Investigation and management of an outbreak of COVID-19 infection in an acute admission unit in a District General Hospital: lessons learnt.

BACKGROUND: SARS-CoV-2 outbreaks are difficult to recognise and control due to its high infectivity and the wide range of clinical manifestations of the infection. An outbreak at Watford general hospital provided an opportunity to recognise the complexity involved in a COVID-19 outbreak investigation. METHODS: An outbreak control team (OCT) was convened. The terms outbreak, a case and a significant exposure were defined as per Public Health England (PHE) Guidance and in the context of the local outbreak. Root cause analyses (RCAs) were carried out on cases to identify possible causes, possible route of transmission and any learning points. All contact patients and staff were screened with RT PCR and genomic sequencing was performed on a set of positive specimens.In addition to active contact tracing, screening and cohorting of patients and staff, standard and transmission-based precautions were reinforced to control the outbreak. FINDINGS: Fifteen patients and four staff members were identified in this outbreak investigation. With contact tracing, screening and through strict infection control measures the outbreak was brought under control. CONCLUSION: We could successfully contain the spread of this outbreak following PHE outbreak control guidelines and our local guidelines. We recognised several challenges in investigating a COVID-19 outbreak in a hospital setting. Problems arising from variable sensitivity of the tests, difficulty in differentiating COVID-19 related symptoms from underlying diseases, problems related to establishing the route of transmission, issues with contact tracing are discussed. Additionally, the importance and limitations of genomic studies in COVID-19 are discussed.

Molecular interactions in cancer cell metastasis.

Metastasis, the process by which cancer cells leave the primary tumour, disseminate and form secondary tumours at anatomically distant sites, is a serious clinical problem as it is disseminated disease, which is often impossible to eradicate successfully, that causes the death of most cancer patients. Metastasis results from a complex molecular cascade comprising many steps, all of which are interconnected through a series of adhesive interactions and invasive processes as well as responses to chemotactic stimuli. In spite of its clinical significance, it remains incompletely understood. This review provides an overview of some of the molecular interactions that are critical to metastasis. It summarises the principle molecular players in the major steps of the metastatic cascade. These are: (1) tumour angiogenesis, (2) disaggregation of tumour cells from the primary tumour mass, mediated by cadherins and catenins, (3) invasion of, and migration through, the basement membrane (BM) and extracellular matrix (ECM) surrounding the tumour epithelium, and subsequent invasion of the BM of the endothelium of local blood vessels. This is mediated through integrins and proteases, including urokinase form of plasminogen activator (uPA), matrix metalloproteinases (MMPs) and cathepsins, (4) intravasation of the tumour cells into the blood vessels prior to hematogeneous dissemination to distant sites, (5) adhesion of the circulating tumour cells to the endothelial cell lining at the capillary bed of the target organ site. This occurs through adhesive interactions between cancer cells and endothelial cells involving selectins, integrins and members of the immunoglobulin superfamily (IgSF), (6) invasion of the tumour cells through the endothelial cell layer and surrounding BM (extravasation) and target organ tissue and (7) the development of secondary tumour foci at the target organ site.

Immunolocalisation of members of the polypeptide N-acetylgalactosaminyl transferase (ppGalNAc-T) family is consistent with biologically relevant altered cell surface glycosylation in breast cancer.

