Pan-human coronavirus and human bocavirus SYBR Green and TaqMan PCR assays; use in studying influenza A viruses co-infection and risk of hospitalization.

PURPOSE: Influenza A viruses, human coronaviruses (hCoV) and human bocavirus (hBoV) are emerging respiratory viruses. This study investigated the association between influenza A viruses co-infection with hBoV and hCoV and severity and the sensitivity of a real-time polymerase chain reaction (RT-PCR) assay for identification of 15 coronaviruses. METHODOLOGY: Published sequences for the 15 human coronaviruses were used to design a consensus PCR targeting the replicase open reading frame 1b. A previously published PCR targeting the NS1 Gene of all known human bocavirus strains was also utilized. A series of 217 samples from patients aged 37.7 (SD ± 30.4)] with seasonal influenza A viruses (SeasFluA) identified between 06/2011 and 06/2012 in NW England were tested for hCoV and hBoV using RT-PCR. Association between co-infection and disease outcome was assessed using logistic regression. RESULTS: The limit of detection of hCoV RT-PCR assay was 2 copies/µl of human coronavirus RNA template, a sensitivity comparable to a previously published SYBR green assay for human coronaviruses. A total of 12 hCoV and 17 hBoV were identified in the 217 influenza A positive samples. A higher proportion (61.5%; 8/13) of SeasFluA/hBoV co-infections were identified in patients that were admitted either to a general ward or the intensive care unit compared to 44.3% (66/149) of single SeasFlu A virus infections (OR 2.5 95% CI 0.67-9.34, p = 0.17). In a stratified analysis, there was a trend towards higher association between FluA, hCoV and hBoV with increasing age (especially in patients aged 24-45 years and >65 year old). CONCLUSION: Our hCoV RT-PCR protocol appeared to be of adequate analytical sensitivity for diagnosis. More and larger studies are needed to confirm the role of hCoV, hBoV in causing severe disease when they co-infect with influenza A viruses.

Single, dual and multiple respiratory virus infections and risk of hospitalization and mortality.

Respiratory virus infections cause a significant number of hospitalization and deaths globally. This study investigated the association between single and multiple respiratory virus infections and risk of admission to a general ward, intensive care unit or death in patients aged 0-105 years (mean ± s.d. = 24·4 ± 24·1 years), from North West England, that were tested for respiratory virus infections between January 2007 and June 2012. The majority of infections were in children aged ⩽5 years. Dual or multiple infections occurred in 10·4% (1214/11 715) of patients, whereas single infection occurred in 89·6% (10 501/11 715). Rhinovirus was the most common co-infecting virus (occurring in 69·5%; 844/1214 of co-infections). In a multivariate logistic regression model, multiple infections were associated with an increased risk of admission to a general ward [odds ratio (OR) 1·43, 95% confidence interval (CI) 1·2-1·7, P < 0·0001]. On the other hand, patients with respiratory syncytial virus (RSV) and human parainfluenza virus types 1-3 (hPIV1-3), as a single infection, had a higher risk of being admitted to a general ward (OR 1·49, 95% CI 1·28-1·73, P < 0·0001 and OR 1·34, 95% CI 1·003-1·8, P = 0·05, respectively); admitted to an intensive-care unit or dying (OR 1·5, 95% CI 1·20-2·0, P = 0·001 and OR 1·60, 95% CI 1·02-2·40, P = 0·04, respectively). This result emphasizes the importance of RSV, hPIV and mixed infections and calls for research on vaccines, drugs and diagnostic tests targeting these respiratory viruses.

Mutations associated with severity of the pandemic influenza A(H1N1)pdm09 in humans: a systematic review and meta-analysis of epidemiological evidence.

