Development of a colorimetric assay for the detection of SARS-CoV-2 3CLpro activity.

Diagnostic testing continues to be an integral component of the strategy to contain the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) global pandemic, the causative agent of Coronavirus Disease 2019 (COVID-19). The SARS-CoV-2 genome encodes the 3C-like protease (3CLpro) which is essential for coronavirus replication. This study adapts an in vitro colorimetric gold nanoparticle (AuNP) based protease assay to specifically detect the activity of SARS-CoV-2 3CLpro as a purified recombinant protein and as a cellular protein exogenously expressed in HEK293T human cells. We also demonstrate that the specific sensitivity of the assay for SARS-CoV-2 3CLpro can be improved by use of an optimised peptide substrate and through hybrid dimerisation with inactive 3CLpro mutant monomers. These findings highlight the potential for further development of the AuNP protease assay to detect SARS-CoV-2 3CLpro activity as a novel, accessible and cost-effective diagnostic test for SARS-CoV-2 infection at the point-of-care. Importantly, this versatile assay could also be easily adapted to detect specific protease activity associated with other viruses or diseases conditions.

Lineage-specific evolution and gene flow in Listeria monocytogenes are independent of bacteriophages.

Listeria monocytogenes is a foodborne pathogen causing systemic infection with high mortality. To allow efficient tracking of outbreaks a clear definition of the genomic signature of a cluster of related isolates is required, but lineage-specific characteristics call for a more detailed understanding of evolution. In our work, we used core genome MLST (cgMLST) to identify new outbreaks combined to core genome SNP analysis to characterize the population structure and gene flow between lineages. Whilst analysing differences between the four lineages of L. monocytogenes we have detected differences in the recombination rate, and interestingly also divergence in the SNP differences between sub-lineages. In addition, the exchange of core genome variation between the lineages exhibited a distinct pattern, with lineage III being the best donor for horizontal gene transfer. Whilst attempting to link bacteriophage-mediated transduction to observed gene transfer, we found an inverse correlation between phage presence in a lineage and the extent of recombination. Irrespective of the profound differences in recombination rates observed between sub-lineages and lineages, we found that the previously proposed cut-off of 10 allelic differences in cgMLST can be still considered valid for the definition of a foodborne outbreak cluster of L. monocytogenes.

Methylation Warfare: Interaction of Pneumococcal Bacteriophages with Their Host.

Virus-host interactions are regulated by complex coevolutionary dynamics. In Streptococcus pneumoniae, phase-variable type I restriction-modification (R-M) systems are part of the core genome. We hypothesized that the ability of the R-M systems to switch between six target DNA specificities also has a key role in preventing the spread of bacteriophages. Using the streptococcal temperate bacteriophage SpSL1, we show that the variants of both the SpnIII and SpnIV R-M systems are able to restrict invading bacteriophage with an efficiency approximately proportional to the number of target sites in the bacteriophage genome. In addition to restriction of lytic replication, SpnIII also led to abortive infection in the majority of host cells. During lytic infection, transcriptional analysis found evidence of phage-host interaction through the strong upregulation of the nrdR nucleotide biosynthesis regulon. During lysogeny, the phage had less of an effect on host gene regulation. This research demonstrates a novel combined bacteriophage restriction and abortive infection mechanism, highlighting the importance that the phase-variable type I R-M systems have in the multifunctional defense against bacteriophage infection in the respiratory pathogen S. pneumoniaeIMPORTANCE With antimicrobial drug resistance becoming an increasing burden on human health, much attention has been focused on the potential use of bacteriophages and their enzymes as therapeutics. However, the investigations into the physiology of the complex interactions of bacteriophages with their hosts have attracted far less attention, in comparison. This work describes the molecular characterization of the infectious cycle of a bacteriophage in the important human pathogen Streptococcus pneumoniae and explores the intricate relationship between phase-variable host defense mechanisms and the virus. This is the first report showing how a phase-variable type I restriction-modification system is involved in bacteriophage restriction while it also provides an additional level of infection control through abortive infection.

Salad Leaf Juices Enhance Salmonella Growth, Colonization of Fresh Produce, and Virulence.