An extensive family of UDP-N-alpha-d-galactosamine: polypeptide N-acetylgalactosaminyltransferases (polypeptide N-acetylgalactosaminyltransferases, ppGalNAc-T's) catalyse the attachment of the first N-acetylgalactosamine (GalNAc) monosaccharide to the polypeptide at the initiation of O-linked glycosylation of proteins. Some members of the family are broadly expressed while others are more restricted in their distribution, their expression and activity being confined to certain cells or tissues, being associated with physiological states or differentiation. Their careful regulation, which is not well understood, may mediate the synthesis of varied glycoforms of cellular proteins with different biological activities. Disruptions in glycosylation are a common feature of cancer and may have functional significance. Immunocytochemistry with confocal scanning laser microscopy was employed to detect members of the ppGalNAc-T family, ppGalNAc-T1, -T2, -T3, -T4 and -T6 in a range of breast cell lines. The cells were chosen to represent a range of phenotypes from 'normal'/benign (HMT 3,522), primary, non-metastatic breast cancer (BT 474), to aggressive, metastatic breast cancer (ZR75-1, T47D, MCF-7, DU 4,475). They stably synthesise varying levels, consistent with origin and phenotype, of aberrantly glycosylated glycoproteins featuring exposed, terminal GalNAc residues, including the cancer-associated Tn antigen, which, in numerous studies, have been associated with metastatic competence and poor cancer prognosis. GalNAc-T1 and -T2 were detectable at low levels in all cell lines studied. ppGalNAc-T4, which has never been described in breast, was very weakly detectable in BT 474, MCF7 and T47D. ppGalNAc-T3 and -T6 were weakly detectable or undetectable, respectively, in the cell line HMT 3,522 derived from 'normal'/benign breast epithelium, but were readily detectable in all malignant cell lines. Thus, a broader range of ppGalNAc-T's were detectable in the malignant cell lines in comparison to the 'normal'/benign cells, where only the 'housekeeping' ppGalNAc-T1 and -T2 were present. Expression of normally tightly restricted ppGalNAc-T's may result in initiation of O-linked glycosylation at normally unoccupied potential glycosylation sites leading to altered glycoforms of proteins with changed biological activity which may contribute to the pathogenesis of cancer.

Detection of aberrant glycosylation in breast cancer using lectin histochemistry.

Lectins are naturally occurring, carbohydrate-binding molecules that can be isolated from diverse biological sources and used in the laboratory to investigate the presence of carbohydrate structures in or on cells, in much the same way as antibodies can be used to probe cells and tissues for the presence of specific antigens. As it is becoming increasingly apparent that subtle alterations in the glycosylation of cancer cells can profoundly influence their biological behavior (with consequent implications for patient outcome and prognosis), lectin histochemistry is a potentially useful modification of the more widely used technique of immunohistochemistry. This chapter provides an introductory background to lectins and their use in breast cancer research, and provides basic protocols for lectin histochemistry that highlight the important technical differences between this approach and immunohistochemistry. The methods given here are broadly applicable and can be modified to investigate virtually any glycosylation change of potential interest in breast cancer research.

Fungal heat-shock proteins in human disease.

Heat-shock proteins (hsps) have been identified as molecular chaperones conserved between microbes and man and grouped by their molecular mass and high degree of amino acid homology. This article reviews the major hsps of Saccharomyces cerevisiae, their interactions with trehalose, the effect of fermentation and the role of the heat-shock factor. Information derived from this model, as well as from Neurospora crassa and Achlya ambisexualis, helps in understanding the importance of hsps in the pathogenic fungi, Candida albicans, Cryptococcus neoformans, Aspergillus spp., Histoplasma capsulatum, Paracoccidioides brasiliensis, Trichophyton rubrum, Phycomyces blakesleeanus, Fusarium oxysporum, Coccidioides immitis and Pneumocystis jiroveci. This has been matched with proteomic and genomic information examining hsp expression in response to noxious stimuli. Fungal hsp90 has been identified as a target for immunotherapy by a genetically recombinant antibody. The concept of combining this antibody fragment with an antifungal drug for treating life-threatening fungal infection and the potential interactions with human and microbial hsp90 and nitric oxide is discussed.

Preclinical assessment of the efficacy of mycograb, a human recombinant antibody against fungal HSP90.