Mutations in the haemagglutinin (HA), non-structural protein 1 (NS1) and polymerase basic protein 2 (PB2) of influenza viruses have been associated with virulence. This study investigated the association between mutations in these genes in influenza A(H1N1)pdm09 virus and the risk of severe or fatal disease. Searches were conducted on the MEDLINE, EMBASE and Web of Science electronic databases and the reference lists of published studies. The PRISMA and STROBE guidelines were followed in assessing the quality of studies and writing-up. Eighteen (18) studies, from all continents, were included in the systematic review (recruiting patients 0 - 77 years old). The mutation D222G was associated with a significant increase in severe disease (pooled RD: 11 %, 95 % CI: 3.0 % - 18.0 %, p = 0.004) and the risk of fatality (RD: 23 %, 95 % CI: 14.0 %-31.0 %, p = < 0.0001). No association was observed between the mutations HA-D222N, D222E, PB2-E627K and NS1-T123V and severe/fatal disease. The results suggest that no virus quasispecies bearing virulence-conferring mutations in the HA, PB2 and NS1 predominated. However issues of sampling bias, and bias due to uncontrolled confounders such as comorbidities, and viral and bacterial coinfection, should be born in mind. Influenza A viruses should continue to be monitored for the occurrence of virulence-conferring mutations in HA, PB2 and NS1. There are suggestions that respiratory virus coinfections also affect virus virulence. Studies investigating the role of genetic mutations on disease outcome should make efforts to also investigate the role of respiratory virus coinfections.

The effect of the weather on pulmonary exacerbations and viral infections among adults with cystic fibrosis.

The effect of changes in the weather on the respiratory health of patients with cystic fibrosis (CF) is unclear. We conducted a prospective study to determine the impact of climate and season on the incidence of viral respiratory infections (VRI) and pulmonary exacerbations (PEx) among adults with CF. Between December 2010 and April 2012, 98 adults with CF were followed for 12 months. Polymerase chain reaction assays for nine viruses were performed on sputum, nose and throat swabs every 2 months and additionally at onset of PEx. Hourly temperature and relative humidity measurements were recorded throughout the study. Statistical analysis utilized generalized estimating equation (GEE) models. Pre-specified criteria for VRI and PEx were met at 29% and 37% of visits, respectively. Rhinovirus accounted for 72% of identified viruses. Incidence of rhinovirus peaked in autumn while non-rhinovirus VRI peaked in winter. Rhinovirus was associated with increased mean temperatures (OR 1.07; p = 0.001), while non-rhinovirus VRI was associated with lower mean temperatures (OR 0.87; p < 0.001). PEx occurred frequently throughout the study with no clear seasonal pattern observed. There was no significant association between climate variables and the incidence of either PEx or antibiotic prescription. There is a seasonal pattern to VRI in adults with CF. The incidence of VRI but not PEx is associated with changes in ambient temperature.

Diagnostic strategy used to establish etiologies of encephalitis in a prospective cohort of patients in England.

The laboratory diagnostic strategy used to determine the etiology of encephalitis in 203 patients is reported. An etiological diagnosis was made by first-line laboratory testing for 111 (55%) patients. Subsequent testing, based on individual case reviews, resulted in 17 (8%) further diagnoses, of which 12 (71%) were immune-mediated and 5 (29%) were due to infection. Seventy-five cases were of unknown etiology. Sixteen (8%) of 203 samples were found to be associated with either N-methyl-d-aspartate receptor or voltage-gated potassium channel complex antibodies. The most common viral causes identified were herpes simplex virus (HSV) (19%) and varicella-zoster virus (5%), while the most important bacterial cause was Mycobacterium tuberculosis (5%). The diagnostic value of testing cerebrospinal fluid (CSF) for antibody was assessed using 139 samples from 99 patients, and antibody was detected in 46 samples from 37 patients. Samples collected at 14 to 28 days were more likely to be positive than samples taken 0 to 6 days postadmission. Three PCR-negative HSV cases were diagnosed by the presence of virus-specific antibody in the central nervous system (CNS). It was not possible to make an etiological diagnosis for one-third of the cases; these were therefore considered to be due to unknown causes. Delayed sampling did not contribute to these cases. Twenty percent of the patients with infections with an unknown etiology showed evidence of localized immune activation within the CNS, but no novel viral DNA or RNA sequences were found. We conclude that a good standard of clinical investigation and thorough first-line laboratory testing allows the diagnosis of most cases of infectious encephalitis; testing for CSF antibodies allows further cases to be diagnosed. It is important that testing for immune-mediated causes also be included in a diagnostic algorithm.