We show in this report that traces of juices released from salad leaves as they become damaged can significantly enhance colonization of salad leaves by Salmonella enterica Salad juices in water increased Salmonella growth by 110% over the level seen with the unsupplemented control and in host-like serum-based media by more than 2,400-fold over control levels. In serum-based media, salad juices induced growth of Salmonella via provision of Fe from transferrin, and siderophore production was found to be integral to the growth induction process. Other aspects relevant to salad leaf colonization and retention were enhanced, such as motility and biofilm formation, which were increased over control levels by >220% and 250%, respectively; direct attachment to salad leaves increased by >350% when a salad leaf juice was present. In terms of growth and biofilm formation, the endogenous salad leaf microbiota was largely unresponsive to leaf juice, suggesting that Salmonella gains a marked growth advantage from fluids released by salad leaf damage. Salad leaf juices also enhanced pathogen attachment to the salad bag plastic. Over 5 days of refrigeration (a typical storage time for bagged salad leaves), even traces of juice within the salad bag fluids increased Salmonella growth in water by up to 280-fold over control cultures, as well as enhancing salad bag colonization, which could be an unappreciated factor in retention of pathogens in fresh produce. Collectively, the study data show that exposure to salad leaf juice may contribute to the persistence of Salmonella on salad leaves and strongly emphasize the importance of ensuring the microbiological safety of fresh produce. IMPORTANCE: Salad leaves are an important part of a healthy diet but have been associated in recent years with a growing risk of food poisoning from bacterial pathogens such as Salmonella enterica Although this is considered a significant public health problem, very little is known about the behavior of Salmonella in the actual salad bag. We show that juices released from the cut ends of the salad leaves enabled the Salmonella cells to grow in water, even when it was refrigerated. Salad juice exposure also helped the Salmonella cells to attach to the salad leaves so strongly that washing could not remove them. Collectively, the results presented in this report show that exposure to even traces of salad leaf juice may contribute to the persistence of Salmonella on salad leaves as well as priming it for establishing an infection in the consumer.

Mutation design and strain background influence the phenotype of Escherichia coli luxS mutants.

Previous analyses of luxS in Escherichia coli have used different strain backgrounds and design formats to produce the luxS mutation, resulting in luxS mutants with confusingly dissimilar phenotypes. This study therefore investigates the roles that strain background and mutational design strategy have upon the phenotype of the pathogenic E. coli luxS mutant. We inactivated luxS in three E. coli backgrounds: enteropathogenic E. coli E2348-69, and enterohaemorrhagic strains Sakai and NCTC12900. To investigate the influence of mutational design strategy, four mutation formats were used: antibiotic resistance insertion methodologies as previously employed, using tetracycline and chloramphenicol resistance cassettes, and non-polar strategies creating deletion and premature termination mutations. Our study showed that the E. coli luxS phenotype was markedly dependent on strain background: in some strains disruption of luxS caused significant metabolic stress or no stress at all. How the luxS mutation was constructed also shaped its phenotype: non-polar mutants were very similar to wild type, while mutations made using the antibiotic insertion methodologies produced phenotypes defective in growth and virulence. Proteomic profiling of our luxS mutants showed only a few proteins were differentially expressed and those that were altered suggested a metabolic rather than communication role for the E. coli luxS gene product.

Role of porin proteins in acquisition of transferrin iron by enteropathogens.

Acquisition of iron from key innate immune defence proteins such as transferrin (Tf) and lactoferrin is an important mechanism by which pathogenic bacteria obtain essential iron for growth within their host. Bacterial species that do not produce siderophores often use specific Tf-binding proteins, the best characterized being the Neisseriaceae-type Tf-binding proteins TbpA and TbpB. Previous work from our laboratory has shown that siderophore-producing enteric species such as Escherichia coli also readily bind Tf, although no genomic evidence exists for Tbp-like Tf-binding proteins. Application of proteomic analyses and molecular mutagenesis strategies to an enteropathogenic E. coli identified the OmpA and OmpC porins as Tf-binding proteins. Mutagenesis of the ompA or ompC genes affected E. coli Tf binding and, furthermore, compromised the ability of the ompA mutant to respond to growth promotion by certain catecholamine stress hormones. Evidence was also found to implicate the OmpA porin as an entry point for catecholamine stress hormones. Further proteomic analyses in other bacterial pathogens revealed wide-scale involvement of porins in Tf binding: Salmonella typhimurium (OmpC), and Shigella sonnei, Shigella flexneri and Shigella boydii (OmpC and/or OmpA). This study shows that in addition to their existing housekeeping functions, the Gram-negative porin family of proteins can also act as Tf-capture proteins for those bacteria that lack the classical Neisseriaceae-type Tf-binding proteins.

Need for effective detection and early warnings for epidemic and pandemic preparedness planning in the context of multi-hazards: Lessons from the COVID-19 pandemic.

