RT-PCR genotyping assays to identify SARS-CoV-2 variants in England in 2021: a design and retrospective evaluation study.

BACKGROUND: Whole-genome sequencing (WGS) is the gold standard diagnostic tool to identify and genetically characterise emerging pathogen mutations (variants), but cost, capacity, and timeliness limit its use when large populations need rapidly assessing. We assessed the potential of genotyping assays to provide accurate and timely variant information at scale by retrospectively examining surveillance for SARS-CoV-2 variants in England between March and September, 2021, when genotyping assays were used widely for variant detection. METHODS: We chose a panel of four RT-PCR genotyping assays to detect circulating variants of SARS-COV-2 in England and developed a decision algorithm to assign a probable SARS-CoV-2 variant to samples using the assay results. We extracted surveillance data from the UK Health Security Agency databases for 115 934 SARS-CoV-2-positive samples (March 1-Sept 6, 2021) when variant information was available from both genotyping and WGS. By comparing the genotyping and WGS variant result, we calculated accuracy metrics (ie, sensitivity, specificity, and positive predictive value [PPV]) and the time difference between the sample collection date and the availability of variant information. We assessed the number of samples with a variant assigned from genotyping or WGS, or both, over time. FINDINGS: Genotyping and an initial decision algorithm (April 10-May 11, 2021 data) were accurate for key variant assignment: sensitivities and PPVs were 0·99 (95% CI 0·99-0·99) for the alpha, 1·00 (1·00-1·00) for the beta, and 0·91 (0·80-1·00) for the gamma variants; specificities were 0·97 (0·96-0·98), 1·00 (1·00-1·00), and 1·00 (1·00-1·00), respectively. A subsequent decision algorithm over a longer time period (May 27-Sept 6, 2021 data) remained accurate for key variant assignment: sensitivities were 0·91 (95% CI 0·74-1·00) for the beta, 0·98 (0·98-0·99) for the delta, and 0·93 (0·81-1·00) for the gamma variants; specificities were 1·00 (1·00-1·00), 0·96 (0·96-0·97), and 1·00 (1·00-1·00), respectively; and PPVs were 0·83 (0·62-1·00), 1·00 (1·00-1·00), and 0·78 (0·59-0·97), respectively. Genotyping produced variant information a median of 3 days (IQR 2-4) after the sample collection date, which was faster than with WGS (9 days [8-11]). The flexibility of genotyping enabled a nine-times increase in the quantity of samples tested for variants by this method (from 5000 to 45 000). INTERPRETATION: RT-PCR genotyping assays are suitable for high-throughput variant surveillance and could complement WGS, enabling larger scale testing for known variants and timelier results, with important implications for effective public health responses and disease control globally, especially in settings with low WGS capacity. However, the choice of panels of RT-PCR assays is highly dependent on database information on circulating variants generated by WGS, which could limit the use of genotyping assays when new variants are emerging and spreading rapidly. FUNDING: UK Health Security Agency and National Institute for Health Research Health Protection Research Unit in Emergency Preparedness and Response.

Surge of lower respiratory tract group A streptococcal infections in England in winter 2022: epidemiology and clinical profile.

