Field study to determine the reliability of HIV viral load results shows minimal impact of delayed testing in South Africa.

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.

Addressing pandemic-wide systematic errors in the SARS-CoV-2 phylogeny.

The SARS-CoV-2 genome occupies a unique place in infection biology - it is the most highly sequenced genome on earth (making up over 20% of public sequencing datasets) with fine scale information on sampling date and geography, and has been subject to unprecedented intense analysis. As a result, these phylogenetic data are an incredibly valuable resource for science and public health. However, the vast majority of the data was sequenced by tiling amplicons across the full genome, with amplicon schemes that changed over the pandemic as mutations in the viral genome interacted with primer binding sites. In combination with the disparate set of genome assembly workflows and lack of consistent quality control (QC) processes, the current genomes have many systematic errors that have evolved with the virus and amplicon schemes. These errors have significant impacts on the phylogeny, and therefore over the last few years, many thousands of hours of researchers time has been spent in "eyeballing" trees, looking for artefacts, and then patching the tree. Given the huge value of this dataset, we therefore set out to reprocess the complete set of public raw sequence data in a rigorous amplicon-aware manner, and build a cleaner phylogeny. Here we provide a global tree of 3,960,704 samples, built from a consistently assembled set of high quality consensus sequences from all available public data as of March 2023, viewable at https://viridian.taxonium.org. Each genome was constructed using a novel assembly tool called Viridian (https://github.com/iqbal-lab-org/viridian), developed specifically to process amplicon sequence data, eliminating artefactual errors and mask the genome at low quality positions. We provide simulation and empirical validation of the methodology, and quantify the improvement in the phylogeny. Phase 2 of our project will address the fact that the data in the public archives is heavily geographically biased towards the Global North. We therefore have contributed new raw data to ENA/SRA from many countries including Ghana, Thailand, Laos, Sri Lanka, India, Argentina and Singapore. We will incorporate these, along with all public raw data submitted between March 2023 and the current day, into an updated set of assemblies, and phylogeny. We hope the tree, consensus sequences and Viridian will be a valuable resource for researchers.

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.

Human Parainfluenza Virus (HPIV) Detection in Hospitalized Children with Acute Respiratory Tract Infection in the Western Cape, South Africa during 2014-2022 Reveals a Shift in Dominance of HPIV 3 and 4 Infections.

The epidemiology of human parainfluenza viruses (HPIV), particularly its role as a cause of acute respiratory infection (ARI) in infants, has not been formally studied in South Africa. We evaluated HPIV prevalence in diagnostic samples from hospitalized children from public sector hospitals in the Western Cape between 2014 and 2022. HPIV infection was detected in 2-10% of patients, with the majority of infections detected in children less than 1 year of age. Prior to 2020, HPIV 4 (40%) and HPIV 3 (34%) were the most prevalent types, with seasonal peaks in late winter/spring for HPIV 3 and autumn/winter for HPIV 4. HPIV 4A and 4B co-circulated during the seasonal activity between 2014 and 2017. Pandemic restrictions in 2020 had a profound effect on HPIV circulation and the rebound was dominated by waves of HPIV 3, accounting for 66% of detections and a sustained decline in the circulation of HPIV 1, 2 and 4. An immunity gap could account for the surge in HPIV 3 infections, but the decline in prior HPIV 4 dominance is unexplained and requires further study.

Acceptability and Feasibility of Providing Adherence Feedback Based on Tenofovir Diphosphate in Dried Blood Spots: Results from a Pilot Study Among Patients and Providers in Cape Town, South Africa.