Mycograb (NeuTec Pharma plc) is a human genetically recombinant antibody against fungal heat shock protein 90 (HSP90). Antibody to HSP90 is closely associated with recovery in patients with invasive candidiasis who are receiving amphotericin B (AMB). Using in vitro assays developed for efficacy assessment of chemotherapeutic antifungal drugs, Mycograb showed activity against a wide range of yeast species (MICs against Candida albicans [fluconazole [FLC]-sensitive and FLC-resistant strains], Candida krusei, Candida tropicalis, Candida glabrata, and Candida parapsilosis, 128 to 256 microg/ml). Mycograb (4 or 8 microg/ml) showed synergy with AMB, the fractional inhibitory index being 0.09 to 0.31. Synergy was not evident with FLC, except for FLC-sensitive C. albicans. Murine kinetics showed that Mycograb at 2 mg/kg produced a maximum concentration of drug in serum of 4.7 microg/ml, a half-life at alpha phase of 3.75 min, a half-life at beta phase of 2.34 h, and an area under the concentration-time curve from 0 to t h of 155 microg. min/ml. Mycograb (2 mg/kg) alone produced significant improvement in murine candidiasis caused by each species: (i). a reduction (Scheffe's test, P < 0.05) in the mean organ colony count for the FLC-resistant strain of C. albicans (kidney, liver, and spleen), C. krusei (liver and spleen), C. glabrata (liver and spleen), C. tropicalis (kidney), and C. parapsilosis (kidney, liver, and spleen) and (ii). a statistically significant increase in the number of negative biopsy specimens (Fisher's exact test, P < 0.05) for C. glabrata (kidney), C. tropicalis (liver and spleen), and C. parapsilosis (liver). AMB (0.6 mg/kg) alone cleared the C. tropicalis infection but failed to clear infections caused by C. albicans, C. krusei, C. glabrata, or C. parapsilosis. Synergy with AMB, defined as an increase (Fisher's exact test, P < 0.05) in the number of negative biopsy specimens compared with those obtained using AMB alone, occurred with the FLC-resistant strain of C. albicans (kidney), C. krusei (spleen), C. glabrata (spleen), and C. parapsilosis (liver and spleen). Only by combining Mycograb with AMB was complete resolution of infection achieved for C. albicans, C. krusei, and C. glabrata.

Identification of ABC transporters in vancomycin-resistant Enterococcus faecium as potential targets for antibody therapy.

The occurrence of an outbreak of septicaemias due to vancomycin-resistant Enterococcus faecium (VRE), in Manchester, UK, provided an opportunity to examine the antibody responses in patients infected by the same strain. Immunoblotting sera from 24 cases, six of whom died, showed an immunodominant cluster of antigens at 34, 54 and 97 kDa, with a statistically significant correlate between survival and immunoglobulin G to the 34 and 97 kDa bands (P<0.05). Screening a genomic expression library of VRE with seropositive serum and peritoneal dialysate from a survivor gave a recombinant clone with two contiguous open reading frames, the derived amino acid sequences of which both showed sequence homologue with ABC transporters, with a Walker A and Walker B motif and the signature sequence LSGGQ. The first open reading frame (putative VRE ABC1) showed 57% homologue with YbxA from Bacillus subtilis. A partial sequence (putative VRE ABC2) was also obtained, in the same recombinant clone, of a second ABC transporter with 72% homologue with ybaE from B. subtilis. Affinity selection with the seropositive serum and peritoneal dialysate used to screen the library showed that the eluted antibody bound to the 97, 54, 34 and 30 kDa bands. Direct amino acid sequencing identified this as a possible ABC transporter. Rabbit antiserum against peptides representing Walker A and an area adjacent to the Walker B site cross-reacted with bands at 34, 54, 97, 110 kDa and at 30, 34 and 54 kDa respectively. This therefore appeared to be an immunodominant complex of ABC transporters of which the smallest was the 30 kDa antigen. Epitope mapping of this antigen with seropositive patients' sera delineated three linear epitopes (KVGIV, FGPKNF and RVAI). The Walker A site represented by peptide 1 (GHNGSGKSTLAKTIN), epitope RVAI represented by peptides 2 (MRRVAIAGVLAMPRE) and 3 (ELSGGQMRRVAIAGV), epitope KVGIV represented by peptide 4 (LKPIRKKVGIVFQFP), and recombinant VRE ABC1 and VRE ABC2 expressed in Escherichia coli pBAD were then used to isolate human genetically recombinant antibodies from a phage antibody display library. An assessment of the protective potential of these antibodies was carried out in a mouse model of the infection. This study suggests that an ABC transporter homologue could be a target for antibody therapy against VRE infections.