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

BackgroundIn 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. MethodsWe 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. FindingsAmong 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). InterpretationThe 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. FundingWellcome Trust, National Health Laboratory Service, the Division of Intramural Research, NIAID, NIH (ADR) and Western Cape Government Health. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSIn low- and middle-income countries where SARS-CoV-2 testing is limited, seroprevalence studies can help describe the true extent of the pandemic. Infection from Omicron was associated with less severe disease in South Africa, but it is unclear whether this was due to a decrease in virulence of the variant or if prior infection provided protection. Added value of this studyThe seroprevalence data nested within a population cohort enabled us to assess differential case ascertainment rates, as well as to examine the contribution of both natural and vaccine-induced immunity in protecting communities against infections and severe disease with different SARS-CoV-2 variants. Implications of the available evidenceInequality and differential access to resources resulted in poorer communities having higher seroprevalence and COVID-19 death rates, with lower case ascertainment rates. Antibody positivity provided strong protection against an immune escape variant like Omicron but came at a high mortality cost.

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

BackgroundEmerging data suggest that SARS-CoV-2 Omicron variant of concern (VOC)is associated with reduced risk of severe disease. The extent to which this reflects a difference in the inherent virulence of Omicron, or just higher levels of population immunity, is currently not clear. MethodsRdRp target delay (RTD: a difference in cycle threshold value of RdRp - E > 3.5) in the Seegene Allplex 2019-nCoV PCR assay is a proxy marker for the Delta VOC. The absence of this proxy marker in the period of transition to Omicron was used to identify suspected Omicron VOC infections. Cox regression was performed for the outcome of hospital admission in those who tested positive for SARS-CoV-2 on the Seegene Allplex assay from 1 November to 14 December 2021 in the Western Cape Province, South Africa, public sector. Vaccination status at time of diagnosis, as well as prior diagnosed infection and comorbidities, were adjusted for. Results150 cases with RTD (proxy for Delta) and 1486 cases without RTD (proxy for Omicron) were included. Cases without RTD had a lower hazard of admission (adjusted Hazard Ratio [aHR] of 0.56, 95% confidence interval [CI] 0.34-0.91). Complete vaccination was protective of admission with an aHR of 0.45 (95%CI 0.26-0.77). ConclusionOmicron has resulted in a lower risk of hospital admission, compared to contemporaneous Delta infection in the Western Cape Province, when using the proxy marker of RTD. Under-ascertainment of reinfections with an immune escape variant like Omicron remains a challenge to accurately assessing variant virulence.

Saliva swabs are the preferred sample for Omicron detection

The Omicron variant is characterised by more than 50 distinct mutations, the majority of which are located in the spike protein. The implications of these mutations for disease transmission, tissue tropism and diagnostic testing are still to be determined. We evaluated the relative performance of saliva and mid-turbinate swabs as RT-PCR samples for the Delta and Omicron variants. The positive percent agreement (PPA) of saliva swabs and mid-turbinate swabs to a composite standard was 71% (95% CI: 53-84%) and 100% (95% CI: 89-100%), respectively, for the Delta variant. However, for the Omicron variant saliva and mid-turbinate swabs had a 100% (95% CI: 90-100%) and 86% (95% CI: 71-94%) PPA, respectively. This finding supports ex-vivo data of altered tissue tropism from other labs for the Omicron variant. Reassessment of the diagnostic testing standard-of-care may be required as the Omicron variant become the dominant variant worldwide.

Higher mortality associated with the SARS-CoV-2 Delta variant in the Western Cape, South Africa, using RdRp target delay as a proxy

A novel proxy for the Delta variant, RNA-dependent RNA polymerase target delay in the Seegene Allplex 2019-nCoV PCR assay, was associated with higher mortality (adjusted Odds Ratio 1.45 [95%CI 1.13-1.86]), compared to presumptive Beta infection, in the Western Cape, South Africa (April-July 2021). Prior diagnosed infection and vaccination were protective.

Reduced amplification efficiency of the RNA-dependent-RNA-polymerase (RdRp) target enables tracking of the Delta SARS-CoV-2 variant using routine diagnostic tests

Routine SARS-CoV-2 surveillance in the Western Cape region of South Africa (January-August 2021) found a reduced PCR amplification efficiency of the RdRp gene target of the Seegene, Allplex 2019-nCoV diagnostic assay when detecting the Delta variant. We propose that this can be used as a surrogate for variant detection.