The need for effective early detection and timely surveillance for a robust pandemic and epidemic early warning and preparedness has been widely discussed amidst the Covid-19 pandemic, which suddenly erupted worldwide. This need is further established by various other hazards reported in many countries amidst the COVID-19 pandemic. In addition, the failure of early detection of pathogens and their source of origin has been largely connected with global transmission and severe outbreaks in many contexts. Therefore, effective early detection , timely surveillance and early warning are key aspects of a successful response to an epidemic or pandemic. . Hence, this paper aims to identify key elements and stages of an effective epidemic and pandemic early warning (EW) and response system. Further, the paper analyses inter-connections of the elements of the early warning system, focusing on the COVID-19 and multi-hazard context. The systematic literature review method was used to collect data from electronic databases. Results suggest that epidemiological surveillance & detection, primary screening of raw data & information, risk and vulnerability assessments, prediction and decision-making, alerts & early warnings are critical components of epidemic and pandemic EW. In addition, response-control & mitigation, preparedness-preventive strategies, and reducing transmission , elimination and eradication of the disease are integrated components of the early warning and response ecosystem that largely depend on effective early warnings. The significance of integrating epidemic and pandemic EW with other EWs to operate as multi-hazard early warning systems is also analysed.

Exhaled SARS-CoV-2 RNA viral load kinetics measured by facemask sampling associates with household transmission.

OBJECTIVES: No studies have examined longitudinal patterns of naturally exhaled SARS-CoV-2 RNA viral load (VL) during acute infection. We report this using facemask sampling (FMS) and assessed the relationship between emitted RNA VL and household transmission. METHODS: Between December 2020 and February 2021, we recruited participants within 24 hours of a positive RT-qPCR on upper respiratory tract sampling (URTS) (day 0). Participants gave FMS (for 1 hour) and URTS (self-taken) on seven occasions up to day 21. Samples were analysed by RT-qPCR (from sampling matrix strips within the mask) and symptom diaries were recorded. Household transmission was assessed through reporting of positive URTS RT-qPCR in household contacts. RESULTS: Analysis of 203 FMS and 190 URTS from 34 participants showed that RNA VL peaked within the first 5 days following sampling. Concomitant URTS, FMS RNA VL, and symptom scores, however, were poorly correlated, but a higher severity of reported symptoms was associated with FMS positivity up to day 5. Of 28 participants who had household contacts, 12 (43%) reported transmission. Frequency of household transmission was associated with the highest (peak) FMS RNA VL obtained (negative genome copies/strip: 0% household transmission; 1 to 1000 copies/strip: 20%; 1001 to 10 000 copies/strip: 57%; >10 000 copies/strip: 75%; p = 0.048; age adjusted OR of household transmission per log increase in copies/strip: 4.97; 95% CI, 1.20-20.55; p = 0.02) but not observed with peak URTS RNA VL. DISCUSSION: Exhaled RNA VL measured by FMS is highest in early infection, can be positive in symptomatic patients with concomitantly negative URTS, and is strongly associated with household transmission.

Activity of a Bacteriophage Cocktail to Control Salmonella Growth Ex Vivo in Avian, Porcine, and Human Epithelial Cell Cultures.

We examined the activity of phages to control the growth of chicken and swine Salmonella strains in avian (CHIC-8E11), porcine (IPEC-1), and human (HT-29) cell cultures. We optimized a six-phage cocktail by selecting the five most effective myoviruses and a siphovirus that have optimal lysis on prevalent serovars. We observed ∼20% of 7 log10 PFU/well phage and 3-6 log10 CFU bacterial adhesions, and 3-5 log10 CFU bacterial invasion per 2 cm2 of the cultured cells at 2 h post-treatment. The invasive bacteria when plated had a variable reduced susceptibility to the phages. After phage application at an MOI of 10, the prophylaxis regimen had better efficacy at controlling bacterial growth with an up to 6 log10 CFU/well reduction as compared with the 1-2 log10 CFU/well bacterial reduction observed in the remedial and coinfection regimens. Our data support the development of these phages to control salmonellosis in chickens, pigs, and humans.

Inducible hydrogen sulfide synthesis in chondrocytes and mesenchymal progenitor cells: is H2S a novel cytoprotective mediator in the inflamed joint?

