Emergence of methicillin resistance predates the clinical use of antibiotics.

The discovery of antibiotics more than 80 years ago has led to considerable improvements in human and animal health. Although antibiotic resistance in environmental bacteria is ancient, resistance in human pathogens is thought to be a modern phenomenon that is driven by the clinical use of antibiotics1. Here we show that particular lineages of methicillin-resistant Staphylococcus aureus-a notorious human pathogen-appeared in European hedgehogs in the pre-antibiotic era. Subsequently, these lineages spread within the local hedgehog populations and between hedgehogs and secondary hosts, including livestock and humans. We also demonstrate that the hedgehog dermatophyte Trichophyton erinacei produces two ß-lactam antibiotics that provide a natural selective environment in which methicillin-resistant S. aureus isolates have an advantage over susceptible isolates. Together, these results suggest that methicillin resistance emerged in the pre-antibiotic era as a co-evolutionary adaptation of S. aureus to the colonization of dermatophyte-infected hedgehogs. The evolution of clinically relevant antibiotic-resistance genes in wild animals and the connectivity of natural, agricultural and human ecosystems demonstrate that the use of a One Health approach is critical for our understanding and management of antibiotic resistance, which is one of the biggest threats to global health, food security and development.

The rapid detection of respiratory pathogens in critically ill children.

PURPOSE: Respiratory infections are the most common reason for admission to paediatric intensive care units (PICU). Most patients with lower respiratory tract infection (LRTI) receive broad-spectrum antimicrobials, despite low rates of bacterial culture confirmation. Here, we evaluated a molecular diagnostic test for LRTI to inform the better use of antimicrobials. METHODS: The Rapid Assay for Sick Children with Acute Lung infection Study was a single-centre, prospective, observational cohort study of mechanically ventilated children (> 37/40 weeks corrected gestation to 18 years) with suspected community acquired or ventilator-associated LRTI. We evaluated the use of a 52-pathogen custom TaqMan Array Card (TAC) to identify pathogens in non-bronchoscopic bronchoalveolar lavage (mini-BAL) samples. TAC results were compared to routine microbiology testing. Primary study outcomes were sensitivity and specificity of TAC, and time to result. RESULTS: We enrolled 100 patients, all of whom were tested with TAC and 91 of whom had matching culture samples. TAC had a sensitivity of 89.5% (95% confidence interval (CI95) 66.9-98.7) and specificity of 97.9% (CI95 97.2-98.5) compared to routine bacterial and fungal culture. TAC took a median 25.8 h (IQR 9.1-29.8 h) from sample collection to result. Culture was significantly slower: median 110.4 h (IQR 85.2-141.6 h) for a positive result and median 69.4 h (IQR 52.8-78.6) for a negative result. CONCLUSIONS: TAC is a reliable and rapid adjunct diagnostic approach for LRTI in critically ill children, with the potential to aid early rationalisation of antimicrobial therapy.

Pathogens that Cause Illness Clinically Indistinguishable from Lassa Fever, Nigeria, 2018.

During the 2018 Lassa fever outbreak in Nigeria, samples from patients with suspected Lassa fever but negative Lassa virus PCR results were processed through custom gene expression array cards and metagenomic sequencing. Results demonstrated no single etiology, but bacterial and viral pathogens (including mixed co-infections) were detected.

Ventilator-associated pneumonia in critically ill patients with COVID-19.

