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The aim of this cluster randomised controlled trial was to test the impact of an infection control education and training programme on meticillin-resistant Staphylococcus aureus (MRSA) prevalence in nursing homes. Nursing homes were randomised to intervention (infection control education and training programme; N=16) or control (usual practice continued; N=16). Staff in intervention homes were educated and trained (0, 3 and 6 months) in the principles and implementation of good infection control practice with infection control audits conducted in all sites (0, 3, 6 and 12 months) to assess compliance with good practice. Audit scores were fed back to nursing home managers in intervention homes, together with a written report indicating where practice could be improved. Nasal swabs were taken from all consenting residents and staff at 0, 3, 6 and 12 months. The primary outcome was MRSA prevalence in residents and staff, and the secondary outcome was a change in infection control audit scores. In all, 793 residents and 338 staff were recruited at baseline. MRSA prevalence did not change during the study in residents or staff. The relative risk of a resident being colonised with MRSA in an intervention home compared with a control home at 12 months was 0.99 (95% confidence interval: 0.69, 1.42) after adjustment for clustering. Mean infection control audit scores were significantly higher in the intervention homes (82%) compared with the control homes (64%) at 12 months (P<0.0001). Consideration should be given to other approaches which may help to reduce MRSA in this setting. [ABSTRACT FROM AUTHOR]

Decolonisation may reduce the risk of methicillin-resistant Staphylococcus aureus (MRSA) infection in individual carriers and prevent transmission to other patients. The aims of this prospective cohort study were to determine the long-term efficacy of a standardised decolonisation regimen and to identify factors associated with failure. Patients colonised with MRSA underwent decolonisation, which was considered to be successful if there was no growth in three consecutive sets of site-specific screening swabs obtained weekly post treatment. If patients were successfully decolonised, follow-up cultures were performed 6 and 12 months later. Of 137 patients enrolled, 79 (58%) were successfully decolonised. Of these 79, 53 (67%) and 44 (56%) remained decolonised at 6 and 12 months respectively. Therefore only 44/137 (32%) patients who completed decolonisation were MRSA negative 12 months later. Outcome was not associated with a particular strain of MRSA. Successful decolonisation was less likely in patients colonised with a mupirocin-resistant isolate (adjusted odds ratio: 0.08; 95% confidence interval: 0.02-0.30), in patients with throat colonisation (0.22; 0.07-0.68) and in patients aged >80 years (0.30; 0.10-0.93) compared with those aged 60-80 years. These findings suggest that although initially successful in some cases, the protocol used did not result in long-term clearance of MRSA carriage for most patients. [ABSTRACT FROM AUTHOR]