Hydrogen sulfide (H(2)S) has recently been proposed as an endogenous mediator of inflammation and is present in human synovial fluid. This study determined whether primary human articular chondrocytes (HACs) and mesenchymal progenitor cells (MPCs) could synthesize H(2)S in response to pro-inflammatory cytokines relevant to human arthropathies, and to determine the cellular responses to endogenous and pharmacological H(2)S. HACs and MPCs were exposed to IL-1ß, IL-6, TNF-α and lipopolysaccharide (LPS). The expression and enzymatic activity of the H(2)S synthesizing enzymes cystathionine-ß-synthase (CBS) and cystathionine-γ-lyase (CSE) were determined by Western blot and zinc-trap spectrophotometry, respectively. Cellular oxidative stress was induced by H(2)O(2), the peroxynitrite donor SIN-1 and 4-hydroxynonenal (4-HNE). Cell death was assessed by 3-(4,5-dimethyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. Mitochondrial membrane potential (DCm) was determined in situ by flow cytometry. Endogenous H(2) S synthesis was inhibited by siRNA-mediated knockdown of CSE and CBS and pharmacological inhibitors D,L-propargylglycine and aminoxyacetate, respectively. Exogenous H(2)S was generated using GYY4137. Under basal conditions HACs and MPCs expressed CBS and CSE and synthesized H(2)S in a CBS-dependent manner, whereas CSE expression and activity was induced by treatment of cells with IL-1ß, TNF-α, IL-6 or LPS. Oxidative stress-induced cell death was significantly inhibited by GYY4137 treatment but increased by pharmacological inhibition of H(2)S synthesis or by CBS/CSE-siRNA treatment. These data suggest CSE is an inducible source of H(2)S in cultured HACs and MPCs. H(2)S may represent a novel endogenous mechanism of cytoprotection in the inflamed joint, suggesting a potential opportunity for therapeutic intervention.

Myeloperoxidase and oxidative stress in rheumatoid arthritis.

OBJECTIVE: To determine whether MPO contributes to oxidative stress and disease activity in RA and whether it produces hypochlorous acid in SF. METHODS: Plasma and where possible SF were collected from 77 RA patients while 120 healthy controls supplied plasma only. MPO and protein carbonyls were measured by ELISAs. 3-Chlorotyrosine in proteins and allantoin in plasma were measured by mass spectrometry. RESULTS: Plasma MPO concentrations were significantly higher in patients with RA compared with healthy controls [10.8 ng/ml, inter-quartile range (IQR): 7.2-14.2; P<0.05], but there was no significant difference in plasma MPO protein concentrations between RA patients with high disease activity (HDA; DAS-28 >3.2) and those with low disease activity (LDA; DAS-28 ≤ 3.2) (HDA 27.9 ng/ml, 20.2-34.1 vs LDA 22.1 ng/ml, 16.9-34.9; P>0.05). There was a significant relationship between plasma MPO and DAS-28 (r=0.35; P=0.005). Plasma protein carbonyls and allantoin were significantly higher in patients with RA compared with the healthy controls. MPO protein was significantly higher in SF compared with plasma (median 624.0 ng/ml, IQR 258.4-2433.0 vs 30.2 ng/ml, IQR 25.1-50.9; P<0.0001). The MPO present in SF was mostly active. 3-Chlorotyrosine, a specific biomarker of hypochlorous acid, was present in proteins from SF and related to the concentration of MPO (r=0.69; P=0.001). Protein carbonyls in SF were associated with MPO protein concentration (r=0.40; P=0.019) and 3-chlorotyrosine (r=0.66; P=0.003). CONCLUSION: MPO is elevated in patients with RA and promotes oxidative stress through the production of hypochlorous acid.

Evaluating the network of stakeholders in Multi-Hazard Early Warning Systems for multiple hazards amidst biological outbreaks: Sri Lanka as a case in point.

Synergized impacts of simultaneous hazards amidst COVID-19 have called for the need for highly collaborative multi-sectoral approaches for disaster preparedness planning. In such a context, this study aims at evaluating the network of stakeholders in the National Early Warning System of Sri Lanka during preparedness planning. Social Network Analysis was used to visualise the network of stakeholders for selected hazard scenarios. Furthermore, a series of key informant interviews were conducted focusing on disaster preparedness planning during the recent multiple hazard scenarios. The findings highlight the need for a framework to guide the stakeholder coordination in preparedness planning for multiple hazards.

A Persistent Tuberculosis Outbreak in the UK Is Characterized by Hydrophobic fadB4-Deficient Mycobacterium tuberculosis That Replicates Rapidly in Macrophages.

