Guidelines for laboratory monitoring of treatment of persistent virus infections.

A growing number of antiviral agents are available for treatment of persistent viral infections. This has increased the requirement for virology laboratories to undertake sophisticated assays for monitoring the efficacy of treatment and identifying drug failure at an early stage. The consensus guidelines within this article address the laboratory requirements for monitoring treatment of the herpes viruses, HIV-1, Hepatitis B and Hepatitis C.

Best Practice No 175. Guidelines for virological and non-viral serological examination of specimens in routine diagnostic microbiological laboratories.

Viral examination is routinely carried out in most routine diagnostic microbiology laboratories. Most often, this comprises the detection of viral antigens and antibodies, and less commonly the isolation of viruses and the detection of viral nucleic acids. However, there are no standards or guidelines available for processing these specimens in routine diagnostic laboratories or for referral to specialist virology centres or units. Clinical Pathology Accreditation (CPA) has defined standards for assessing the quality of service provided by laboratories, but these do not include the scientific and technical aspects of provision of service. The Association of Medical Microbiologists has recently published Standards for Laboratory practice in medical microbiology, which covers scientific and technical aspects of provision of microbiology service, mainly bacteriological examination of specimens in routine diagnostic microbiology laboratories. These guidelines are complementary to the CPA guidelines and aim to ensure a consistent and high quality service. This article presents guidelines for the examination of specimens for the diagnosis of viral infections.

Binding of bacteria to HEp-2 cells infected with influenza A virus.

Epidemiological studies indicate influenza virus infection increases susceptibility to bacterial respiratory pathogens and to meningococcal disease. Because density of colonisation is an important factor in the development of bacterial disease, the objectives of the study were to use flow cytometry methods for assessment of bacterial binding and detection of cell surface antigens to determine: (1) if HEp-2 cells infected with human influenza A virus bind greater numbers of bacteria than uninfected cells; (2) if influenza infection alters expression of cell surface antigens which act as receptors for bacterial binding; (3) if neuraminidase affects binding of bacteria to HEp-2 cells. There was significantly increased binding of all isolates tested regardless of surface antigen characteristics. There were no significant differences between virus-infected and -uninfected Hep-2 cells in binding of monoclonal antibodies to Lewisb, Lewisx or H type 2. There were significant increases in binding of monoclonal antibodies to CD14 (P < 0.05) and CD18 (P < 0.01). Treatment of cells with monoclonal antibodies significantly reduced binding of Neisseria meningitidis strain C:2b:P1.2, CD14 (P < 0.001) and CD18 (P < 0.001). No reduction in binding of a strain of Streptococcus pneumoniae (12F) was observed in these experiments. Neuraminidase treatment of HEp-2 cells increased binding of monoclonal antibodies to CD14 (P < 0.01) and CD18 (P < 0.01). In three experiments, the increase in binding of meningococcal strain C:2b:P1.2 to neuraminidase-treated cells was not significant, but binding of Staphylococcus aureus strain NCTC 10655 was significant (P < 0.05).

Evidence for the role of glycoprotein G of respiratory syncytial virus in binding of Neisseria meningitidis to HEp-2 cells.

Viral glycoproteins G and F are expressed on the surface of cells infected with respiratory syncytial virus (RSV). We investigated the role of these proteins in the previously reported enhanced binding of Neisseria meningitidis to RSV-infected HEp-2 cells. Virus particles attached to bacteria were detected by immunofluorescence with flow cytometry. Binding of FITC-labelled bacteria to RSV-infected cells was significantly inhibited by monoclonal antibody against glycoprotein G. Unlabelled bacteria interfered with binding of the anti-G monoclonal antibody to these cells. These interactions were not found with a monoclonal antibody against glycoprotein F. We propose that glycoprotein G of RSV expressed on the surface of infected cells might act as an additional receptor for meningococci.

The effect of cigarette smoke on adherence of respiratory pathogens to buccal epithelial cells.

