Bacterial surface lipoproteins mediate epithelial microinvasion by Streptococcus pneumoniae.

Streptococcus pneumoniae, a common colonizer of the upper respiratory tract, invades nasopharyngeal epithelial cells without causing disease in healthy participants of controlled human infection studies. We hypothesized that surface expression of pneumococcal lipoproteins, recognized by the innate immune receptor TLR2, mediates epithelial microinvasion. Mutation of lgt in serotype 4 (TIGR4) and serotype 6B (BHN418) pneumococcal strains abolishes the ability of the mutants to activate TLR2 signaling. Loss of lgt also led to the concomitant decrease in interferon signaling triggered by the bacterium. However, only BHN418 lgt::cm but not TIGR4 lgt::cm was significantly attenuated in epithelial adherence and microinvasion compared to their respective wild-type strains. To test the hypothesis that differential lipoprotein repertoires in TIGR4 and BHN418 lead to the intraspecies variation in epithelial microinvasion, we employed a motif-based genome analysis and identified an additional 525 a.a. lipoprotein (pneumococcal accessory lipoprotein A; palA) encoded by BHN418 that is absent in TIGR4. The gene encoding palA sits within a putative genetic island present in ~10% of global pneumococcal isolates. While palA was enriched in the carriage and otitis media pneumococcal strains, neither mutation nor overexpression of the gene encoding this lipoprotein significantly changed microinvasion patterns. In conclusion, mutation of lgt attenuates epithelial inflammatory responses during pneumococcal-epithelial interactions, with intraspecies variation in the effect on microinvasion. Differential lipoprotein repertoires encoded by the different strains do not explain these differences in microinvasion. Rather, we postulate that post-translational modifications of lipoproteins may account for the differences in microinvasion.IMPORTANCEStreptococcus pneumoniae (pneumococcus) is an important mucosal pathogen, estimated to cause over 500,000 deaths annually. Nasopharyngeal colonization is considered a necessary prerequisite for disease, yet many people are transiently and asymptomatically colonized by pneumococci without becoming unwell. It is therefore important to better understand how the colonization process is controlled at the epithelial surface. Controlled human infection studies revealed the presence of pneumococci within the epithelium of healthy volunteers (microinvasion). In this study, we focused on the regulation of epithelial microinvasion by pneumococcal lipoproteins. We found that pneumococcal lipoproteins induce epithelial inflammation but that differing lipoprotein repertoires do not significantly impact the magnitude of microinvasion. Targeting mucosal innate immunity and epithelial microinvasion alongside the induction of an adaptive immune response may be effective in preventing pneumococcal colonization and disease.

Tuberculosis-Associated Hemophagocytic Lymphohistiocytosis: Diagnostic Challenges and Determinants of Outcome.

Background: Tuberculosis (TB) can induce secondary hemophagocytic lymphohistiocytosis (HLH), a severe inflammatory syndrome with high mortality. We integrated all published reports of adult HIV-negative TB-associated HLH (TB-HLH) to define clinical characteristics, diagnostic strategies, and therapeutic approaches associated with improved survival. Methods: PubMed, Embase, and Global Index Medicus were searched for eligible records. TB-HLH cases were categorized into (1) patients with a confirmed TB diagnosis receiving antituberculosis treatment while developing HLH and (2) patients presenting with HLH of unknown cause later diagnosed with TB. We used a logistic regression model to define clinical and diagnostic parameters associated with survival. Results: We identified 115 individual cases, 45 (39.1%) from countries with low TB incidence (<10/100 000 per year). When compared with patients with HLH and known TB (n = 21), patients with HLH of unknown cause (n = 94) more often had extrapulmonary TB (66.7% vs 88.3%), while the opposite was true for pulmonary disease (91.5% vs 59.6%). Overall, Mycobacterium tuberculosis was identified in the bone marrow in 78.4% of patients for whom examination was reported (n = 74). Only 10.5% (4/38) of patients tested had a positive result upon a tuberculin skin test or interferon-γ release assay. In-hospital mortality was 28.1% (27/96) in those treated for TB and 100% (18/18) in those who did not receive antituberculosis treatment (P < .001). Conclusions: Tuberculosis should be considered a cause of unexplained HLH. TB-HLH is likely underreported, and the diagnostic workup of patients with HLH should include bone marrow investigations for evidence of Mycobacerium tuberculosis. Prompt initiation of antituberculosis treatment likely improves survival in TB-HLH.