BACKGROUND: Pandemic COVID-19 caused by the coronavirus SARS-CoV-2 has a high incidence of patients with severe acute respiratory syndrome (SARS). Many of these patients require admission to an intensive care unit (ICU) for invasive ventilation and are at significant risk of developing a secondary, ventilator-associated pneumonia (VAP). OBJECTIVES: To study the incidence of VAP and bacterial lung microbiome composition of ventilated COVID-19 and non-COVID-19 patients. METHODS: In this retrospective observational study, we compared the incidence of VAP and secondary infections using a combination of microbial culture and a TaqMan multi-pathogen array. In addition, we determined the lung microbiome composition using 16S RNA analysis in a subset of samples. The study involved 81 COVID-19 and 144 non-COVID-19 patients receiving invasive ventilation in a single University teaching hospital between March 15th 2020 and August 30th 2020. RESULTS: COVID-19 patients were significantly more likely to develop VAP than patients without COVID (Cox proportional hazard ratio 2.01 95% CI 1.14-3.54, p = 0.0015) with an incidence density of 28/1000 ventilator days versus 13/1000 for patients without COVID (p = 0.009). Although the distribution of organisms causing VAP was similar between the two groups, and the pulmonary microbiome was similar, we identified 3 cases of invasive aspergillosis amongst the patients with COVID-19 but none in the non-COVID-19 cohort. Herpesvirade activation was also numerically more frequent amongst patients with COVID-19. CONCLUSION: COVID-19 is associated with an increased risk of VAP, which is not fully explained by the prolonged duration of ventilation. The pulmonary dysbiosis caused by COVID-19, and the causative organisms of secondary pneumonia observed are similar to that seen in critically ill patients ventilated for other reasons.

Performance Evaluation of the SAMBA II SARS-CoV-2 Test for Point-of-Care Detection of SARS-CoV-2.

Nucleic acid amplification for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in respiratory samples is the standard method for diagnosis. The majority of this testing is centralized and therefore has turnaround times of several days. Point-of-care (POC) testing with rapid turnaround times would allow more effective triage in settings where patient management and infection control decisions need to be made rapidly. The inclusivity and specificity of the Simple AMplification-Based Assay (SAMBA) II SARS-CoV-2 test were determined by both in silico analyses of the primers and probes and wet testing. The SAMBA II SARS-CoV-2 test was evaluated for performance characteristics. Clinical performance was evaluated in residual combined throat/nose swabs and compared to that of the Public Health England real-time PCR assay targeting the RdRp gene. The SAMBA II SARS-CoV-2 test has an analytical sensitivity of 250 copies/ml for detecting two regions of the genome (open reading frame 1ab [ORF1ab] and nucleocapsid protein [N]). The clinical performance was evaluated in 172 residual combined nose/throat swabs provided by the Clinical Microbiology and Public Health Laboratory, Addenbrooke's Hospital, Cambridge (CMPHL), which showed an estimated positive percent agreement of 98.9% (95% confidence interval [CI], 93.83 to 99.97) and negative percent agreement of 96.4% (95% CI, 89.92 to 99.26) compared to testing by the CMPHL. The data show that the SAMBA II SARS-CoV-2 test performs equivalently to the centralized testing methods, but with a shorter turnaround time of 86 to 101 min. Point-of-care tests such as SAMBA should enable rapid patient management and effective implementation of infection control measures.

Towards accurate exclusion of neonatal bacterial meningitis: a feasibility study of a novel 16S rDNA PCR assay.

BACKGROUND: PCRctic is an innovative assay based on 16S rDNA PCR technology that has been designed to detect a single intact bacterium in a specimen of cerebro-spinal fluid (CSF). The assay's potential for accurate, fast and inexpensive discrimination of bacteria-free CSF makes it an ideal adjunct for confident exclusion of bacterial meningitis in newborn babies where the negative predictive value of bacterial culture is poor. This study aimed to stress-test and optimize PCRctic in the "field conditions" to attain a clinically useful level of specificity. METHODS: The specificity of PCRctic was evaluated in CSF obtained from newborn babies investigated for meningitis on a tertiary neonatal unit. Following an interim analysis, the method of skin antisepsis was changed to increase bactericidal effect, and snap-top tubes (Eppendorf™) replaced standard universal containers for collection of CSF to reduce environmental contamination. RESULTS: The assay's specificity was 90.5% in CSF collected into the snap-top tubes - up from 60% in CSF in the universal containers. The method of skin antisepsis had no effect on the specificity. All CSF cultures were negative and no clinical cases of neonatal bacterial meningitis occurred during the study. CONCLUSIONS: A simple and inexpensive optimization of CSF collection resulted in a high specificity output. The low prevalence of neonatal bacterial meningitis means that a large multi-centre study will be required to validate the assay's sensitivity and its negative predictive value.

Molecular epidemiology and socio-demographic risk factors of sexually transmitted infections among women in Lebanon.