Sentinel laboratory surveillance of hepatitis C antibody testing in England: understanding the epidemiology of HCV infection.

This paper describes sentinel laboratory surveillance of hepatitis C antibody testing in England. Demographic and test result data were supplemented by follow-up questionnaires sent to the requesting clinician. Between October 2002 and September 2003 almost 75000 anti-HCV tests were performed in eight sentinel centres. More males were tested than females and over half of those tested were aged 25-44 years. Overall 5.7% (3333/58144, range 2.8-7.7%) individuals tested positive. Follow-up questionnaire data showed that 82% (1043/1277) of the positives had injecting drug use reported as the main risk exposure. The majority of negative individuals were undergoing routine screening as recommended for specific patient groups. Most individuals were asymptomatic. Antibody prevalence was estimated to be 34% in current injecting drug users and 42% in former injectors. Comparing positives to routine national surveillance suggests that only 53% (1782/3333) of diagnosed cases were reported. Sentinel laboratory data can provide valuable supplementary data to national surveillance.

Respiratory viral infections after bone marrow/peripheral stem-cell transplantation: the Christie hospital experience.

Respiratory virus infections are an important cause of morbidity and mortality in bone marrow transplant patients. A retrospective study was performed on the bone marrow transplant unit at the Christie Hospital Manchester. The aim of this study was to determine the frequency, clinical presentation, laboratory diagnosis, types of intervention (eg antiviral agents used) and the outcome of such infections in this cohort of transplant recipients. Data were collected from a total of 626 adult patients and showed 27 patients with 29 confirmed episodes of viral respiratory tract infections. The viruses present were rhinovirus (40%), respiratory syncytial virus (RSV) (22.2%), influenza A (18.5%), parainfluenza (PIV) (14.8%) and enteroviruses (7.4%). The overall frequency of documented respiratory virus infections was 4.3% during the 5-year period of the study. The prevalence of respiratory viral infections was 7.8% among allogeneic and 2.3% among autologous transplant recipients. The frequency of lower respiratory tract infection (LRTI) was 3.0% among allogeneic and 1.3% among autologous transplant recipients. Eight patients died (seven had allogeneic transplants). Three of these deaths were directly attributable to a respiratory viral infection (two rhinoviruses; one PIV 3). This study further supports the role played by human respiratory viruses in transplant-associated morbidity and mortality, and particularly highlights the significance of rhinovirus infections.

Screening for cytomegalovirus (CMV) infection in allogeneic bone marrow transplantation using a quantitative whole blood polymerase chain reaction (PCR) method: analysis of potential risk factors for CMV infection.

Potential risk factors for CMV infection and the use of quantitative CMV PCR screening to guide pre-emptive anti-CMV therapy were reviewed retrospectively in 32 allogeneic bone marrow transplant patients accrued over a 2-year period. Significant CMV PCR positivity (an indicator of CMV infection) developed in 34% of patients. When analysed by recipient CMV IgG serostatus, 69% of seropositive recipients developed significant CMV PCR positivity while none of the seronegative recipients did so (P = 0.00007). Considering only the seropositive recipients, 100% of those who received the low intensity campath-1H/fludarabine/melphalan 'mini-allograft' conditioning regimen developed significant CMV PCR positivity, while only 44% of those who had received cyclophosphamide/TBI did so (P = 0.0337). The mean time to first episode of significant CMV PCR positivity for those who had received campath/fludarabine/melphalan was 25 days while for those who had received cyclophosphamide/TBI, this was 66 days (P = 0.0372). For the first episode of significant CMV PCR positivity, the mean index and peak CMV PCR counts for those who had received campath/fludarabine/melphalan were 4.54 and 5.22 log copies/ml respectively, while for cyclophosphamide/TBI, the corresponding figures were 3.85 and 4.12 log copies/ml respectively (P = 0.2986 and P = 0.0472 for index and peak values). 85% of those who had significant CMV PCR positivity with the campath/fludarabine/melphalan regimen developed more than one such episode, while 50% of those receiving cyclophosphamide/TBI regimen did so (P = 0.491). Significant CMV PCR positivity was associated with symptoms in a proportion of patients (pyrexia 45%, cough 18%, rise in AST 72%). No patient developed overt CMV disease. CMV PCR is useful for guiding pre-emptive anti-CMV therapy and for monitoring response.