Dysfunctional effector memory CD8 T cells in the bronchoalveolar compartment of people living with HIV.

Mechanisms by which HIV causes susceptibility to respiratory pathogens remain incompletely understood. We obtained whole blood and bronchoalveolar lavage (BAL) from people with latent TB infection in the presence or absence of antiretroviral-naïve HIV co-infection. Transcriptomic and flow cytometric analyses demonstrated HIV-associated cell proliferation plus type I interferon activity in blood and effector memory CD8 T-cells in BAL. Both compartments displayed reduced induction of CD8 T-cell-derived IL-17A in people with HIV, associated with elevated T-cell regulatory molecule expression. The data suggest that dysfunctional CD8 T-cell responses in uncontrolled HIV contribute to susceptibility to secondary bacterial infections, including tuberculosis.

T2Candida assay: diagnostic performance and impact on antifungal prescribing.

Objectives: To assess the performance of T2Candida for the diagnosis of invasive candidiasis (IC) against gold standards of candidaemia or consensus IC definitions, and to evaluate the impact of T2Candida on antifungal drug prescribing. Methods: A retrospective review was undertaken of all T2Candida (T2MR technology, T2 Biosystems) performed from October 2020 to February 2022. T2Candida performance was evaluated against confirmed candidaemia or against proven/probable IC within 48 hours of T2Candida, and its impact on antifungal drug prescriptions. Results: T2Candida was performed in 61 patients, with 6 (9.8%) positive results. Diagnostic performance of T2Candida against candidaemia had a specificity of 85.7% and negative predictive value (NPV) of 96.8%. When comparing T2Candida results with consensus definitions of IC, the specificity and NPV of T2Candida was respectively 90% (54/60) and 98.2% (54/55) for proven IC, and 91.4% (53/58) and 96.4% (53/55) for proven/probable IC. Antifungals were initiated in three of six patients (50%) with a positive T2Candida result. Thirty-three patients were receiving empirical antifungals at the time of T2Candida testing, and a negative result prompted cessation of antifungals in 11 (33%) patients, compared with 6 (25%) antifungal prescriptions stopped following negative beta-d-glucan (BDG) testing in a control population (n = 24). Conclusions: T2Candida shows high specificity and NPV compared with evidence of Candida bloodstream infection or consensus definitions for invasive Candida infection, and may play an adjunctive role as a stewardship tool to limit unnecessary antifungal prescriptions.

Genomic investigations of unexplained acute hepatitis in children.

Since its first identification in Scotland, over 1,000 cases of unexplained paediatric hepatitis in children have been reported worldwide, including 278 cases in the UK1. Here we report an investigation of 38 cases, 66 age-matched immunocompetent controls and 21 immunocompromised comparator participants, using a combination of genomic, transcriptomic, proteomic and immunohistochemical methods. We detected high levels of adeno-associated virus 2 (AAV2) DNA in the liver, blood, plasma or stool from 27 of 28 cases. We found low levels of adenovirus (HAdV) and human herpesvirus 6B (HHV-6B) in 23 of 31 and 16 of 23, respectively, of the cases tested. By contrast, AAV2 was infrequently detected and at low titre in the blood or the liver from control children with HAdV, even when profoundly immunosuppressed. AAV2, HAdV and HHV-6 phylogeny excluded the emergence of novel strains in cases. Histological analyses of explanted livers showed enrichment for T cells and B lineage cells. Proteomic comparison of liver tissue from cases and healthy controls identified increased expression of HLA class 2, immunoglobulin variable regions and complement proteins. HAdV and AAV2 proteins were not detected in the livers. Instead, we identified AAV2 DNA complexes reflecting both HAdV-mediated and HHV-6B-mediated replication. We hypothesize that high levels of abnormal AAV2 replication products aided by HAdV and, in severe cases, HHV-6B may have triggered immune-mediated hepatic disease in genetically and immunologically predisposed children.

Altered IL-6 signalling and risk of tuberculosis disease: a meta-analysis and Mendelian randomisation study.