The genetic diversity of Mycobacterium tuberculosis can influence disease severity and transmissibility. To better understand how this diversity influences individuals and communities, we phenotyped M. tuberculosis that was causing a persistent outbreak in the East Midlands, United Kingdom. Compared to nonoutbreak isolates, bacilli had higher lipid contents and more hydrophobic cell surfaces. In macrophage infection models, the bacteria increased more rapidly, provoked the enhanced accumulation of macrophage lipid droplets and enhanced the secretion of IL-1ß. Natural deletions in fadB4, nrdB, and plcC distinguished the outbreak isolates from other lineage 3 isolates in the region. fadB4 is annotated with a putative role in cell envelope biosynthesis, so the loss of this gene has the potential to alter the interactions of bacteria with immune cells. Reintroduction of fadB4 to the outbreak strain led to a phenotype that more closely resembled those of nonoutbreak strains. The improved understanding of the microbiological characteristics and the corresponding genetic polymorphisms that associate with outbreaks have the potential to inform tuberculosis control. IMPORTANCE Tuberculosis (TB) killed 1.5 million people in 2020 and affects every country. The extent to which the natural genetic diversity of Mycobacterium tuberculosis influences disease manifestation at both the individual and epidemiological levels remains poorly understood. Insights into how pathogen polymorphisms affect patterns of TB have the potential to translate into clinical and public health practice. Two distinct lineage 3 strains isolated from local TB outbreaks, one of which (CH) was rapidly terminated and the other of which (Lro) persistently transmitted for over a decade, provided us with an opportunity to study these issues. We compared genome sequences, microbiological characteristics, and early immune responses that were evoked upon infection. Our results indicate that the natural lack of fadB4 in the Lro strain contributes to its unique features.

Measurement and meaning of markers of reactive species of oxygen, nitrogen and sulfur in healthy human subjects and patients with inflammatory joint disease.

Reactive species of oxygen, nitrogen and sulfur play cell signalling roles in human health, e.g. recent studies have shown that increased dietary nitrate, which is a source of RNS (reactive nitrogen species), lowers resting blood pressure and the oxygen cost of exercise. In such studies, plasma nitrite and nitrate are readily determined by chemiluminescence. At sites of inflammation, such as the joints of RA (rheumatoid arthritis) patients, the generation of ROS (reactive oxygen species) and RNS overwhelms antioxidant defences and one consequence is oxidative/nitrative damage to proteins. For example, in the inflamed joint, increased RNS-mediated protein damage has been detected in the form of a biomarker, 3-nitrotyrosine, by immunohistochemistry, Western blotting, ELISAs and MS. In addition to NO•, another cell-signalling gas produced in the inflamed joint is H2S (hydrogen sulfide), an RSS (reactive sulfur species). This gas is generated by inflammatory induction of H2S-synthesizing enzymes. Using zinc-trap spectrophotometry, we detected high (micromolar) concentrations of H2S in RA synovial fluid and levels correlated with clinical scores of inflammation and disease activity. What might be the consequences of the inflammatory generation of reactive species? Effects on inflammatory cell-signalling pathways certainly appear to be crucial, but in the current review we highlight the concept that ROS/RNS-mediated protein damage creates neoepitopes, resulting in autoantibody formation against proteins, e.g. type-II collagen and the complement component, C1q. These autoantibodies have been detected in inflammatory autoimmune diseases.

Extracellular calreticulin is present in the joints of patients with rheumatoid arthritis and inhibits FasL (CD95L)-mediated apoptosis of T cells.

OBJECTIVE: The binding of FasL (CD95L) to its receptor, Fas (CD95), induces apoptosis. Studies have shown that in patients with rheumatoid arthritis (RA), T lymphocytes are resistant to FasL-induced apoptosis in vivo but are susceptible to FasL-induced apoptosis in vitro. Dysfunction in this mechanism may be an important contributor to the pathophysiology of RA. Thus, the present study was undertaken to determine which factors might inhibit FasL-Fas binding in vivo and those that would inhibit apoptosis of T lymphocytes in an in vitro model system. METHODS: Human Jurkat T cells rendered apoptotic by FasL exposure were analyzed by flow cytometry. Necrosis was determined according to measurement of lactate dehydrogenase release. Quantification of calreticulin in plasma and synovial fluid and of calreticulin-FasL binding was performed by enzyme-linked immunosorbent assay. Measurement of nitrite/nitrate in the plasma and synovial fluid was carried out by chemiluminescence assay. RESULTS: Extracellular calreticulin was present at a significantly higher concentration in the plasma (median 10.3 ng/ml, interquartile range [IQR] 14.8 ng/ml) and synovial fluid (median 10.3 ng/ml, IQR 12.0 ng/ml) of RA patients (each P < 0.05) compared with the plasma (median 3.1 ng/ml, IQR 1.3 ng/ml) and synovial fluid (median 2.9 ng/ml, IQR 0.9 ng/ml) of patients with psoriatic arthritis and the plasma of healthy control subjects (median 2.9 ng/ml, IQR 0.9 ng/ml). Calreticulin concentrations in the synovial fluid correlated with the tender and swollen joint counts and the activity scores on the 28-joint Disease Activity Score assessment. Calreticulin also bound directly to FasL. In vitro, calreticulin (2-16 ng/ml) inhibited FasL-induced apoptosis of Jurkat T cells. CONCLUSION: Calreticulin was present at higher concentrations in the plasma and synovial fluid of RA patients. Calreticulin had the capacity to bind directly to FasL and to inhibit FasL-mediated apoptosis of Jurkat T cells, and thus might play a role in inhibiting apoptosis of inflammatory T cells in RA.