Tenofovir diphosphate (TFV-DP) concentrations in dried blood spots (DBS) predict viral breakthrough, but their use remains understudied in real-world clinic settings. This pilot study examined acceptability, feasibility, and initial adherence outcomes of providing adherence feedback using TFV-DP concentrations on patient- and provider-levels in Cape Town, South Africa. We enrolled 60 persons with HIV (PWH) receiving tenofovir-containing ART attending a primary health clinic. They were randomized 1:1 to an intervention receiving TFV-DP concentration feedback by research staff vs. no feedback at monthly visits for 4 months. Acceptability among medical providers and level of clinical follow-up of TFV-DP results was examined. Patient acceptability was assessed descriptively. Mean electronic adherence (EA), as measured by WisePill device, and TFV-DP in DBS were compared between the two arms. All participants in the intervention group (100%) reported finding TFV-DP feedback helpful and 86% reported changing adherence behaviors. Medical providers indicated high acceptability of incorporating TFV-DP concentration feedback into the clinic, yet among 29 results < 1000 fmol/punch, only 2 were reviewed with no follow-up actions performed. In the intervention arm, mean TFV-DP concentrations were significantly higher (t = 2.5, p < .01) during follow-up and EA in upper quartile (96-100%) was greater compared to controls (x2 = 7.8, p ≤ .05). This study found high acceptability among patients for receiving adherence feedback based on TFV-DP concentrations. TFV-DP and EA data demonstrated greater adherence in the intervention group. Providers indicated high acceptability of incorporating TFV-DP feedback into the clinic, but few providers reviewed results, which could impact clinic-level feasibility.

Comparing Predictive Ability of Two Objective Adherence Measures in a Community-Based Cohort on Antiretroviral Therapy in South Africa: Tenofovir Diphosphate Concentrations and Electronic Adherence Monitors.

BACKGROUND: Electronic adherence (EA) and tenofovir diphosphate (TFV-DP) in dried blood spots (DBS) are objective measures of antiretroviral therapy (ART) adherence. We characterized the association between these measures in a prospective cohort of persons with HIV (PWH) on ART. SETTING: Four primary health clinics in Cape Town, South Africa. METHODS: We enrolled 250 virally suppressed PWH receiving tenofovir-based ART. We collected EA data, monthly viral load, and TFV-DP in DBS for 12 months. We used logistic regression to estimate the adjusted odds ratio (aOR) and 95% confidence intervals (CIs) for future viral breakthrough (VB) (>400 copies/mL) for each adherence measure. Receiver operating characteristics (ROCs) provided the predictive power of these measures. RESULTS: Participants had a median (IQR) age of 34 (27-42); 78% were women. Twenty-one (8%) developed VB. Logistic regression showed that when percent EA and TFV-DP concentrations increased, the odds of VB decreased. This relationship was consistent at the time of VB (aOR of 0.41 [95% CI: 0.25 to 0.66] for TFV-DP and aOR of 0.64 [95% CI: 0.54 to 0.76] for EA) and for up to 2 months before VB. Both adherence measures predicted future VB at both 1 month and 2 months before viral load measurement. CONCLUSION: We established that 2 objective adherence measures, EA and TFV-DP in DBS, have a positive association with, and are both strongly predictive of, VB in a community-based South African cohort on ART. Future research is needed to determine the feasibility of implementing these adherence measures in resource-limited settings to facilitate adherence interventions.

Mobility during the post-partum period and viraemia in women living with HIV in South Africa.

BACKGROUND: We investigated the association between travel and viraemia in post-partum women with human immunodeficiency virus on antiretroviral therapy (ART). METHODS: Data are from a trial of post-partum ART delivery strategies. Women who initiated ART during pregnancy, were clinically stable with a viral load (VL) <400 copies/ml and were <10 weeks post-partum were enrolled at a primary care antenatal clinic in Cape Town, South Africa. Study visits at 3, 6, 12, 18 and 24 months post-partum included questions about travel, defined as ≥1 night spent outside of the city, and VL testing. Generalised mixed effects models assessed the association between travel and subsequent VL ≥400 copies/ml. RESULTS: Among 402 women (mean age 29 y, 35% born in the Western Cape), 69% reported one or more travel events over 24 months. Being born beyond the Western Cape (adjusted odds ratio [aOR] 2.03 [95% confidence interval {CI} 1.49 to 2.77]), duration post-partum in months (aOR 1.03 [95% CI 1.02 to 1.05]) and living with the child (aOR 0.60 [95% CI 0.38 to 0.93]) were associated with travel. In multivariable analyses, a travel event was associated with a 92% increase in the odds of a VL ≥400 copies/ml (aOR 1.92 [95% CI 1.19 to 3.10]). CONCLUSIONS: Interventions to support women on ART who travel are urgently required.