BACKGROUND: Sexually transmitted infections (STIs) cause a major public health problem that affect both men and women in developing and developed countries. The aim of the study was to estimate the prevalence of 11 STIs among women who voluntarily participated in the study, while seeking gynecological checkup. The existence of an association between the presence of pathogens and symptoms and various sociodemographic risk factors was assessed. METHODS: A total of 505 vaginal and cervical specimens were collected from women above 18 years of age, with or without symptoms related to gynecological infections. Nucleic acid was extracted and samples were tested by real-time PCR for the following pathogens: Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Ureaplasma urealyticum, Urealplasma parvum, Trichomonas vaginalis, Mycoplasma hominis, Mycoplasma girerdii, Gardnerella vaginalis, Candida albicans and Human Papillomavirus (HPV). Positive HPV samples underwent genotyping using a microarray system. RESULTS: Of the 505 samples, 312 (62%) were screened positive for at least one pathogen. Of these, 36% were positive for Gardnerella vaginalis, 35% for Ureaplasma parvum, 8% for Candida albicans, 6.7% for HPV, 4.6% for Ureaplasma urealyticum, 3.6% for Mycoplasma hominis, 2% for Trichomonas vaginalis, 0.8% for Chlamydia trachomatis, 0.4% for Mycoplasma girerdii, 0.2% for Mycoplasma genitalium and 0.2% for Neisseria gonorrhoeae. Lack of symptoms was reported in 187 women (37%), among whom 61% were infected. Thirty-four samples were HPV positive, with 17 high risk HPV genotypes (HR-HPV); the highest rates being recorded for types 16 (38%), 18 (21%) and 51 (18%). Out of the 34 HPV positives, 29 participants had HR-HPV. Association with various risk factors were reported. CONCLUSIONS: This is the first study that presents data about the presence of STIs among women in Lebanon and the MENA region by simultaneous detection of 11 pathogens. In the absence of systematic STI surveillance in Lebanon, concurrent screening for HPV and PAP smear is warranted.

Epidemiology and clinical impact of viral, atypical, and fungal respiratory pathogens in symptomatic immunocompromised patients: a two-center study using a multi-parameter customized respiratory Taqman® array card.

The prevalence of respiratory viruses in immunocompromised adult patients and the association with clinical outcomes is still underexplored. Our goal was to assess the epidemiology and the potential clinical impact of respiratory viral infections in a high-risk patient population. Two large hospitals performed a respiratory Taqman array card (TAC), targeting 24 viruses, 8 bacteria, and 2 fungi simultaneously, on 435 samples from 397 symptomatic immunocompromised patients. Clinical details were collected retrospectively using a structured case report form. An overall positivity rate of 68% was found (51% mono- and 17% co-infections). Pathogen distribution was as follows: influenza A (20.7%), rhinoviruses (15.2%), coronaviruses (7.8%), Pneumocystis jirovecii (7.4%), RSV (7.1%), and CMV (6.0%) were the most frequently encountered, followed by HSV (5.5%), hMPV (4.4%), parainfluenza viruses (3.9%), influenza B (3.7%), and Aspergillus species (3.7%). Other pathogens were not detected or detected only in ≤ 1% of samples. Hospital and ICU admission rates were 84% and 11%, respectively. The presence of a pathogen was strongly associated with higher need for supplemental oxygen (p = 0.001), but it had no impact on ICU admission, mechanical ventilation requirement, antibacterial therapy, or mortality. In conclusion, our study described the epidemiology of respiratory pathogens in a large group of symptomatic immunocompromised patients and provides evidence of a relationship between pathogen detection and the need for supplemental oxygen. This association was still found after the exclusion of the results positive for influenza viruses, suggesting that non-influenza viruses contribute to severe respiratory illness in patients with compromised immunity.

Hepatitis E Virus Genotype 1 Cases Imported to Portugal from India, 2016.

