Changes to an intensive care unit Acinetobacter baumannii population following COVID-19 disruptions and targeted infection prevention interventions. (preprint)

Carbapenem-resistant Acinetobacter baumannii (CRAB) is a persistent nosocomial pathogen that poses a significant threat to global public health. A three-month cross-sectional observational study was conducted in a 28-bed ICU in Hangzhou, China. The same ICU was sampled for the same duration and with a similar methodology in 2019, 20 months prior to the outset of this study. Following COVID-19-associated delays, a series of IPC measures targeting patients, staff and the ICU environment were implemented for 8 months prior to and throughout this study. A total of 5,341 samples were collected from the ICU environment (n = 4450) and patients (n = 891). A. baumannii isolates were obtained from 9.5% of these samples (n = 505). Most A. baumannii isolated in this ICU were CRAB (419/518; 80.9%). Fewer CRAB were isolated here (407 from 363 sampling occasions) than in 2019 (502 from 336 sampling occasions). However, MIC50/MIC90 values for imipenem increased from 32/64 mg/L in the 2019 study to 64/128 mg/L here. This was accompanied by the proportion of global clone 2 (GC2) isolates falling from 99.5% in 2019 to 50.8% (213/419) in 2021. The phylogenetic diversity of GC2 increased, apparently driven by regular introductions of distinct clusters in association with patients. The remaining CRAB (40.2%; 206/419) were a highly clonal population of ST164, which appears to have persisted in the ICU since an introduction in mid-2020. We found clusters of GC2 and ST164 isolates with identical core genomes in different room or bed unit environments, and in multiple patients, indicative of transmission in the ICU. Changes to IPC procedures in this ICU were associated with a reduction in the total prevalence of CRAB, and in the number of CRAB isolated from clinical specimens. At the phenotypic level, the CRAB population exhibited increased resistance to carbapenems, and this may be the result of increased antibiotic prescribing over the COVID-19 period. The increased diversity of this CRAB population appears to have been the result of repeated introductions to the ICU with patients, which have continued despite interventions.

SARS-CoV-2 testing in the community: Testing positive samples with the TaqMan SARS-CoV-2 Mutation Panel to find variants in real-time (preprint)

Genome sequencing is a powerful tool for identifying SARS-CoV-2 variant lineages, however there can be limitations due to sequence drop-out when used to identify specific key mutations. Recently, Thermo Fisher Scientific have developed genotyping assays to help bridge the gap between testing capacity and sequencing capability to generate real-time genotyping results based on specific variants. Over a 6-week period during the months of April and May 2021, we set out to assess the Thermo Fisher TaqMan Mutation Panel Genotyping Assay, initially for three mutations of concern and then an additional two mutations of concern, against SARS-CoV-2 positive clinical samples and the corresponding COG-UK sequencing data. We demonstrate that genotyping is a powerful in-depth technique for identifying specific mutations, an excellent complement to genome sequencing and has real clinical health value potential allowing laboratories to report and action variants of concern much quicker.

The mechanism of SARS-CoV-2 nucleocapsid protein recognition by the human 14-3-3 proteins (preprint)

The coronavirus nucleocapsid protein (N) controls viral genome packaging and contains numerous phosphorylation sites located within unstructured regions. Phosphorylated SARS-CoV N was shown to bind to the host 14-3-3 protein in the cytoplasm. Proteomic data indicate that seven human 14-3-3 proteins are highly abundant in human tissues vulnerable to SARS-CoV-2 infection, collectively reaching ~1.8% of all proteins in the lungs, ~1.4% in the gastrointestinal system, ~2.3% in the nervous system. Although the association between 14-3-3 and SARS-CoV-2 N proteins can represent one of the key host-pathogen interactions, its mechanism and the specific critical phosphosites were unknown. Here, we show that phosphorylated SARS-CoV-2 N protein (pN) dimers, reconstituted via bacterial co-expression with protein kinase A, directly associate, in a phosphorylation-dependent manner, with the dimeric 14-3-3 protein hub, but not with its monomeric mutant. We demonstrate that pN is recognized by all seven human 14-3-3 isoforms with various efficiencies and determine the apparent KD to selected isoforms in a low micromolar range. Serial truncations pinpointed a critical phosphorylation site to Ser197, located within the SR-rich region of N. The tight 14-3-3/pN association suggests it could regulate nucleocytoplasmic shuttling of N, while hijacking cellular pathways by 14-3-3 sequestration. As such, the assembly may represent a valuable target for therapeutic intervention.