Smoking is associated with an increased risk of respiratory tract infection in adults. In children, exposure to cigarette smoke is a risk factor for respiratory tract infection and bacterial meningitis: Active smoking and passive exposure to cigarette smoke is also associated with carriage of some potentially pathogenic species of bacteria in both adults and children. The aims of the study were to determine the effect of active smoking on: (1) bacterial binding to epithelial cells; (2) expression of host cell antigens that act as receptors for some species; and (3) the effects of passive exposure to water-soluble components of cigarette smoke on bacterial binding. Flow cytometry was used to assess binding to buccal epithelial cells of the following species labelled with fluorescein isothiocyanate: Neisseria meningitidis, Neisseria lactamica, Streptococcus pneumoniae, Bordetella pertussis, Haemophilus influenzae, Moraxella catarrhalis, Staphylococcus aureus. Flow cytometry was also used to assess expression of host cell antigens which have been identified as bacterial receptors. For each species, binding to cells of smokers was significantly higher than to cells of non-smokers; however, expression of host cell antigens was similar on epithelial cells of both groups. Non-dilute cigarette smoke extract reduced binding of bacteria to epithelial cells, but dilutions between 1 in 10 and 1 in 320 enhanced binding. We conclude that smokers might be more densely colonised by a variety of potentially pathogenic bacteria. The enhanced bacterial binding to epithelial cells of smokers is not related to enhanced expression of host cell antigens that can act as receptors for some species, but possibly to components in the smoke that alter charge or other properties of the epithelial cell surface. Passive coating of mucosal surfaces with components of cigarette smoke might enhance binding of potentially pathogenic bacteria.

Infection with respiratory syncytial virus enhances expression of native receptors for non-pilate Neisseria meningitidis on HEp-2 cells.

Respiratory virus infections have been suggested to be predisposing factors for meningococcal disease. Respiratory syncytial virus (RSV) affects young children in the age range at greatest risk of disease caused by Neisseria meningitidis. It has been previously shown that glycoprotein G expressed on the surface of RSV-infected HEp-2 cells (a human epithelial cell line) contributed to higher levels of binding of meningococci compared with uninfected cells. The aim of the present study was to examine the effect of RSV infection on expression of surface molecules native to HEp-2 cells and their role in bacterial binding. Flow cytometry and fluorescence microscopy were used to assess bacterial binding and expression of host cell antigens. Some molecules analysed in this study have not been reported previously on epithelial cells. RSV infection significantly enhanced the expression of CD15 (P < 0.05), CD14 (P < 0.001) and CD18 (P < 0.01), and the latter two contributed to increased binding of meningococci to cells but not the Gram-positive Streptococcus pneumoniae.

Infection with respiratory syncytial virus and water-soluble components of cigarette smoke alter production of tumour necrosis factor alpha and nitric oxide by human blood monocytes.

Cigarette smoke and virus infections contribute to the pathogenesis and exacerbation of chronic obstructive pulmonary disease and asthma. The objective of this study was to examine the effects of a water-soluble cigarette smoke extract (CSE) and/or respiratory syncytial virus (RSV) infection on release from monocytes of the blood from donors of tumour necrosis factor alpha (TNF-alpha) and nitric oxide (NO). Both RSV infection and CSE stimulated TNF-alpha release from monocytes and there was an additive effect if both the agents were present. There was a decrease in NO release, but the effect was significant only with CSE or a combination of CSE and RSV infection. Interferon gamma significantly increased TNF-alpha release and cotinine significantly increased NO release. Nicotine decreased both TNF-alpha and NO responses. The general pattern observed for individual donors was increased TNF-alpha and decreased NO. The proportion of extreme responses with very high TNF-alpha and very low NO in the presence of both RSV and CSE increased to 20% compared with 5% observed with CSE or RSV alone. The results show that RSV infection and components of cigarette smoke elicit inflammatory responses that could contribute to damage to the respiratory tract and these individual factors could be more harmful in combination.

Developmental and environmental factors that enhance binding of Bordetella pertussis to human epithelial cells in relation to sudden infant death syndrome (SIDS).

Asymptomatic infection due to Bordetella pertussis has been suggested to be one cause of sudden infant death syndrome (SIDS). We examined developmental and environmental factors previously found to affect binding of another toxigenic species, Staphylococcus aureus, to human epithelial cells: expression of the Lewis(a) antigen; infection with respiratory syncytial virus (RSV); exposure to cigarette smoke; and the inhibitory effect of breast milk on bacterial binding. Binding of two strains of B. pertussis (8002 and 250825) to buccal epithelial cells was significantly reduced by treating the cells with monoclonal antibodies to Lewis(a) (P < 0.05) and Lewis(x) (P < 0.01) antigens. Both strains bound in significantly greater numbers to cells from smokers compared with cells from non-smokers (P < 0.05). HEp-2 cells infected with RSV subtypes A or B had higher binding indices for both 8002 (P < 0.001) and 250825 (P < 0.01). On RSV-infected cells, there was significantly enhanced binding of monoclonal antibodies to Lewis(x) (P < 0.05), CD14 (P < 0.001) and CD18 (P < 0.01); and pre-treatment of cells with anti-CD14 or CD18 also significantly reduced binding of both strains of B. pertussis. Pre-treatment of the bacteria with human milk significantly reduced their binding to epithelial cells. The results are discussed in relation to our three-year survey of bacterial carriage among 253 healthy infants, their mothers and local SIDS cases between 1993-1995 and in relation to the change to an earlier immunisation schedule for infants and the recent decline in SIDS in Britain.