Hepatitis E in industrialized countries is mainly associated with genotype 3 hepatitis E virus (HEV) and normally causes a sporadic self-limiting disease in immunocompetent individuals. Unlike genotype 3, genotypes 1 and 2 circulate in developing countries, produce severe disease and occur in the epidemic form. Hepatitis E occurring in travellers returning from endemic areas in developing countries is not a novel epidemiological occurrence, however the vast majority of cases remain to be genetically studied. The present study describes two cases of severe acute hepatitis E that required hospitalization for 6 and 9 days in two individuals of Indian nationality that had recently migrated to Portugal to work. The retrieved HEV sequences both belonged to genotype 1 and had a high degree of nucleotide sequence identity, clustering with strains isolated in India and Nepal, in 2013 and 2014. Confirmed HEV genotypes of increased pathogenicity like genotype 1 are being introduced into otherwise naïve populations of industrialized countries such as European countries with consequences difficult to predict. As far as we know the present study is the first in Portugal to describe and genetically characterize imported cases of hepatitis E infection caused by HEV genotype 1.

Etiology, seasonality, and clinical characterization of viral respiratory infections among hospitalized children in Beirut, Lebanon.

Acute respiratory tract viral infections occur worldwide and are one of the major global burdens of diseases in children. The aim of this study was to determine the viral etiology of respiratory infections in hospitalized children, to understand the viral seasonality in a major Lebanese hospital, and to correlate disease severity and the presence of virus. Over a 1-year period, nasal and throat swabs were collected from 236 pediatric patients, aged 16-year old or less and hospitalized for acute respiratory illness. Samples collected were tested for the presence of 17 respiratory viruses using multiplex real-time RT-PCR. Pathogens were identified in 165 children (70%) and were frequently observed during fall and winter seasons. Co-infection was found in 37% of positive samples. The most frequently detected pathogens were human Rhinovirus (hRV, 23%), Respiratory Syncytial Virus (RSV, 19%), human Bocavirus (hBov, 15%), human Metapneumovirus (hMPV, 10%), and human Adenovirus (hAdV, 10%). A total of 48% of children were diagnosed with bronchiolitis and 25% with pneumonia. While bronchiolitis was often caused by RSV single virus infection and hAdV/hBoV coinfection, pneumonia was significantly associated with hBoV and HP1V1 infections. No significant correlation was observed between a single viral etiology infection and a specific clinical symptom. This study provides relevant facts on the circulatory pattern of respiratory viruses in Lebanon and the importance of using PCR as a useful tool for virus detection. Early diagnosis at the initial time of hospitalization may reduce the spread of the viruses in pediatric units. J. Med. Virol. 88:1874-1881, 2016. © 2016 Wiley Periodicals, Inc.

The role of chronic norovirus infection in the enteropathy associated with common variable immunodeficiency.

OBJECTIVES: A severe enteropathy of unknown etiology can be associated with common variable immunodeficiency (CVID). METHODS: S tool and archived small intestinal mucosal biopsies from patients with CVID enteropathy were analyzed by PCR for the presence of Norovirus RNA. The PCR products were sequenced to determine the relationship of viral isolates. Stool samples from 10 patients with CVID but no enteropathy served as controls. RESULTS: All eight patients in our CVID cohort with enteropathy showed persistent fecal excretion of Norovirus. Analysis of archived duodenal biopsies revealed a strong association between the presence of Norovirus and villous atrophy over a period of up to 8 years. Analysis of the viral isolates from each patient revealed distinct strains of genogroup II.4. Sequence analysis from consecutive biopsy specimens of one patient demonstrated persistence of the same viral strain over a 6-year period. CVID patients without enteropathy showed no evidence of Norovirus carriage. Viral clearance occurred spontaneously in one patient and followed oral Ribavirin therapy in two further patients, and resulted in complete symptomatic and histological recovery. However, Ribavirin treatment in two further patients was unsuccessful. CONCLUSIONS: Norovirus is an important pathogen for patients with CVID and a cause of CVID enteropathy, as viral clearance, symptom resolution, and histological recovery coincide. Ribavirin requires further evaluation as a potential therapy.

C-reactive protein level and microbial aetiology in patients hospitalised with acute exacerbation of COPD.

