An internet-delivered handwashing intervention to modify influenza-like illness and respiratory infection transmission (PRIMIT): a primary care randomised trial.

BACKGROUND: Handwashing to prevent transmission of respiratory tract infections (RTIs) has been widely advocated, especially during the H1N1 pandemic. However, the role of handwashing is debated, and no good randomised evidence exists among adults in non-deprived settings. We aimed to assess whether an internet-delivered intervention to modify handwashing would reduce the number of RTIs among adults and their household members. METHODS: We recruited individuals sharing a household by mailed invitation through general practices in England. After consent, participants were randomised online by an automated computer-generated random number programme to receive either no access or access to a bespoke automated web-based intervention that maximised handwashing intention, monitored handwashing behaviour, provided tailored feedback, reinforced helpful attitudes and norms, and addressed negative beliefs. We enrolled participants into an additional cohort (randomised to receive intervention or no intervention) to assess whether the baseline questionnaire on handwashing would affect handwashing behaviour. Participants were not masked to intervention allocation, but statistical analysis commands were constructed masked to group. The primary outcome was number of episodes of RTIs in index participants in a modified intention-to-treat population of randomly assigned participants who completed follow-up at 16 weeks. This trial is registered with the ISRCTN registry, number ISRCTN75058295. FINDINGS: Across three winters between Jan 17, 2011, and March 31, 2013, we enrolled 20,066 participants and randomly assigned them to receive intervention (n=10,040) or no intervention (n=10,026). 16,908 (84%) participants were followed up with the 16 week questionnaire (8241 index participants in intervention group and 8667 in control group). After 16 weeks, 4242 individuals (51%) in the intervention group reported one or more episodes of RTI compared with 5135 (59%) in the control group (multivariate risk ratio 0·86, 95% CI 0·83-0·89; p<0·0001). The intervention reduced transmission of RTIs (reported within 1 week of another household member) both to and from the index person. We noted a slight increase in minor self-reported skin irritation (231 [4%] of 5429 in intervention group vs 79 [1%] of 6087 in control group) and no reported serious adverse events. INTERPRETATION: In non-pandemic years, an effective internet intervention designed to increase handwashing could have an important effect in reduction of infection transmission. In view of the heightened concern during a pandemic and the likely role of the internet in access to advice, the intervention also has potential for effective implementation during a pandemic. FUNDING: Medical Research Council.

Longevity and determinants of protective humoral immunity after pandemic influenza infection.

RATIONALE: Antibodies to influenza hemagglutinin are the primary correlate of protection against infection. The strength and persistence of this immune response influences viral evolution and consequently the nature of influenza epidemics. However, the durability and immune determinants of induction of humoral immunity after primary influenza infection remain unclear. OBJECTIVES: The spread of a novel H1N1 (A[H1N1]pdm09) virus in 2009 through an unexposed population offered a natural experiment to assess the nature and longevity of humoral immunity after a single primary influenza infection. METHODS: We followed A(H1N1)pdm09-seronegative adults through two influenza seasons (2009-2011) as they developed A(H1N1)pdm09 influenza infection or were vaccinated. Antibodies to A(H1N1)pdm09 virus were measured by hemagglutination-inhibition assay in individuals with paired serum samples collected preinfection and postinfection or vaccination to assess durability of humoral immunity. Preexisting A(H1N1)pdm09-specific multicytokine-secreting CD4 and CD8 T cells were quantified by multiparameter flow cytometry to test the hypothesis that higher frequencies of CD4(+) T-cell responses predict stronger antibody induction after infection or vaccination. MEASUREMENTS AND MAIN RESULTS: Antibodies induced by natural infection persisted at constant high titer for a minimum of approximately 15 months. Contrary to our initial hypothesis, the fold increase in A(H1N1)pdm09-specific antibody titer after infection was inversely correlated to the frequency of preexisting circulating A(H1N1)pdm09-specific CD4(+)IL-2(+)IFN-γ(-)TNF-α(-) T cells (r = -0.4122; P = 0.03). CONCLUSIONS: The longevity of protective humoral immunity after influenza infection has important implications for influenza transmission dynamics and vaccination policy, and identification of its predictive cellular immune correlate could guide vaccine development and evaluation.