Background and Objective: This study explored the relationship between total bacterial density, airway microbiota composition and clinical parameters in bronchiectasis. We determined changes with time during clinical stability and following antibiotic treatment of a pulmonary exacerbation., Methods: We conducted a multicentre longitudinal cohort study of UK participants with CT confirmed bronchiectasis. Sputum samples and clinical parameters [FEV 1 % predicted, lung clearance index, C-reactive protein, white cell count and Quality of Life] were collected when participants were clinically stable and pre/post-antibiotic treatment of an exacerbation. Total bacterial density and microbiota community composition was measured by quantitative polymerase chain reaction and sequencing of the V4 region of bacterial 16S rRNA, respectively., Results: Among 105 participants at baseline, 65 (62 %) were female with a mean age of 65 years and FEV 1 at 69 % predicted. In participants who remained clinically stable (n = 15), no significant changes were observed in bacterial density, microbiota diversity, richness, evenness, and dominance (p = 0.30, 0.45, 0.54, 0.23 and 0.43; respectively) across four time points over a 1-year period. Similarly, for participants with paired pre/post-antibiotic treatment samples (n = 19), no significant changes were observed (p = 0.30, 0.46, 0.44, 0.71 and 0.58; respectively). However, considerable fluctuation in community composition between samples was apparent for most patients. Total bacterial density and microbiota composition did not correlate with clinical parameters at baseline (n = 75)., Conclusions: Stability in bacterial density and microbiota diversity, richness, evenness and dominance was observed over time at a population level but considerable fluctuation was apparent in samples from individual patients., Competing Interests: Declaration of competing interest Z.A, G.E., D.G., K.O., K.F., A.H., M.C., T.G., C.J., J.B.: no conflict of interest. J.S.E.: reports grants from European Union IMI Grant (in collaboration with Novartis) and grants from Novartis, and personal fees from Vertex, Galapagos and Ionis, outside the submitted work. M.L.: reports funding from by the Innovative Medicines Initiative (IMI) and EFPIA companies under the European Commission funded project, iABC (grant 115721). Consulting fees from Armata, 30T, Astra Zeneca, Parion, Insmed, Chiesi, Zambon, Electromed, Recode, AN2, Boehringer Ingelheim and Mannkind. Payment or honoraria for lectures by Insmed and Unpaid ERS infection group chair. A.D.S.: received grants from AstraZeneca, Pfizer, GSK and Novartis for research into Bronchiectasis and consulting fees from AstraZeneca, Insmed, GSK, Boehringer, 30T and Bayer. J.C.: received funding from Astrazeneca, Boehringer Ingelheim, Chiesi, Glaxosmithkline, Insmed, Novartis, Gilead Sciences, Trudell, Genentech and consulting fees from Astrazeneca Boehringer Ingelheim, Chiesi, Glaxosmithkline, Insmed, Novartis, Pfizer, Zambon, Janssen, Antabio. J.H.: received funding from Astra Zeneca and consulting fees from Astra Zeneca and GSK. Payment for honorary lectures from Astra Zeneca, Boehringer Ingelheim, Chiesi, Sanofi, Takeda and equipment from Nonin. J.M.B.: received funding Northern Ireland Clinical Research Facility, Health and Social Care (Northern Ireland) and the National Institute for Health and Care Research. M.M.T: received funding from the European Union Innovative Medicines Initiative (Grant Agreement number 115721), Novartis, Spexis and Antabio., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Influenza A viruses (IAVs) are significant pathogens of humans and other animals. Although endemic in humans and birds, novel IAV strains can emerge, jump species, and cause epidemics, like the latest variant of H5N1. Wastewater-based epidemiology (WBE) has been shown capable of detecting human IAVs. We aimed to assess whether whole-genome sequencing (WGS) of IAVs from wastewater is possible and can be used to discriminate between circulating strains of human and any non-human IAVs, such as those of avian origin., Methods: Using a pan-IAV RT-quantitative PCR assay, six wastewater treatment works (WWTWs) across Northern Ireland were screened from Aug 1 to Dec 5, 2022. A nanopore WGS approach was used to sequence RT-qPCR-positive samples. Phylogenetic analysis of sequences relative to currently circulating human and non-human IAVs was performed. For comparative purposes, clinical data (PCR test results) were supplied by The Regional Virus Laboratory, Belfast Health and Social Care Trust (Belfast, Northern Ireland, UK)., Findings: We detected a dynamic IAV signal in wastewater from Sept 5, 2022, onwards across Northern Ireland, which did not show a clear positive relationship with the clinical data obtained for the region. Meta (mixed strain) whole-genome sequences were generated from wastewater samples displaying homology to only human and avian IAV strains. The relative proportion of IAV reads of human versus avian origin differed across time and sample site. A diversity in subtypes and lineages was detected (eg, H1N1, H3N2, and several avian). Avian segment 8 related to those found in recent H5N1 clade 2.3.4.4b was identified., Interpretation: WBE affords a means to monitor circulating human and avian IAV strains and provide crucial genetic information. As such, WBE can provide rapid, cost-effective, year-round One Health surveillance to help control IAV epidemic and pandemic-related threats. However, optimisation of WBE protocols are necessary to ensure observed wastewater signals not only correlate with clinical case data, but yield information on the wider environmental pan-influenz-ome., Funding: Department of Health for Northern Ireland., Competing Interests: Declaration of interests DFG and JWM report grants from the Northern Ireland Department of Health and Public Health Agency. All other authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Respiratory syncytial virus (RSV) causes severe infections in infants, immunocompromised or elderly individuals resulting in annual epidemics of respiratory disease. Currently, limited clinical surveillance and the lack of predictable seasonal dynamics limit the public health response. Wastewater-based epidemiology (WBE) has recently been used globally as a key metric in determining prevalence of severe acute respiratory syndrome coronavirus 2 in the community, but its application to other respiratory viruses is limited. In this study, we present an integrated genomic WBE approach, applying reverse-transcription quantitative polymerase chain reaction and partial G-gene sequencing to track RSV levels and variants in the community. We report increasing detection of RSV in wastewater concomitant with increasing numbers of positive clinical cases. Analysis of wastewater-derived RSV sequences permitted identification of distinct circulating lineages within and between seasons. Altogether, our genomic WBE platform has the potential to complement ongoing global surveillance and aid the management of RSV by informing the timely deployment of pharmaceutical and nonpharmaceutical interventions., Competing Interests: Potential conflicts of interest . All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Background: Whether there is any benefit in integrating culture-independent molecular analysis of the lower airway microbiota of people with cystic fibrosis into clinical care is unclear. This study determined the longitudinal trajectory of the microbiota and if there were microbiota characteristics that corresponded with response to treatment or predicted a future pulmonary exacerbation., Methods: At least one sputum sample was collected from 149 participants enrolled in this prospective longitudinal multi-centre study and total bacterial density and microbiota community measurements were determined and compared with clinical parameters., Results: In 114 participants with paired samples when clinically stable, ∼8 months apart, the microbiota remained conserved between timepoints, regardless of whether participants received acute intravenous antibiotic treatment or not. In 62 participants, who presented with an acute exacerbation, a decrease in community richness correlated best with patient response to antibiotic treatment. Analysis of baseline samples from 30 participants who exacerbated within 4 months of their stable sample being collected and 72 participants who remained stable throughout the study showed that community characteristics such as lower richness at baseline may be predictive of an exacerbation in addition to several clinical parameters. However, lasso regression analysis indicated that only lung function (p = 0.014) was associated with a future exacerbation., Conclusions: The airway microbiota remains stable over periods <1 year with modest shifts related to treatment apparent which might provide some additional insights to patient-level measurements., Competing Interests: Declaration of Competing Interest MMT and JSE report grants from Northern Ireland Health and Social Care Research and Development Office and MSM reports grants from National Institutes of Health HL084934, during the conduct of the study. NGMcE reports grants from US-Ireland partnership/Science Foundation Ireland/Health Research Board, during the conduct of the study. JSE and MMT also reports grants from the EU Innovative Medicines Initiative, outside the submitted work. MMT reports grants from Novartis, Basilea Pharmaceutica, Alaxia SAS, and Polyphor outside the submitted work. JSE reports grants, personal fees and clinical trial involvement with Vertex and clinical trial involvement with Celtaxsys and Corbus Pharmaceuticals, outside the submitted work. RCB reports personal fees from and has a patent pending with Parion Sciences, outside the submitted work. All other authors report no conflicts of interest., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Wastewater Based Epidemiology (WBE) has become an integral part of the public health effort to track the levels of SARS-CoV-2 within communities. Detection of SARS-CoV-2 in wastewater can be challenging due to relatively low levels of virus within the sample. The wastewater matrix is also comprised of commercial and domestically derived contaminants, as well as RNases, all of which can adversely affect RT-qPCR analysis. To improve SARS-CoV-2 detection within wastewater samples we investigated both the effect of template dilution (as a means to reduce RT-qPCR inhibition) and sample stabilisation via addition of DNA/RNA Shield™ and/or RNA Later™ (to prevent RNA degradation via RNases) as a means to improve viral fragment detection. Using both methodologies, a significant improvement in SARS-CoV-2 detection from wastewater samples was observed. No adverse effects of stabilising agent addition on downstream Next-Generation Sequencing workflows were detected., (© The Author(s) 2023. Published by Oxford University Press on behalf of Applied Microbiology International.)

