Gert Marais

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.

Carbapenem-resistant Enterobacterales (CRE) are a substantial problem in Cape Town. CRE epidemiology is largely unknown and mortality remains high. To describe and characterize the clinical and microbiological epidemiology of CRE within Cape Town hospitals to better inform therapy with regard to current and novel antibiotics, as well as improve antimicrobial stewardship (AMS), and infection prevention and control (IPC). This prospective, multicentre study performed between 1 November 2020 and 30 November 2022, across three public and three private hospitals included hospitalized participants with CRE from clinical cultures. Participant demographics, clinical information and microbiology results were collected and analysed. Ninety percent of participants were from public hospitals. The age distribution ranged from 7 days to 88 years. Notable risk factors for CRE infection included recent exposure to antibiotics, medical devices and surgery. The most prevalent species was Klebsiella pneumoniae. However, a higher proportion of Serratia marcescens compared with previous reports was identified. The detected carbapenemases were bla_OXA-48-like (80%) and bla_NDM (11%). With the exception of amikacin (63%), tigecycline (65%), colistin (95%) and ceftazidime/avibactam (87%), susceptibility to antibiotics was low. This study identified common risk factors for CRE infection and generated a description of carbapenemase enzymes, species distribution and antibiograms, enabling a better understanding of CRE epidemiology. This provides insights into transmission patterns and resistance determinants of CREs, beneficial to informing data-driven regional patient management, AMS and IPC strategies.

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.

Infections are an underappreciated cause of stroke, particularly in young and immunocompromised individuals. Varicella‐zoster virus (VZV) reactivation, particularly ophthalmic zoster, has been linked to increased risk of stroke but diagnosing VZV‐associated cerebral vasculopathy is challenging as neither a recent zoster rash, nor detectable levels of VZV DNA are universally present at stroke presentation. Detection of VZV IgG in cerebrospinal fluid (CSF‐VZVG) presents a promising alternative, but requires evaluation of individual blood‐CSF dynamics, particularly in the setting of chronic inflammatory states such as HIV infection. Consequently, its use has not been broadly adopted as simple diagnostic algorithms are not available. In this study looking at young adults presenting with acute stroke, we used an algorithm that includes testing for both VZV nucleic acids and CSF‐VZVG which was corrected for blood‐CSF barrier dynamics and poly‐specific immune activation. We found that 13 of 35 (37%), including 7 with a positive CSF VZV PCR, young HIV‐infected adults presenting with stroke, 3 of 34 (9%) young HIV‐uninfected adults presenting with stroke, and 1 of 18 (6%) HIV‐infected nonstroke controls demonstrated evidence of central nervous system reactivation of VZV.

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 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.

Introduction: We report on the first documented cluster of Coronavirus Disease 2019 cases amongst diagnostic laboratory staff and outline some of the initial and ongoing steps that are being implemented to manage and prevent the spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in our laboratory. Case presentation: On 24 April 2020, three staff members of a tertiary diagnostic laboratory in Groote Schuur Hospital, Cape Town, South Africa, tested positive for SA RS-CoV-2. Within seven days, a further nine cases were identified, which suggested an outbreak and prompted a full investigation. Management and outcome: A multifaceted strategic approach was adopted to halt the spread of SARS-CoV-2 in our laboratory. Interventions focused on simultaneously establishing appropriate risk mitigation and stratification strategies through the upscaling of infection prevention and control measures, whilst minimising disruption to service delivery. Conclusion: Laboratory Coronavirus Disease 2019 outbreaks have the potential to cripple a laboratory's testing capacity. Contingency planning and risk assessments should occur early, and interventions should be modified according to each laboratory's available resources and infrastructure.

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.