Hugh J. O'Neill

Respiratory viral infection in exacerbations of COPD

Summary Background Patients with COPD have frequent exacerbations. The role of respiratory viral infection is just emerging. We wished to determine prospectively the incidence of viral infection in exacerbated and stable COPD patients as well as smokers who do not have airways obstruction. Methods Stable and exacerbated COPD patients were recruited along with a group of patients who had smoked but who did not have any airways obstruction. Spirometry was performed and sputum specimens were tested for a range of 12 different respiratory viruses using PCR. Results One hundred and thirty-six patients with exacerbations of COPD, 68 stable COPD patients and 16 non-obstructed smokers were recruited. A respiratory virus was detected in 37% of exacerbations, 12% of stable COPD patients and 12% of non-obstructed smokers, p <0.0005. Rhinovirus was most frequently detected. The symptom of fever was associated with virus detection, p <0.05. Infection with more than one virus was only found in the exacerbated COPD patients. Conclusion Respiratory viral infection is associated with exacerbations of COPD. Rhinovirus was the most common infecting agent identified and in two cases human metapneumovirus was also detected. Dual infections were only seen amongst those patients admitted to hospital with acute exacerbations of COPD. Viruses were more commonly detected in those with more severe airways disease.

Coxiella burnetii (Q fever) seroprevalence in cattle

Human cases of Q fever appear to be common in Northern Ireland compared to the rest of the British Isles. The purpose of this study was to describe the seroepidemiology of Coxiella burnetii infection in cattle in Northern Ireland in terms of seroprevalence and determinants of infection. A total of 5182 animals (from a stratified systematic random sample of 273 herds) were tested with a commercial C. burnetii phase 2 IgG ELISA. A total of 6.2% of animals and 48.4% of herds tested positively. Results from a multilevel logistic regression model indicated that the odds of cattle being infected with Q fever increased with age, Friesian breed, being from large herds and from dairy herds. Large dairy herd animal prevalence was 12.5% compared to 2.1% for small beef herds. Preliminary seroprevalence in sheep (12.3%), goats (9.3%), pigs (0%) rats (9.7%) and mice (3.2%) using indirect immunofluorescence is reported.

Human Seroprevalence to Coxiella burnetii (Q fever) in Northern Ireland

Despite the widespread prevalence of infection with Coxiella burnetii, there have been few large population‐based studies examining the epidemiology of this infection. The aim of this study was to examine the distribution and determinants of C. burnetii past infection in Northern Ireland (NI). Coxiella burnetii phase II specific IgG antibodies were measured by enzyme‐linked immunosorbent assay in stored serum from 2394 randomly selected subjects, aged 12–64, who had participated in population‐based surveys of cardiovascular risk factors performed in 1986 and 1987. The overall prevalence of C. burnetii antibody positivity was 12.8%. The prevalence of sero‐positivity was slightly higher in males than that in females (14.3% versus 11.2%, P = 0.02). Sero‐positivity was low in children (<10%), increasing to 19.5% and 16.4% in males and females, respectively, in the 25–34 age group and subsequently remaining fairly steady with increasing age. Sero‐positivity among farmers, at 48.8%, was significantly higher than the general population. More sero‐positive than sero‐negative women had a history of a miscarriage or still‐birth (19.5% versus 9.8%, P < 0.001). In conclusion, this study demonstrated a high prevalence of evidence of past C. burnetii infection in NI. Associations between past C. burnetii infection and age, sex, social class, occupation and reproductive history were seen. We estimate that 20% of Q fever infections in NI occur in farmers.

Non-detection of Chlamydia species in carotid atheroma using generic primers by nested PCR in a population with a high prevalence of Chlamydia pneumoniae antibody