As of 8 July 2022, the World Health Organization (WHO) have reported 1010 probable cases of acute hepatitis of unknown aetiology in children worldwide, including approximately 250 cases in the United Kingdom (UK). Clinical presentations have often been severe, with liver transplantation a frequent clinical outcome. Human adenovirus F41 (HAdV-F41) has been detected in most children with acute hepatitis, but its role in the pathogenesis of this infection has yet to be established. Wastewater-based epidemiology (WBE) has become a well-established tool for monitoring the community spread of SARS-CoV-2, as well as other pathogens and chemicals. In this study, we adopted a WBE approach to monitoring levels of HAdV-F40/41 in wastewater before and during an acute hepatitis outbreak in Northern Ireland. We report increasing detection of HAdV-F40/41 in wastewater, concomitant with increasing numbers of clinical cases. Amplicon whole genome sequencing further classified the wastewater-derived HAdV as belonging to the F41 genotype which in turn was homologous to clinically derived sequences. We propose that WBE has the potential to inform community surveillance of HAdV-F41 and can further contribute to the ongoing global discussion supporting HAdV-F41 involvement in acute hepatitis cases., Competing Interests: Declaration of competing interest None., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Although urinary tract infections (UTIs) are extremely common, isolation of causative uropathogens is not always routinely performed, with antibiotics frequently prescribed empirically. This study determined the susceptibility of urinary isolates from two Health and Social Care Trusts (HSCTs) in Northern Ireland to a range of antibiotics commonly used in the treatment of UTIs. Furthermore, we determined if detection of trimethoprim resistance genes (dfrA) could be used as a potential biomarker for rapid detection of phenotypic trimethoprim resistance in urinary pathogens and from urine without culture., Methods: Susceptibility of E. coli and Klebsiella spp. isolates (n = 124) to trimethoprim, amoxicillin, ceftazidime, ciprofloxacin, co-amoxiclav and nitrofurantoin in addition to susceptibility of Proteus mirabilis (n = 61) and Staphylococcus saprophyticus (n = 17) to trimethoprim was determined by ETEST® and interpreted according to EUCAST breakpoints. PCR was used to detect dfrA genes in bacterial isolates (n = 202) and urine samples(n = 94)., Results: Resistance to trimethoprim was observed in 37/124 (29.8%) E. coli and Klebsiella spp. isolates with an MIC 90  > 32 mg/L. DfrA genes were detected in 29/37 (78.4%) trimethoprim-resistant isolates. Detection of dfrA was highly sensitive (93.6%) and specific (91.4%) in predicting phenotypic trimethoprim resistance among E. coli and Klebsiella spp. isolates. The dfrA genes analysed were detected using a culture-independent PCR method in 16/94 (17%) urine samples. Phenotypic trimethoprim resistance was apparent in isolates cultured from 15/16 (94%) dfrA-positive urine samples. There was a significant association (P < 0.0001) between the presence of dfrA and trimethoprim resistance in urine samples containing Gram-negative bacteria (Sensitivity = 75%; Specificity = 96.9%; PPV = 93.8%; NPV = 86.1%)., Conclusions: This study demonstrates that molecular detection of dfrA genes is a good indicator of trimethoprim resistance without the need for culture and susceptibility testing., (© 2022. The Author(s).)

To date, investigations of the microbiota in the lungs of people with Cystic Fibrosis (PWCF) have primarily focused on microbial community composition in luminal mucus, with fewer studies observing the microbiota in tissue samples from explanted lung tissue. Here, we analysed both tissue and airway luminal mucus samples extracted from whole explanted lungs of PWCF and unused donor lungs. We determined if the lung microbiota in end-stage CF varied within and between patients, was spatially heterogeneous and related to localized structural damage. Microbial community composition was determined by Illumina MiSeq sequencing and related to the CF-Computed Tomography (CT) score and features of end-stage lung disease on micro-CT. Ninety-eight CF tissue (n=11 patients), 20 CF luminal mucus (n=8 patients) and 33 donor tissue (n=4 patients) samples were analysed. Additionally, we compared 20 paired CF tissue and luminal mucus samples that enabled a direct "geographical" comparison of the microbiota in these two niches. Significant differences in microbial communities were apparent between the 3 groups. However, overlap between the three groups, particularly between CF and donor tissue and CF tissue and CF luminal mucus was also observed. Microbial diversity was lower in CF luminal mucus compared to CF tissue, with dominance higher in luminal mucus. For both CF and donor tissue, intra- and inter-patient variability in ecological parameters was observed. No relationships were observed between ecological parameters and CF-CT score, or features of end-stage lung disease. The end-stage CF lung is characterised by a low diversity microbiota, differing within and between individuals. No clear relationship was observed between regional microbiota variation and structural lung damage., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Einarsson, Vanaudenaerde, Spence, Lee, Boon, Verleden, Elborn, Dupont, Van Raemdonck, Gilpin, Vos, Verleden and Tunney.)

The COVID-19 pandemic has put unprecedented pressure on public health resources around the world. From adversity, opportunities have arisen to measure the state and dynamics of human disease at a scale not seen before. In the United Kingdom, the evidence that wastewater could be used to monitor the SARS-CoV-2 virus prompted the development of National wastewater surveillance programmes. The scale and pace of this work has proven to be unique in monitoring of virus dynamics at a national level, demonstrating the importance of wastewater-based epidemiology (WBE) for public health protection. Beyond COVID-19, it can provide additional value for monitoring and informing on a range of biological and chemical markers of human health. A discussion of measurement uncertainty associated with surveillance of wastewater, focusing on lessons-learned from the UK programmes monitoring COVID-19 is presented, showing that sources of uncertainty impacting measurement quality and interpretation of data for public health decision-making, are varied and complex. While some factors remain poorly understood, we present approaches taken by the UK programmes to manage and mitigate the more tractable sources of uncertainty. This work provides a platform to integrate uncertainty management into WBE activities as part of global One Health initiatives beyond the pandemic., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Treatment with Ivacaftor provides a significant clinical benefit in people with cystic fibrosis (PWCF) with the class III G551D-CFTR mutation. This study determined the effect of CFTR modulation with ivacaftor on the lung microbiota in PWCF., Methods: Using both extended-culture and culture-independent molecular methods, we analysed the lower airway microbiota of 14 PWCF, prior to commencing ivacaftor treatment and at the last available visit within the following year. We determined total bacterial and Pseudomonas aeruginosa densities by both culture and qPCR, assessed ecological parameters and community structure and compared these with biomarkers of inflammation and clinical outcomes., Results: Significant improvement in FEV 1 , BMI, sweat chloride and levels of circulating inflammatory biomarkers were observed POST-ivacaftor treatment. Extended-culture demonstrated a higher density of strict anaerobic bacteria (p = 0.024), richness (p = 1.59*10 -4 ) and diversity (p = 0.003) POST-treatment. No significant difference in fold change was observed by qPCR for either total bacterial 16S rRNA copy number or P. aeruginosa density for oprL copy number with treatment. Culture-independent (MiSeq) analysis revealed a significant increase in richness (p = 0.03) and a trend towards increased diversity (p = 0.07). Moreover, improvement in lung function, richness and diversity displayed an inverse correlation with the main markers of inflammation (p < 0.05)., Conclusions: Following treatment with ivacaftor, significant improvements in clinical parameters were seen. Despite modest changes in overall microbial community composition, there was a shift towards a bacterial ecology associated with less severe CF lung disease. Furthermore, a significant correlation was observed between richness and diversity and levels of circulating inflammatory markers., Competing Interests: Declaration of Competing Interest None declared., (Copyright © 2021 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

The advent of high-throughput multi-omics technologies has underpinned the expansion in lung microbiome research, increasing our understanding of the nature, complexity and significance of the polymicrobial communities harbored by people with CF (PWCF). Having established that structurally complex microbial communities exist within the airways, the focus of recent research has now widened to investigating the function and dynamics of the resident microbiota during disease as well as in health. With further refinement, multi-omics approaches present the opportunity to untangle the complex interplay between microbe-microbe and microbe-host interactions in the lung and the relationship with respiratory disease progression, offering invaluable opportunities to discover new therapeutic approaches for our management of airway infection in CF., (Copyright © 2020 Lee, Einarsson, Gilpin and Tunney.)