Outcomes of laboratory-confirmed SARS-CoV-2 infection during resurgence driven by Omicron lineages BA.4 and BA.5 compared with previous waves in the Western Cape Province, South Africa.

OBJECTIVES: We aimed to compare the clinical severity of Omicron BA.4/BA.5 infection with BA.1 and earlier variant infections among laboratory-confirmed SARS-CoV-2 cases in the Western Cape, South Africa, using timing of infection to infer the lineage/variant causing infection. METHODS: We included public sector patients aged ≥20 years with laboratory-confirmed COVID-19 between May 01-May 21, 2022 (BA.4/BA.5 wave) and equivalent previous wave periods. We compared the risk between waves of (i) death and (ii) severe hospitalization/death (all within 21 days of diagnosis) using Cox regression adjusted for demographics, comorbidities, admission pressure, vaccination, and previous infection. RESULTS: Among 3793 patients from the BA.4/BA.5 wave and 190,836 patients from previous waves, the risk of severe hospitalization/death was similar in the BA.4/BA.5 and BA.1 waves (adjusted hazard ratio [aHR] 1.12; 95% confidence interval [CI] 0.93; 1.34). Both Omicron waves had a lower risk of severe outcomes than previous waves. Previous infection (aHR 0.29, 95% CI 0.24; 0.36) and vaccination (aHR 0.17; 95% CI 0.07; 0.40 for at least three doses vs no vaccine) were protective. CONCLUSION: Disease severity was similar among diagnosed COVID-19 cases in the BA.4/BA.5 and BA.1 periods in the context of growing immunity against SARS-CoV-2 due to previous infection and vaccination, both of which were strongly protective.

Changing character and waning impact of COVID-19 at a tertiary centre in Cape Town, South Africa.

Background: The emergence of genetic variants of SARS-CoV-2 was associated with changing epidemiological characteristics throughout coronavirus disease 2019 (COVID-19) pandemic in population-based studies. Individual-level data on the clinical characteristics of infection with different SARS-CoV-2 variants in African countries is less well documented. Objectives: To describe the evolving clinical differences observed with the various SARS-CoV-2 variants of concern and compare the Omicron-driven wave in infections to the previous Delta-driven wave. Method: We performed a retrospective observational cohort study among patients admitted to a South African referral hospital with COVID-19 pneumonia. Patients were stratified by epidemiological wave period, and in a subset, the variants associated with each wave were confirmed by genomic sequencing. Outcomes were analysed by Cox proportional hazard models. Results: We included 1689 patients were included, representing infection waves driven predominantly by ancestral, Beta, Delta and Omicron BA1/BA2 & BA4/BA5 variants. Crude 28-day mortality was 25.8% (34/133) in the Omicron wave period versus 37.1% (138/374) in the Delta wave period (hazard ratio [HR] 0.68 [95% CI 0.47-1.00] p = 0.049); this effect persisted after adjustment for age, gender, HIV status and presence of cardiovascular disease (adjusted HR [aHR] 0.43 [95% CI 0.28-0.67] p < 0.001). Hospital-wide SARS-CoV-2 admissions and deaths were highest during the Delta wave period, with a decoupling of SARS-CoV-2 deaths and overall deaths thereafter. Conclusion: There was lower in-hospital mortality during Omicron-driven waves compared with the prior Delta wave, despite patients admitted during the Omicron wave being at higher risk. Contribution: This study summarises clinical characteristics associated with SARS-CoV-2 variants during the COVID-19 pandemic at a South African tertiary hospital, demonstrating a waning impact of COVID-19 on healthcare services over time despite epidemic waves driven by new variants. Findings suggest the absence of increasing virulence from later variants and protection from population and individual-level immunity.

