Search

Previous exposure to Epstein-Barr virus (EBV) is strongly associated with the development of multiple sclerosis (MS). By contrast, past cytomegalovirus (CMV) infection may have no association, or be negatively associated with MS. This study aimed to investigate the associations of herpesvirus infections with MS in an Italian population. Serum samples ( n = 200) from Italian people with multiple sclerosis (PwMS) classified as the relapsing-and-remitting clinical phenotype and ( n = 137) healthy controls (HCs) were obtained from the CRESM Biobank, Orbassano, Italy. Both PwMS and HCs samples were selected according to age group (20-39 years, and 40 or more years) and sex. EBV virus capsid antigen (VCA) IgG, EBV nucleic acid-1 antigen (EBNA-1) IgG, CMV IgG, herpes simplex virus (HSV) IgG, and varicella zoster virus (VZV) IgG testing was undertaken using commercial ELISAs. EBV VCA IgG and EBNA-1 IgG seroprevalences were 100% in PwMS and 93.4% and 92.4%, respectively, in HCs. EBV VCA IgG and EBNA-1 IgG levels were higher ( p < 0.001) in PwMS compared with HCs. For PwMS, the EBNA-1 IgG levels decreased with age, particularly in females. The CMV IgG seroprevalence was 58.7% in PwMS and 62.9% in HCs. CMV IgG seroprevalence increased with age. The HSV IgG seroprevalence was 71.2% in PwMS and 70.8% in HCs. HSV IgG levels were lower ( p = 0.0005) in PwMS compared with HCs. VZV IgG seroprevalence was 97.5% in PwMS and 98.5% in HCs. In the population studied, several herpesvirus infections markers may have been influenced by the age and sex of the groups studied. The lack of a negative association of MS with CMV infection, and the observation of lower levels of HSV IgG in PwMS compared with HCs are findings worthy of further investigation.

There is increasing evidence suggesting that Epstein-Barr virus infection is a causative factor of multiple sclerosis (MS). Epstein-Barr virus (EBV) is a human herpesvirus, Human Gammaherpesvirus 4. EBV infection shows two peaks: firstly, during early childhood and, secondly during the teenage years. Approximately, 90-95% of adults have been infected with EBV and for many this will have been a subclinical event. EBV infection can be associated with significant morbidity and mortality; for example, primary infection in older children or adults is the leading cause of infectious mononucleosis (IM). A disrupted immune response either iatrogenically induced or through genetic defects can result in lymphoproliferative disease. Finally, EBV is oncogenic and is associated with several malignancies. For these reasons, vaccination to prevent the damaging aspects of EBV infection is an attractive intervention. No EBV vaccines have been licensed and the prophylactic vaccine furthest along in clinical trials contains the major virus glycoprotein gp350. In a phase 2 study, the vaccine reduced the rate of IM by 78% but did not prevent EBV infection. An EBV vaccine to prevent IM in adolescence or young adulthood is the most likely population-based vaccine strategy to be tested and adopted. National registry studies will need to be done to track the incidence of MS in EBV-vaccinated and unvaccinated people to see an effect of the vaccine on MS. Assessment of vaccine efficacy with MS being a delayed consequence of EBV infection with the average age of onset being approximately 30 years of age represents multiple challenges., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Maple, Ascherio, Cohen, Cutter, Giovannoni, Shannon-Lowe, Tanasescu and Gran.)

Healthcare workers (HCWs) reporting no history of varicella frequently receive varicella vaccination (vOka) if they test varicella-zoster virus (VZV) immunoglobulin G (IgG) negative. In this study, the utilities of VZV-IgG time-resolved fluorescence immunoassay (VZV-TRFIA) and a commercial VZV-IgG purified glycoprotein enzyme immunoassay (gpEIA) currently used in England for confirming VZV immunity have been compared to the fluorescent-antibody-to-membrane-antigen assay (FAMA). A total of 110 HCWs received two doses of vOka vaccine spaced 6 weeks apart and sera collected pre-vaccination (n = 100), at 6 weeks post-completion of vaccination (n = 86) and at 12-18 months follow-up (n = 73) were analysed. Pre-vaccination, by FAMA, 61·0% sera were VZV IgG negative, and compared to FAMA the sensitivities of VZV-TRFIA and gpEIA were 74·4% [95% confidence interval (CI) 57·9-87·0] and 46·2% (95% CI 30·1-62·8), respectively. Post-completion of vaccination the seroconversion rate by FAMA was 93·7% compared to rates of 95·8% and 70·8% determined by VZV-TRFIA and gpEIA, respectively. At 12-18 months follow-up seropositivity rates by FAMA, VZV-TRFIA and gpEIA were 78·1%, 74·0% and 47·9%, respectively. Compared to FAMA the sensitivities of VZV-TRFIA and gpEIA for measuring VZV IgG following vaccination were 96·4% (95% CI 91·7-98·8) and 74·6% (95% CI 66·5-81·6), respectively. Using both FAMA and VZV-TRFIA to identify healthy adult VZV susceptibles and measure seroconversion showed that vOka vaccination of HCWs is highly immunogenic.