Hepatitis G virus infection in drug users in Liverpool.

OBJECTIVES: To establish the prevalence of hepatitis G (HGV) in drug users in Liverpool; to explore the risk factors for, and the effects of, HGV infection. METHODS: Serum samples from 129 drug users who had attended the Infectious Diseases Unit at Fazakerley Hospital, Liverpool, between January 1995 and June 1996 were examined for HGV RNA using PCR, HGV RNA results were collated with demographic data, information on drug-use behaviour, hepatitis B (HBV) and C (HCV) serology, and the results of serum bilirubin and aspartate amino-transferase (AST) measurements. RESULTS: Overall, 37 (29%) of patients were HGV RNA positive, 89 (69%) were negative, and equivocal results were obtained in three (2%) cases. Direct sequencing of PCR products of the 5' non-translated region for 13 patients showed that these were generally more closely related to the HGV than the GB virus C (GBV-C) sequence. HGV co-infection with HCV and HBV was common: of HGV-positive patients, 28 (76%) and 16 (44%) had antibodies to HCV (anti-HCV) and hepatitis B core protein (anti-HBc), respectively. Increasing duration of injecting drug use was associated with a decreasing seroprevalence of HGV RNA, dropping from 39% for 0-4 years of injecting to 14% for > 12 years injecting. Serum bilirubin and AST values were frequently elevated, but statistical analysis showed no differences between HGV-positive and HGV-negative patient groups. CONCLUSIONS: HGV infection is common in drug users in Liverpool, but HGV RNA prevalence falls with increasing duration of injecting drug use, probably as a result of viral clearance and the development of protective immunity. HGV infection does not appear to be a significant cause of hepatic dysfunction in Liverpool drug users.

Hepatitis B and hepatitis C virus infections: risk factors among drug users in Northwest England.

OBJECTIVES: to identify the risk factors for hepatitis B (HBV) and hepatitis C (HCV) virus infections in drug users attending two drug treatment centres in Northwest England, and to evaluate the effect of both needle exchange and hepatitis B vaccination on the prevalence of hepatitis B and hepatitis C infections. METHODS: a retrospective, cross-sectional study performed at the Regional Infectious Disease Unit and a Primary Care Centre for drug users in Liverpool. The study population included 773 drug users who had hepatitis serology performed between January 1992 and April 1996. Information on risk factors was obtained from clinical records; hepatitis serology data were obtained from the Liverpool Public Health Laboratory database. RESULTS: the overall seroprevalences of exposure markers for HBV (anti-HBc antibody) and HCV (anti-HCV antibody) were 48% and 67%, respectively. Duration of injecting drug use was the strongest predictor of HCV infection, with a crude odds ratio of 8.9 (95% confidence interval (CI): 4.5-17) for >10 compared to <3 years of injecting, and was also a strong predictor of HBV infection, with an adjusted odds ratio (controlled for the effects of HBV vaccination) of 5.7 (95% CI: 3.2-10) for >10 compared to <3 years' injecting. Vaccination against HBV was associated with greatly reduced HBV seroprevalence (crude odds ratio 0.11, 95% CI: 0.06-0.18). Overall, HCV was acquired earlier in the injecting career than HBV, but drug users who were not vaccinated against HBV acquired markers for HBV even more rapidly than for HCV. We found no independent protective effect for either anti-HBc or anti-HCV acquisition after the introduction of a needle-exchange scheme. CONCLUSIONS: hepatitis C is highly prevalent among Merseyside drug users and is likely to prove difficult to control because of rapid acquisition early in the injecting career. Vaccination against hepatitis B is the best means of protecting drug users from hepatitis B, and should be offered before injecting is commenced.