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.

Routine Antiretroviral Pharmacy Refill Information Can Predict Failure Postpartum in Previously Suppressed South African Women With HIV.

Background: Detection of antiretrovirals (ARVs) in biological specimens is a reliable, objective way to measure adherence. However, routine ARV testing is not feasible in many high-burden settings. This study explored if pharmacy data could accurately predict HIV viremia postpartum in previously virally suppressed women. Methods: South African women with HIV who initiated antiretroviral therapy (ART) during pregnancy and achieved viral suppression (VS; viral load [VL]≤50 copies/mL) were followed postpartum; during follow-up, plasma VL was measured and ARV adherence self-reported. A portion of samples were tested for the presence of ARV using mass spectrometry. Patient-level routine pharmacy data were used to classify if women should have the drug in hand for the past 7 days before the visit date. Logistic regressions were used to calculate associations between adherence and viral nonsuppression (VNS; VL > 50) or failure (VF; VL > 1000) at the first study visit of women who had ARV measured. Data for all women were examined for associations of self-reported adherence and drug in hand with VS and VF at 2, 6, and 12 months postpartum. Results: Women with no ARV detected were significantly more likely to have VNS (odds ratio [OR], 26.4). Having no drug in hand for 7 days was also predictive of VNS in these same women (OR, 7.0) and the full cohort (n = 572) at 3 (OR, 2.9), 6 (OR, 8.7), and 12 months (OR, 14.5). Similar results were seen for VF. Conclusions: These data show that routine pharmacy data can act as a highly predictive mechanism for identifying patients at risk of VNS and VF due to nonadherence.

Clinical severity of SARS-CoV-2 Omicron BA.4 and BA.5 lineages compared to BA.1 and Delta in South Africa.

Omicron lineages BA.4 and BA.5 drove a fifth wave of COVID-19 cases in South Africa. Here, we use the presence/absence of the S-gene target as a proxy for SARS-CoV-2 variant/lineage for infections diagnosed using the TaqPath PCR assay between 1 October 2021 and 26 April 2022. We link national COVID-19 individual-level data including case, laboratory test and hospitalisation data. We assess severity using multivariable logistic regression comparing the risk of hospitalisation and risk of severe disease, once hospitalised, for Delta, BA.1, BA.2 and BA.4/BA.5 infections. After controlling for factors associated with hospitalisation and severe outcome respectively, BA.4/BA.5-infected individuals had a similar odds of hospitalisation (aOR 1.24, 95% CI 0.98-1.55) and severe outcome (aOR 0.72, 95% CI 0.41-1.26) compared to BA.1-infected individuals. Newly emerged Omicron lineages BA.4/BA.5 showed similar severity to the BA.1 lineage and continued to show reduced clinical severity compared to the Delta variant.

Low prevalence of hepatitis B virus infection in HIV-uninfected pregnant women in Cape Town, South Africa: implications for oral pre-exposure prophylaxis roll out.