Incidence of influenza A(H1N1)pdm09 infection, United Kingdom, 2009-2011.

We conducted a longitudinal community cohort study of healthy adults in the UK. We found significantly higher incidence of influenza A(H1N1)pdm09 infection in 2010-11 than in 2009-10, a substantial proportion of subclinical infection, and higher risk for infection during 2010-11 among persons with lower preinfection antibody titers.

Origin and fate of A/H1N1 influenza in Scotland during 2009.

The spread of influenza has usually been described by a 'density' model, where the largest centres of population drive the epidemic within a country. An alternative model emphasizing the role of air travel has recently been developed. We have examined the relative importance of the two in the context of the 2009 H1N1 influenza epidemic in Scotland. We obtained genome sequences of 70 strains representative of the geographical and temporal distribution of H1N1 influenza during the summer and winter phases of the pandemic in 2009. We analysed these strains, together with another 128 from the rest of the UK and 292 globally distributed strains, using maximum-likelihood phylogenetic and bayesian phylogeographical methods. This revealed strikingly different epidemic patterns within Scotland in the early and late parts of 2009. The summer epidemic in Scotland was characterized by multiple independent introductions from both international and other UK sources, followed by major local expansion of a single clade that probably originated in Birmingham. The winter phase, in contrast, was more diverse genetically, with several clades of similar size in different locations, some of which had no particularly close phylogenetic affinity to strains sampled from either Scotland or England. Overall there was evidence to support both models, with significant links demonstrated between North American sequences and those from England, and between England and East Asia, indicating that major air-travel routes played an important role in the pattern of spread of the pandemic, both within the UK and globally.

Development and assay of RNA transcripts of enterovirus species A to D, rhinovirus species a to C, and human parechovirus: assessment of assay sensitivity and specificity of real-time screening and typing methods.

Nucleic acid amplification methods such as the PCR have had a major impact on the diagnosis of viral infections, often achieving greater sensitivities and shorter turnaround times than conventional assays and an ability to detect viruses refractory to conventional isolation methods. Their effectiveness is, however, significantly influenced by assay target sequence variability due to natural diversity and rapid sequence changes in viruses that prevent effective binding of primers and probes. This was investigated for a diverse range of enteroviruses (EVs; species A to D), human rhinoviruses (HRVs; species A to C), and human parechovirus (HPeV) in a multicenter assay evaluation using a series of full-length prequantified RNA transcripts. RNA concentrations were quantified by absorption (NanoDrop) and fluorescence methods (RiboGreen) prior to dilution in buffer supplemented with RNase inhibitors and carrier RNA. RNA transcripts were extremely stable, showing minimal degradation after prolonged storage at temperatures between ambient and -20°C and after multiple freeze-thaw cycles. Transcript dilutions distributed to six referral laboratories were screened by real-time reverse transcriptase PCR assays using different primers and probes. All of the laboratories reported high assay sensitivities for EV and HPeV transcripts approaching single copies and similar amplification kinetics for all four EV species. HRV detection sensitivities were more variable, often with substantially impaired detection of HRV species C. This could be accounted for in part by the placement of primers and probes to genetically variable target regions. Transcripts developed in this study provide reagents for the ongoing development of effective diagnostics that accommodate increasing knowledge of genetic heterogeneity of diagnostic targets.

Epidemiology of physical-mental multimorbidity and its impact among Aboriginal and Torres Strait Islander in Australia: a cross-sectional analysis of a nationally representative sample.