Enzyme-linked immunosorbent assays for monitoring TNF-alpha inhibitors and antibody levels in people with rheumatoid arthritis: a systematic review and economic evaluation.

BACKGROUND: Rheumatoid arthritis is a chronic autoimmune disease that primarily causes inflammation, pain and stiffness in the joints. People with severe disease may be treated with biological disease-modifying anti-rheumatic drugs, including tumour necrosis factor-α inhibitors, but the efficacy of these drugs is hampered by the presence of anti-drug antibodies. Monitoring the response to these treatments typically involves clinical assessment using response criteria, such as Disease Activity Score in 28 joints or European League Against Rheumatism. Enzyme-linked immunosorbent assays can also be used to measure drug and antibody levels in the blood. These tests may inform whether or not adjustments to treatment are required or help clinicians to understand the reasons for treatment non-response or a loss of response. METHODS: Systematic reviews were conducted to identify studies reporting on the clinical effectiveness and cost-effectiveness of using enzyme-linked immunosorbent assays to measure drug and anti-drug antibody levels to monitor the response to tumour necrosis factor-α inhibitors [adalimumab (Humira®; AbbVie, Inc., North Chicago, IL, USA), etanercept (Enbrel®; Pfizer, Inc., New York, NY, USA), infliximab (Remicade®, Merck Sharp & Dohme Limited, Hoddesdon, UK), certolizumab pegol (Cimzia®; UCB Pharma Limited, Slough, UK) and golimumab (Simponi®; Merck Sharp & Dohme Limited)] in people with rheumatoid arthritis who had either achieved treatment target (remission or low disease activity) or shown primary or secondary non-response to treatment. A range of bibliographic databases, including MEDLINE, EMBASE and CENTRAL (Cochrane Central Register of Controlled Trials), were searched from inception to November 2018. The risk of bias was assessed using the Cochrane ROBINS-1 (Risk Of Bias In Non-randomised Studies - of Interventions) tool for non-randomised studies, with adaptations as appropriate. Threshold and cost-utility analyses that were based on a decision tree model were conducted to estimate the economic outcomes of adding therapeutic drug monitoring to standard care. The costs and resource use were considered from the perspective of the NHS and Personal Social Services. No discounting was applied to the costs and effects owing to the short-term time horizon of 18 months that was adopted in the economic analysis. The impact on the results of variations in testing and treatment strategies was explored in numerous clinically plausible sensitivity analyses. RESULTS: Two studies were identified: (1) a non-randomised controlled trial, INGEBIO, that compared standard care with therapeutic drug monitoring using Promonitor® assays [Progenika Biopharma SA (a Grifols-Progenika company), Derio, Spain] in Spanish patients receiving adalimumab who had achieved remission or low disease activity; and (2) a historical control study. The economic analyses were informed by INGEBIO. Different outcomes from INGEBIO produced inconsistent results in both threshold and cost-utility analyses. The cost-effectiveness of therapeutic drug monitoring varied, from the intervention being dominant to the incremental cost-effectiveness ratio of £164,009 per quality-adjusted life-year gained. However, when the frequency of testing was assumed to be once per year and the cost of phlebotomy appointments was excluded, therapeutic drug monitoring dominated standard care. LIMITATIONS: There is limited relevant research evidence and much uncertainty about the clinical effectiveness and cost-effectiveness of using enzyme-linked immunosorbent assay-based testing for therapeutic drug monitoring in rheumatoid arthritis patients. INGEBIO had serious limitations in relation to the National Institute for Health and Care Excellence scope: only one-third of participants had rheumatoid arthritis, the analyses were mostly not by intention to treat and the follow-up was 18 months only. Moreover, the outcomes might not be generalisable to the NHS. CONCLUSIONS: Based on the available evidence, no firm conclusions could be made about the cost-effectiveness of therapeutic drug monitoring in England and Wales. FUTURE WORK: Further controlled trials are required to assess the impact of using enzyme-linked immunosorbent assays for monitoring the anti-tumour necrosis factors in people with rheumatoid arthritis. STUDY REGISTRATION: This study is registered as PROSPERO CRD42018105195. FUNDING: This project was funded by the National Institute for Health Research (NIHR) Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 25, No. 8. See the NIHR Journals Library website for further project information.

