Biomedical subjects
Publications and source records attributed to P V Coyle.
We report a herpes simplex virus type 1 mutant which failed to amplify with a commonly used glycoprotein D primer set. The virus contained a nine-base deletion in the gene's 5' nontranslated region. The altered amplicon was clearly distinguishable on a 4% high-resolution agarose gel.
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Hantaviruses do not produce cytopathic effects (CPE) in cell culture. However, a syncytial CPE can be induced in 7-day cultures of hantavirus growing in Vero E6 cells by reduction of the pH to approximately 6.2 using a HEPES based buffer. The appearance of this acid induced CPE was examined for seven different hantavirus strains. The differences noted were striking and reflected the taxonomic differences between hantaviruses. At 10-100 TCID50% the size of syncytial foci was very large for Seoul type viruses and smallest for Puumala viruses. The size of syncytia for Hantaan (HTN) virus was intermediate between Puumala (PUU) and Seoul (SEO) type viruses.
134 swabs in viral transport medium were received from 126 patients with suspected clinical HSV-1 and HSV-2 infections. They were tested by (i) nested multiplex polymerase chain reaction NMPCR (strongly positive specimens had visible bands on both rounds of PCR) without prior extraction, (ii) culture in primary rhesus monkey kidney, E6-Vero, RD and HEp-2 cells and (iii) antigen detection by immunofluorescence (IF). Antigen detection employed four novel pools (A-D) of monoclonal antibodies (Mab): A was HSV-1 specific, B was HSV-2 specific while C and D were generic. In comparison to NMPCR the sensitivity and specificity of (i) culture was 59% (22/37) and 100% (134/134), (ii) IF by Pool A was 59% (16/27) and 100% (117/117), (iii) IF by Pool B was 40% (4/10) and 100% (130/130) and (iv) IF by Pools C and D were 60% (18/30) and 100% (96/96). Specimens positive by culture were more likely to be strongly positive by NMPCR (chi2 P = 0.004). Typing by each method concurred on all occasions. NMPCR was cost effective, easier to perform and was the most sensitive method for HSV detection. It should become the method of choice for HSV diagnosis.
Respiratory viruses are an extremely common cause of childhood morbidity. However, the current seroprevalence of viruses in infant populations is difficult to establish because invasive venipuncture may be technically and ethically unacceptable. This prospective study aimed to establish the seroprevalence of respiratory viruses in an infant population by use of a novel multiantigen fluorescence immunoassay against common respiratory viruses, using heel-prick blood samples collected on filter paper. Mothers and babies were recruited in the immediate peripartum period in the Royal Maternity Hospital, Belfast. Cord blood samples at birth and heel-prick filter paper blood samples at 7 mo were collected for measurement of virus-specific IgG to respiratory syncytial virus, influenza A virus, adenovirus, and parainfluenza virus type 1, type 2, and type 3 by indirect immunofluorescence using a multiviral assay developed for this purpose. Of 386 mothers approached, 325 (84%) permitted follow-up at 7 mo, and of these, 256 (79%) agreed to the heel prick. From 234 paired samples, 125 infections were documented. Adenovirus infections were commonest, 53 (22.6%), followed by respiratory syncytial virus, 32 (13.7%); influenza A virus, 22 (9.4%); parainfluenza virus type 3, 14 (6%); parainfluenza virus type 1, 2 (0.85%); and parainfluenza virus type 2, 2 (0.85%). These results demonstrate the seroprevalence of a range of respiratory viruses in an infant population, using a novel multiviral immunoassay. The filter paper collection of blood samples and multiantigen assay format has implications for easy, widespread viral serodiagnosis in both seroepidemiology studies and in the diagnosis of pediatric viral illnesses. Filter paper permits recovery of respiratory virus-specific IgG and can be used as a simple and acceptable epidemiologic and diagnostic tool.
A study was set up to investigate the effect of consistency of routine faecal specimens on the diagnostic yield by electron microscopy (EM) and virus isolation. A total of 3078 specimens were characterized as solid, semisolid, or liquid. Of 2568 specimens processed by EM a virus was demonstrated in 8.6% of liquid, 19.9% of semisolid and 25.2% of solid specimens (Chi-squared for linear trend, P value <0.0001). This observation was valid for both adenovirus (2.4%, 5.0% and 6.6%) and rotavirus (5.2%, 13.6% and 16.6%). Virus isolation was positive in 3.6% of liquid, 17.4% of semisolid and 18.1% of solid specimens. (Chi-squared for linear trend, P value <0.0001). We suggest that solid faecal specimens at the end of an episode of diarrhoea will have a higher diagnostic yield than liquid specimens at the peak of symptoms. Our findings repudiate the commonly held dogma that viruses of gastroenteritis are more likely to be found in liquid than in solid faecal specimens. This finding has important implications for those establishing diagnostic algorithms for the investigation of viral gastroenteritis.
We report six cases of recrudescent intraoral herpes simplex infection clinically indistinguishable from primary herpetic gingivostomatitis. All infections occurred in healthy children or young adults. Serological analysis demonstrated herpes simplex virus (HSV)-specific IgG at initial presentation, indicating that the infection was not a primary infection. Convalescent sera exhibited HSV-specific IgM and a rising HSV-specific IgG titre. These findings demonstrate that the initial clinical diagnosis of primary herpetic gingivostomatitis was erroneous and that what was actually being observed was widespread recrudescent intraoral herpes simplex infection.