Both viruses and bacteria are thought to cause exacerbations of chronic obstructive pulmonary disease (COPD); however, the relative importance of each remains uncertain. C-reactive protein (CRP) levels increase during exacerbations but the relationship with aetiology is not established. We aimed to explore the relationship between serum CRP and the rate of detection of viruses and bacteria. This was a prospectively recruited, observational study of patients hospitalised with exacerbations of COPD. Nasopharyngeal swabs were tested for respiratory viruses by reverse transcriptase-PCR. Sputum and blood were collected for bacterial culture and urine tested for pneumococcal antigen. CRP levels were measured on sera. CRP and other factors associated with viral, bacterial or mixed detection were assessed using multiple logistic regression analysis. 264 patients with exacerbations of COPD were studied: 26% tested positive for respiratory viruses only, 13% had bacteria only, 12% had mixed viral/bacterial detection, and 49% had no pathogens detected. CRP level and temperature were strongly associated with viral detection rate (p<0.001 and p=0.004, respectively) and mixed viral/bacterial detection rate (p=0.02 and p=0.03, respectively) on multivariate analysis. Bacterial detection rate was not associated with CRP level or body temperature. This study supports the role of viruses as important aetiological agents causing exacerbations of COPD.

Whole-genome sequencing to identify transmission of Mycobacterium abscessus between patients with cystic fibrosis: a retrospective cohort study.

BACKGROUND: Increasing numbers of individuals with cystic fibrosis are becoming infected with the multidrug-resistant non-tuberculous mycobacterium (NTM) Mycobacterium abscessus, which causes progressive lung damage and is extremely challenging to treat. How this organism is acquired is not currently known, but there is growing concern that person-to-person transmission could occur. We aimed to define the mechanisms of acquisition of M abscessus in individuals with cystic fibrosis. METHOD: Whole genome sequencing and antimicrobial susceptibility testing were done on 168 consecutive isolates of M abscessus from 31 patients attending an adult cystic fibrosis centre in the UK between 2007 and 2011. In parallel, we undertook detailed environmental testing for NTM and defined potential opportunities for transmission between patients both in and out of hospital using epidemiological data and social network analysis. FINDINGS: Phylogenetic analysis revealed two clustered outbreaks of near-identical isolates of the M abscessus subspecies massiliense (from 11 patients), differing by less than ten base pairs. This variation represents less diversity than that seen within isolates from a single individual, strongly indicating between-patient transmission. All patients within these clusters had numerous opportunities for within-hospital transmission from other individuals, while comprehensive environmental sampling, initiated during the outbreak, failed to detect any potential point source of NTM infection. The clusters of M abscessus subspecies massiliense showed evidence of transmission of mutations acquired during infection of an individual to other patients. Thus, isolates with constitutive resistance to amikacin and clarithromycin were isolated from several individuals never previously exposed to long-term macrolides or aminoglycosides, further indicating cross-infection. INTERPRETATION: Whole genome sequencing has revealed frequent transmission of multidrug resistant NTM between patients with cystic fibrosis despite conventional cross-infection measures. Although the exact transmission route is yet to be established, our epidemiological analysis suggests that it could be indirect. FUNDING: The Wellcome Trust, Papworth Hospital, NIHR Cambridge Biomedical Research Centre, UK Health Protection Agency, Medical Research Council, and the UKCRC Translational Infection Research Initiative.

New methods for identifying infectious diseases.

BACKGROUND: The goal of clinical microbiology is to identify the cause of infection, aiding rapid treatment initiation or altering empirically chosen anti-microbial regimens. Automation and molecular techniques have brought about a revolution in the clinical laboratory, ensuring ever faster and more accurate diagnoses. In the last few years however, there have been a number of developments that radically alter the way that microbiology and other diagnostic laboratories are advancing. In particular, clinical microbiology will have the opportunity to intervene at the public health level as well as at the individual patient. SOURCES OF DATA, AREAS OF AGREEMENT AND CONTROVERSY: Experts in the new technologies discuss the advances and some of the key literature that has been published to-date. They touch upon both the potential benefits and some of the hurdles that must be overcome before the technologies are embraced fully into the clinical laboratory. GROWING POINTS: This review discusses a number of technologies that may alter the way in which clinical microbiology is used to investigate infectious disease. Diagnostic services in the UK are currently undergoing a process of rationalization, which involves a shift towards laboratory amalgamation, adoption of 24/7 working patterns and greater automation in order to reduce costs. This review explores technologies that are already or are expected to be important in this on-going transition because they simplify or accelerate the complex workflows that are required for pathogen identification.