Background: Alterations in the lung microbiota may drive disease development and progression in patients with chronic respiratory diseases. Following lung transplantation (LTx), azithromycin is used to both treat and prevent chronic lung allograft dysfunction (CLAD). The objective of this study was to determine the association between azithromycin use, CLAD, acute rejection, airway inflammation, and bacterial microbiota composition and structure after LTx., Methods: Bronchoalveolar lavage samples (n = 219) from 69 LTx recipients (azithromycin, n = 32; placebo, n = 37) from a previously conducted randomized placebo-controlled trial with azithromycin were analyzed. Samples were collected at discharge, 1, and 2 years following randomization and at CLAD diagnosis. Bacterial microbial community composition and structure was determined using 16S ribosomal RNA gene sequencing and associated with clinically important variables., Results: At discharge and following 1 and 2 years of azithromycin therapy, no clear differences in microbial community composition or overall diversity were observed. Moreover, no changes in microbiota composition were observed in CLAD phenotypes. However, acute rejection was associated with a reduction in community diversity (p = 0.0009). Significant correlations were observed between microbiota composition, overall diversity, and levels of inflammatory cytokines in bronchoalveolar lavage, particularly CXCL8., Conclusions: Chronic azithromycin usage did not disturb the bacterial microbiota. However, acute rejection episodes were associated with bacterial dysbiosis., (Copyright © 2019 International Society for Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.)

The objective was to determine the in vitro antimicrobial susceptibility of Pseudomonas aeruginosa isolates cultured from cystic fibrosis (CF) patients and explore associations between strain sequence type and susceptibility. Fourteen antibiotics and antibiotic combinations, including the novel antibacterial peptide murepavadin, were tested for activity against 414 Pseudomonas aeruginosa isolates cultured from respiratory samples of CF patients. The complete genomes of the isolates were sequenced, and minimum spanning trees were constructed based on the sequence types (STs). Percentages of resistance according to CLSI 2019 breakpoints were as follows: cefepime, 14%; ceftazidime, 11%; ceftazidime-avibactam, 7%; ceftolozane-tazobactam, 3%; piperacillin-tazobactam, 12%; meropenem, 18%; imipenem, 32%; aztreonam, 23%; ciprofloxacin, 30%; gentamicin, 30%; tobramycin, 12%; amikacin, 18%; and colistin, 4%. Murepavadin MIC 50 and MIC 90 were 0.12 mg/liter and 2 mg/liter, respectively. There were no apparent clonal clusters associated with resistance, but higher MICs did appear to occur more often in STs with multiple isolates than in single ST isolates. In general, the CF isolates showed a wide genetic distribution. P. aeruginosa CF isolates exhibited the lowest resistance rates against ceftolozane-tazobactam, ceftazidime-avibactam, and colistin. Murepavadin demonstrated the highest activity on a per-weight basis and may therefore become a valuable addition to the currently available antibiotics for treatment of respiratory infection in people with CF., (Copyright © 2020 American Society for Microbiology.)

Introduction: Bacteria have been extensively implicated in the development of smoking related diseases, such as COPD, by either direct infection or bacteria-mediated inflammation. In response to the health risks associated with tobacco exposure, the use of electronic cigarettes (e-cigs) has increased. This study compared the effect of e-cig vapour (ECV) and cigarette smoke (CSE) on the virulence and inflammatory potential of key lung pathogens (Haemophilus influenzae, Streptococcus pneumoniae, Staphylococcus aureus and Pseudomonas aeruginosa)., Methods: Biofilm formation, virulence in the Galleria mellonella infection model, antibiotic susceptibility and IL-8/TNF-α production in A549 cells, were compared between bacteria exposed to ECV, CSE and non-exposed bacteria., Results: Statistically significant increases in biofilm and cytokine secretion were observed following bacterial exposure to either ECV or CSE, compared to non-exposed bacteria; the effect of exposure to ECV on bacterial phenotype and virulence was comparable, and in some cases greater, than that observed following CSE exposure. Treatment of A549 cells with cell signaling pathway inhibitors prior to infection, did not suggest that alternative signaling pathways were being activated following exposure of bacteria to either ECV or CSE., Conclusions: These findings therefore suggest that ECV and CSE can induce changes in phenotype and virulence of key lung pathogens, which may increase bacterial persistence and inflammatory potential.

Background: Routine clinical culture detects a subset of the cystic fibrosis (CF) airways microbiota based on culture-independent (molecular) methods. This study aimed to determine how extended sputum culture of viable bacteria changes over time in relation to clinical status and predicts exacerbations., Methods: Sputa from patients at a baseline stable and up to three subsequent time-points were analysed by extended-quantitative culture; aerobe/anaerobe densities, ecological indexes and community structure were assessed together with clinical outcomes., Results: Eighty patients were prospectively recruited. Sputa were successfully collected and cultured at 199/267 (74.5%) study visits. Eighty-two sputa from 25 patients comprised a complete sample-set for longitudinal analyses. Bacterial density, ecological indexes and clinical outcomes were unchanged in 18 patients with three sequential stable visits. Conversely, in 7 patients who had an exacerbation, total bacterial and aerobe densities differed over four study visits (P < .001) with this difference particularly apparent between the baseline visit and completion of acute antibiotic treatment where a decrease in density was observed. Bacterial communities were more similar within than between patients but stable patients had the least variation in community structure over time. Using logistic regression in a further analysis, baseline features in 37 patients without compared to 15 patients with a subsequent exacerbation showed that clinical measures rather than bacterial density or ecological indexes were independent predictors of an exacerbation., Conclusions: Greater fluctuation in the viable bacterial community during treatment of an exacerbation than between stable visits was observed. Extended-quantitative culture did not provide prognostic information of a future exacerbation., (Copyright © 2019 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