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Bone marrow of 61 HIV-1-infected patients and 23 control patients was examined to determine the incidence of B19 infection and its clinical impact in HIV-1-infected persons. Of the 61 HIV-infected patients studied, ages ranged from 22-47 years with a mean of 33.2 years. There was a man:woman ratio of 3.8:1. With regard to staging of HIV disease at the time of bone marrow sampling, 52 patients were CDC group 4, 5 patients were CDC group 3, and 4 patients were CDC group 2. Control patients, were not known to be HIV-1-infected, and had one of the following conditions: lymphoma, leukaemia, thrombocytopenia, thrombocytosis, anaemia, multiple myeloma, raised serum IgM. Thirteen of 61 HIV-infected patients and 0 of 23 control patients were positive for B19 DNA in bone marrow (two-tailed P value = 0.016). Within the HIV-infected group, the average haemoglobin among persons testing B19 DNA positive (n = 13) was 11.1 g/dl, compared with 11.5 g/dl among persons testing B19 DNA negative (n = 48). In conclusion, B19 persistence may be common and frequently subclinical in AIDS patients.
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Previous evidence has suggested that Northern Ireland is a low seroprevalence area for HIV infection. The Unlinked Anonymous HIV Prevalence Monitoring Programme initiated in England and Wales in 1990 was extended to Northern Ireland in 1992. Patients attending the Genitourinary Medicine Clinic at the Royal Victoria Hospital have, with informed consent, been tested anonymously for HIV infection since that time. The results of the survey between 1992 and 1995 have shown an overall seroprevalence rate 3.01% for homosexual/bisexual men, 0.08% for heterosexual men, and 0.05% for heterosexual women. These results confirm the previous impression of low HIV seroprevalence in Northern Ireland and the survey provides an excellent longitudinal study by which changes may be monitored.
This study was undertaken to examine the natural history of parvovirus B19 infection in persons without a known immune defect in terms of both clinical symptoms and immune responsiveness to the virus. Fifty-three patients with acute B19 infection (positive for serum anti-B19 IgM) were studied; symptoms at acute infection were rash and arthralgia (n = 26), rash (n = 7), arthralgia (n = 16), aplastic crisis (n = 3), and intrauterine fetal death (n = 1). Patients were followed for 26-85 months (mean 57 months) and reassessed for persistent symptoms, anti-B19 antibodies, and antibodies to the unique region of B19 VP1. There were 23 cases of arthralgia persisting for longer than 1 year after acute infection. One of these patients, a 48-year-old woman at follow-up, had had persistent arthralgia for 4 years following acute B19 infection, had rheumatoid factor at a titre of 1920 IU/ml detected at follow-up, and had been independently diagnosed as having rheumatoid arthritis at the time of follow-up. All 53 patients were positive for serum anti-B19 IgG compared to 45 of 53 age- and sex-matched control patients, a significant difference (two-tailed P value = 0.008). All test patients at follow-up and control patients were negative for serum anti-B19 IgM and antibodies to the unique region of B19 VP1. Serum from acute infection from 33 of 53 test patients was tested for antibodies to the unique region of VP1, and 16 of these were positive. The presence of this antibody did not correlate with subsequent duration of symptoms but did correlate with a short interval between symptom onset and blood sampling. The unique region of B19 VP1 is known to be crucial for a successful humoral response to the virus, and it seems that the antigenic role played by this region is important only during the acute phase of B19 infection.
Virus isolation is essential for the provision of a full diagnostic virology service. Present methods are time consuming, expensive and relatively inflexible for routine use. Our objective was to audit our existing virus isolation system and to develop a sensitive, flexible virus isolation system which could be adapted for use in a busy routine laboratory which is required to provide a service for a wide range of clinical situations. We carried out a pilot study which compared conventional roller tube monolayer cultures to a microplate system using cells inoculated in suspension and showed that the microplate method using extra cell lines could provide a more sensitive system for virus isolation. This system was adapted for routine use using six cell lines inoculated in suspension and the results are presented for 2610 specimens for virus isolation and 972 for Clostridium difficile toxin (CDT) detection. There were 516 viruses isolated and 229 specimens positive for CDT using this system. Polioviruses (92), echoviruses (35), coxsackieviruses (15) and untyped enteroviruses (13) were isolated in RMK, E6-vero and RD cells. Adenoviruses (137) were isolated in HEp2 and E6-vero cells. Herpes simplex virus (HSV) was isolated from 149 specimens in E6-vero, FCL and HFF9 cells. Myxoviruses (38) and paramyxoviruses were isolated in RMK cells. HEp2 was the only cell line necessary to isolate the 33 respiratory syncytial viruses (RSV). Cytomegaloviruses (CMV) (2) and varicella zoster (1) virus (VZV) were isolated only in the human fibroblast cell line HFF9. Rubella virus was isolated from a baby with congenital rubella in RMK, E6-vero and additionally in BGM cells. In conclusion, the use of cells inoculated in suspension in microtitre plates for virus isolation was sensitive and convenient. It allowed the use of six cell lines for routine virus isolation without using additional laboratory staff time. It improved turnaround times. It was also safer microbiologically than conventional isolation in tube monolayers. The precise identification of virus isolates was simplified.
A survey of evidence of rodent hantavirus infection in County Down, Northern Ireland was carried out by using immunofluorescence to detect virus antigen and antibody. Antibodies to hantavirus (R22 strain of Seoul virus and Hantaan 76-118) were found in 11/51 (21.6%) brown rats (Rattus norvegicus), 1/31 (3.2%) field mice (Apodemus sylvaticus) and 17/59 (28.8%) house mice (Mus domesticus). Seven rodents had evidence of hantavirus antigen in lung tissues. Antibody positive animals were significantly more likely to be adults than juveniles (P = 0.04) but and there was no sex difference between antibody positive and negative animals. House mice were more likely to be antibody positive if captured inside farm outbuildings (P = 0.08). Attempts to culture virus from the rodent material were unsuccessful. This work demonstrates a substantial rodent reservoir for hantavirus in Northern Ireland.
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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.
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