Improving influenza surveillance in Portuguese preschool children by parents' report.

UNLABELLED: Influenza surveillance is usually based on nationally organized sentinel networks of physicians and on hospital reports. This study aimed to test a different report system, based on parents' phone contact to the research team and in home collection of samples by a dedicated team. The identification of influenza and other respiratory viruses in children who attended a Hospital Emergency Department was also recorded. Real-time PCR and reverse transcription PCR were performed for influenza A and B, parainfluenza 1-4, adenovirus, human metapneumovirus, respiratory syncytial virus A and B, rhinovirus, enterovirus, group 1 coronaviruses, group 2 coronaviruses, and human bocavirus. One hundred children were included, 64 from the day care centers and 36 from the Hospital. Overall, 79 samples were positive for at least one respiratory virus. Influenza A (H3) was the virus most frequently detected: 25 cases, 20 of these in children under 5 years of age (ten from day care centers and ten who went to the hospital) which was higher than those reported by the National Influenza Surveillance Programme for this age. CONCLUSION: The results obtained in this study suggest that a surveillance system based on parents' reports could complement the implanted system of the National Influenza Surveillance Programme.

Enhanced diagnosis of severe bacterial and fungal respiratory infection in children using a rapid syndromic array-case report.

Background: A microbiological cause of infection is infrequently identified in critically unwell children with a respiratory infection. Molecular diagnostic arrays provide an alternative. These tests are becoming more broadly available, but little is known about how clinicians interpret the results to impact clinical decision making. Case Description: Here we describe three cases of bacterial and fungal lower respiratory tract infection (LRTI) diagnosed in the paediatric intensive care unit (PICU) using a custom 52 respiratory pathogen TaqMan array card (TAC). Firstly, an early diagnosis of Candida albicans pneumonia was made with the support of the TAC in a trauma patient who received prolonged mechanical ventilation. The pathogen was only identified on microbiological cultures after further clinical deterioration had occurred. Secondly, a rare case of psittacosis was identified in an adolescent with acute respiratory distress, initially suspected to have multisystem inflammatory syndrome in children (MIS-C). Finally, Haemophilus influenzae pneumonia was identified in an infant with recurrent apnoeas, initially treated for meningitis. Two diagnoses would not have been established using commercially available arrays, and pathogen-specific diagnoses were established faster than that of routine microbiological culture. Conclusions: The pathogens included on molecular arrays and interpretation by a multidisciplinary team are crucial in providing value to PICU diagnostic services. Molecular arrays have the potential to enhance early pathogen-specific diagnosis of LRTI in the PICU.

Duplex molecular assay intended for point-of-care diagnosis of influenza A/B virus infection.

Early diagnosis and management of influenza virus infection directly correlates with the effectiveness in disease control. Current molecular influenza virus tests were designed for use in diagnostic testing facilities, where sophisticated equipment and highly trained technicians are available. A longer turnaround time for the centralized testing than when testing near the sample source could delay the initiation of medical intervention, thereby reducing the efficacy of antiviral treatment. The new assay, the SAMBA (simple amplification-based assay) Flu duplex test, is a dipstick-based molecular assay developed to provide a simple, accurate, and cost-effective solution for the diagnosis of influenza A/B viruses intended for near-patient testing. The test presents an alternative format of influenza virus molecular testing that utilizes isothermal amplification and visual detection of nucleic acid on a test strip. The entire test procedure (extraction, amplification, and detection) is integrated into an enclosed semiautomated system. Analytically, the SAMBA Flu duplex test detects 95 and 85 copies of viral genomes for influenza A and B viruses, respectively, with no cross-reactivity observed against other common respiratory pathogens. The clinical performance was established by blind testing of 328 nasal/throat and nasopharyngeal swab specimens from the United Kingdom and Belgium and comparing the results with the quantitative reverse transcription-PCR method routinely used in two public health laboratories. The SAMBA Flu duplex test showed a clinical sensitivity and specificity of 100% and 97.9% for influenza virus A and 100% and 100% for influenza virus B. The test provides a new technology that could facilitate simple and timely identification of influenza virus infection, potentially resulting in more efficient control measures.