BACKGROUND: Oral daily preexposure prophylaxis (PrEP) using emtricitabine (FTC)/tenofovir disoproxil fumarate (TDF) is recommended as standard of care for prevention in individuals at high risk for HIV infection, including pregnant and postpartum cisgender women. FTC/TDF is also active against hepatitis B virus (HBV); however, concern has been raised that providing PrEP to individuals infected with HBV could lead to hepatitis flares and liver injury, especially in the setting of suboptimal PrEP use. METHODS: We conducted a cross-sectional analysis of baseline data from the PrEP in pregnant and postpartum women (PrEP-PP) cohort study from February 2020-March 2022 in one antenatal care clinic in Cape Town, South Africa (SA) to evaluate: (1) the field performance of a point of care test (POCT) (Determine II, Abbott Inc., Japan) for diagnosis of hepatitis B surface antigen (HBsAg) in a maternity setting, (2) the prevalence of HBV in a cohort of pregnant women not living with HIV. RESULTS: We enrolled 1194 HIV sero-negative pregnant women at their first antenatal visit. Median age was 26 years (IQR = 22-31 years); 52% were born before 1995 (before universal HBV vaccination had started in South Africa). Median gestational age was 22 weeks (IQR = 16-30 weeks). There were 8 POCT and laboratory confirmed HBV cases among 1194 women. The overall prevalence of 0.67% (95% CI = 0.34-1.32%). In women born before 1995, 8 of 622 women were diagnosed with HBsAg; the prevalence was 1.29% (95% CI = 0.65-2.52%), and in women born in 1995 or after (n = 572); the prevalence was 0% (95% CI = 0.0-0.67%). We confirmed the test results in 99.8% of the rapid HBsAg (Determine II). Sensitivity was 100% (95% CI = 68-100%). Specificity was 100% (95% CI = 99.67-100%). CONCLUSION: The prevalence of HBV was very low in pregnant women not living with HIV and was only in women born before the HBV vaccine was included in the Expanded Program of Immunization. The Determine II POCT HBsAg showed excellent performance against the laboratory assay. HBV screening should not be a barrier to starting PrEP in the context of high HIV risk communities.

Improved virologic outcomes in postpartum women living with HIV referred to differentiated models of care.

OBJECTIVES: Differentiated service delivery (DSD) models are used to deliver antiretroviral therapy (ART) but data are limited in postpartum women, who are at high risk of non-adherence and elevated viral load (VL) over the extended postpartum period. DESIGN: Randomized controlled trial. METHODS: We enrolled consecutive postpartum women who initiated ART during pregnancy and met local DSD eligibility (clinically stable, VL less than 400 copies/ml) at a large primary healthcare (PHC) clinic. Women were randomized to a community-based 'adherence club' (AC, the local DSD model: community health worker-led groups of 20-30 patients with ART dispensing at a community venue) or routine PHC clinics (local standard of care with nurse/doctor-led services). Follow-up visits with VL separate from routine care took place at 3, 6, 12, 18 and 24 months postpartum. Endpoints were time to VL of at least 1000 copies/ml (primary) and VL of at least 50 copies/ml (secondary) by intention-to-treat. RESULTS: At enrolment ( n  = 409), the median duration postpartum was 10 days, all women had a VL less than 1000 copies/ml and 88% had a VL less than 50 copies/ml; baseline characteristics did not differ by arm. Twenty-four-month retention was 89%. Sixteen and 29% of women in AC experienced a VL of at least 1000 copies/ml by 12 and 24 months, compared to 23 and 37% in PHC, respectively (hazard ratio [HR] = 0.71; 95% confidence interval [CI] = 0.50-1.01). Thirty-two and 44% of women in ACs had a VL of at least 50 copies/ml by 12 and 24 months, compared to 42 and 56% in PHC, respectively (HR = 0.68; 95% CI = 0.51-0.91). CONCLUSIONS: Early DSD referral was associated with reduced viraemia through 24 months postpartum and may be an important strategy to improve maternal virologic outcomes.

Outcomes of laboratory-confirmed SARS-CoV-2 infection during resurgence driven by Omicron lineages BA.4 and BA.5 compared with previous waves in the Western Cape Province, South Africa.