S-variant SARS-CoV-2 is associated with significantly higher viral loads in samples tested by ThermoFisher TaqPath RT-QPCR (preprint)

Birmingham University Turnkey laboratory is part of the Lighthouse network responsible for testing clinical samples under the UK government ‘ Test & Trace’ scheme. Samples are analysed for the presence of SARS-CoV-2 in respiratory samples using the Thermofisher TaqPath RT-QPCR test, which is designed to co-amplify sections of three SARS-CoV-2 viral genes. Since more recent information became available regarding the presence of SARS-CoV-2 variants of concern (S-VoC), which can show a suboptimal profile in RT-QPCR tests such as the ThermoFisher TaqPath used at the majority of Lighthouse laboratories, we analysed recently published data for trends and significance of the S-gene ‘dropout’ variant. Results: showed that: the population of S-gene dropout samples had significantly lower median Ct values of ORF and N-gene targets compared to samples where S-gene was detected on a population basis, S-gene dropout samples clustered around very low Ct values for ORF and N targets linked Ct values for individual samples showed that a low Ct for ORF and N were clearly associated with an S-dropout characteristic when conservatively inferring relative viral load from Ct values, approximately 35% of S-dropout samples had high viral loads between 10 and 10,000-fold greater than 1 × 10 6 , compared to 10% of S-positive samples. This analysis suggests that patients whose samples exhibit the S-dropout profile in the TaqPath test are more likely to have high viral loads at the time of sampling. The relevance of this to epidemiological reports of fast spread of the SARS-CoV-2 in regions of the UK is discussed.

Disruption of nasal bacteria enhances protective immune responses to influenza A virus and SARS-CoV-2 infection in mice (preprint)

Gut microbiota plays a critical role in the induction of adaptive immune responses to influenza virus infection. However, the role of nasal bacteria in the induction of the virus-specific adaptive immunity is less clear. Here we demonstrate that while intranasal administration of influenza virus hemagglutinin vaccine alone was insufficient to induce the vaccine-specific antibody responses, disruption of nasal bacteria by lysozyme or addition of culturable oral bacteria from a healthy human volunteer rescued inability of the nasal bacteria to generate antibody responses to intranasally administered the split-virus vaccine. Myd88-depdnent signaling in the hematopoietic compartment was required for adjuvant activity of intranasally administered oral bacteria. In addition, we found that the oral bacteria-combined intranasal vaccine induced protective antibody response to influenza virus and SARS-CoV-2 infection. Our findings here have identified a previously unappreciated role for nasal bacteria in the induction of the virus-specific adaptive immune responses.

Extrapolation of United Kingdom Pillar 2 Care home Covid-19 test data to ascertain effectiveness of lateral flow testing in low prevalence settings (preprint)

Lateral flow devices are quickly being implemented for use in large scale population surveillance programs for SARS-CoV-2 infection in the United Kingdom. These programs have been piloted in city wide screening in the city of Liverpool, and are now being rolled out to support care home visits and the return home of University students for the Christmas break. Very little data exists comparing the performance of the UK lateral flow tests with gold standard PCR diagnostics, especially against comparable test populations such as the national Pillar 2 testing program in the United Kingdom. Here we utilise thousands of pillar 2 test data from our University of Birmingham test lab, and by extrapolation against the validate limit-of-detection of the lateral flow assay, provide a potential sensitivity for the test in a comparable low prevalence population captured in the pillar 2 program. Our data suggests the lateral flow assay should successfully capture around 85% of all PCR positive tests performed in our pillar 2 laboratory, and that a fully designed comparative study of lateral flow versus PCR testing is merited in a real life testing environment