Antiviral prophylaxis and treatment in chickenpox. A review prepared for the UK Advisory Group on Chickenpox on behalf of the British Society for the Study of Infection.

Prophylactic intervention with varicella-zoster immunoglobulin early in the incubation period can prevent or attenuate the disease manifestations of varicella in susceptible contacts at high risk from this infection. Detailed guidelines are issued in the UK Department of Health publication on Immunization against Infectious Disease. Sensitive immunoassays are available for investigation of antibody status and subclinical seroconversion. Live attenuated varicella vaccine, which has been used successfully post-exposure as well as electively elsewhere, is at present not generally available in the UK. Effective protocols for prophylaxis against varicella with the antiviral agent aciclovir are not yet established. The nucleoside analogue aciclovir (syn: acyclovir, Zovirax) is effective in inhibiting replication of VZV when given at a dosage higher than that required for treatment of HSV, and is currently the only available and approved treatment for varicella in the U.K. Intravenous aciclovir therapy for 5-10 days is effective for varicella in neonates and the immunocompromised, and for varicella pneumonia or other complications in adults and children, if begun early. Oral aciclovir is only effective if begun with 24 h of onset of rash. With that proviso. it is recommended for treatment of varicella in otherwise healthy adults and adolescents, but not for routine use in children under 13 years of age unless they are sibling contacts or have other medical conditions. Aciclovir has a high therapeutic index and good safety profile, but caution is advised with use in pregnancy.

Association between secretor status and respiratory viral illness.

OBJECTIVE: To determine whether non-secretion of blood group antigens is associated with respiratory virus diseases. DESIGN: Study of secretor status in patients with respiratory virus diseases determined by an enzyme linked immunosorbent assay (ELISA) developed to identify Lewis (Le) blood group antigen phenotypes (Le(a) non-secretor; Le(b) secretor). SUBJECTS: Patients aged 1 month to 90 years in hospital with respiratory virus diseases (584 nasal specimens). MAIN OUTCOME MEASURES: Criteria for validation of ELISA (congruence between results on ELISA testing of 1155 saliva samples from a previous study and previously established results on haemagglutination inhibition (HAI) testing, proportions of Le(a), Le(b), and Le- phenotypes in 872 samples of nasal washings from a previous study compared with the normal population). Secretor status of patients determined by ELISA and viruses isolated. RESULTS: Agreement between HAI and ELISA for 1155 saliva samples was 97%. Lewis antigens were detected by ELISA in 854 (97.9%) of nasal washings (Le(a) 233 (26.7%), Le(b) 621 (71.2%), and Le- 18 (2.1%)) in proportions predicted for a northern European population. Secretors were significantly overrepresented among patients from whom influenza viruses A and B (55/64, 86%; p less than 0.025), rhinoviruses (63/72, 88%; p less than 0.01), respiratory syncytial virus (97/109, 89%; p less than 0.0005), and echoviruses (44/44, p less than 0.0005) had been isolated compared with the distribution of secretors in the local population. CONCLUSION: Secretion of blood group antigens is associated with respiratory virus diseases.

Serological studies with human papova (wart) virus.

Methods for demonstrating antibody to wart virus by complement fixation and passive haemagglutination tests are described and compared with the precipitin test of Almeida & Goffe (1965). The results reveal the much greater sensitivity of the passive haemagglutination method, particularly in the detection of the immunoglobulin M class of antibody. Both complement-fixing and precipitating antibody were detected in sera from patients whose warts had undergone a spontaneous resolution.The presence of antibody to wart virus was demonstrated in sera from persons who had had warts up to 10 years previously, and in a few cases from those who thought they had never had warts.The antigenic identity of virus from hand warts and plantar warts of the simple and mosaic types was revealed, and some evidence was obtained for similar identity of the virus from genital warts.

DNA polymerase activity in rheumatoid synovial membranes.

RNAase-sensitive DNA polymerase activity was demonstrated in synovial membrane preparations from 23 out of 25 rheumatoid arthritis patients. Control groups consisted of twelve patients with osteoarthrosis, four with secondary osteoarthrosis, and twelve with other conditions. The last group showed no activity, while the results with the other two groups were varied. The properties of the polymerase enzyme, such as its stimulation by synthetic templates and inhibition by actinomycin D, were not consistent with it being associated with an oncogenic virus; it seems to be more like that found in stimulated normal human lymphocytes, described as an RNA-primed DNA-directed DNA polymerase.

Effect of low dose acyclovir (5 mg/kg) on virus shedding, interferon and humoral immunity in herpes zoster.