OBJECTIVES: This study aimed to examine the differences in multimorbidity between Aboriginal and Torres Strait Islander people and non-Indigenous Australians, and the effect of multimorbidity on health service use and work productivity. SETTING: Cross-sectional sample of the Household, Income and Labour Dynamics in Australia wave 17. PARTICIPANTS: A nationally representative sample of 16 749 respondents aged 18 years and above. OUTCOME MEASURES: Multimorbidity prevalence and pattern, self-reported health, health service use and employment productivity by Indigenous status. RESULTS: Aboriginal respondents reported a higher prevalence of multimorbidity (24.2%) compared with non-Indigenous Australians (20.7%), and the prevalence of mental-physical multimorbidity was almost twice as high (16.1% vs 8.1%). Multimorbidity pattern varies significantly among the Aboriginal and non-Indigenous Australians. Multimorbidity was associated with higher health service use (any overnight admission: adjusted OR=1.52, 95% CI=1.46 to 1.58), reduced employment productivity (days of sick leave: coefficient=0.25, 95% CI=0.19 to 0.31) and lower perceived health status (SF6D score: coefficient=-0.04, 95% CI=-0.05 to -0.04). These associations were found to be comparable in both Aboriginal and non-Indigenous populations. CONCLUSIONS: Multimorbidity prevalence was significantly greater among Aboriginal and Torres Strait Islanders compared with the non-Indigenous population, especially mental-physical multimorbidity. Strategies are required for better prevention and management of multimorbidity for the aboriginal population to reduce health inequalities in Australia.

Rapid simultaneous detection of enterovirus and parechovirus RNAs in clinical samples by one-step real-time reverse transcription-PCR assay.

Enteroviruses (EVs) are recognized as the major etiological agent in meningitis in children and young adults. The use of molecular techniques, such as PCR, has substantially improved the sensitivity of enterovirus detection compared to that of virus culture methods. PCR-based methods also can detect a much wider range of EV variants, including those within species A, as well as human parechoviruses (HPeVs) that often grow poorly in vitro and which previously have been underdiagnosed by traditional methods. To exploit these developments, we developed a real-time one-step reverse transcription-PCR (RT-PCR) for the rapid and sensitive detection of EV and HPeV in clinical specimens. Two commercially available RT-PCR kits were used (method I, Platinum one-step kit; method II, Express qPCR one-step kit) with primers and probes targeting the EV and HPeV 5'-untranslated regions (5'UTR). Amplification dynamics (threshold cycle [C(T)]values and efficiencies) of absolutely quantified full-length RNA transcripts representative of EV species A to D and HPeV were similar, demonstrating the effectiveness of both assays across the range of currently described human EV and HPeV variants. Probit analysis of multiple endpoint replicates demonstrated comparable sensitivities of the assays for EV and HPeV (method I, approximately 10 copies per reaction for both targets; method II, 20 copies per reaction). C(T) values were highly reproducible on repeat testing of positive controls within assays and between assay runs. Considering the sample turnaround time of less than 3 h, the multiplexed one-step RT-PCR method provides rapid diagnostic testing for EV and HPeV in cases of suspected central nervous system infections in a clinically relevant time frame.

During the summer 2009 outbreak of "swine flu" in Scotland what respiratory pathogens were diagnosed as H1N1/2009?

BACKGROUND: During the April-July 2009 outbreak of H1N1/2009 in scotland the West of Scotland Specialist Virology Centre (WoSSVC) in Glasgow tested > 16,000 clinical samples for H1N1/2009. Most were from patients clinically diagnosed with H1N1/2009. Out of these, 9% were positive. This study sought to determine what respiratory pathogens were misdiagnosed as cases of H1N1/2009 during this time. METHODS: We examined the results from 3247 samples which were sent to the laboratory during April-July 2009. All were from patients clinically diagnosed as having H1N1/2009 (based on accepted criteria) and all were given a full respiratory screen using real time reverse transcriptase polymerase chain reaction (rtRT-PCR) assays. RESULTS: In total, respiratory pathogens were detected in 27.9% (95% confidence interval, 26.3-29.5%) of the samples submitted. Numerous pathogens were detected, the most common of which were rhinovirus (8.9% (95% confidence interval, 7.9-9.9%)), parainfluenza 1 (1.9% (95% confidence interval, 1.4-2.4%)) and 3 (4.1% (95% confidence interval, 3.3-4.9%)), and adenovirus ((3.5% (95% confidence interval, 2.9-4.2%)). CONCLUSIONS: This study highlights the problems of using a clinical algorithm to detect H1N1/2009. Clinicians frequently misdiagnosed common respiratory pathogens as H1N1/2009 during the spring/summer outbreak in Scotland. Many undesirable consequences would have resulted, relating to treatment, infection control, and public health surveillance.