IL-6 responses are ubiquitous in Mycobacterium tuberculosis (Mtb) infections, but their role in determining human tuberculosis (TB) disease risk is unknown. We used single nucleotide polymorphisms (SNPs) in and near the IL-6 receptor (IL6R) gene, focusing on the non-synonymous variant, rs2228145, associated with reduced classical IL-6 signalling, to assess the effect of altered IL-6 activity on TB disease risk. We identified 16 genome wide association studies (GWAS) of TB disease collating 17,982 cases of TB disease and 972,389 controls across 4 continents. Meta-analyses and Mendelian randomisation analyses revealed that reduced classical IL-6 signalling was associated with lower odds of TB disease, a finding replicated using multiple, independent SNP instruments and 2 separate exposure variables. Our findings establish a causal relationship between IL-6 signalling and the outcome of Mtb infection, suggesting IL-6 antagonists do not increase the risk of TB disease and should be investigated as adjuncts in treatment.

Rapid synchronous type 1 IFN and virus-specific T cell responses characterize first wave non-severe SARS-CoV-2 infections.

Effective control of SARS-CoV-2 infection on primary exposure may reveal correlates of protective immunity to future variants, but we lack insights into immune responses before or at the time virus is first detected. We use blood transcriptomics, multiparameter flow cytometry, and T cell receptor (TCR) sequencing spanning the time of incident non-severe infection in unvaccinated virus-naive individuals to identify rapid type 1 interferon (IFN) responses common to other acute respiratory viruses and cell proliferation responses that discriminate SARS-CoV-2 from other viruses. These peak by the time the virus is first detected and sometimes precede virus detection. Cell proliferation is most evident in CD8 T cells and associated with specific expansion of SARS-CoV-2-reactive TCRs, in contrast to virus-specific antibodies, which lag by 1-2 weeks. Our data support a protective role for early type 1 IFN and CD8 T cell responses, with implications for development of universal T cell vaccines.

C-reactive protein-guided use of procalcitonin in COVID-19.

BACKGROUND: A low procalcitonin (PCT) concentration facilitates exclusion of bacterial co-infections in COVID-19, but high costs associated with PCT measurements preclude universal adoption. Changes in inflammatory markers, including C-reactive protein (CRP), can be concordant, and predicting low PCT concentrations may avoid costs of redundant tests and support more cost-effective deployment of this diagnostic biomarker. OBJECTIVES: To explore whether, in COVID-19, low PCT values could be predicted by the presence of low CRP concentrations. METHODS: Unselected cohort of 224 COVID-19 patients admitted to hospital that underwent daily PCT and CRP measurements as standard care. Both 0.25 ng/mL and 0.5 ng/mL were used as cut-offs for positive PCT test results. Geometric mean was used to define high and low CRP values at each timepoint assessed. RESULTS: Admission PCT was <0.25 ng/mL in 160/224 (71.4%), 0.25-0.5 ng/mL in 27 (12.0%) and >0.5 ng/mL in 37 (16.5%). Elevated PCT was associated with increased risk of death (P = 0.0004) and was more commonly associated with microbiological evidence of bacterial co-infection (P < 0.0001). For high CRP values, significant heterogeneity in PCT measurements was observed, with maximal positive predictive value of 50% even for a PCT cut-off of 0.25 ng/mL. In contrast, low CRP was strongly predictive of low PCT concentrations, particularly <0.5 ng/mL, with a negative predictive value of 97.6% at time of hospital admission and 100% 48 hours into hospital stay. CONCLUSIONS: CRP-guided PCT testing algorithms can reduce unnecessary PCT measurement and costs, supporting antimicrobial stewardship strategies in COVID-19.

Blood transcriptional biomarkers of acute viral infection for detection of pre-symptomatic SARS-CoV-2 infection: a nested, case-control diagnostic accuracy study.