Population seroprevalence can be estimated from serosurveys by classifying quantitative measurements into positives (past infection/vaccinated) or negatives (susceptible) according to a fixed assay cut-off. The choice of assay cut-offs has a direct impact on seroprevalence estimates. A time-resolved fluorescence immunoassay (TRFIA) was used to test exposure to human parvovirus 4 (HP4). Seroprevalence estimates were obtained after applying the diagnostic assay cut-off under different scenarios using simulations. Alternative methods for estimating assay cut-offs were proposed based on mixture modelling with component distributions for the past infection/vaccinated and susceptible populations. Seroprevalence estimates were compared to those obtained directly from the data using mixture models. Simulation results showed that when there was good distinction between the underlying populations all methods gave seroprevalence estimates close to the true one. For high overlap between the underlying components, the diagnostic assay cut-off generally gave the most biased estimates. However, the mixture model methods also gave biased estimates which were a result of poor model fit. In conclusion, fixed cut-offs often produce biased estimates but they also have advantages compared to other methods such as mixture models. The bias can be reduced by using assay cut-offs estimated specifically for seroprevalence studies.

Detection of specific viral antibody or nucleic acid produced by infection or immunization, using oral fluid samples, offers increased potential for wider population uptake compared to blood sampling. This methodology is well established for the control of HIV and measles infections, but can also be applied to the control of other vaccine preventable infections, and this review describes the application of oral fluid assays in support of mumps, rubella and varicella national immunization programs. In England and Wales individuals with suspected mumps or rubella, based on clinical presentation, can have an oral fluid swab sample taken for case confirmation. Universal varicella immunization of children has led to a drastic reduction of chickenpox in those countries where it is used; however, in England and Wales such a policy has not been instigated. Consequently, in England and Wales most children have had chickenpox by age 10 years; however, small, but significant, numbers of adults remain susceptible. Targeted varicella zoster virus (VZV) immunization of susceptible adolescents offers the potential to reduce the pool of susceptible adults and oral fluid determination of VZV immunity in adolescents is a potential means of identifying susceptible individuals in need of VZV vaccination. The main application of oral fluid testing is in those circumstances where blood sampling is deemed not necessary, or is undesirable, and when the documented sensitivity and specificity of the oral fluid assay methodology to be used is considered sufficient for the purpose intended.

Children treated for acute leukemia are at increased risk of severe infection with varicella zoster virus (VZV). We studied the VZV sero-status of children with acute leukemia prior to starting chemotherapy and after completion of chemotherapy. VZV sero-status was assessed using time resolved fluorescence immunoassay (TRFIA) before starting treatment and 6 months after completion of treatment. Prior to starting treatment for acute leukemia, a significant proportion of children (35%) are VZV seronegative. On completion of treatment most patients maintained protective VZV antibody levels; however, 35% had reduced/loss VZV antibody to a level considered non-protective and susceptible to VZV infection., (© 2014 Wiley Periodicals, Inc.)

Parvovirus B19 (B19V) is a member of the family Parvoviridae, genus Erythrovirus. B19V-specific IgG and IgM react differently against conformational and linear epitopes of VP1 and VP2 antigens, leading to the development of IgG avidity and epitope type specificity (ETS) enzyme immunoassays (EIAs) for distinguishing past from recent infection. Additionally, B19V viral load determination (by quantitative PCR [qPCR]) is increasingly used in the staging of B19V infection. In this study, the utility of these methods is compared. A panel of 78 sera was jointly tested by the Virus Reference Department (VRD), London, United Kingdom, and the Haartman Institute (HI), Helsinki, Finland, using a number of EIAs, e.g., B19V-specific IgG and IgM, IgG avidity, and ETS EIAs. At VRD, the sera were also tested by a B19V viral load PCR (qPCR). By consensus analysis, 43 (55.1%) sera represented past infection, 28 (35.9%) sera represented recent infection, and 7 (9.0%) sera were indeterminate. Both VRD B19V qPCR and HI B19V VP2 IgM EIA gave the highest agreement with consensus interpretation for past or recent infection, with an overall agreement of 99% (95% confidence interval [CI], 92 to 100) and positive predictive value (PPV) of 100% (95% CI, 87 to 100). Nine sera designated as representing past infection by consensus analysis were B19V IgM positive by a commercial VRD B19V IgM EIA and B19V IgM negative by a new HI in-house B19V VP2 IgM EIA. A new VRD B19V IgG avidity EIA showed good (>95%) agreement (excluding equivocal results) with consensus interpretations for past or recent infection. Correct discrimination of past from recent B19V infection was achieved through application of qPCR or by appropriate selection of EIAs.