Killer Immunoglobulin-like Receptors (KIR) haplogroups A and B track with Natural Killer Cells and Cytokine Profile in Aged Subjects: Observations from Octo/Nonagenarians in the Belfast Elderly Longitudinal Free-living Aging STudy (BELFAST).

BACKGROUND: Natural Killer Cells (NK) play an important role in detection and elimination of virus-infected, damaged or cancer cells. NK cell function is guided by expression of Killer Immunoglobulin-like Receptors (KIRs) and contributed to by the cytokine milieu. KIR molecules are grouped on NK cells into stimulatory and inhibitory KIR haplotypes A and B, through which NKs sense and tolerate HLA self-antigens or up-regulate the NK-cytotoxic response to cells with altered HLA self-antigens, damaged by viruses or tumours. We have previously described increased numbers of NK and NK-related subsets in association with sIL-2R cytokine serum levels in BELFAST octo/nonagenarians. We hypothesised that changes in KIR A and B haplotype gene frequencies could explain the increased cytokine profiles and NK compartments previously described in Belfast Elderly Longitudinal Free-living Aging STudy (BELFAST) octo/nonagenarians, who show evidence of ageing well. RESULTS: In the BELFAST study, 24% of octo/nonagenarians carried the KIR A haplotype and 76% KIR B haplotype with no differences for KIR A haplogroup frequency between male or female subjects (23% v 24%; p=0.88) or for KIR B haplogroup (77% v 76%; p=0.99). Octo/nonagenarian KIR A haplotype carriers showed increased NK numbers and percentage compared to Group B KIR subjects (p=0.003; p=0.016 respectively). There were no KIR A/ B haplogroup-associated changes for related CD57+CD8 (high or low) subsets. Using logistic regression, KIR B carriers were predicted to have higher IL-12 cytokine levels compared to KIR A carriers by about 3% (OR 1.03, confidence limits CI 0.99-1.09; p=0.027) and 14% higher levels for TGF-ß (active), a cytokine with an anti-inflammatory role, (OR 1.14, confidence limits CI 0.99-1.09; p=0.002). CONCLUSION: In this observational study, BELFAST octo/nonagenarians carrying KIR A haplotype showed higher NK cell numbers and percentage compared to KIR B carriers. Conversely, KIR B haplotype carriers, with genes encoding for activating KIRs, showed a tendency for higher serum pro-inflammatory cytokines compared to KIR A carriers. While the findings in this study should be considered exploratory they may serve to stimulate debate about the immune signatures of those who appear to age slowly and who represent a model for good quality survivor-hood.

Overlapping signals for translational regulation and packaging of influenza A virus segment 2.

Influenza A virus segment 2 mRNA expresses three polypeptides: PB1, PB1-F2 and PB1-N40, from AUGs 1, 4 and 5 respectively. Two short open reading frames (sORFs) initiated by AUGs 2 and 3 are also present. To understand translational regulation in this system, we systematically mutated AUGs 1-4 and monitored polypeptide synthesis from plasmids and recombinant viruses. This identified sORF2 as a key regulatory element with opposing effects on PB1-F2 and PB1-N40 expression. We propose a model in which AUGs 1-4 are accessed by leaky ribosomal scanning, with sORF2 repressing synthesis of downstream PB1-F2. However, sORF2 also up-regulates PB1-N40 expression, most likely by a reinitiation mechanism that permits skipping of AUG4. Surprisingly, we also found that in contrast to plasmid-driven expression, viruses with improved AUG1 initiation contexts produced less PB1 in infected cells and replicated poorly, producing virions with elevated particle:PFU ratios. Analysis of the genome content of virus particles showed reduced packaging of the mutant segment 2 vRNAs. Overall, we conclude that segment 2 mRNA translation is regulated by a combination of leaky ribosomal scanning and reinitiation, and that the sequences surrounding the PB1 AUG codon are multifunctional, containing overlapping signals for translation initiation and for segment-specific packaging.