Objectives: To determine the antimicrobial activity of ALX-009, a combination of bovine lactoferrin and hypothiocyanite, in sputum against Pseudomonas aeruginosa and Burkholderia cepacia complex (Bcc), key pathogens causing infection in the lungs of cystic fibrosis (CF) patients., Methods: The antimicrobial activity of ALX-009 against clinical respiratory P. aeruginosa isolates was determined by time-kill assay. Sputum from CF patients was treated with ALX-009, either alone or in combination with tobramycin, and the effect on P. aeruginosa, Bcc and total sputum density was determined., Results: Time-kill assay indicated that ALX-009 was bactericidal at 24 h against 4/4 P. aeruginosa isolates under aerobic conditions, and against 3/4 isolates under anaerobic conditions. ALX-009 was also bactericidal against P. aeruginosa in sputum samples at 6 h (n = 22/24 samples) and 24 h (n = 14/24 samples), and demonstrated significantly greater activity than tobramycin at both timepoints. Activity against Bcc in sputum samples (n = 9) was also demonstrated, but the magnitude of change in Bcc density was less than for P. aeruginosa. To determine the effect of treating sputum with two doses of ALX-009, similar to current regimens for inhaled antibiotics, aliquots of a further 10 sputum samples positive for P. aeruginosa were treated with one (t = 0 h) or two doses (t = 0 h, t = 12 h) of ALX-009; treatment with two doses resulted in bactericidal activity in 7/10 samples at 34 h compared with only 3/10 samples when treatment was with one dose., Conclusions: ALX-009 demonstrates promise as a novel antimicrobial that could be used to decrease P. aeruginosa density in the lungs of people with CF.

Background: Urinary Tract Infections (UTIs) are common bacterial infections, second only to respiratory tract infections and particularly prevalent within primary care. Conventional detection of UTIs is culture, however, return of results can take between 24 and 72 hours. The introduction of a point of care (POC) test would allow for more timely identification of UTIs, facilitating improved, targeted treatment. This study aimed to obtain consensus on the criteria required for a POC UTI test, to meet patient need within primary care., Methods: Criteria for consideration were compiled by the research team. These criteria were validated through a two-round Delphi process, utilising an expert panel of healthcare professionals from across Europe and United States of America. Using web-based questionnaires, panellists recorded their level of agreement with each criterion based on a 5-point Likert Scale, with space for comments. Using median response, interquartile range and comments provided, criteria were accepted/rejected/revised depending on pre-agreed cut-off scores., Results: The first round questionnaire presented thirty-three criteria to the panel, of which 22 were accepted. Consensus was not achieved for the remaining 11 criteria. Following response review, one criterion was removed, while after revision, the remaining 10 criteria entered the second round. Of these, four were subsequently accepted, resulting in 26 criteria considered appropriate for a POC test to detect urinary infections., Conclusion: This study generated an approved set of criteria for a POC test to detect urinary infections. Criteria acceptance and comments provided by the healthcare professionals also supports the development of a multiplex point of care UTI test., Competing Interests: M A Crockard is an employee of Randox Laboratories Ltd (Northern Ireland), a manufacturer of molecular diagnostic tests. NJ Weir, S Pattison, D Gilpin, M Tunney and C Hughes are working in collaboration with Randox Laboratories Ltd (Northern Ireland). This does not alter our adherence to PLOS ONE policies on sharing data and materials. The other authors have no conflicts of interest to declare.

Purpose. Prevotella spp. represent a diverse genus of bacteria, frequently identified by both culture and molecular methods in the lungs of patients with chronic respiratory infection. However, their role in the pathogenesis of chronic lung infection is unclear; therefore, a more complete understanding of their molecular epidemiology is required. Methodology. Pulsed Field Gel Electrophoresis (PFGE) and Random Amplified Polymorphic DNA (RAPD) assays were developed and used to determine the degree of similarity between sequential isolates ( n =42) from cystic fibrosis (CF) patients during periods of clinical stability and exacerbation. Results. A wide diversity of PFGE and RAPD banding patterns were observed, demonstrating considerable within-genus heterogeneity. In 8/12 (66.7 %) cases, where the same species was identified at sequential time points, pre- and post-antibiotic treatment of an exacerbation, PFGE/RAPD profiles were highly similar or identical. Congruence was observed between PFGE and RAPD (adjusted Rand coefficient, 0.200; adjusted Wallace RAPD->PFGE 0.459, PFGE->RAPD 0.128). Furthermore, some isolates could not be adequately assigned a species name on the basis of 16S rRNA analysis: these isolates had identical PFGE/RAPD profiles to Prevotella histicola . Conclusion. The similarity in PFGE and RAPD banding patterns observed in sequential CF Prevotella isolates may be indicative of the persistence of this genus in the CF lung. Further work is required to determine the clinical significance of this finding, and to more accurately distinguish differences in pathogenicity between species.

Purpose: The purpose of this study was to develop an alternative, more clinically relevant approach to susceptibility reporting for implant-associated infections. Using 20 staphylococcal isolates, isolated from clinical implant infections, the majority (85 %) demonstrated biofilm-forming capabilities. A significantly increased minimum biofilm eradication concentration (MBEC) compared to minimum inhibitory concentration (MIC) breakpoint was obtained, with MBEC values greater than 256 µg ml-1 for the majority of bacteria. Such a vast increase was also demonstrated for isolates defined as negligible biofilm formers via crystal violet staining, likely due to the high protein content of biofilms, as confirmed by proteinase-K treatment., Methodology: This study employed a variety of techniques to assess MIC and MBEC of the isolates tested. In addition, the nature of bacterial biofilm across a range of clinical isolates was investigated using crystal violet staining, sodium metaperiodate and proteinase-K treatment, and PCR analysis.Results/Key findings. Infection of medical implants is associated with increased rates of infection and increased bacterial tolerance to antibiotic strategies. Clinical significance is due to the presence of pathogens attached to biomaterial surfaces enclosed in an extracellular polymeric matrix termed the biofilm. This article highlights the importance of defining the clinical susceptibility of implant-associated infections in vitro using methods that are relevant to the biofilm phenotype in vivo, and highlights how current planktonic-based antimicrobial susceptibility tests are often misleading., Conclusion: The use of biofilm-relevant susceptibility tests would improve patient outcomes by enabling correct antimicrobial regimens to be rapidly identified, reducing treatment failure and halting the spread of antimicrobial-resistant strains.