A new multiplex real-time PCR test for HSV1/2 and syphilis: an evaluation of its impact in the laboratory and clinical setting.

OBJECTIVES: To develop, evaluate and implement a new multiplex real-time PCR test for the detection of herpes simplex virus (HSV)1, HSV2 and syphilis in a single sample using a single test. METHODS: A multiplex real-time PCR test detecting HSV1, HSV2 and Treponema pallidum was designed, validated and evaluated for a period of 6 months on patients attending the Sandyford Initiative (a series of genitourinary medicine clinics in and around Glasgow). A total of 692 samples were tested, and T pallidum PCR positives were confirmed by a second PCR at the Scottish Reference Laboratory (SBSTIRL). All PCR results were aligned with dark ground microscopy findings and serological results where available and compared. RESULTS: The laboratory validation of the multiplex assay showed the test to be sensitive, specific and robust. Of the 692 samples, 139 were positive for HSV1, 136 for HSV2, 15 for syphilis, one for both syphilis and HSV1, and 401 were negative; the reference laboratory confirmed all T pallidum PCR-positive samples. The PCR test was more sensitive than both dark ground microscopy and serological testing for the diagnosis of primary syphilis. CONCLUSIONS: The introduction of this new test has led to a better turnaround time for the diagnosis of genital ulcer disease, better detection of primary syphilis infection, and the detection of unexpected cases of syphilis where the aetiological agent suspected was HSV.

Genotypic analysis of two hypervariable human cytomegalovirus genes.

Most human cytomegalovirus (HCMV) genes are highly conserved in sequence among strains, but some exhibit a substantial degree of variation. Two of these genes are UL146, which encodes a CXC chemokine, and UL139, which is predicted to encode a membrane glycoprotein. The sequences of these genes were determined from a collection of 184 HCMV samples obtained from Africa, Australia, Asia, Europe, and North America. UL146 is hypervariable throughout, whereas variation in UL139 is concentrated in a sequence encoding a potentially highly glycosylated region. The UL146 sequences fell into 14 genotypes, as did all previously reported sequences. The UL139 sequences grouped into 8 genotypes, and all previously reported sequences fell into a subset of these. There were minor differences among continents in genotypic frequencies for UL146 and UL139, but no clear geographical separation, and identical nucleotide sequences were represented among communities distant from each other. The frequent detection of multiple genotypes indicated that mixed infections are common. For both genes, the degree of divergence was sufficient to preclude reliable sequence alignments between genotypes in the most variable regions, and the mode of evolution involved in generating the genotypes could not be discerned. Within genotypes, constraint appears to have been the predominant mode, and positive selection was detected marginally at best. No evidence was found for linkage disequilibrium. The emerging scenario is that the HCMV genotypes developed in early human populations (or even earlier), becoming established via founder or bottleneck effects, and have spread, recombined and mixed worldwide in more recent times.

Erratum to 'The management of chronic hepatitis B in the immunocompromised patient: recommendations from a single topic meeting' [J. Clin. Virol. 41 (4) 2008 243-254].

Patients with chronic hepatitis B virus (HBV) infection have a substantial risk of reactivation and jaundice following the use of immunosuppressant therapy. A single topic conference was convened to discuss the management of HBV patients undergoing chemotherapy for haematological malignancy, liver and renal transplantation and with HIV co-infection. In advance of the meeting a draft guideline was prepared and circulated to a participating expert panel. Presentations and consensus views were obtained on the day of conference to allow pragmatic algorithms to be established on each of these topics. Use of lamivudine prophylaxis for HBV patients undergoing chemotherapy and renal transplantation is strongly supported with good evidence. Patients with HBV cirrhosis who are candidates for transplantation should be started on nucleos(t)ide therapy prior to surgery and, in addition, hepatitis B immune globulin given from the time of transplantation onward. Co-infection with HBV and HIV offers unique challenges. If the patient is a candidate for highly active retroviral therapy then dual nucleos(t)ide analogues which are also active against HBV must be used to prevent immune reconstitution hepatitis. In all these conditions, awareness of possible HBV resistance to therapy must be kept in mind and HBV DNA levels monitored.