Introduction: In the UK, primary varicella is usually a mild infection in children, but can cause serious illness in susceptible pregnant women and adults. The UK Joint Committee on Vaccination and Immunisation is considering an adolescent varicella vaccination programme. Cost-effectiveness depends upon identifying susceptibles and minimising vaccine wastage, and chickenpox history is one method to screen for eligibility. To inform this approach, we estimated the proportion of adolescents with varicella antibodies by reported chickenpox history., Methods: Recruitment occurred through secondary schools in England from February to September 2012. Parents were asked about their child's history of chickenpox, explicitly setting the context in terms of the implications for vaccination. 247 adolescents, whose parents reported positive (120), negative (77) or uncertain (50) chickenpox history provided oral fluid for varicella zoster virus-specific immunoglobulin-G (VZV-IgG) testing., Results: 109 (90.8% [85.6-96.0%]) adolescents with a positive chickenpox history, 52 (67.5% [57.0-78.1%]) with a negative history and 42 (84.0% [73.7-94.3%]) with an uncertain history had VZV-IgG suggesting prior infection. Combining negative and uncertain histories, 74% had VZV-IgG (best-case). When discounting low total-IgG samples and counting equivocals as positive (worst-case), 84% had VZV-IgG. We also modelled outcomes by varying the negative predictive value (NPV) for the antibody assay, and found 74-87% under the best-case and 84-92% under the worst-case scenario would receive vaccine unnecessarily as NPV falls to 50%., Conclusion: Reported chickenpox history discriminates between varicella immunity and susceptibility in adolescents, but significant vaccine wastage would occur if this approach alone were used to determine vaccine eligibility. A small but important proportion of those with positive chickenpox history would remain susceptible. These data are needed to determine whether reported history, with or without oral fluid testing in those with negative and uncertain history, is sufficiently discriminatory to underpin a cost-effective adolescent varicella vaccination programme., (Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Human parvovirus 4 (ParV4), a newly described member of the family Parvoviridae, like B19V, has been found in pooled plasma preparations. The extent, and significance, of ParV4 exposure in UK blood donors remain to be determined and reliable detection of ParV4 immunoglobulin (Ig)G, using validated methods, is needed., Study Design and Methods: With ParV4 virus-like particles a ParV4 IgG time-resolved fluorescence immunoassay (TRFIA) was developed. There is no gold standard or reference assay for measuring ParV4 IgG and the utility of the TRFIA was first examined using a panel of sera from people who inject drugs (PWIDS)--a high-prevalence population for ParV4 infection. Western blotting was used to confirm the specificity of TRFIA-reactive sera. Two cohorts of UK blood donor sera comprising 452 sera collected in 1999 and 156 sera collected in 2009 were tested for ParV4 IgG. Additional testing for B19V IgG, hepatitis C virus antibodies (anti-HCV), and ParV4 DNA was also undertaken., Results: The rate of ParV4 IgG seroprevalence in PWIDS was 20.7% and ParV4 IgG was positively associated with the presence of anti-HCV with 68.4% ParV4 IgG-positive sera testing anti-HCV-positive versus 17.1% ParV4 IgG-negative sera. Overall seropositivity for ParV4 IgG, in 608 UK blood donors was 4.76%. The ParV4 IgG seropositivity for sera collected in 1999 was 5.08%, compared to 3.84% for sera collected in 2009. No ParV4 IgG-positive blood donor sera had detectable ParV4 DNA., Conclusion: ParV4 IgG has been found in UK blood donors and this finding needs further investigation., (© 2013 American Association of Blood Banks.)

Background: Recently, a commercial, standardised VZV IgG glycoprotein EIA, Binding Site VaccZyme™VZV glycoprotein IgG low level EIA (VaccZyme™EIA) has become available. The VaccZyme™EIA is more robust and user friendly than the reference VZV time-resolved fluorescence immunoassay (VZV TRFIA)., Objectives: To assess the usefulness of the VaccZyme™EIA in the diagnostic laboratory by comparing VZV IgG levels generated by both assays on serum panels representing, non-vaccinated, and vOka vaccinated populations., Study Design: Sera from non-vaccinated individuals were tested; 248 from pregnant women, 117 from various patient groups referred to the Virus Reference Department for confirmatory VZV IgG testing and 102 from healthcare workers enrolled in a study (ROVE) of antibody/IgG response to vOka. From the ROVE study, 282 post vaccination sera were tested; 108 and 101 collected at six weeks post first and second doses of vOka, respectively, and 73 collected at 18 month follow-up., Results: Sensitivities and specificities (equivocals treated as negatives) of the VaccZyme™EIA for sera from pregnant women were 97.8% (95% CI: [94.6%, 99.4%]) and 96.8% (95% CI: [89.0%, 99.6%]), respectively, and for sera referred for confirmatory testing were 81.2% (95% CI: [71.2%, 88.8%]) and 96.9% (95% CI: [83.8%, 99.9%]), respectively, and for ROVE baseline sera were 54.2% (95% CI: [32.8%, 74.4%]) and 100% (95% CI: [95.4%, 100.0%]), respectively. For the post vOka serum panels sensitivities of the VaccZyme™EIA ranged from 65.3% (95% CI: [50.4%, 78.3%]) to 80.4% (95% CI: [71.1%, 87.8%]). Specificities were all 100%. Correlation with VZV TRFIA was high and agreement varied between the serum panels tested., Conclusions: VaccZyme™EIA is recommended for detecting VZV IgG in sera from non-vaccinated populations; however, caution is advised when measuring post vOka VZV IgG levels., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The purpose of this study was to validate through natural exposure a cut-off level of varicella zoster IgG as protective against infection with varicella zoster virus (VZV). Laboratory testing to determine VZV immune status of pregnant women exposed to varicella is recommended. Quantitative assays are now available which are sensitive and specific. More than 200 consecutive requests for screening in pregnant patients with recent varicella contacts were followed-up by questionnaire. DiaSorin LIAISON and VZV time resolved fluorescence immuno assay (VZV TRFIA) were used to measure VZV antibody level. One hundred fifty out of 209 (72%) questionnaires were returned; 14 patients developed varicella, 129 did not and seven were not known. Patients who had been given VZIG and developed varicella on follow-up had a mean antibody level before VZIG of 28 mIU/ml and 62 mIU/ml, by LIAISON and TRFIA, respectively. The mean IgG level of those that did not develop varicella was 885 and 866 mIU/ml by LIAISON and TRFIA, respectively. Those with levels <100 mIU/ml were more likely to develop chicken pox than those with levels >100 mIU/ml (relative risk of 10.4 for LIAISON and 8.8 for TRFIA). On the basis of the relatively small numbers in this study, quantitative assays, using a 100mIU/ml cut-off, can differentiate between those who are susceptible and those who are protected against exposure, however follow-up studies should include sampling for VZV DNA and IgM.