Extended-spectrum β-lactamase (ESBL) production and the prevalence of the β-lactamase-encoding gene blaTEM were determined in Prevotella isolates (n=50) cultured from the respiratory tract of adults and young people with cystic fibrosis (CF). Time-kill studies were used to investigate the concept of passive antibiotic resistance and to ascertain whether a β-lactamase-positive Prevotella isolate can protect a recognised CF pathogen from the action of ceftazidime in vitro. The results indicated that approximately three-quarters (38/50; 76%) of Prevotella isolates produced ESBLs. Isolates positive for ESBL production had higher minimum inhibitory concentrations (MICs) of β-lactam antibiotics compared with isolates negative for production of ESBLs (P<0.001). The blaTEM gene was detected more frequently in CF Prevotella isolates from paediatric patients compared with isolates from adults (P=0.002), with sequence analysis demonstrating that 21/22 (95%) partial blaTEM genes detected were identical to blaTEM-116. Furthermore, a β-lactamase-positive Prevotella isolate protected Pseudomonas aeruginosa from the antimicrobial effects of ceftazidime (P=0.03). Prevotella isolated from the CF respiratory microbiota produce ESBLs and may influence the pathogenesis of chronic lung infection via indirect methods, including shielding recognised pathogens from the action of ceftazidime., (Copyright © 2016 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.)

Objectives: To investigate mechanisms of reduced susceptibility to commonly used antibiotics in Prevotella cultured from patients with cystic fibrosis (CF), patients with invasive infection and healthy control subjects and to determine whether genotype can be used to predict phenotypic resistance., Methods: The susceptibility of 157 Prevotella isolates to seven antibiotics was compared, with detection of resistance genes (cfxA-type gene, ermF and tetQ), mutations within the CfxA-type β-lactamase and expression of efflux pumps., Results: Prevotella isolates positive for a cfxA-type gene had higher MICs of amoxicillin and ceftazidime compared with isolates negative for this gene (P < 0.001). A mutation within the CfxA-type β-lactamase (Y239D) was associated with ceftazidime resistance (P = 0.011). The UK CF isolates were 5.3-fold, 2.7-fold and 5.7-fold more likely to harbour ermF compared with the US CF, UK invasive and UK healthy control isolates, respectively. Higher concentrations of azithromycin (P < 0.001) and clindamycin (P < 0.001) were also required to inhibit the growth of the ermF-positive isolates compared with ermF-negative isolates. Furthermore, tetQ-positive Prevotella isolates had higher MICs of tetracycline (P = 0.001) and doxycycline (P < 0.001) compared with tetQ-negative isolates. Prevotella spp. were also shown, for the first time, to express resistance nodulation division (RND)-type efflux pumps., Conclusions: This study has demonstrated that Prevotella isolated from various sources harbour a common pool of resistance genes and possess RND-type efflux pumps, which may contribute to tetracycline resistance. The findings indicate that antibiotic resistance is common in Prevotella spp., but the genotypic traits investigated do not reflect phenotypic antibiotic resistance in every instance., (© The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

The activity of aminoglycosides, which are used to treat Pseudomonas aeruginosa respiratory infection in cystic fibrosis (CF) patients, is reduced under the anaerobic conditions that reflect the CF lung in vivo. In contrast, a 4:1 (wt/wt) combination of fosfomycin and tobramycin (F:T), which is under investigation for use in the treatment of CF lung infection, has increased activity against P. aeruginosa under anaerobic conditions. The aim of this study was to elucidate the mechanisms underlying the increased activity of F:T under anaerobic conditions. Microarray analysis was used to identify the transcriptional basis of increased F:T activity under anaerobic conditions, and key findings were confirmed by microbiological tests, including nitrate utilization assays, growth curves, and susceptibility testing. Notably, growth in subinhibitory concentrations of F:T, but not tobramycin or fosfomycin alone, significantly downregulated (P < 0.05) nitrate reductase genes narG and narH, which are essential for normal anaerobic growth of P. aeruginosa. Under anaerobic conditions, F:T significantly decreased (P < 0.001) nitrate utilization in P. aeruginosa strains PAO1, PA14, and PA14 lasR::Gm, a mutant known to exhibit increased nitrate utilization. A similar effect was observed with two clinical P. aeruginosa isolates. Growth curves indicate that nitrate reductase transposon mutants had reduced growth under anaerobic conditions, with these mutants also having increased susceptibility to F:T compared to the wild type under similar conditions. The results of this study suggest that downregulation of nitrate reductase genes resulting in reduced nitrate utilization is the mechanism underlying the increased activity of F:T under anaerobic conditions.

Objectives: To compare the antimicrobial susceptibility of Prevotella spp. isolated from cystic fibrosis (CF) and non-CF patients and analyse the impact of antibiotic prescribing in the preceding year on resistance amongst CF isolates., Methods: The susceptibility of 80 CF Prevotella isolates to 12 antibiotics was compared with that of 50 Prevotella isolates from invasive infections in people who did not have CF and 27 Prevotella isolates from healthy controls., Results: All isolates were susceptible to chloramphenicol, meropenem and piperacillin/tazobactam, with only four isolates resistant to metronidazole. However, resistance to amoxicillin, ceftazidime and tetracycline was apparent in all groups. Significant differences in clindamycin resistance (UK CF, 56%; UK invasive, 10%) and co-amoxiclav non-susceptibility (UK CF, 32%; UK invasive, 12%) were observed between UK CF and UK invasive isolates. The likelihood of non-susceptibility to clindamycin and co-amoxiclav in UK CF isolates was 5.5-fold and 2.5-fold higher relative to that in UK invasive isolates, respectively. Azithromycin MICs were also significantly higher for CF isolates (P < 0.001), which was associated with current prescription of azithromycin. More than 50% of clinical isolates tested in this study were β-lactamase positive., Conclusions: This study profiles antibiotic susceptibility in Prevotella spp. in CF and demonstrates that meropenem, piperacillin/tazobactam, chloramphenicol and metronidazole are likely to be the most effective antibiotics if treatment is indicated.

Cystic fibrosis (CF) is characterized by defective mucociliary clearance and chronic airway infection by a complex microbiota. Infection, persistent inflammation and periodic episodes of acute pulmonary exacerbation contribute to an irreversible decline in CF lung function. While the factors leading to acute exacerbations are poorly understood, antibiotic treatment can temporarily resolve pulmonary symptoms and partially restore lung function. Previous studies indicated that exacerbations may be associated with changes in microbial densities and the acquisition of new microbial species. Given the complexity of the CF microbiota, we applied massively parallel pyrosequencing to identify changes in airway microbial community structure in 23 adult CF patients during acute pulmonary exacerbation, after antibiotic treatment and during periods of stable disease. Over 350,000 sequences were generated, representing nearly 170 distinct microbial taxa. Approximately 60% of sequences obtained were from the recognized CF pathogens Pseudomonas and Burkholderia, which were detected in largely non-overlapping patient subsets. In contrast, other taxa including Prevotella, Streptococcus, Rothia and Veillonella were abundant in nearly all patient samples. Although antibiotic treatment was associated with a small decrease in species richness, there was minimal change in overall microbial community structure. Furthermore, microbial community composition was highly similar in patients during an exacerbation and when clinically stable, suggesting that exacerbations may represent intrapulmonary spread of infection rather than a change in microbial community composition. Mouthwash samples, obtained from a subset of patients, showed a nearly identical distribution of taxa as expectorated sputum, indicating that aspiration may contribute to colonization of the lower airways. Finally, we observed a strong correlation between low species richness and poor lung function. Taken together, these results indicate that the adult CF lung microbiome is largely stable through periods of exacerbation and antibiotic treatment and that short-term compositional changes in the airway microbiota do not account for CF pulmonary exacerbations.