Development of working reference materials for clinical virology.

Nucleic acid amplification technique (NAT)-based assays are replacing traditional diagnostic methods in clinical laboratories. However, many of these assays are developed in-house and the lack of standardised reference materials has hindered assay implementation and control. Consequently, in the UK, the Clinical Virology Network (CVN), the National Institute for Biological Standards and Control (NIBSC), and the Health Protection Agency (HPA), are working in collaboration to develop working standards or 'run controls' for diagnostic NAT-based assays, particularly real-time PCR. These run controls are intended for use in microbiology laboratories and are designed to be extracted and amplified in the same way as clinical samples and included in each assay run. The aim is to enable clinical laboratories to continuously monitor the performance of their diagnostic NAT assays on a run-by-run basis allowing inter-laboratory comparisons, and ultimately improving the consistency of results. At present, eight candidate run controls representing clinically relevant viral targets have been prepared for evaluation by CVN laboratories. Data have been returned on the performance of each run control in routine diagnostic assays. Preliminary results presented here indicate a high level of variability in intra- and inter-assay detection of these targets, highlighting the need for standardisation of assays within molecular diagnostics.

Comparison of mumps-IgM ELISAs in acute infection.

BACKGROUND: In Scotland there has been an outbreak of mumps with over 4000 confirmed cases in the last 2 years. As laboratory diagnosis is usually requested on patients with symptoms, a prompt diagnosis early in the illness is required. OBJECTIVES: To assess the performance of the five different commercial IgM-ELISAs used in Scottish Virus laboratories. STUDY DESIGN: The Specialist Virology Laboratory (SVC) Edinburgh distributed a serum panel to all Scottish laboratories that diagnose mumps by IgM-ELISA. The panel consisted of 45 sera from patients with confirmed mumps (date of onset known for 44/45) and 11 sera from patients with alternative diagnoses. Each laboratory performed their own commercial IgM-ELISAs blindly and reported results to the SVC Edinburgh. RESULTS: Sensitivity ranged from 24% to 51%. Assays performed better on samples taken later than 10 days after onset of symptoms. Specificity was about 82% for most assays. CONCLUSION: The sensitivity of commercial mumps IgM-ELISAs varied greatly. The Microimmune mumps-IgM ELISA had the best overall sensitivity in acute serum specimens. Diagnostic laboratories should be developing the means to perform direct detection of mumps virus for acute presentation and requesting convalescent bloods, if acute blood samples have no detectable IgM.

Differential reactivity of mouse monoclonal anti-HBs antibodies with recombinant mutant HBs antigens.

AIM: To investigate the reactivity of a panel of 8 mouse anti-hepatitis B surface antigen (HBsAg) monoclonal antibodies (mAbs) using a collection of 9 recombinant HBsAg mutants with a variety of amino acid substitutions mostly located within the "a" region. METHODS: The entire HBs genes previously cloned into a mammalian expression vector were transiently transfected into COS7 cells. Two standard unmutated sequences of the ayw and adw subtypes served as controls. Secreted mutant proteins were collected and measured by three commercial diagnostic immunoassays to assess transfection efficiency. Reactivity of anti-HBs mAbs with mutated HBsAgs was determined by sandwich enzyme-linked immunosorbent assay (ELISA). RESULTS: Reactivity of anti-HBs mAbs with mutated HBsAgs revealed different patterns. While three mutants reacted strongly with all mAbs, two mutants reacted weakly with only two mAbs and the remaining proteins displayed variable degrees of reactivity towards different mAbs. Accordingly, four groups of mAbs with different but overlapping reactivity patterns could be envisaged. One group consisting of two mAbs (37C5-S7 and 35C6-S11) was found to recognize stable linear epitopes conserved in all mutants. Mutations outside the "a" determinant at positions 120 (P-->S), 123(T-->N) and 161 (M-->T) were found to affect reactivity of these mAbs. CONCLUSION: Our findings could have important implications for biophysical studies, vaccination strategies and immunotherapy of hepatitis B virus (HBV) mutants.