BackgroundThe association of Chlamydia pneumoniae with atherosclerosis is controversial. We investigated the presence of C. pneumoniae and other Chlamydia spp. in atheromatous carotid artery tissue.MethodsForty elective carotid endarterectomy patients were recruited (27 males, mean age 65 and 13 females mean age 68), 4 had bilateral carotid endarterectomies (n= 44 endarterectomy specimens). Control specimens were taken from macroscopically normal carotid artery adjacent to the atheromatous lesions (internal controls), except in 8 cases where normal carotid arteries from post mortem (external controls) were used. Three case-control pairs were excluded when the HLA DRB gene failed to amplify from the DNA. Genus specific primers to the major outer membrane protein (MOMP) gene were used in a nested polymerase chain reaction (nPCR) in 41 atheromatous carotid specimens and paired controls. PCR inhibition was monitored by spiking with target C. trachomatis. Atheroma severity was graded histologically. Plasma samples were tested by microimmunofluorescence (MIF) for antibodies to C. pneumoniae, C. trachomatis and C. psittaci and the corresponding white cells were tested for Chlamydia spp. by nPCR.ResultsC. pneumoniae was not detected in any carotid specimen. Twenty-five of 38 (66%) plasma specimens were positive for C. pneumoniae IgG, 2/38 (5%) for C. trachomatis IgG and 1/38 (3%) for C. psittaci IgG.ConclusionsWe were unable to show an association between the presence of Chlamydia spp. and atheroma in carotid arteries in the presence of a high seroprevalence of C. pneumoniae antibodies in Northern Ireland.

A touchdown nucleic acid amplification protocol as an alternative to culture backup for immunofluorescence in the routine diagnosis of acute viral respiratory tract infections.

BackgroundImmunofluorescence and virus culture are the main methods used to diagnose acute respiratory virus infections. Diagnosing these infections using nucleic acid amplification presents technical challenges, one of which is facilitating the different optimal annealing temperatures needed for each virus. To overcome this problem we developed a diagnostic molecular strip which combined a generic nested touchdown protocol with in-house primer master-mixes that could recognise 12 common respiratory viruses.ResultsOver an 18 month period a total of 222 specimens were tested by both immunofluorescence and the molecular strip. The specimens came from 103 males (median age 3.5 y), 80 females (median age 9 y) and 5 quality assurance scheme specimens. Viruses were recovered from a number of specimen types including broncho-alveolar lavage, nasopharyngeal secretions, sputa, post-mortem lung tissue and combined throat and nasal swabs. Viral detection by IF was poor in sputa and respiratory swabs. A total of 99 viruses were detected in the study from 79 patients and 4 quality control specimens: 31 by immunofluorescence and 99 using the molecular strip. The strip consistently out-performed immunofluorescence with no loss of diagnostic specificity.ConclusionsThe touchdown protocol with pre-dispensed primer master-mixes was suitable for replacing virus culture for the diagnosis of respiratory viruses which were negative by immunofluorescence. Results by immunofluorescence were available after an average of 4–12 hours while molecular strip results were available within 24 hours, considerably faster than viral culture. The combined strip and touchdown protocol proved to be a convenient and reliable method of testing for multiple viruses in a routine setting.

Rising incidence of Pneumocystis jirovecii pneumonia suggests iatrogenic exposure of immune-compromised patients may be becoming a significant problem

Against a background of point-source outbreaks of Pneumocystis pneumonia (PCP) in renal transplant units in Europe, we undertook a retrospective 3 year observational review of PCP in Northern Ireland. This showed an unexpected increase in incidence, with a mortality rate of 30 %. Fifty-one cases were confirmed compared to 10 cases confirmed in the preceding 7 years. Where undiagnosed HIV infection had previously been the main risk factor for PCP, this was now equally matched by chemotherapy for haematological and non-haematological malignancy and immune suppression for a range of autoimmune conditions. Congenital immunodeficiency and transplantation were less common predisposing factors, but renal grafts also showed a rising incidence. Asymptomatic carriage was uncommon. At presentation both upper and lower respiratory samples were of equal use in establishing the diagnosis, and treatment resulted in rapid clearance. These data suggest the need for considering PCP in at-risk patients, reviewing its mode of acquisition and whether iatrogenic colonization is a treatable pre-condition.

High levels of Epstein–Barr virus in COPD

Latent viral infection has been implicated in the pathophysiology of chronic obstructive pulmonary disease (COPD). Epstein–Barr virus (EBV) is known to be important in pulmonary fibrosis. The current authors hypothesised that EBV is associated with the pathogenesis of COPD. Sputum samples were collected from patients both during exacerbations of COPD and when stable. A control group of smokers who did not have airways obstruction also had their sputum examined. The presence of EBV DNA was established and quantified using a real-time nucleic acid amplification assay. A total of 136 patients with COPD were recruited during an acute exacerbation and a total of 68 when stable. EBV was detected in 65 (48%) exacerbation cases and 31 (46%) stable patients. In the comparison group of 16 nonobstructed smokers, EBV was demonstrated in only one (6%) case. Risk of COPD in patients with EBV and who are smokers confers an odds ratio of 12.6. Epstein–Barr virus DNA is more frequently identified in the respiratory tract of chronic obstructive pulmonary disease patients in comparison with unaffected smokers. It is present both during exacerbation and when stable, suggesting that infection is persistent. Smokers who do not develop chronic obstructive pulmonary disease rarely have Epstein–Barr virus in their sputum. This finding may be of importance in the pathogenesis of chronic obstructive pulmonary disease.