Background: Anaerobic bacteria are increasingly regarded as important in cystic fibrosis (CF) pulmonary infection. The aim of this study was to determine the effect of antibiotic treatment on aerobic and anaerobic microbial community diversity and abundance during exacerbations in patients with CF., Methods: Sputum was collected at the start and completion of antibiotic treatment of exacerbations and when clinically stable. Bacteria were quantified and identified following culture, and community composition was also examined using culture-independent methods., Results: Pseudomonas aeruginosa or Burkholderia cepacia complex were detected by culture in 24/26 samples at the start of treatment, 22/26 samples at completion of treatment and 11/13 stable samples. Anaerobic bacteria were detected in all start of treatment and stable samples and in 23/26 completion of treatment samples. Molecular analysis showed greater bacterial diversity within sputum samples than was detected by culture; there was reasonably good agreement between the methods for the presence or absence of aerobic bacteria such as P aeruginosa (κ=0.74) and B cepacia complex (κ=0.92), but agreement was poorer for anaerobes. Both methods showed that the composition of the bacterial community varied between patients but remained relatively stable in most individuals despite treatment. Bacterial abundance decreased transiently following treatment, with this effect more evident for aerobes (median decrease in total viable count 2.3×10(7) cfu/g, p=0.005) than for anaerobes (median decrease in total viable count 3×10(6) cfu/g, p=0.046)., Conclusion: Antibiotic treatment targeted against aerobes had a minimal effect on abundance of anaerobes and community composition, with both culture and molecular detection methods required for comprehensive characterisation of the microbial community in the CF lung. Further studies are required to determine the clinical significance of and optimal treatment for these newly identified bacteria.

Objective: This study aimed to investigate antimicrobial treatment of an infected cochlear implant, undertaken in an attempt to salvage the infected device., Methods: We used the broth microdilution method to assess the susceptibility of meticillin-sensitive Staphylococcus aureus isolate, cultured from an infected cochlear implant, to common antimicrobial agents as well as to novel agents such as tea tree oil. To better simulate in vivo conditions, where bacteria grow as microcolonies encased in glycocalyx, the bactericidal activity of selected antimicrobial agents against the isolate growing in biofilm were also compared., Results: When grown planktonically, the S aureus isolate was susceptible to 17 of the 18 antimicrobials tested. However, when grown in biofilm, it was resistant to all conventional antimicrobials. In contrast, 5 per cent tea tree oil completely eradicated the biofilm following exposure for 1 hour., Conclusion: Treatment of infected cochlear implants with novel agents such as tea tree oil could significantly improve salvage outcome.

Objectives: To determine the prevalence of, and factors associated with, methicillin-resistant Staphylococcus aureus (MRSA) colonization in residents and staff in nursing homes in one geographically defined health administration area of Northern Ireland., Design: Point prevalence study., Setting: Nursing homes., Participants: Residents and staff in nursing homes., Measurements: Nasal swabs were taken from all consenting residents and staff. If relevant, residents also provided urine samples, and swabs were taken from wounds and indwelling devices., Results: A total of 1,111 residents (66% of all residents) and 553 staff (86% of available staff) in 45 nursing homes participated. The combined prevalence rate of MRSA in the resident population was 23.3% (95% confidence interval (CI)=18.8-27.7%) and 7.5% in staff (95% CI=5.1-9.9%). Residents who lived in nursing homes that were part of a chain were more likely to be colonized with MRSA (odds ratio (OR)=1.91, 95% CI=1.21-3.02) than those living in independently owned facilities. Residents were also more likely to be colonized if they lived in homes in which more than 12.5% of all screened healthcare staff (care assistants and nurses) were colonized with MRSA (OR=2.46, 95% CI=1.41-4.29) or if they lived in homes in which more than 15% of care assistants were colonized with MRSA (OR=2.64, 95% CI=1.58-4.42)., Conclusion: The findings suggest that there is substantial colonization of MRSA in nursing home residents and staff in this one administrative health area. Implementation of infection control strategies should be given high priority in nursing homes.

Rapid detection of MRSA may be important for the control of MRSA spread in hospitals. The aim of this investigation was to compare the use of a rapid polymerase chain reaction (PCR) screening method with standard culture for the detection of meticillin-resistant Staphylococcus aureus (MRSA) colonisation and to determine its impact on the incidence of MRSA in two hospital wards. During the first phase of the investigation (four months), patients in a surgical ward were screened using the rapid PCR technique and patients in a medical/cardiology ward were screened with standard culture methods. During the second phase of the investigation (four months), MRSA screening methods were switched between the two wards. An audit of infection control practices on each ward was made at the end of each phase in order to check whether any changes had occurred that might influence the risks of MRSA transmission. Use of the rapid PCR method significantly reduced the median time between swabs being taken, to the results being telephoned to the wards (excluding weekends), from 47 to 21 h (P<0.001). However, comparison of MRSA incidence during use of PCR (20/1000 bed-days) and culture methods (22.1/1000 bed-days) revealed no significant difference in incidence on the surgical ward (P=0.69). Regarding the medical/cardiology ward, analysis of data was complicated by an increase in the detection of MRSA during the PCR phase (P<0.05). The study demonstrated that rapid PCR can significantly reduce the turnaround times but reducing the time between swabs being taken to results being telephoned to the ward is still not sufficient to limit the transmission of MRSA.