Determination of varicella zoster virus (VZV) immunity in healthcare workers without a history of chickenpox is important for identifying those in need of vOka vaccination. Post immunisation, healthcare workers in the UK who work with high risk patients are tested for seroconversion. To assess the performance of the time-resolved fluorescence immunoassay (TRFIA) for the detection of antibody in vaccinated as well as unvaccinated individuals, a cut-off was first calculated. VZV-IgG specific avidity and titres six weeks after the first dose of vaccine were used to identify subjects with pre-existing immunity among a cohort of 110 healthcare workers. Those with high avidity (≥ 60%) were considered to have previous immunity to VZV and those with low or equivocal avidity (<60%) were considered naive. The former had antibody levels ≥ 400 mIU/mL and latter had levels < 400 mIU/mL. Comparison of the baseline values of the naive and immune groups allowed the estimation of a TRFIA cut-off value of > 130 mIU/mL which best discriminated between the two groups and this was confirmed by ROC analysis. Using this value, the sensitivity and specificity of TRFIA cut-off were 90% (95% CI 79-96), and 78% (95% CI 61-90) respectively in this population. A subset of samples tested by the gold standard Fluorescence Antibody to Membrane Antigen (FAMA) test showed 84% (54/64) agreement with TRFIA., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Following a laboratory audit, a significant number of Treponema pallidum particle agglutination assay (TPPA)-negative sera were identified when TPPA was used as a confirmatory assay of syphilis enzyme immunoassay (EIA) screening-reactive sera (SSRS). Sera giving such discrepant results were further characterized to assess their significance. A panel of 226 sera was tested by the Abbott Murex ICE Syphilis EIA and then by the Newmarket Syphilis EIA II. TPPA testing was performed on 223 sera. Further testing by the Venereal Disease Research Laboratory (VDRL) test, the Mercia Syphilis IgM EIA, the fluorescent treponemal antibody (FTA-ABS) assay, and INNO-LIA immunoblotting was undertaken in discrepant cases. One hundred eighty-seven of 223 (83.8%) SSRS were TPPA reactive, while 26 (11.6%) sera which were reactive in both the ICE and Newmarket EIAs were nonreactive by TPPA. The majority (68%) of the TPPA-discrepant sera were from HIV-positive patients and did not represent early acute cases, based on previous or follow-up samples, which were available for 22/26 samples. FTA-ABS testing was performed on 24 of these sera; 14 (58.3%) were FTA-ABS positive, and 10 (41.7%) were FTA-ABS negative. Twenty-one of these 26 sera were tested by INNO-LIA, and an additional 4 FTA-ABS-negative samples were positive. In this study, significant numbers (18/26) of SSRS- and TPPA-negative sera were shown by further FTA-ABS and LIA (line immunoblot assay) testing to be positive. The reason why certain sera are negative by TPPA but reactive by treponemal EIA and other syphilis confirmatory assays is not clear, and these initial findings should be further explored.

Infection by Varicella Zoster virus (VZV) during pregnancy has been associated with adverse foetal development and more severe disease in the mother. Accurate determination of VZV immunity in pregnant women exposed to VZV, with no history of chickenpox, guides therapeutic interventions. The accepted gold standard assay for the determination of immunity/protection against Varicella Zoster virus was for many years the fluorescent antibody to membrane antigen (FAMA) assay which is labour intensive and subjective. A validated alternative is the Merck glycoprotein EIA (Merck Sharp & Dohme Research Laboratories, West Point, PA, USA) which reports VZV IgG levels in enzyme units per ml (EU/ml) because an internal, non-international reference serum is used as calibrator. Comparison of different VZV IgG detection assays is hampered by a lack of an agreed cut-off in standardised units. A time resolved fluorescence immunoassay (TRFIA) for VZV IgG using British Standard VZV antibody has been developed and standardised. The limit of detection of VZV IgG by this assay was of the order 39-78mIU/ml. Following comparison with the Merck glycoprotein EIA and the application of the USA Advisory Committee on Immunization Practices recommended 5.0EU/ml cut-off the following standardised cut-offs in mIU/ml are proposed. A VZV TRFIA IgG cut-off of less than 100mIU/ml VZV IgG equates with susceptibility and an equivocal range of 100mIU/ml to less than 150mIU/ml is proposed. VZV IgG levels of 150mIU/ml, or greater, are indicative of natural infection at some time and the ability to mount a protective immune response is inferred.

The performance of fifteen, commercially available, VZV IgG assays and an "in house" indirect immunofluorescence (IF) assay has been compared to a reference VZV IgG time resolved immunofluorescence assay (VZV TRFIA). A panel of 273 VZV TRFIA IgG positive serum samples and 136 VZV TRFIA IgG susceptible sera, collected from a number of UK hospitals was used. Irrespective of the interpretation of equivocal results the most sensitive assays were Dade Behring EIA (97.4%), "in house" IF (95.2%), Human EIA (92.3%) and Becton Dickinson latex agglutination (94.1%). The least sensitive assays were Virion EIA (69.6%), Diesse EIA (68.9%) and Diasys EIA (68.5%). The least sensitive (<70%) assays all had >99.0% specificity whereas the most sensitive assays had lower specificities; for example, Dade Behring EIA had a specificity of 69.9% when equivocals were treated as VZV IgG negative. For some assays e.g. Dade Behring EIA there were major discrepancies between our findings and those reported by the manufacturer which may reflect the constitution of the panel(s) of sera used for evaluation or the reference method adopted or the choice of cut-off criteria (particularly relevant to our findings for the Behring EIA). Care must be taken to choose an assay with high specificity in order to accurately assess the need for vaccination or immunoprophylaxis; however, high sensitivity is preferable to prevent inappropriate and expensive treatment.