Nested multiplex polymerase chain reaction for the diagnosis of cutaneous herpes simplex and herpes zoster infections and a comparison with electronmicroscopy

Herpes simplex virus (HSV) and varicella zoster virus (VZV) are common causes of cutaneous and mucocutaneous vesicular eruptions. Laboratory diagnostic techniques include Tzanck smears, electronmicroscopy, antigen detection and viral culture. This paper describes a nested multiplex polymerase chain reaction with respective sensitivities of 0.0001, 0.01 and 0.1 TCID50 for VZV, HSV‐1 and HSV‐2. The assay was used in (a) a salvage capacity for slides already processed for electronmicroscopy, and (b) as a front‐line assay for prospectively processed specimens. Sixty‐two glass slides with vesicle lymph/scrapings from 58 patients with suspected cutaneous herpetic lesions were examined. The clinical presentations were described as atypical/not specified (24), VZV (20) or HSV (18), and involved eruptions from diverse anatomical sites, including the genitalia. Of the 62 specimens, 6 and 38 were positive by electronmicroscopy and multiplex PCR respectively, giving a comparative sensitivity of 16% for electronmicroscopy. Nested multiplex PCR identified 15 VZV and 20 HSV‐1 infections. Where the clinical details indicated either HSV or VZV (38/62), nested multiplex PCR was statistically likely to be reactive (26/38 vs. 9/24) (χ2 P = 0.000004) whereas electronmicroscopy was not (4/38 vs. 2/24) (χ2 P = 0.77). Where the clinical details indicated VZV (20/62) or HSV (18/62), nested multiplex PCR was statistically more likely to confirm VZV (10/20 vs. 5/42) (χ2 P = 0.001) or HSV (9/18 vs. 11/44) (χ2 P = 0.05) respectively. Two suspected HSV and 6 suspected VZV infections were shown to be VZV and HSV respectively by nested multiplex PCR. J. Med. Virol. 63:52–56, 2001.

A simple standardised protocol for the production of monoclonal antibodies against viral and bacterial antigens.

A simple standardised protocol for making monoclonal antibodies against a range of human bacteria and viruses is described. The protocol was designed to reduce the number of steps to a minimum. A one step footpad immunisation was followed by the fusion schedule 10-15 days later. A vital step in the technique was the use of the immunised mouses spleen to provide a feeder layer post fusion. This simplified the protocol and more importantly greatly accelerated the growth of the hybridomas produced. Immunisation, fusion and clonal expansion of specific antibody secreting hybridomas was complete within 5 weeks. The percentage of hybridomas secreting specific antibody ranged from 6% to 28%, the majority of which were of the IgG isotypes. The method was economical in the use of tissue culture medium and simple to perform.

Design and production of a target-specific monoclonal antibody to parvovirus B19 capsid proteins

Native parvovirus B19 was used as antigen to produce a mouse monoclonal antibody, R92F6, which reacted with B19 VP1 and VP2, neutralised the virus in bone marrow culture, and labelled infected cells in paraffin-embedded tissues from cases of B19-related fetal hydrops. The B19 epitope recognised by R92F6 (amino acids 328-344 from the amino terminal region of B19 VP2) appears to be highly conserved, since these tissue specimens were obtained over a 13 year period from widely spaced locations in the UK. This epitope was synthesised as a peptide (S7b) which was used as antigen to produce a mouse monoclonal antibody, 3H8, which specifically reacted with the B19 capsid proteins VP1 and VP2 in immunofluorescence and immunoblot assays. 3H8 was also capable of labelling formalin-fixed, paraffin-embedded, B19-infected fetal tissue and was shown to be of the same isotype as R92F6 (IgG1). Highly conserved epitopes derived from conserved amino acid sequences are valuable in the diagnosis of infectious disease. If these can be recognised and accurately synthesised, the production of specific mouse monoclonal antibodies may be possible for many human pathogens. Considering the vast amount of sequence data available in the literature, this approach seems to be both feasible and of wide potential.