Aims: To investigate the effect of sub-lethal challenge with tea tree oil (TTO) on the antibiotic resistance profiles of staphylococci., Methods and Results: Isolates of methicillin-resistant/-sensitive Staphylococcus aureus (MRSA and MSSA) and coagulase-negative staphylococci (CONS) were habituated to sub-lethal concentrations of TTO (72 h). Following habituation, the minimum inhibitory concentrations (MIC) of antibiotics and TTO were determined. Habituated MRSA/MSSA cultures had higher (P < 0.05) MIC values than control cultures for the examined antibiotics. Habituated MRSA/MSSA cultures also displayed decreased susceptibility to TTO. Although the MIC of habituated MRSA/MSSA for the examined antibiotics reverted to control values after subsequent culture in the absence of TTO, the increased MIC against TTO were maintained. When compared with control cultures, habituated CoNS cultures had higher (P < 0.05) MIC values against three-fifths of the antibiotics examined; no changes in TTO MIC were observed., Conclusions: TTO habituation 'stress-hardens' MRSA and MSSA, evidenced by transient decreased antibiotic susceptibility and stable decreased TTO susceptibility. Although TTO habituation did not decrease susceptibility of CoNS to TTO, such cultures showed transient decreased antibiotic susceptibility., Significance and Impact of the Study: Application of TTO at sub-lethal concentrations may reduce the efficacy of topical antibiotics used with TTO in combination therapies.

Rationale: Pulmonary infection in cystic fibrosis (CF) is polymicrobial and it is possible that anaerobic bacteria, not detected by routine aerobic culture methods, reside within infected anaerobic airway mucus., Objectives: To determine whether anaerobic bacteria are present in the sputum of patients with CF., Methods: Sputum samples were collected from clinically stable adults with CF and bronchoalveolar lavage fluid (BALF) samples from children with CF. Induced sputum samples were collected from healthy volunteers who did not have CF. All samples were processed using anaerobic bacteriologic techniques and bacteria within the samples were quantified and identified., Measurements and Main Results: Anaerobic species primarily within the genera Prevotella, Veillonella, Propionibacterium, and Actinomyces were isolated in high numbers from 42 of 66 (64%) sputum samples from adult patients with CF. Colonization with Pseudomonas aeruginosa significantly increased the likelihood that anaerobic bacteria would be present in the sputum. Similar anaerobic species were identified in BALF from pediatric patients with CF. Although anaerobes were detected in induced sputum samples from 16 of 20 volunteers, they were present in much lower numbers and were generally different species compared with those detected in CF sputum. Species-dependent differences in the susceptibility of the anaerobes to antibiotics with known activity against anaerobes were apparent with all isolates susceptible to meropenem., Conclusions: A range of anaerobic species are present in large numbers in the lungs of patients with CF. If these anaerobic bacteria are contributing significantly to infection and inflammation in the CF lung, informed alterations to antibiotic treatment to target anaerobes, in addition to the primary infecting pathogens, may improve management.

Immunoreactive T lymphocyte epitopes within the ORF1, ORF2, and ORF 3 products of porcine circovirus type 2 (PCV2) were mapped. For this, overlapping linear 20-mer peptides were synthesized and tested for their ability to induce T lymphocyte proliferation in porcine peripheral blood mononuclear cells (PBMCs) isolated from experimentally PCV2-infected pigs. After a preliminary screening of 31 (ORF1), 23 (ORF2), and 10 (ORF3) peptides using PBMCs from 4 PCV2-infected pigs, none of the peptides appeared to be immunoreactive (stimulation index [SI] : 2) in all four pigs. Only 14 peptides appeared to be immunoreactive in 3 of the 4 pigs. These peptides were designated as immunodominant in the preliminary screening and selected for further analysis. The immunodominant peptides were resynthesized and purified by high-performance liquid chromatography and tested for their ability to induce T lymphocyte proliferation in PBMCs from another three PCV2-infected pigs. None of the immunodominant peptides appeared to be immunoreactive in all three pigs of the second screening. Only three peptides appeared to be immunoreactive in two of three pigs, two encoded by PCV2 ORF1 (amino acid residues 81-100 and 201-220) and one encoded by PCV2 ORF3 (amino acid residues 31-50), and were therefore considered to be immunodominant in both screenings. Although peptides encoded by ORF2 appeared to show the highest immunoreactivity in some pigs, none of these peptides displayed immunodominance in both screenings. In summary, the present study indicates that the T lymphocyte responses to PCV2 are primarily directed toward epitopes of the nonstructural proteins of ORF1 and ORF3.

The purpose of this study was to determine serum profiles of cytokines at a protein level and Creactive protein (CRP) during the development of postweaning multisystemic wasting syndrome (PMWS) in experimentally inoculated pigs. Levels of serum IFN-alpha, IL-6, IL-10, and CRP were examined for a 35-day period in 10 piglets experimentally infected with PCV2 at 3 weeks of age. Four of the infected piglets developed severe PMWS at 14 to 21 days post-infection (d.p.i.) and died prior to termination of the experiment. The remaining six PCV2-infected piglets experienced transient fever, but did not display overt clinical signs of PMWS and were considered as subclinically infected. A bioassay was used to detect IL-6 and ELISAs were used to detect IFN-alpha, IL-10, and CRP. There were no significant differences in cytokine or CRP expression from 0 to 7 d.p.i. between the PMWS-affected and the subclinically infected piglets. Levels of IL-10 and CRP were elevated from 10 and 14 d.p.i. respectively in the PMWS-affected piglets compared to the subclinically infected piglets. There were no significant differences in IFN-alpha and IL-6 expression between the PMWS-affected piglets and the subclinically infected piglets. The present study shows that elevated levels of serum CRP and IL-10 were associated with PCV2-infected piglets that subsequently developed severe PMWS. This may help to provide further insight into the immunoaetiogenesis of this syndrome.

Porcine circovirus type 2 (PCV2) nucleic acid and/or antigens are consistently observed in cells of monocytic morphology in lesions of pigs affected by post-weaning multisystemic wasting syndrome (PMWS). In this study, PCV2 antigen was detected in the cytoplasm of monocytes, pulmonary macrophages (PMs) and monocyte-derived macrophages exposed to the virus in vitro, by immunofluorescence analysis (IFA) and the phenotype of these cells confirmed by detection of monocytic cell surface markers using flow cytometry. Viral antigen was not observed in lymphocytic cells. Replication of the virus in PMs was investigated further by comparison to that observed in the continuous pig kidney cell line (PK15A) using quantitative virus titration, quantitative PCR and by the detection of double stranded DNA intermediates of viral replication by Southern blotting analyses. Although increases in viral DNA and levels of infectious virus progeny and the presence of replicative intermediates, indicative of viral replication, were observed in PK15A cells, no such changes were observed in PMs in spite of the fact that infectious virus, viral antigen and viral DNA persisted in the cells for at least the duration of the experiment. These results suggest that in vivo, monocytic cells may not represent the primary target for PCV2 replication.