BACKGROUND: Following low incidence of invasive group A streptococcal (iGAS) infections during the COVID-19 pandemic, marked increases were noted in many countries during 2022, particularly in children. In November 2022, severe presentations of lower respiratory tract infections (LRTIs), including empyema, were notified by clinicians across the UK. UKHSA investigated this rise with the aim of informing clinical management and public health response. METHODS: We undertook a case-series analysis using multiple routine data sources, exempted from ethics approval or patient consent. We identified iGAS cases in England in children younger than 15 years with an LRTI reported between Oct 1 and Dec 21, 2022, using UKHSA laboratory surveillance data (GAS detected in LRT specimens) and notifications by clinicians and Health Protection Teams (HPTs). Symptoms, diagnoses, health-care interactions, and outcome (death or recovery) data were obtained from HPT case management notes, the National Child Mortality Database, and the NHS Digital Emergency Care Dataset. FINDINGS: We identified 147 cases of LRTI iGAS in children across England (77 [52%] male, 70 [48%] female; median age 4 years [IQR 2-6]). Predominant ethnicities were White (74 [65%] of 113 with known ethnicity) and Asian (18 [16%] of 113). Most reported symptoms were fever (90 [75%] of 120 children with ≥1 symptom) and cough (60 [50%] of 120), and 71 (48%) of all 147 children had a diagnosed respiratory viral coinfection (most commonly hMPV and RSV). 127 (86%) of children attended an emergency department, 31% (n=36/114 with onset date) at least twice within 21 days after symptom onset. 37 (25%) of 147 children died, with a median time from symptom onset to death of 4 days (IQR 3-7). Of 32 children with sample dates, 16 (84%) were tested for GAS on or after the day they died. Over half of deaths (21 [57%] of 37 deaths) occurred in the community after rapid deterioration, of whom 18 had previous contact with health-care services documented. INTERPRETATION: The UK saw an unusual rise in iGAS LRTIs in children in late 2022. One in four cases died, over half in the community. Non-specific symptoms, viral symptoms, or positive virology might have lowered suspicion of bacterial infection. Although the use of multiple available data sources expedited the analysis, varying data completeness limited interpretation. Our study highlights the need for earlier detection and identification of effective measures to prevent death. FUNDING: None.

Infections in travellers returning to the UK: a retrospective analysis (2015-2020).

BACKGROUND: Every year, many thousands of travellers return to the United Kingdom (UK) from visits to other countries and some will become unwell due to infections acquired abroad. Many imported infections have similar clinical presentations, such as fever and myalgia, so diagnostic testing is an important tool to improve patient management and outcomes. The aim of this study was to examine the demographics, travel history, presenting symptoms and diagnostic outcomes of referrals to the UK's specialist diagnostic Rare & Imported Pathogens Laboratory (RIPL) for the period 2015-2020. METHODS: Anonymised clinical and laboratory data were extracted from RIPL's Laboratory Information Management System and cleaned prior to descriptive analysis of the data. Travel history data were mapped to one of eight world regions, whereas symptom data were categorised into presenting syndromes. Diagnostic data were categorised as either positive, equivocal or negative. RESULTS: During the period 2015-2020, RIPL received 73 951 samples from 53 432 patients suspected of having infections that are rare in the UK. The most common age group for unwell returning travellers was 30-39 years and the most commonly reported travel destination was Southern and SE Asia. Dengue virus was the most diagnosed infection overall, followed by chikungunya, Zika, leptospirosis and spotted fever group Rickettsia. Dengue virus was among the top three most frequent diagnoses for all world regions except Europe and represented 62.5% of all confirmed/probable diagnoses. CONCLUSIONS: None of the top five infections diagnosed by RIPL in travellers are vaccine-preventable, therefore understanding traveller demographics, destination-specific risk factors and encouraging preventative behaviours is the best available strategy to reduce the number of returning travellers who become infected. Prompt referral of acute samples with a detailed travel history, including purpose of travel and activities undertaken as well as dates and destinations can be a valuable tool in designing public health interventions and diagnostic algorithms.

Processing and Structure of the Lantibiotic Peptide Nso From the Human Gut Bacterium Blautia obeum A2-162 analysed by Mass Spectrometry.

A previously reported gene cluster encoding four nisin-like peptides, three with the same sequence (NsoA1-3) and the unique NsoA4, produced antimicrobial activity in the presence of trypsin after heterologous expression in Lactococcus lactis. Protein extracts were separated by SDS gel electrophoresis or immunoprecipitation using an antibody to the NsoA2 leader. Tryptic peptides observed by LC-MS/MS covered the complete sequence of preNsoA1-3 and part of the leader sequence of preNsoA4 and confirmed the expression and the predicted sequences of the preNsoA peptides. Further, the data revealed that the preNsoA1-3 peptides were partly modified with dehydrations and formation of lanthionine rings. A certain amount of fully modified preNsoA1-3 was observed. Details of modifications of the core peptide and the C-terminal tryptic peptide TATCGCHITGK covering rings D and E indicated that 22% of these preNsoA1-3 peptides were completely modified. A lower amount of ring formation is estimated for rings A-C. Intact masses of immunoprecipitation-derived peptides determined by LC-MS accurately matched the expected preNsoA precursor peptides. The most abundant peptides detected were preNsoA2-3-8H2O followed by preNsoA1-8H2O and other states of dehydration. The results confirm incomplete processing of preNsoA peptides in the heterologous system, with the formation of a certain amount of fully modified peptides.