BACKGROUND: We hypothesised that host-response biomarkers of viral infections might contribute to early identification of individuals infected with SARS-CoV-2, which is critical to breaking the chains of transmission. We aimed to evaluate the diagnostic accuracy of existing candidate whole-blood transcriptomic signatures for viral infection to predict positivity of nasopharyngeal SARS-CoV-2 PCR testing. METHODS: We did a nested case-control diagnostic accuracy study among a prospective cohort of health-care workers (aged ≥18 years) at St Bartholomew's Hospital (London, UK) undergoing weekly blood and nasopharyngeal swab sampling for whole-blood RNA sequencing and SARS-CoV-2 PCR testing, when fit to attend work. We identified candidate blood transcriptomic signatures for viral infection through a systematic literature search. We searched MEDLINE for articles published between database inception and Oct 12, 2020, using comprehensive MeSH and keyword terms for "viral infection", "transcriptome", "biomarker", and "blood". We reconstructed signature scores in blood RNA sequencing data and evaluated their diagnostic accuracy for contemporaneous SARS-CoV-2 infection, compared with the gold standard of SARS-CoV-2 PCR testing, by quantifying the area under the receiver operating characteristic curve (AUROC), sensitivities, and specificities at a standardised Z score of at least 2 based on the distribution of signature scores in test-negative controls. We used pairwise DeLong tests compared with the most discriminating signature to identify the subset of best performing biomarkers. We evaluated associations between signature expression, viral load (using PCR cycle thresholds), and symptom status visually and using Spearman rank correlation. The primary outcome was the AUROC for discriminating between samples from participants who tested negative throughout the study (test-negative controls) and samples from participants with PCR-confirmed SARS-CoV-2 infection (test-positive participants) during their first week of PCR positivity. FINDINGS: We identified 20 candidate blood transcriptomic signatures of viral infection from 18 studies and evaluated their accuracy among 169 blood RNA samples from 96 participants over 24 weeks. Participants were recruited between March 23 and March 31, 2020. 114 samples were from 41 participants with SARS-CoV-2 infection, and 55 samples were from 55 test-negative controls. The median age of participants was 36 years (IQR 27-47) and 69 (72%) of 96 were women. Signatures had little overlap of component genes, but were mostly correlated as components of type I interferon responses. A single blood transcript for IFI27 provided the highest accuracy for discriminating between test-negative controls and test-positive individuals at the time of their first positive SARS-CoV-2 PCR result, with AUROC of 0·95 (95% CI 0·91-0·99), sensitivity 0·84 (0·70-0·93), and specificity 0·95 (0·85-0·98) at a predefined threshold (Z score >2). The transcript performed equally well in individuals with and without symptoms. Three other candidate signatures (including two to 48 transcripts) had statistically equivalent discrimination to IFI27 (AUROCs 0·91-0·95). INTERPRETATION: Our findings support further urgent evaluation and development of blood IFI27 transcripts as a biomarker for early phase SARS-CoV-2 infection for screening individuals at high risk of infection, such as contacts of index cases, to facilitate early case isolation and early use of antiviral treatments as they emerge. FUNDING: Barts Charity, Wellcome Trust, and National Institute of Health Research.

Exaggerated IL-17A activity in human in vivo recall responses discriminates active tuberculosis from latent infection and cured disease.

Host immune responses at the site of Mycobacterium tuberculosis infection can mediate pathogenesis of tuberculosis (TB) and onward transmission of infection. We hypothesized that pathological immune responses would be enriched at the site of host-pathogen interactions modeled by a standardized tuberculin skin test (TST) challenge in patients with active TB compared to those without disease, and interrogated immune responses by genome-wide transcriptional profiling. We show exaggerated interleukin-17A (IL-17A) and T helper 17 (TH17) responses among 48 individuals with active TB compared to 191 with latent TB infection, associated with increased neutrophil recruitment and matrix metalloproteinase-1 expression, both involved in TB pathogenesis. Curative antimicrobial treatment reversed these observed changes. Increased IL-1ß and IL-6 responses to mycobacterial stimulation were evident both in circulating monocytes and in molecular changes at the site of TST in individuals with active TB, supporting a model in which monocyte-derived IL-1ß and IL-6 promote TH17 differentiation within tissues. Modulation of these cytokine pathways may provide a rational strategy for host-directed therapy in active TB.

Persistent T Cell Repertoire Perturbation and T Cell Activation in HIV After Long Term Treatment.

