SARS-CoV-2 variants that escape neutralization and potentially affect vaccine efficacy have emerged. T cell responses play a role in protection from reinfection and severe disease, but the potential for spike mutations to affect T cell immunity is incompletely understood. We assessed neutralizing antibody and T cell responses in 44 South African COVID-19 patients either infected with the Beta variant (dominant from November 2020 to May 2021) or infected before its emergence (first wave, Wuhan strain) to provide an overall measure of immune evasion. We show that robust spike-specific CD4 and CD8 T cell responses were detectable in Beta-infected patients, similar to first-wave patients. Using peptides spanning the Beta-mutated regions, we identified CD4 T cell responses targeting the wild-type peptides in 12 of 22 first-wave patients, all of whom failed to recognize corresponding Beta-mutated peptides. However, responses to mutated regions formed only a small proportion (15.7%) of the overall CD4 response, and few patients (3 of 44) mounted CD8 responses that targeted the mutated regions. Among the spike epitopes tested, we identified three epitopes containing the D215, L18, or D80 residues that were specifically recognized by CD4 T cells, and their mutated versions were associated with a loss of response. This study shows that despite loss of recognition of immunogenic CD4 epitopes, CD4 and CD8 T cell responses to Beta are preserved overall. These observations may explain why several vaccines have retained the ability to protect against severe COVID-19 even with substantial loss of neutralizing antibody activity against Beta.

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.

The molecular epidemiology of carbapenem-resistant Enterobacterales in Cape Town remains largely unknown. This study aimed to describe the molecular epidemiology, resistome, virulome and mobilome of carbapenem-resistant Klebsiella pneumoniae (CRKP) within Cape Town to guide therapy, antimicrobial stewardship and infection prevention and control practices. Eighty-five CRKP isolates from hospitalized patients underwent WGS as part of a prospective, multicentre, cross-sectional study, conducted between 1 November 2020 and 30 November 2022, across public-sector and private-sector hospitals in Cape Town, South Africa. MLST revealed three novel types, ST6785, ST6786 and ST6787, while the most common were ST219, ST307, ST17, ST13 and ST2497. Different predominant clones were noted in each hospital. The most common carbapenemase gene was bla_OXA-48-like, detected in 71% of isolates, with bla_NDM detected in 5%. Notably, co-detection of two carbapenemase genes (bla_OXA-48-like and bla_NDM) occurred in 13% of isolates. The yersiniabactin siderophore was detected in 73% of isolates, and was most commonly associated with the ICEKp5 mobile element. All carbapenemases were located on plasmids. The genes bla_OXA-181 and bla_OXA-232 colocalized with a ColKP3 replicon type on assembled contigs in 83% and 100% of cases, respectively. CRKP epidemiology in Cape Town reflects institutionally dominant, rather than regional, clones. The most prevalent carbapenemase gene was bla_OXA-48-like, in keeping with CRKP epidemiology in South Africa in general. Emerging clones harbouring both bla_OXA-48-like and bla_NDM, such as ST17, ST2497 and the novel ST6787, are a concern due to the limited availability of appropriate antimicrobial agents in South Africa.

Clinical metagenomics is the diagnostic approach with the broadest capacity to detect both known and novel pathogens. Clinical metagenomics is costly to run and requires infrastructure, but the use of next-generation sequencing for SARS-CoV-2 molecular epidemiology in low-income and middle-income countries (LMICs) offers an opportunity to direct this infrastructure to the establishment of clinical metagenomics programmes. Local implementation of clinical metagenomics is important to create relevant systems and evaluate cost-effective methodologies for its use, as well as to ensure that reference databases and result interpretation tools are appropriate to local epidemiology. Rational implementation, based on the needs of LMICs and the available resources, could ultimately improve individual patient care in instances in which available diagnostics are inadequate and supplement emerging infectious disease surveillance systems to ensure the next pandemic pathogen is quickly identified.

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.

The consequences of falsely reactive HIV test results can be significant, for patients and healthcare providers. This case describes a diagnostic investigation of a patient with pronounced discordant HIV serological results, to determine HIV status. The fourth generation serological screening assay (Roche COBAS Elecsys HIV combiPT) had high positive results but confirmatory testing was negative (Abbott HIV Ag/Ab Combo). Five separate samples over 13 days were tested using multiple fourth generation HIV immunoassays and molecular tests for HIV-1 and HIV-2. Potential causes of falsely reactive serological results were investigated. Samples were sent to the manufacturer for analysis. The screening assay was positive on all samples with a very high signal to cut-off ratio (S/CO) of greater than 400. However, multiple serological and molecular assays did not detect HIV-1 or HIV-2 specific antibodies, antigen or nucleic acid. A recombinant immunochromatographic assay had faint reactivity to gp41 peptide and the manufacturer investigation reported cross-reactivity to one of the screening assay's synthetic peptides. Possible causes of the false positive result include cross reactivity to other antigens, including prior schistosomiasis infection, or the patient's previously excised ameloblastoma (a rare germ cell tumor of the jaw). This is a rare case of false high positive results on fourth-generation HIV serology testing due to high level non-specific reactivity to an isolated synthetic peptide component of the assay. It highlights the need for confirmatory testing even in settings with HIV high prevalence and awareness that false-positive serological results may have a high S/CO.