Objective: We aimed to compare clinical severity of Omicron BA.4/BA.5 infection with BA.1 and earlier variant infections among laboratory-confirmed SARS-CoV-2 cases in the Western Cape, South Africa, using timing of infection to infer the lineage/variant causing infection. Methods: We included public sector patients aged ≥20 years with laboratory-confirmed COVID-19 between 1-21 May 2022 (BA.4/BA.5 wave) and equivalent prior wave periods. We compared the risk between waves of (i) death and (ii) severe hospitalization/death (all within 21 days of diagnosis) using Cox regression adjusted for demographics, comorbidities, admission pressure, vaccination and prior infection. Results: Among 3,793 patients from the BA.4/BA.5 wave and 190,836 patients from previous waves the risk of severe hospitalization/death was similar in the BA.4/BA.5 and BA.1 waves (adjusted hazard ratio [aHR] 1.12; 95% confidence interval [CI] 0.93; 1.34). Both Omicron waves had lower risk of severe outcomes than previous waves. Prior infection (aHR 0.29, 95% CI 0.24; 0.36) and vaccination (aHR 0.17; 95% CI 0.07; 0.40 for boosted vs. no vaccine) were protective. Conclusion: Disease severity was similar amongst diagnosed COVID-19 cases in the BA.4/BA.5 and BA.1 periods in the context of growing immunity against SARS-CoV-2 due to prior infection and vaccination, both of which were strongly protective.

Improved oral detection is a characteristic of Omicron infection and has implications for clinical sampling and tissue tropism.

BACKGROUND: The Omicron variant of concern is characterised by more than 50 distinct mutations, most in the spike protein. The implications of these for disease transmission, tissue tropism and diagnostic testing needs study. OBJECTIVES: We evaluated the performance of RT-PCR on saliva (SA) swabs and antigen testing on mid-turbinate MT samples relative to RT-PCR on MT swabs. Patients (n = 453) presenting for outpatient testing at the Groote Schuur Hospital COVID-19 testing centre in Cape Town South Africa were recruited. Participants were recruited during the Delta (n = 304) and Omicron (n = 149) waves. RESULTS: In 30 confirmed Delta infections, positive percent agreement (PPA) of RT-PCR on saliva was only 73% compared to a composite standard of either MT or SA RT-PCR positivity, with rapid decay by day 3 after symptom onset. In contrast, in the 70 Omicron infections, SA performed as well as, or better than, MT samples up to day 5, with an overall PPA of SA swabs of 96% and MT of 93%. A change in antigen test performance was noted, with PPA of 93% in Delta, but only 68% for Omicron. CONCLUSIONS: Altered shedding kinetics appear to be present in Omicron-infected patients with more viral RNA detectable in saliva. Saliva swabs are a promising alternative to nasal samples, especially early in infection when sampling of both sites could improve detection. Lower sensitivity of antigen tests in Omicron is a concern and requires further study.

Outcomes of laboratory-confirmed SARS-CoV-2 infection in the Omicron-driven fourth wave compared with previous waves in the Western Cape Province, South Africa.

OBJECTIVES: The objective was to compare COVID-19 outcomes in the Omicron-driven fourth wave with prior waves in the Western Cape, assess the contribution of undiagnosed prior infection to differences in outcomes in a context of high seroprevalence due to prior infection and determine whether protection against severe disease conferred by prior infection and/or vaccination was maintained. METHODS: In this cohort study, we included public sector patients aged ≥20 years with a laboratory-confirmed COVID-19 diagnosis between 14 November and 11 December 2021 (wave four) and equivalent prior wave periods. We compared the risk between waves of the following outcomes using Cox regression: death, severe hospitalisation or death and any hospitalisation or death (all ≤14 days after diagnosis) adjusted for age, sex, comorbidities, geography, vaccination and prior infection. RESULTS: We included 5144 patients from wave four and 11,609 from prior waves. The risk of all outcomes was lower in wave four compared to the Delta-driven wave three (adjusted hazard ratio (aHR) [95% confidence interval (CI)] for death 0.27 [0.19; 0.38]. Risk reduction was lower when adjusting for vaccination and prior diagnosed infection (aHR: 0.41, 95% CI: 0.29; 0.59) and reduced further when accounting for unascertained prior infections (aHR: 0.72). Vaccine protection was maintained in wave four (aHR for outcome of death: 0.24; 95% CI: 0.10; 0.58). CONCLUSIONS: In the Omicron-driven wave, severe COVID-19 outcomes were reduced mostly due to protection conferred by prior infection and/or vaccination, but intrinsically reduced virulence may account for a modest reduction in risk of severe hospitalisation or death compared to the Delta-driven wave.