Objective: In people living with HIV (PLHIV), we sought to test the hypothesis that long term anti-retroviral therapy restores the normal T cell repertoire, and investigate the functional relationship of residual repertoire abnormalities to persistent immune system dysregulation. Methods: We conducted a case-control study in PLHIV and HIV-negative volunteers, of circulating T cell receptor repertoires and whole blood transcriptomes by RNA sequencing, complemented by metadata from routinely collected health care records. Results: T cell receptor sequencing revealed persistent abnormalities in the clonal T cell repertoire of PLHIV, characterized by reduced repertoire diversity and oligoclonal T cell expansion correlated with elevated CD8 T cell counts. We found no evidence that these expansions were driven by cytomegalovirus or another common antigen. Increased frequency of long CDR3 sequences and reduced frequency of public sequences among the expanded clones implicated abnormal thymic selection as a contributing factor. These abnormalities in the repertoire correlated with systems level evidence of persistent T cell activation in genome-wide blood transcriptomes. Conclusions: The diversity of T cell receptor repertoires in PLHIV on long term anti-retroviral therapy remains significantly depleted, and skewed by idiosyncratic clones, partly attributable to altered thymic output and associated with T cell mediated chronic immune activation. Further investigation of thymic function and the antigenic drivers of T cell clonal selection in PLHIV are critical to efforts to fully re-establish normal immune function.

Exclusion of bacterial co-infection in COVID-19 using baseline inflammatory markers and their response to antibiotics.

BACKGROUND: COVID-19 is infrequently complicated by bacterial co-infection, but antibiotic prescriptions are common. We used community-acquired pneumonia (CAP) as a benchmark to define the processes that occur in bacterial pulmonary infections, testing the hypothesis that baseline inflammatory markers and their response to antibiotic therapy could distinguish bacterial co-infection from COVID-19. METHODS: Retrospective cohort study of CAP (lobar consolidation on chest radiograph) and COVID-19 (PCR detection of SARS-CoV-2) patients admitted to Royal Free Hospital (RFH) and Barnet Hospital (BH), serving as independent discovery and validation cohorts. All CAP and >90% COVID-19 patients received antibiotics on hospital admission. RESULTS: We identified 106 CAP and 619 COVID-19 patients at RFH. Compared with COVID-19, CAP was characterized by elevated baseline white cell count (WCC) [median 12.48 (IQR 8.2-15.3) versus 6.78 (IQR 5.2-9.5) ×106 cells/mL, P < 0.0001], C-reactive protein (CRP) [median 133.5 (IQR 65-221) versus 86.0 (IQR 42-160) mg/L, P < 0.0001], and greater reduction in CRP 48-72 h into admission [median ΔCRP -33 (IQR -112 to +3.5) versus +14 (IQR -15.5 to +70.5) mg/L, P < 0.0001]. These observations were recapitulated in the independent validation cohort at BH (169 CAP and 181 COVID-19 patients). A multivariate logistic regression model incorporating WCC and ΔCRP discriminated CAP from COVID-19 with AUC 0.88 (95% CI 0.83-0.94). Baseline WCC >8.2 × 106 cells/mL or falling CRP identified 94% of CAP cases, and excluded bacterial co-infection in 46% of COVID-19 patients. CONCLUSIONS: We propose that in COVID-19, absence of both elevated baseline WCC and antibiotic-related decrease in CRP can exclude bacterial co-infection and facilitate antibiotic stewardship efforts.

Vitamin D3 replacement enhances antigen-specific immunity in older adults.

Introduction: Ageing is associated with increased number of infections, decreased vaccine efficacy and increased systemic inflammation termed inflammageing. These changes are reflected by reduced recall responses to varicella zoster virus (VZV) challenge in the skin of older adults. Vitamin D deficiency is more common in the old and has been associated with frailty and increased inflammation. In addition, vitamin D increases immunoregulatory mechanisms and therefore has the potential to inhibit inflammageing. Objectives: We investigated the use of vitamin D3 replacement to enhance cutaneous antigen-specific immunity in older adults (≥65 years). Methods: Vitamin D insufficient older adults (n = 18) were administered 6400IU of vitamin D3/day orally for 14 weeks. Antigen-specific immunity to VZV was assessed by clinical score assessment of the injection site and transcriptional analysis of skin biopsies collected from challenged injection sites pre- and post-vitamin D3 replacement. Results: We showed that older adults had reduced VZV-specific cutaneous immune response and increased non-specific inflammation as compared to young. Increased non-specific inflammation observed in the skin of older adults negatively correlated with vitamin D sufficiency. We showed that vitamin D3 supplementation significantly increased the response to cutaneous VZV antigen challenge in older adults. This enhancement was associated with a reduction in inflammatory monocyte infiltration with a concomitant enhancement of T cell recruitment to the site of antigen challenge in the skin. Conclusion: Vitamin D3 replacement can boost antigen-specific immunity in older adults with sub-optimal vitamin D status.

