diagnosed per clinic per month between the study arms. Intervention clinics diagnosed 6,777 patients with TB, 20.7 patients with TB per clinic month (95% CI 16.7, 24.8) versus 6,750, 18.8 patients with TB per clinic month (95% CI 15.3, 22.2) in control clinics during study months. A direct comparison, adjusting for province and clinic TB case volume strata, did not show a significant difference in the number of TB cases between the 2 arms, incidence rate ratio (IRR) 1.14 (95% CI 0.94, 1.38, p = 0.46). However, prespecified difference-in-differences analyses showed that while the rate of TB diagnoses in control clinics decreased over time, intervention clinics had a 17% relative increase in TB patients diagnosed per month compared to the prior year, interaction IRR 1.17 (95% CI 1.14, 1.19, p < 0.001). Trial limitations were the premature stop due to COVID-19 lockdowns and the absence of between-arm comparisons of initiation and outcomes of TB treatment in those diagnosed with TB. CONCLUSIONS: Our trial suggests that the implementation of TUTT in these 3 groups at extreme risk of TB identified more TB patients than SoC and could assist in reducing undiagnosed TB patients in settings of high TB prevalence. TRIAL REGISTRATION: South African National Clinical Trials Registry DOH-27-092021-4901.

Epidemiology and outcomes of SARS-CoV-2 infection associated with anti-nucleocapsid seropositivity in Cape Town, South Africa.

Background: In low- and middle-income countries where SARS-CoV-2 testing is limited, seroprevalence studies can characterise the scale and determinants of the pandemic, as well as elucidate protection conferred by prior exposure. Methods: We conducted repeated cross-sectional serosurveys (July 2020 - November 2021) using residual plasma from routine convenient blood samples from patients with non-COVID-19 conditions from Cape Town, South Africa. SARS-CoV-2 anti-nucleocapsid antibodies and linked clinical information were used to investigate: (1) seroprevalence over time and risk factors associated with seropositivity, (2) ecological comparison of seroprevalence between subdistricts, (3) case ascertainment rates, and (4) the relative protection against COVID-19 associated with seropositivity and vaccination statuses, to estimate variant disease severity. Findings: Among the subset sampled, seroprevalence of SARS-CoV-2 in Cape Town increased from 39.2% in July 2020 to 67.8% in November 2021. Poorer communities had both higher seroprevalence and COVID-19 mortality. Only 10% of seropositive individuals had a recorded positive SARS-CoV-2 test. Antibody positivity before the start of the Omicron BA.1 wave (28 November 2021) was strongly protective for severe disease (adjusted odds ratio [aOR] 0.15; 95%CI 0.05-0.46), with additional benefit in those who were also vaccinated (aOR 0.07, 95%CI 0.01-0.35). Interpretation: The high population seroprevalence in Cape Town was attained at the cost of substantial COVID-19 mortality. At the individual level, seropositivity was highly protective against subsequent infections and severe COVID-19. Funding: Wellcome Trust, National Health Laboratory Service, the Division of Intramural Research, NIAID, NIH (ADR) and Western Cape Government Health.

HIV false positive screening serology due to sample contamination reduced by a dedicated sample and platform in a high prevalence environment.

Automated testing of HIV serology on clinical chemistry analysers has become common. High sample throughput, high HIV prevalence and instrument design could all contribute to sample cross-contamination by microscopic droplet carry-over from seropositive samples to seronegative samples resulting in false positive low-reactive results. Following installation of an automated shared platform at our public health laboratory, we noted an increase in low reactive and false positive results. Subsequently, we investigated HIV serology screening test results for a period of 21 months. Of 485 initially low positive or equivocal samples 411 (85%) tested negative when retested using an independently collected sample. As creatinine is commonly requested with HIV screening, we used it as a proxy for concomitant clinical chemistry testing, indicating that a sample had likely been tested on a shared high-throughput instrument. The contamination risk was stratified between samples passing the clinical chemistry module first versus samples bypassing it. The odds ratio for a false positive HIV serology result was 4.1 (95% CI: 1.69-9.97) when creatinine level was determined first, versus not, on the same sample, suggesting contamination on the chemistry analyser. We subsequently issued a notice to obtain dedicated samples for HIV serology and added a suffix to the specimen identifier which restricted testing to a dedicated instrument. Low positive and false positive rates were determined before and after these interventions. Based on measured rates in low positive samples we estimate that before the intervention, of 44 117 HIV screening serology samples, 753 (1.71%) were false positive, declining to 48 of 7 072 samples (0.68%) post-intervention (p<0.01). Our findings showed that automated high throughput shared diagnostic platforms are at risk of generating false-positive HIV test results, due to sample contamination and that measures are required to address this. Restricting HIV serology samples to a dedicated platform resolved this problem.

Incidence of acetylcholine receptor-antibody-positive myasthenia gravis in South Africa.

INTRODUCTION: The aim of this study was to assess age- and gender-specific incidence rates (IRs) of acetylcholine receptor (AChR)-antibody-positive myasthenia gravis (MG) in South Africa, and geographical variations in incidence. METHODS: IRs were calculated from positive AChR-antibody laboratory data between 2011 and 2012, using 2011 population census data. RESULTS: 890 individuals were seropositive, for an annual IR of 8.5 per million. Age-standardized IR for early-onset (<50) and late-onset (≥50) MG were 4.1 and 24 per million, respectively, and 4.3 per million for juveniles. The IR between provinces ranged from 1 to 19 per million. CONCLUSIONS: In this Southern Hemisphere African population, the overall IR and peak IR (in older men) for seropositive MG is comparable to that in Europe and North America, arguing against environmental factors. However, IRs may be higher among children with African genetic ancestry. Geographical variation in incidence underscores the importance of outreach programs for regions with limited resources.