Rheumatoid arthritis is a long-term condition that causes pain, swelling and stiffness in the joints. People with severe disease may be treated with drugs called tumour necrosis factor-α inhibitors [adalimumab (Humira^®; AbbVie Inc., North Chicago, IL, USA), etanercept (Enbrel^®; Pfizer, Inc., New York, NY, USA), infliximab (Remicade^®; Merck Sharp & Dohme Limited, Hoddesdon, UK), certolizumab pegol (Cimzia^®; UCB Pharma Limited, Slough, UK) and golimumab (Simponi^®; Merck Sharp & Dohme Limited)]. Some people taking these drugs find that their disease improves, whereas others do not respond to the treatment or improve initially and then experience loss of response. One cause of lost response is that individuals develop antibodies (i.e. protective proteins) against the drug, which hamper the effect of treatment. Various tests have been developed to measure the level of drugs and antibodies against these drugs in patient's blood samples. This kind of monitoring would allow treatment to be adjusted in response to the test outcomes to optimise benefit for the patient, and help clinicians to better understand the reasons for an absence or a loss of response to treatment. The aim of this study was to find out whether or not it would be clinically effective (i.e. good for patients) and cost-effective (i.e. a good use of NHS resources) to use these tests for monitoring drug and antibody levels, as a means of assessing treatment response in rheumatoid arthritis patients who are controlled, have not responded or have lost response. Results from a systematic review showed that, because of the limited and poor-quality evidence, there was much uncertainty in the clinical effectiveness of testing. A simple mathematical model drew on evidence from one poorly reported study, which was heavily supplemented by data from other studies and expert advice. Results from the model were inconclusive and suggest that there is considerable uncertainty in the cost-effectiveness of testing. Therefore, the results presented here should be considered with caution. Further studies are needed to assess the impact of tumour necrosis factor testing in patients with rheumatoid arthritis.

Exhaled SARS-CoV-2 quantified by face-mask sampling in hospitalised patients with COVID-19.

BACKGROUND: Human to human transmission of SARS-CoV-2 is driven by the respiratory route but little is known about the pattern and quantity of virus output from exhaled breath. We have previously shown that face-mask sampling (FMS) can detect exhaled tubercle bacilli and have adapted its use to quantify exhaled SARS-CoV-2 RNA in patients admitted to hospital with Coronavirus Disease-2019 (COVID-19). METHODS: Between May and December 2020, we took two concomitant FMS and nasopharyngeal samples (NPS) over two days, starting within 24 h of a routine virus positive NPS in patients hospitalised with COVID-19, at University Hospitals of Leicester NHS Trust, UK. Participants were asked to wear a modified duckbilled facemask for 30 min, followed by a nasopharyngeal swab. Demographic, clinical, and radiological data, as well as International Severe Acute Respiratory and emerging Infections Consortium (ISARIC) mortality and deterioration scores were obtained. Exposed masks were processed by removal, dissolution and analysis of sampling matrix strips fixed within the mask by RT-qPCR. Viral genome copy numbers were determined and results classified as Negative; Low: ≤999 copies; Medium: 1000-99,999 copies and High ≥ 100,000 copies per strip for FMS or per 100 µl for NPS. RESULTS: 102 FMS and NPS were collected from 66 routinely positive patients; median age: 61 (IQR 49 - 77), of which FMS was positive in 38% of individuals and concomitant NPS was positive in 50%. Positive FMS viral loads varied over five orders of magnitude (<10-3.3 x 106 genome copies/strip); 21 (32%) patients were asymptomatic at the time of sampling. High FMS viral load was associated with respiratory symptoms at time of sampling and shorter interval between sampling and symptom onset (FMS High: median (IQR) 2 days (2-3) vs FMS Negative: 7 days (7-10), p = 0.002). On multivariable linear regression analysis, higher FMS viral loads were associated with higher ISARIC mortality (Medium FMS vs Negative FMS gave an adjusted coefficient of 15.7, 95% CI 3.7-27.7, p = 0.01) and deterioration scores (High FMS vs Negative FMS gave an adjusted coefficient of 37.6, 95% CI 14.0 to 61.3, p = 0.002), while NPS viral loads showed no significant association. CONCLUSION: We demonstrate a simple and effective method for detecting and quantifying exhaled SARS-CoV-2 in hospitalised patients with COVID-19. Higher FMS viral loads were more likely to be associated with developing severe disease compared to NPS viral loads. Similar to NPS, FMS viral load was highest in early disease and in those with active respiratory symptoms, highlighting the potential role of FMS in understanding infectivity.

Identification and characterization of a new ferric enterobactin receptor, CfrB, in Campylobacter.