Background: Determination of Varicella Zoster virus (VZV) immune status in pregnant women without history of chickenpox is important in identifying those who genuinely need VZV immune globulin prophylaxis following significant exposure to chickenpox or shingles. Immune status testing requires highly sensitive and specific immunoassays for timely and accurate results., Objectives: To compare the performance of DiaSorin LIAISON and Biomerieux VIDAS VZV-IgG assays with reference to a VZV-IgG time-resolved fluorescence immunoassay (TRFIA)., Study Design: A panel of sera collected from 65 pregnant contacts of VZV and 62 individuals tested for VZV immunity was tested in all three assays. Dose-response curves were generated using International Standards W1044 and 90/690., Results: Sensitivity and specificity of VIDAS compared to VZV-TRFIA was 54.5% and 97.9% respectively and for LIAISON compared to VZV-TRFIA was 67% and 100% respectively. Both assays correlated well with TRFIA with R2 correlation coefficients of 0.79 and 0.76 respectively. Dose-response curves showed both Standards behaved in a similar manner in each assay. For VIDAS, the test cut-off value of 0.9 correlated with 275-280mIU/ml and for LIAISON a cut-off value of 150mIU/ml correlated with 208-219mIU/ml., Conclusions: By dose-response data and in comparison with TRFIA, LIAISON is more sensitive and specific than VIDAS.

Background: In clinical trials, maternal tetanus toxoid (TT) vaccination is effective in protecting newborns against tetanus infection, but inadequate placental transfer of tetanus antibodies may contribute to lower-than-expected rates of protection in routine practice. We studied the effect of placental malaria and maternal human immunodeficiency virus (HIV) infection on placental transfer of antibodies to tetanus., Methods: A total of 704 maternal-cord paired serum samples were tested by ELISA for antibodies to tetanus. The HIV status of all women was determined by an immunoglobulin G antibody-capture particle-adherence test, and placental malaria was determined by placental biopsy. Maternal history of TT vaccination was recorded., Results: Tetanus antibody levels were reduced by 52% (95% confidence interval [CI], 30%-67%) in newborns of HIV-infected women and by 48% (95% CI, 26%-62%) in newborns whose mothers had active-chronic or past placental malaria. Thirty-seven mothers (5.3%) and 55 newborns (7.8%) had tetanus antibody levels <0.1 IU/mL (i.e., were seronegative). Mothers' self-reported history of lack of tetanus immunization was the strongest predictor of seronegativity and of tetanus antibody levels in maternal and cord serum., Conclusion: Malarial and HIV infections may hinder efforts to eliminate maternal and neonatal tetanus, making implementation of the current policy for mass vaccination of women of childbearing age an urgent priority.

The performance of a time-resolved fluorescence immunoassay (TRFIA) for detection of treponemal IgG from oral fluid specimens has been assessed in a predominantly HIV-infected population. Serological investigation is the method of choice for confirming clinical suspicion of syphilis; however, in the primary stage of disease, direct detection of treponemes in lesion fluid or Treponema pallidum DNA is recommended because of the reduced sensitivity of serological tests. There may be occasions when blood for serological investigation is difficult to obtain due to individual patient preference or logistical necessity to improve participation in screening initiatives, particularly in outreach situations. Collection of oral fluid for detection of treponemal antibody may prove an attractive alternative and, with this in mind, an oral fluid assay for detection of treponemal IgG was developed. Time-resolved fluorescence was used to detect treponemal IgG extracted from commercially available oral fluid collection devices. Paired serum and saliva samples were obtained from 210 individuals, 101 of whom were diagnosed with syphilis on the grounds of medical examination confirmed by serological testing. Oral fluid specimens from 14 subjects were rejected because they contained insufficient control antibody or were inhibitory. The population tested was predominantly men who have sex with men, many of whom were HIV infected. The overall sensitivity and specificity of the oral fluid assay was 95.8 and 86.1%, respectively, based on the 5th percentile of the positive results, and 93.7 and 91.1%, respectively, based on a cutoff derived by mixture model analysis. For individuals with primary syphilis, the optimum sensitivity of the oral fluid assay was 87.5%, whereas in those with disease classified as secondary syphilis and early latent syphilis, the sensitivity of the oral fluid assay was 100 and 94.7%, respectively. The oral fluid assay is a useful alternative to serological testing in certain situations, and further development of this technology to enable detection of treponemal IgM should increase its sensitivity for detecting cases of primary syphilis.

Highly sensitive and specific, quantitative assays are needed to detect varicella-zoster virus (VZV) immunoglobulin G in human sera, particularly for determining immune status and response following vaccination. A time-resolved fluorescence immunoassay (TRFIA) has been developed, and its performance was compared to that of two commercial enzyme immunoassays (EIAs) and Merck glycoprotein EIA (gpEIA). The TRFIA had equivalent sensitivity (97.8%) and high specificity (93.5%) in relation to gpEIA. A commercial (Behring) EIA compared favorably with TRFIA in terms of sensitivity (98.4%) but had lower specificity (80.7%). Another commercial EIA (Diamedix) had high specificity (97.1%) but low sensitivity (76.4%) compared to TRFIA if equivocal test results were treated as negative for VZV antibody. A novel feature of the TRFIA was that the cutoff was generated using population mixture modeling and was expressed in mIU/ml, as the assay was calibrated using the British standard VZV antibody.