In a double-blind randomized trial, virological studies were carried out in non-immunocompromised patients with herpes zoster who received either acyclovir (5 mg/kg) or placebo intravenously three times daily for five days. Mean duration of virus shedding was not significantly different in the two groups and all patients developed high titres of antibody to varicella-zoster virus. Interferon levels in vesicle fluid reached a peak significantly sooner (P less than 0.025) and at a lower level in eight treated patients compared with eight given placebo, corresponding with significantly earlier cessation of vesicle formation in the treated group (P less than 0.05). Pretreatment virus isolates tested for sensitivity to the drug showed ED50 values from 4 +/- 0.7 to 17.5 +/- 1 microM acyclovir. Treatment with a dose of acyclovir greater than 5 mg/kg, but not exceeding 10 mg/kg, is recommended for herpes zoster.

Antibody response to varicella-zoster virus surface glycoproteins in chickenpox and shingles.

Varicella-zoster virus (VZV)-infected cell surface proteins were investigated using extrinsic radiolabelling of the cell surface, immunoprecipitation of detergent-solubilized extract of the same cell surface and fractionation of the immunoprecipitates using SDS-PAGE. Glycosylated proteins were identified by their affinity for Ricinus communis lectin. Six glycoproteins with apparent mol. wt. of 170K, 105K, 93K, 81K, 53K and 45K were identified. The 170K glycoprotein was shown to be disulphide-linked since under reducing conditions for SDS-PAGE it was cleaved to a protein of 63K mol. wt. The IgG responses to these glycoproteins during various clinical circumstances are described. In acute sera from all chickenpox patients and in the majority of acute shingles sera, antibodies reactive with glycoproteins could not be detected. In chickenpox convalescence, antibodies reactive with glycoproteins of mol. wt. 170K, 105K, 53K and 45K were identified, whilst during zoster convalescence antibodies to all six were prominent. Antibodies to the disulphide-linked glycoprotein persisted for many years following both the primary disease and its reactivation. Disseminated zoster was associated with significantly low levels of antibodies to these surface glycoproteins.

Herpes simplex infections in pemphigus: an indication for urgent viral studies and specific antiviral therapy.

Four cases of herpes simplex virus infection in exacerbations of pemphigus are described. Three of these patients had severe generalized infection requiring systemic anti-viral therapy. The presenting features, methods for rapid viral diagnosis and appropriate treatment are discussed.

Antibodies to respiratory syncytial virus polypeptides and their significance in human infection.

The human antibody response to respiratory syncytial (RS) virus infection was investigated using radioimmunoprecipitation analysis (RIPA). A total of nine RS virus-specific proteins, VP200, VGP95, VP68, VGP48, VPN41, VP35, VP27, VP23 and VGP20 were identified by comparing 35S- or 3H-labelled extracts of infected and uninfected HEp-2 cells, and by radioimmunoprecipitation using a hyperimmune human serum. Three glycopeptides, VGP95, VGP48 and VGP20, were identified by incorporation of [3H]glucosamine, and two of these (VGP48 and VGP20) were assumed to be part of a single disulphide-bonded polypeptide since they were precipitated by a monoclonal antibody raised against a surface protein. Human serum antibodies to three major RS virus proteins, VGP95, VGP48/VGP20 and VPN41 were measured by RIPA using radioiodinated RS virus antigens. Sera from a group of mothers whose babies escaped RS virus infection during a local epidemic showed increased antibody levels to VPN41 when compared to sera from mothers whose babies had become infected with RS virus within the first 6 months of life. In infants who remained uninfected with RS virus during the first 12 months of life the maternal gift of antibody decayed to about 50% at 3 months with traces of antibodies detected in a few sera at 12 months. The antibody levels detected in the sera of infants less than 3 months old convalescent from primary RS virus infection did not exceed the mean levels present in the serum of uninfected babies. Infants between the ages of 6 and 12 months were able to mount an IgG response to VPN41 and VGP48 but, unlike adults and older children, a particularly striking finding was their failure to produce antibodies to VGP95.

Maternal antibody and respiratory syncytial virus infection in infancy.

One hundred newborn infants were studied prospectively for 1 year for evidence of infection with respiratory syncytial virus (RSV). The indirect membrane fluorescence technique was used to determine specific antibody in sera. Infection was shown in 29 cases. In 31 infants exposed to an RSV epidemic season, there was no evidence of infection. Maternal antenatal sera were also tested, and wide range of IgG antibody to RSV was found. Mean titre of maternal IgG antibody to RSV was significantly higher (P less than 0.001) in those mothers whose babies remained uninfected than in those whose babies had proved RSV infection before 6 months of age. Babies born to mothers with high levels of IgG antibody to respiratory syncytial virus were protected against infection with this virus during the first months of life when the risk of severe disease was greatest.