Improved performance of saliva for the detection of the B.1.351 variant in South Africa

Assessment of the unknown performance of saliva for the detection of the novel SARS-CoV-2 variant of concern (VOC) B.1.351 (501Y.V2) lineage is essential as saliva has been shown to be an equivalent, less invasive and a less costly alternative to nasopharyngeal swabs for the molecular detection of SARS-CoV-2 infection in pre-variant studies. Between 1st August 2020 and 16th January 2021, we enrolled 410 eligible ambulatory participants who presented to Groote Schuur Hospital (GSH) in Cape Town, South Africa for SARS-CoV-2 testing. Of these, 300 were enrolled prior to, and 110 after, the initial detection and replacement of wild-type by the B.1.351 variant. All participants provided a supervised self-collected mid-turbinate (MT) and saliva (SA) swab, in addition to the standard HCW collected NP swab which were all tested by RT-PCR in an accredited diagnostic laboratory. Positive percent agreement to NP swab for SA swabs pre- and post-variant were 51.5% and 72.5% respectively while these values for MT swabs were 75.8% and 77.5%. The negative percent agreement for all swab types during all periods was >98%. The basis for this marked improvement of SA swabs as a diagnostic sample for B.1.351 virus is still being investigated.

Field performance evaluation of the PanBio rapid SARS-CoV-2 antigen assay in an epidemic driven by 501Y.v2 (lineage B.1.351) in the Eastern Cape, South Africa

BackgroundSouth Africa was the African country most severely affected by the SARS-CoV-2 pandemic during 2020, experiencing 2 waves of infection. During the first wave, diagnostics were largely based on reverse transcription-linked PCR (RT-PCR). The Abbott PanBio antigen test was deployed during the 2nd wave which was driven by emergence of the 501Y.v2 variant. At the time of evaluation in mid-November 2020, 501Y.v2 was the dominant circulating virus in Nelson Mandela Bay, in the Eastern Cape Province. MethodsA prospective diagnostic evaluation study was undertaken, during a period of high community transmission, to evaluate the field performance of the PanBio antigen RTD. Testing was conducted at mobile community testing centres on 677 ambulant patients seeking SARS-CoV-2 testing. RT-PCR was performed on the original naso-pharyngeal antigen swabs to evaluate test performance. ResultsOf 146 RT-PCR positive individuals, 101 were RTD positive in the clinic. The antigen RTD had an overall sensitivity of 69.2% (95%CI 61.4, 75.8) and specificity of 99.0% (95%CI 98.8, 99.3) in this clinical context. Sensitivity was strongly dependent on the amount of virus in clinical samples, as reflected by the PCR cycle threshold (CT) value, with 100% detection in samples where the CT was <20, 96% with CT between 20-25, 89% with CT between 26-30 and 64% when CT was 31-35. ConclusionsThe assay reliably detected 501Y.v2 infections in ambulatory ill patients. Assay sensitivity was >90% in patients with high viral loads who are expected to be most infectious. Negative and positive predictive values were also >90%.

The implementation of a rapid sample preparation method for the detection of SARS-CoV-2 in a diagnostic laboratory in South Africa

The SARS-CoV-2 pandemic has resulted in shortages of both critical reagents for nucleic acid purification and highly trained staff as supply chains are strained by high demand, public health measures and frequent quarantining and isolation of staff. This created the need for alternate workflows with limited reliance on specialised reagents, equipment and staff. We present here the validation and implementation of such a workflow for preparing samples for downstream SARS-CoV-2 RT-PCR using liquid handling robots. The rapid sample preparation technique evaluated, which included sample centrifugation and heating prior to RT-PCR, showed a 97.37% (95% CI: 92.55-99.28%) positive percent agreement and 97.30% (95% CI: 90.67-99.52%) negative percent agreement compared to nucleic acid purification-based testing. This method was subsequently adopted as the primary sample preparation method in the Groote Schuur Hospital Virology Diagnostic Laboratory in Cape Town, South Africa.