We have developed a time-resolved fluorescence immunoassay to detect antibodies to Treponema pallidum recombinant antigens in oral fluid specimens. Using an 'Oracol' swab, oral fluid was collected from 34 subjects with a serological diagnosis of syphilis and 97 seronegative controls. Using a cut-off of three standard deviations over control mean, the sensitivity and specificity of the assay in all subjects with positive syphilis serology was 76.5% and 97.9%, respectively. In those with early syphilis, the sensitivity and specificity of the assay was 100% and 97.9%. In a non-outbreak situation, screening clinic attendees for syphilis using oral fluid specimens is potentially useful when collection of blood is not practicable. In addition, it may have much to offer in outreach projects and epidemiological investigations.

High titres of pertussis toxin (PT) antibody have been shown to be predictive of recent infection with Bordetella pertussis. The seroprevalence of standardized anti-PT antibody was determined in six Western European countries between 1994 and 1998 and related to historical surveillance and vaccine programme data. Standardized anti-PT titres were calculated for a series of whole-cell and acellular pertussis vaccine trials. For the serological surveys, high-titre sera (> 125 units/ml) were distributed throughout all age groups in both high- (> 90%) and low-coverage (< 90%) countries. High-titre sera were more likely in infants in countries using high-titre-producing vaccines in their primary programme (Italy, 11.5%; Western Germany, 13.3%; France, 4.3%; Eastern Germany, 4.0%) compared to other countries (The Netherlands, 0.5%; Finland, 0%). Recent infection was significantly more likely in adolescents (10-19 years old) and adults in high-coverage countries (Finland, The Netherlands, France, East Germany), whereas infection was more likely in children (3-9 years old) than adolescents in low-coverage (< 90%; Italy, West Germany, United Kingdom) countries. The impact and role of programmatic changes introduced after these surveys aimed at protecting infants from severe disease by accelerating the primary schedule or vaccinating older children and adolescents with booster doses can be evaluated with this approach.

The incidence of pertussis infection can be estimated in the population by defining a single high titre of anti pertussis toxin (PT) immunoglobulin G (IgG) antibody predictive of recent infection. Sera samples collected in 1986, 1996 or annually between 1987 and 1998 were tested for anti-PT IgG antibody. In 1996, the age-adjusted prevalence of pertussis infection was 1.2% and was higher in children than in adults. Amongst samples collected annually, older age and female sex, but none of the temporal variables, were associated with a serologically defined pertussis infection. There is an important incidence of infection in the population, which is greater amongst children than adults, but there is only limited evidence of a correlation with epidemic cycles.

A standardisation process was developed in order to compare and harmonize serological results of pertussis toxin (PT) antibody measurements performed by laboratories using different technical procedures for detection. This involved the development of a common panel, of sera by a designed reference centre, the distribution of the panel to each participating laboratory for testing with their routine methods, the comparison of the obtained results to those of the reference centre, and the calculation of standardisation equations by regressing the quantitative results against those of the reference centre. As a cut-off indicative of protection against pertussis has not yet been defined, a particular emphasis was laid upon achieving standardisation of high titre results that would allow epidemiological evaluations based on the estimation of the incidence of recent infections rather than on the traditional approach of determining the population immunity profile. A generally good agreement was achieved between the participating laboratories, all using ELISA procedures very similar in many crucial aspects, and standardisation equations were produced useful to enable inter-country comparison during the next stages of the European Sero-Epidemiology Network (ESEN) project concerning the serological surveillance of immunity to pertussis in Europe.

Background: Screening for Chlamydia trachomatis specific antibodies is valuable in diagnosing asymptomatic pelvic inflammatory disease (PID) and tubal damage following repeated episodes of PID. The assays in current use are unsuitable for screening large numbers of samples so there is a need to develop more suitable assays., Aims: To compare the performance of several commercial C trachomatis enzyme immunoassays (EIAs) (SeroCT, C tracho(pep), Medac p-EIA, Vircell and Labsystems C trachomatis IgG EIAs) using major outer membrane protein (MOMP), an inactivated organism EIA (Genzyme Virotech EIA), and a genus specific EIA (Platelia Chlamydia IgG) with the whole cell inclusion immunofluorescence (WIF) assay. In addition, to adapt, using time resolved fluorescence technology, the assay showing the highest correlation with WIF., Methods: Ninety sera from patients presenting with ectopic pregnancies, 187 sera from those with a variety of types of infertility, 33 sera from cases of PID where a fourfold rise in WIF titre occurred, and 90 sera from antenatal clinic attenders were tested. A panel of 36 sera from laboratory diagnosed cases of Chlamydia psittaci/Chlamydia pneumoniae infection was also tested., Results: The Genzyme Virotech EIA showed the highest rank correlation coefficient (0.82) with WIF, particularly at high WIF titres. The MOMP specific assays varied in their correlation with WIF, with rank correlation coefficients ranging from 0.70 (Medac p-EIA) to 0.80 (Vircell EIA). The Genzyme Virotech assay showed poor specificity (5.6%; 95% confidence interval (CI), 0.68% to 18.7%)--it was reactive with 34 of the panel of 36 C psittaci/C pneumoniae positive sera. The MOMP based EIAs showed high specificity, particularly the Medac p-ELISA (97.2%; 95% CI, 85.5% to 99.9%)--only one serum was reactive. In view of the good correlation between WIF and the Genzyme Virotech EIA, a time resolved fluorescence immunoassay (TRFIA) was developed using the Genzyme Virotech antigen. Using an appropriate cut off the TRFIA assay showed excellent correlation with WIF., Conclusions: The TRFIA assay may be useful as a screening assay, possibly in conjunction with one of the highly specific EIAs studied (for example, Medac p-EIA) to confirm the antibody specificity of sera selected by the screening assay.