Transcriptional response modules characterize IL-1ß and IL-6 activity in COVID-19.

Dysregulated IL-1ß and IL-6 responses have been implicated in the pathogenesis of severe Coronavirus Disease 2019 (COVID-19). Innovative approaches for evaluating the biological activity of these cytokines in vivo are urgently needed to complement clinical trials of therapeutic targeting of IL-1ß and IL-6 in COVID-19. We show that the expression of IL-1ß or IL-6 inducible transcriptional signatures (modules) reflects the bioactivity of these cytokines in immunopathology modelled by juvenile idiopathic arthritis (JIA) and rheumatoid arthritis. In COVID-19, elevated expression of IL-1ß and IL-6 response modules, but not the cytokine transcripts themselves, is a feature of infection in the nasopharynx and blood but is not associated with severity of COVID-19 disease, length of stay, or mortality. We propose that IL-1ß and IL-6 transcriptional response modules provide a dynamic readout of functional cytokine activity in vivo, aiding quantification of the biological effects of immunomodulatory therapies in COVID-19.

Transcriptional response modules characterise IL-1ß and IL-6 activity in COVID-19.

Dysregulated IL-1ß and IL-6 responses have been implicated in the pathogenesis of severe Coronavirus Disease 2019 (COVID-19). Innovative approaches for evaluating the biological activity of these cytokines in vivo are urgently needed to complement clinical trials of therapeutic targeting of IL-1ß and IL-6 in COVID-19. We show that the expression of IL-1ß or IL-6 inducible transcriptional signatures (modules) reflects the bioactivity of these cytokines in immunopathology modelled by juvenile idiopathic arthritis (JIA) and rheumatoid arthritis. In COVID-19, elevated expression of IL-1ß and IL-6 response modules, but not the cytokine transcripts themselves, is a feature of infection in the nasopharynx and blood, but is not associated with severity of COVID-19 disease, length of stay or mortality. We propose that IL-1ß and IL-6 transcriptional response modules provide a dynamic readout of functional cytokine activity in vivo, aiding quantification of the biological effects of immunomodulatory therapies in COVID-19.

Keep calm and carry on learning: using Microsoft Teams to deliver a medical education programme during the COVID-19 pandemic.

The outbreak of COVID-19 in the UK in March 2020 required a radical remodelling of the medical workforce at Royal Free London NHS Foundation Trust to prepare for the anticipated surge of hospital admissions. The provision of relevant teaching and training was immediately identified as a priority, particularly for staff due to work outside their regular medical specialty. Rather than deliver face-to-face teaching, doctors at the Trust utilised Microsoft Teams, an online communications and collaboration platform, to deliver a multi-disciplinary Trust-wide education programme responsive to the needs of surveyed medical staff. To date members of 18 departments across the Trust have delivered 51 virtual teaching sessions which have been viewed 3,814 times. During this pandemic the virtual education programme has facilitated rapid dissemination of new information and provided a platform for discussion and unity amongst colleagues with overwhelmingly positive feedback from both learners and teachers.

Antiviral treatment for COVID-19: the evidence supporting remdesivir.

The emergence of the novel beta coronavirus SARS-CoV-2 and the ensuing COVID-19 pandemic has generated a rapidly evolving research landscape in the search for new therapeutic agents. The intravenous antiviral drug remdesivir has in vitro activity against SARS-CoV-2 and now studies have reported its clinical efficacy, demonstrating shorter time to recovery in hospitalised patients with severe COVID-19. Adverse event rates were low and remdesivir has now received conditional marketing authorisation from the European Medicines Agency. An interim clinical commissioning policy is in place in the UK. These studies make remdesivir the first antiviral drug able to alter the natural history of severe COVID-19, and a benchmark for the comparison of new therapies in the future. Ongoing studies are investigating its use in early mild/moderate COVID-19, alternative formulations, and the combination of remdesivir with immunomodulatory agents.