Emergence and phenotypic characterization of the global SARS-CoV-2 C.1.2 lineage.

Global genomic surveillance of SARS-CoV-2 has identified variants associated with increased transmissibility, neutralization resistance and disease severity. Here we report the emergence of the PANGO lineage C.1.2, detected at low prevalence in South Africa and eleven other countries. The initial C.1.2 detection is associated with a high substitution rate, and includes changes within the spike protein that have been associated with increased transmissibility or reduced neutralization sensitivity in SARS-CoV-2 variants of concern or variants of interest. Like Beta and Delta, C.1.2 shows significantly reduced neutralization sensitivity to plasma from vaccinees and individuals infected with the ancestral D614G virus. In contrast, convalescent donors infected with either Beta or Delta show high plasma neutralization against C.1.2. These functional data suggest that vaccine efficacy against C.1.2 will be equivalent to Beta and Delta, and that prior infection with either Beta or Delta will likely offer protection against C.1.2.

Assessing the clinical severity of the Omicron variant in the Western Cape Province, South Africa, using the diagnostic PCR proxy marker of RdRp target delay to distinguish between Omicron and Delta infections - a survival analysis.

BACKGROUND: At present, it is unclear whether the extent of reduced risk of severe disease seen with SARS-Cov-2 Omicron variant infection is caused by a decrease in variant virulence or by higher levels of population immunity. METHODS: RdRp target delay (RTD) in the Seegene AllplexTM 2019-nCoV PCR assay is a proxy marker for the Delta variant. The absence of this proxy marker in the transition period was used to identify suspected Omicron infections. Cox regression was performed for the outcome of hospital admission in those who tested positive for SARS-CoV-2 on the Seegene AllplexTM assay from November 1 to December 14, 2021 in the Western Cape Province, South Africa, in the public sector. Adjustments were made for vaccination status and prior diagnosis of infection. RESULTS: A total of 150 cases with RTD and 1486 cases without RTD were included. Cases without RTD had a lower hazard of admission (adjusted hazard ratio [aHR], 0.56; 95% confidence interval [CI], 0.34-0.91). Complete vaccination was protective against admission, with an aHR of 0.45 (95% CI, 0.26-0.77). CONCLUSION: Omicron has resulted in a lower risk of hospital admission compared with contemporaneous Delta infection, when using the proxy marker of RTD. Under-ascertainment of reinfections with an immune escape variant remains a challenge to accurately assessing variant virulence.

Selection Analysis Identifies Clusters of Unusual Mutational Changes in Omicron Lineage BA.1 That Likely Impact Spike Function.

Among the 30 nonsynonymous nucleotide substitutions in the Omicron S-gene are 13 that have only rarely been seen in other SARS-CoV-2 sequences. These mutations cluster within three functionally important regions of the S-gene at sites that will likely impact (1) interactions between subunits of the Spike trimer and the predisposition of subunits to shift from down to up configurations, (2) interactions of Spike with ACE2 receptors, and (3) the priming of Spike for membrane fusion. We show here that, based on both the rarity of these 13 mutations in intrapatient sequencing reads and patterns of selection at the codon sites where the mutations occur in SARS-CoV-2 and related sarbecoviruses, prior to the emergence of Omicron the mutations would have been predicted to decrease the fitness of any virus within which they occurred. We further propose that the mutations in each of the three clusters therefore cooperatively interact to both mitigate their individual fitness costs, and, in combination with other mutations, adaptively alter the function of Spike. Given the evident epidemic growth advantages of Omicron overall previously known SARS-CoV-2 lineages, it is crucial to determine both how such complex and highly adaptive mutation constellations were assembled within the Omicron S-gene, and why, despite unprecedented global genomic surveillance efforts, the early stages of this assembly process went completely undetected.