Chloride Intracellular Channel 4 Overexpression in the Proximal Tubules of Kidneys from the Spontaneously Hypertensive Rat: Insight from Proteomic Analysis.

BACKGROUND: Hypertensive nephropathy, a leading cause of declining kidney function, is a multifactorial process not well understood. In order to elucidate biological processes and identify novel macromolecular components crucially involved in the process of kidney damage, the application of system biology approaches, like proteomics, is required. METHODS: Proteomic studies were performed using the renal parenchyma of spontaneously hypertensive rats (SHR) and their normotensive Wistar Kyoto controls. Animals were sacrificed at early time intervals (6, 13, and 20 weeks after birth), the renal tissue extract was subjected to two-dimensional gel electrophoresis, differential expressed proteins were identified, and altered pathways were evaluated. One specific protein, chloride intracellular channel 4 (CLIC4), not implicated so far in the development of hypertension and nephrosclerosis, was further studied by Western blotting, immunohistochemistry and immunofluorescence. RESULTS: Proteomic analysis identified several pathways/processes and organelles (mitochondria) as being affected from the early stages of hypertension. CLIC4 was overexpressed in SHR at all 3 time intervals examined. This finding was confirmed by Western blotting and by immunohistochemistry and immunofluorescence; these morphological techniques demonstrated that CLIC4 was almost exclusively localized at the apical surface of the proximal tubular epithelial cells. CONCLUSIONS: Our studies provide evidence that major changes occur in the renal parenchyma from early stages of the development of hypertension. The overexpression of CLIC4 suggests that alterations in the proximal tubular compartment during hypertension should be further examined and that CLIC4 may be a useful early marker of renal tubular alterations due to elevated blood pressure.

Discovery of a novel lantibiotic nisin O from Blautia obeum A2-162, isolated from the human gastrointestinal tract.

A novel lanC-like sequence was identified from the dominant human gut bacterium Blautia obeum strain A2-162. This sequence was extended to reveal a putative lantibiotic operon with biosynthetic and transport genes, two sets of regulatory genes, immunity genes, three identical copies of a nisin-like lanA gene with an unusual leader peptide, and a fourth putative lanA gene. Comparison with other nisin clusters showed that the closest relationship was to nisin U. B. obeum A2-162 demonstrated antimicrobial activity against Clostridium perfringens when grown on solid medium in the presence of trypsin. Fusions of predicted nsoA structural sequences with the nisin A leader were expressed in Lactococcus lactis containing the nisin A operon without nisA. Expression of the nisA leader sequence fused to the predicted structural nsoA1 produced a growth defect in L. lactis that was dependent upon the presence of biosynthetic genes, but failed to produce antimicrobial activity. Insertion of the nso cluster into L. lactis MG1614 gave an increased immunity to nisin A, but this was not replicated by the expression of nsoI. Nisin A induction of L. lactis containing the nso cluster and nisRK genes allowed detection of the NsoA1 pre-peptide by Western hybridization. When this heterologous producer was grown with nisin induction on solid medium, antimicrobial activity was demonstrated in the presence of trypsin against C. perfringens, Clostridium difficile and L. lactis. This research adds to evidence that lantibiotic production may be an important trait of gut bacteria and could lead to the development of novel treatments for intestinal diseases.

A representative of the dominant human colonic Firmicutes, Roseburia faecis M72/1, forms a novel bacteriocin-like substance.

During screening of human gut isolates in search of novel antimicrobials, the butyrate-producing strain Roseburia faecis M72/1 was found to produce an inhibitory substance active against Bacillus subtilis. Partial purification of the antimicrobial was achieved and activity found to be heat labile. Our findings suggest that R. faecis M72/1 produces a proteinaceous inhibitor whose production may be triggered by trypsin in the gastrointestinal tract.