Hepatitis G virus RNA and its relation to hepatitis C infection in adult haemophilic patients.

The prevalence of hepatitis C virus (HCV) infection and hepatitis G virus (HGV) RNA were studied in 50 adult haemophilic patients who had received commercial clotting factors prior to 1980. HGV RNA was detectable in 6/ 50 patients (12%); 49/50 (98%) had antibody to HCV and 40/49 (82%) of these were viraemic with detectable HCV RNA; 5/6 patients with detectable HGV RNA had co-existing HCV infection and viraemia. The HGV PCR products from all six patients were directly sequenced and all were shown to be similar to that of HGV but more diverse from that of GB virus C. One patient who had persistent abnormal liver function tests had detectable HGV RNA but no evidence of hepatitis B or C. The presence of HGV RNA in the absence of hepatitis B and C infection indicates that this virus is capable of independent transmission. Independent response to interferon was demonstrated in one patient with co-infection who lost HGV but not HCV after interferon therapy.

Early anti-HBs antibody response to accelerated and to conventional hepatitis B vaccination regimens in healthy persons.

A significantly higher prevalence of a protective concentration of anti-HBs was achieved at 1 and 2 months among 132 healthy medical students who received an accelerated vaccination schedule with a recombinant hepatitis B vaccine (Engerix-B) than among 120 medical students who received a conventional regimen. With the accelerated schedule, however, 60% students at 1 month and 27% at 2 months were still unprotected (anti-HBs titre less than 10.0 IU/l). Additional passive immunisation should therefore continue to be offered if the accelerated schedule is adopted for post-exposure prophylaxis.

PIP: In 1989-1990, researchers placed healthy 1st year medical students either into the standard vaccine scheduled group (120) or the accelerated vaccine scheduled group (132) to compare early hepatitis B antibody (anti-HBs) titers to determine if accelerated postexposure prophylaxis without hepatitis B immune globulin (HBIG) can induce as effective protection as conventional postexposure prophylaxis with HBIG. The students attended the Liverpool Medical School in Liverpool, England. Fewer students in the accelerated group used oral contraceptives and smoked cigarettes than the conventional group (p.05). Among students who were on the accelerated schedule, many more Asian than European students and many more students with a body mass index 20 developed anti-Hbs 10 IU/1 at the 1st month (p.05). Other than the differences at 1 month, the differences did not affect anti-Hbs titers. considerably more students in the accelerated group attained anti-HBs titers 10 IU/1 at 1 month (p.001) and 2 months (p.05) than those in the standard group. Nevertheless 60% of the students in the accelerated group at 1 month and 27% at 2 months were still susceptible to hepatitis B infection since they had anti-HBs titers -or= 10 IU/1. Students on the accelerated schedule experienced similar side effects at a similar rate as those on the conventional schedule. The reactions usually lasted for 24 hours and included pain and/or redness and/or swelling and headache and/or fever. In conclusion, health practitioners should continue to suggest passive immunization with HBIG in concert with an accelerated hepatitis B vaccine schedule.

Cold agglutinins in haemophiliac boys infected with HIV.

Eleven haemophiliac boys infected with HIV were screened for irregular red cell antibodies and were compared with nine haemophiliac boys who did not have antibodies to HIV. Seven (64%) of the children who had antibodies to HIV also had cold agglutinins, mostly of anti-I specificity, compared with one (11%) of those who did not have antibodies to HIV. The children with antibodies to HIV and cold agglutinins had a significantly increased mean IgM concentration. The presence of cold agglutinins was not correlated with T4 lymphocyte count, symptoms of HIV infection, serum beta 2 microglobulin concentrations, concentrations of IgG or IgA, or with the evidence of past infection with cytomegalovirus or Epstein-Barr virus.