Rubella antibody (IgG) has been measured in females reporting for antenatal screening using single radial haemolysis (SRH) and time-resolved fluorescence immunoassay (TRFIA). We have shown TRFIA to be a simple, specific, highly sensitive, quantitative assay for rubella IgG with a lower limit of detection of 0.2IU/ml. Out of 506 sera tested by SRH, 18 (3.6%) had low levels of antibody (< 15IU/ml) compared to 83 (16.4%) tested by TRFIA and, of these, 32 (6.3%) had rubella antibody concentrations < 10IU/ml. The lowest level (3.1%) of rubella susceptibility (antibody levels < 10IU/ml) was found in females aged 25-29 and the highest level of susceptibility (23.5%) occurred in females aged 40 years, and over. Geometric mean rubella antibody concentrations (IU/ml) were 26.8, 34.4, 34.8, 29.7, 27.5 and 20.0 for age groups <20, 20-24, 25-29, 30-34, 35-39 and > or =40 years, respectively. Our rubella vaccination policies have built up good levels of rubella immunity in women of childbearing age in our locality, and using TRFIA technology we can accurately monitor changes over time.

A time resolved fluorometric immunoassay (TRFIA) has been developed and compared with an in house enzyme linked immunosorbent assay (ELISA) and commercial ELISA (Bindazyme) for the detection of tetanus antitoxin in human sera. A panel of 132 sera submitted for routine testing was used. Scatterplots showed a high degree of correlation between all three assays, although some divergence of results was apparent for low titre sera when comparing in house ELISA results with Bindazyme ELISA and TRFIA results. The TRFIA appeared to be more sensitive than the in house ELISA, and the Bindazyme assay compared well with the TRFIA. The intra-assay precision of all three assays, in terms of percentage coefficient of variation (%CV), was between 2.0% and 4.0%. The interassay precision ranged from 5% to 8% for the in house ELISA, 13% to 19% for the Bindazyme assay, and 11% to 13% for TRFIA. Both Bindazyme and TRFIA assays were simple to perform, accurate, reproducible, and amenable to automation. A particular benefit of the TRFIA was its large dynamic range, enabling tetanus antitoxin values of 0.01 IU/ml to 50 IU/ml to be measured with just one dilution of serum. TRFIA appears to be a useful serological technique worthy of further development.

Today, tetanus is a rare disease in England and Wales although in less developed and less affluent areas of the World it is still a major cause of morbidity and mortality, particularly in neonates and women following childbirth. An estimated 400,000 deaths worldwide occur annually from neonatal tetanus and in 1989 the World Health Organization adopted the goal of eliminating neonatal tetanus as a public health problem by 1995-2000. This goal has not yet been achieved worldwide; however in England and Wales tetanus in neonates and young women has been extremely rare for the last 50 years. We can attribute the success in eliminating tetanus in the UK to the adoption of highly effective preventative measures as we know that the causative organism is still ubiquitous in the environment. This article outlines how we have achieved the elimination of tetanus in the UK through the development and introduction of appropriately targeted immunisation, combined with an appreciation of the importance of adequate wound toilet measures. The discovery of tetanus toxoid and its applicability for vaccination has been of great benefit and the new challenges which we face relate to the introduction of new multi-component vaccines and how we might better protect the elderly. In other parts of the world, for the cost of a few pence, one dose of tetanus toxoid can still mean the difference between life and death.

The immunity profile of the English and Welsh population to diphtheria and tetanus has been determined by measuring diphtheria and tetanus antitoxin levels for 3088 and 3142 sera, respectively. Time-resolved fluorimetric immunoassay - DELFIA was used to measure diphtheria antitoxin levels and an in-house, indirect ELISA to measure tetanus antitoxin levels. More than 80% of those aged between 2 and 20-24 years had protective diphtheria antitoxin levels of 0.01 IU/ml, or greater, and more than 80% of those aged between 4 and 35-39 years had protective tetanus antitoxin levels of 0.1 IU/ml, or greater. Only 29% and 53% of those aged 60 and over were protected against diphtheria and tetanus. Two increases of diphtheria antitoxin levels greater than 0.1 IU/ml and tetanus antitoxin levels greater than 1.0 IU/ml were apparent, starting at 4 and 14 years of age, which correspond with the policy of giving a diphtheria and tetanus toxoid booster on school entry and a tetanus plus low-dose diphtheria toxoid (recently introduced) booster to school leavers. This is the first comprehensive study of diphtheria and tetanus immunity in the English and Welsh population and shows that the accelerated schedule of immunisation, introduced in 1990, has effectively primed immunological memory against both these antigens and that boosting at school entry and at school leaving is effective in increasing levels of immunity.

A European Sero-Epidemiological Network (ESEN) was established with the aim to co-ordinate and harmonise serological surveillance of immunity to communicable diseases in Europe. In this study the inter-laboratory standardisation of diphtheria toxin antibody measurements is reported. A standard panel of 162 sera was tested by the participating laboratories using an in vitro assay of their choice: VERO cell toxin neutralisation assay (NT), double-antigen delayed time-resolved fluorescence immuno-assay (DA-DELFIA), double-antigen enzyme-linked immunosorbent assay (DAE), toxin binding inhibition test (ToBI) and an indirect enzyme-linked immunosorbent assay (ELISA). The results were standardised using regression against the NT. The variations due to inter-laboratory and inter-assay variation, which would otherwise make it difficult directly to compare the main serum bank results by the different laboratories and the various assays were successfully minimised by the standardisation. The regression equations obtained will be used to transform the respective local results of testing the main serum bank into the reference test unitages. This study also gave the opportunity to compare the various assays within and between laboratories. This demonstrated a very high correlation between DA-DELFIA, DAE, ToBI and the NT. The ELISA showed a good correlation, too, however sera below some 0.1 IU/ml were overestimated.