The ferric enterobactin (FeEnt) receptor CfrA is present in the majority of Campylobacter jejuni isolates and is responsible for high-affinity iron acquisition. Our recent work and that of others strongly suggested the existence of another FeEnt uptake system in Campylobacter. Here we have identified and characterized a new FeEnt receptor (designated CfrB) using both in vitro and in vivo systems. CfrB, a homolog of C. jejuni NCTC 11168 Cj0444, shares approximately 34% of amino acid identity with CfrA. Alignment of complete CfrB sequences showed that the CfrB is highly conserved in Campylobacter. Immunoblotting analysis using CfrB-specific antiserum demonstrated that CfrB was dramatically induced under iron-restricted conditions and was produced in the majority of Campylobacter coli (41 out of 45) and in some C. jejuni (8 out of 32) primary strains from various sources and from geographically diverse areas. All of the CfrB-producing C. coli strains also produced CfrA, which was rarely observed in the tested C. jejuni strains. Isogenic cfrB, cfrA, and cfrA cfrB double mutants were constructed in 43 diverse Campylobacter strains. Growth promotion assays using these mutants demonstrated that CfrB has a major role in FeEnt iron acquisition in C. coli. Chicken colonization experiments indicated that inactivation of the cfrB gene alone greatly reduced and even abolished Campylobacter colonization of the intestines. A growth assay using CfrB-specific antiserum strongly suggested that specific CfrB antibodies could block the function of CfrB and diminish FeEnt-mediated growth promotion under iron-restricted conditions. Together, this work reveals the complexity of FeEnt systems in the two closely related Campylobacter species and demonstrates the important role of the new FeEnt receptor CfrB in Campylobacter iron acquisition and in vivo colonization.

Elucidation of the mechanism by which catecholamine stress hormones liberate iron from the innate immune defense proteins transferrin and lactoferrin.

The ability of catecholamine stress hormones and inotropes to stimulate the growth of infectious bacteria is now well established. A major element of the growth induction process has been shown to involve the catecholamines binding to the high-affinity ferric-iron-binding proteins transferrin (Tf) and lactoferrin, which then enables bacterial acquisition of normally inaccessible sequestered host iron. The nature of the mechanism(s) by which the stress hormones perturb iron binding of these key innate immune defense proteins has not been fully elucidated. The present study employed electron paramagnetic resonance spectroscopy and chemical iron-binding analyses to demonstrate that catecholamine stress hormones form direct complexes with the ferric iron within transferrin and lactoferrin. Moreover, these complexes were shown to result in the reduction of Fe(III) to Fe(II) and the loss of protein-complexed iron. The use of bacterial ferric iron uptake mutants further showed that both the Fe(II) and Fe(III) released from the Tf could be directly used as bacterial nutrient sources. We also analyzed the transferrin-catecholamine interactions in human serum and found that therapeutically relevant concentrations of stress hormones and inotropes could directly affect the iron binding of serum-transferrin so that the normally highly bacteriostatic tissue fluid became significantly more supportive of the growth of bacteria. The relevance of these catecholamine-transferrin/lactoferrin interactions to the infectious disease process is considered.

Changes in apoptotic gene expression in lymphocytes from rheumatoid arthritis and systemic lupus erythematosus patients compared with healthy lymphocytes.

INTRODUCTION: Systemic lupus erythematosus (SLE) and rheumatoid arthritis have complex genetic traits, but in both autoimmune diseases, dysfunctional apoptosis appears to play a part in disease pathology. This study examined the levels of in vitro apoptosis in lymphocytes from healthy, rheumatoid arthritis (RA) and SLE individuals and related observed differences to their lymphocyte apoptosis gene profiles. MATERIALS AND METHODS: Lymphocytes were assessed for cell death by nuclear pyknosis and DNA fragmentation. Control, SLE and RA apoptosis gene profiles were obtained by quantitative real-time polymerase chain reaction (QRT-PCR) analysis. RESULTS AND DISCUSSION: The mean levels of pyknosis in RA and SLE freshly isolated lymphocytes were significantly higher than in control lymphocytes. Ninety-three apoptosis genes were analysed by QRT-PCR of mRNA from RA, SLE and healthy lymphocytes. We identified significant differences (p < 0.05) in the expression of the same 11 of 93 and two of 93 apoptotic genes in individual SLE and RA patients tested as compared with controls. CONCLUSION: We propose that similarly altered expression of specific apoptotic regulatory genes (e.g., the death effector domain-containing DNA-binding protein and apoptosis-associated speck-like protein containing a CARD) occurs in the lymphocytes of individual patients with SLE or RA that may influence the extent and rate of spontaneous apoptosis in these autoimmune conditions.