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.

Varicella-zoster virus reactivation is frequently detected in HIV-infected individuals presenting with stroke.

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.

Reduced amplification efficiency of the RNA-dependent-RNA-polymerase 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 RT-PCR amplification efficiency of the RdRp-gene target of the Seegene, Allplex 2019-nCoV diagnostic assay from June 2021 when detecting the Delta variant. We investigated whether the reduced amplification efficiency denoted by an increased RT-PCR cycle threshold value (RΔE) can be used as an indirect measure of SARS-CoV-2 Delta variant prevalence. We found a significant increase in the median RΔE for patient samples tested from June 2021, which coincided with the emergence of the SARS-CoV-2 Delta variant within our sample set. Whole genome sequencing on a subset of patient samples identified a highly conserved G15451A, non-synonymous mutation exclusively within the RdRp gene of Delta variants, which may cause reduced RT-PCR amplification efficiency. While whole genome sequencing plays an important in identifying novel SARS-CoV-2 variants, monitoring RΔE value can serve as a useful surrogate for rapid tracking of Delta variant prevalence.

MicroRNA-1205 Regulation of FRYL in Prostate Cancer.

High mortality rates of prostate cancer (PCa) are associated with metastatic castration-resistant prostate cancer (CRPC) due to the maintenance of androgen receptor (AR) signaling despite androgen deprivation therapies (ADTs). The 8q24 chromosomal locus is a region of very high PCa susceptibility that carries genetic variants associated with high risk of PCa incidence. This region also carries frequent amplifications of the PVT1 gene, a non-protein coding gene that encodes a cluster of microRNAs including, microRNA-1205 (miR-1205), which are largely understudied. Herein, we demonstrate that miR-1205 is underexpressed in PCa cells and tissues and suppresses CRPC tumors in vivo. To characterize the molecular pathway, we identified and validated fry-like (FRYL) as a direct molecular target of miR-1205 and observed its overexpression in PCa cells and tissues. FRYL is predicted to regulate dendritic branching, which led to the investigation of FRYL in neuroendocrine PCa (NEPC). Resistance toward ADT leads to the progression of treatment related NEPC often characterized by PCa neuroendocrine differentiation (NED), however, this mechanism is poorly understood. Underexpression of miR-1205 is observed when NED is induced in vitro and inhibition of miR-1205 leads to increased expression of NED markers. However, while FRYL is overexpressed during NED, FRYL knockdown did not reduce NED, therefore revealing that miR-1205 induces NED independently of FRYL.

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.

Response to a cluster of Severe Acute Respiratory Syndrome Coronavirus 2 cases at a diagnostic laboratory.

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 SARS-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.

Fibronectin Regulation of Integrin B1 and SLUG in Circulating Tumor Cells.

Metastasis is the leading cause of cancer death worldwide. Circulating tumor cells (CTCs) are a critical step in the metastatic cascade and a good tool to study this process. We isolated CTCs from a syngeneic mouse model of hepatocellular carcinoma (HCC) and a human xenograft mouse model of castration-resistant prostate cancer (CRPC). From these models, novel primary tumor and CTC cell lines were established. CTCs exhibited greater migration than primary tumor-derived cells, as well as epithelial-to-mesenchymal transition (EMT), as observed from decreased E-cadherin and increased SLUG and fibronectin expression. Additionally, when fibronectin was knocked down in CTCs, integrin B1 and SLUG were decreased, indicating regulation of these molecules by fibronectin. Investigation of cell surface molecules and secreted cytokines conferring immunomodulatory advantage to CTCs revealed decreased major histocompatibility complex class I (MHCI) expression and decreased endostatin, C-X-C motif chemokine 5 (CXCL5), and proliferin secretion by CTCs. Taken together, these findings indicate that CTCs exhibit distinct characteristics from primary tumor-derived cells. Furthermore, CTCs demonstrate enhanced migration in part through fibronectin regulation of integrin B1 and SLUG. Further study of CTC biology will likely uncover additional important mechanisms of cancer metastasis.

miR-1207-3p regulates the androgen receptor in prostate cancer via FNDC1/fibronectin.

Prostate cancer (PCa) is frequently diagnosed in men, and dysregulation of microRNAs is characteristic of many cancers. MicroRNA-1207-3p is encoded at the non-protein coding gene locus PVT1 on the 8q24 human chromosomal region, an established PCa susceptibility locus. However, the role of microRNA-1207-3p in PCa is unclear. We discovered that microRNA-1207-3p is significantly underexpressed in PCa cell lines in comparison to normal prostate epithelial cells. Increased expression of microRNA-1207-3p in PCa cells significantly inhibits proliferation, migration, and induces apoptosis via direct molecular targeting of FNDC1, a protein which contains a conserved protein domain of fibronectin (FN1). FNDC1, FN1, and the androgen receptor (AR) are significantly overexpressed in PCa cell lines and human PCa, and positively correlate with aggressive PCa. Prostate tumor FN1 expression in patients that experienced PCa-specific death is significantly higher than in patients that remained alive. Furthermore, FNDC1, FN1 and AR are concomitantly overexpressed in metastatic PCa. Consequently, these studies have revealed a novel microRNA-1207-3p/FNDC1/FN1/AR regulatory pathway in PCa.

A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis.

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of cancer metastasis are still unclear. Animal models are essential for elucidating the mechanisms and for evaluating novel strategies for the treatment of metastatic cancers. Here, an in-depth description of a "patient-like" orthotopic syngeneic mouse model for exploring the mechanisms of metastasis of solid organ tumors is provided. The survival surgical implantation of BNL 1ME A.7R.1 mouse hepatocellular carcinoma cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c is described. The success and reproducibility of this methodology recommends it for widespread use in elucidating the biological mechanisms of solid organ cancer metastasis.

Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model.

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer metastasis. Indeed, the number of CTCs correlates with tumor size. Here, a detailed description is provided of a methodology for isolation and propagation of CTCs from a syngeneic mouse model of hepatocellular carcinoma (HCC) which allows for downstream analysis of potentially important molecular mechanisms of solid organ tumor metastasis. This method is efficient and reproducible. It is a non-invasive technique and, therefore, has potential to replace the invasive biopsy of tissues from humans which may be associated with complications. Therefore, the method discussed here allows for the isolation and propagation of CTCs from whole blood samples such that they can be examined and characterized. This has potential for future adaptation for clinical applications such as diagnosis, and personalized targeted therapy.

Response to a cluster of severe acute respiratory syndrome coronavirus 2 cases at a diagnostic laboratory

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 SARS-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