The use of the OraSure collection device for hepatitis virus testing in health care settings.

Oral mucosal transudate (OMT) has levels of IgG and IgA that, although lower than in serum, are of the same specificity as serum. Blood can be a difficult sample to take especially from persons with an aversion to the process or with poor veins. There is also a small exposure risk to the phlebotomist. OMT has been shown to be useful in screening for a range of bloodborne viruses, however, the ELISA assays need to be modified, usually by increasing test volumes and incubation times and/or by modifying the substrate. A number of commercial serum kits have been modified successfully in this way. The major roles for these assays are for epidemiology or for screening high risk populations. If used for screening, it is imperative that any positive is confirmed by a serum test. The use of OMT samples using the OraSure(R) collection device, which is FDA approved and with which we have the most experience, has increased in recent years in community health care settings, with positive feedback from users.

Optimisation of PCR reactions using primer chessboarding.

In-house polymerase chain reaction (PCR) assays are now an integral part of the work of most diagnostic microbiological laboratories. Despite the availability of commercial reagent 'master-mixes' of some PCR reagents, the optimisation of primers still poses a significant problem. Here, we describe a simple method to assess the concentration of primer needed in single round, multiplex, nested and 'real-time' PCR procedures.

Virological surveillance of influenza-like illness in the community using PCR and serology.

BACKGROUND: Surveillance of winter respiratory viral illness has been carried out for nearly 30 years using a clinical diagnosis by general practitioners as part of the Scottish Sentinel General Practice (SSGP) network. Contemparaneous laboratory diagnosis has not been available previously. OBJECTIVES: To assess the proportion of influenza-like illness (ILI) attributable to influenza, respiratory syncytial virus (RSV) and picornavirus infection during the winter season. To compare the influenza PCR data with serology of paired blood samples. STUDY DESIGN: Combined nose and throat swabs, from patients with ILI attending 15 general practices across Scotland, were submitted to the laboratory in virus PCR sample solution (VPSS). The extracted nucleic acid was tested using a multiplex reverse-transcription polymerase chain reaction (RT-PCR) assay. Serological analysis was performed on paired serum samples using complement fixation assays. The rate of influenza virus positivity was compared with reports of ILI obtained from the SSGP network. RESULTS: Of 240 samples received at the laboratory, 132 (55%) were PCR positive for influenza A virus. There were nine (3.8%) picornavirus and three (1.2%) RSV PCR positives, two (0.8%) were dual influenza A/picornavirus infections. Ninety four (39.2%) were negative for all viruses tested. Results on paired sera from 89 patients showed a rising titre to influenza A in 48 of the 57 PCR positive samples (84.2%). One PCR negative patient displayed a significant rising titre to influenza A. Virological data paralleled the SSGP data but was available at least a week earlier. CONCLUSIONS: Influenza A infection was detected in the majority of patients with ILI; picornavirus infection was also shown to be an important cause of illness. PCR is a rapid and sensitive method for respiratory virus surveillance. Serology is slow, insensitive and difficult to interpret at low titres.

The development of a multiplex real-time PCR for the detection of herpes simplex virus 1 and 2, varizella zoster virus, adenovirus and Chlamydia trachomatis from eye swabs.

Infectious conjunctivitis can be difficult to distinguish clinically due to the considerable overlap in clinical presentation so clinical diagnosis of conjunctivitis is often insufficient. It is therefore necessary to have a rapid diagnostic test that differentiates between the different causes of infectious conjunctivitis. Screening clinical samples by sample type/syndrome based multiplex real time PCR would allow for rapid detection of a variety of pathogens simultaneously, which will in turn aid in the treatment and clinical management of the patient. A multiplex real-time PCR assay for rapid and simultaneous detection of HSV 1 and 2, VZV, adenovirus and Chlamydia trachomatis (C. trachomatis) from eye swabs was developed and evaluated. The multiplex assay was shown to be sensitive, specific and robust. Reductions in sample turn around times have been achieved by reducing the amount of separate tests needed to be carried out.