Seven countries in Western Europe collected large, representative serum banks across the entire age range and tested them for diphtheria anti-toxin (sample size ranged from 2991 to 7715). Although a variety of assays were used, the results were all standardized to those of a reference laboratory and expressed in international units. The standardization process, and the availability of similar, large data sets allowed comparative analyses to be performed in which a high degree of confidence could be ascribed to observed epidemiological differences. The results showed that there were large differences in the proportion of adults with insufficient levels of protection amongst different countries. For instance, roughly 35% of 50- to 60-year-olds were found to be seronegative (titre < or = 0.01 IU/ml) in Finland compared with 70-75% in the United Kingdom. Furthermore, the proportion of seronegative adults would be expected to increase in some countries, notably Italy and the western part of Germany. In those countries with vaccination of military recruits there was a marked sex-related difference in the proportion of seropositive individuals. All countries have high levels of infant vaccine coverage (> 90%) but the accelerated schedule in the United Kingdom appears to result in lower anti-toxin titres than elsewhere. In Sweden, booster doses are not offered until 10 years of age which results in large numbers of children with inadequate levels of protection. Although the United Kingdom and Sweden both have higher proportions of seronegative children than elsewhere the likelihood of a resurgence of diphtheria in these countries seems remote.

Immunity to diphtheria was determined in serum samples from 1000 UK-born blood donors at the North London Blood Transfusion Centre during a three-month period in 1993; 125 women and 125 men were stratified in 10-year age groups, from 20 to 59. A tissue (vero cell)-culture toxin-neutralisation assay was used to measure serum diphtheria antitoxin concentrations. According to internationally accepted definitions (antitoxin < 0.01 IU/mL = susceptibility, 0.01-0.09 IU/mL = basic protection, and > or = 0.1 IU/mL = full protection), 37.6% of donors were susceptible to diphtheria, 31.5% had basic protection, and 30.9% were fully protected. Log-linear modelling of the influence of age and sex on population immunity showed a significant trend (p < 0.001) of decreasing immunity with increasing age: 25.2% of donors aged 20-29 were susceptible compared with 52.8% of those aged 50-59. There was a small sex effect (p = 0.052); similar proportions of men and women were susceptible, but fewer women had full protection. There was no age-sex interaction on immunity (p = 0.454). Our results suggest that booster immunisation of adults is necessary to increase herd immunity of the adult population.

The in-vitro activities of ten antibiotics against 83 toxigenic strains of Corynebacterium diphtheriae recently isolated in northwestern Russia and surrounding areas were determined by an agar dilution method. All of the strains were susceptible to erythromycin, penicillin, ampicillin, cefuroxime, chloramphenicol, ciprofloxacin, gentamicin and tetracycline. Trimethoprim and rifampicin were each active against 81 isolates, the two strains resistant to the latter agent having been isolated from two members of the same family.

Hepatitis C virus (HCV) is transmitted by organs of HCV antibody-positive donors to transplant recipients. This study investigated the serological and virological responses of 14 initially HCV antibody-negative transplant patients who received organs from four HCV antibody-positive donors (A-D) (before donor screening for HCV infection was introduced in 1991). Second generation HCV enzyme immunoassay (Abbott HCV EIA) was used to detect anti-HCV antibody. Recombinant immunoblot (RIBA-2; Chiron Corporation) and Wellcozyme Western blot (Wwb) assays were compared as confirmatory assays of positive EIA results. Reverse transcription (RT) followed by "nested" polymerase chain reaction (PCR) was performed to detect viral RNA. HCV RNA was only found in the sera of donors B and C, however, transplantation of organs from all donors resulted in infection of all recipients. HCV RNA was found in recipient sera within 30 days after transplant and remained detectable throughout the period of sampling. An anti-HCV antibody response was found in only 6 (of the 14) recipients and only after 300 days. Much longer periods passed before detection of HCV antibody in six recipients. For detection of HCV infection in transplant recipients it is essential that testing for HCV RNA by RT-PCR is carried out.

68 strains of methicillin- and gentamicin-resistant Staphylococcus aureus (MRSA) have been characterized by four different methods. First, by their production of lecithinase, lipase, pigment and sheep haemolysin. Second, by API Staph code. Third, by their sensitivity to 9 antibiotics. Fourth, by phage typing using the International Set and Supplementary phages. The third method was the most discriminatory. The combination of the first three techniques provides a highly effective, cheap and simple system to type MRSA. 80 separate MRSA strains from 26 countries were found to belong to a wide variety of phage types. Most were of group III. The most commonly found types were 85 (6 strains), 84 (4 strains) and 47 (3 strains).

The activities of six fluoroquinolones were determined for 117 separate strains of Shigella sonnei. The order of increasing activity (MICs for 90% of strains tested) was enoxacin (0.25 micrograms/ml), temafloxacin (0.032 micrograms/ml), sparfloxacin (0.016 micrograms/ml), CI-960 (0.008 micrograms/ml), ciprofloxacin (0.008 micrograms/ml), and PD-131628-2 (0.008 micrograms/ml). These data, along with results of killing and mutational rate studies, showed that all six fluoroquinolones were highly inhibitory against S. sonnei and five fluoroquinolones were rapidly and persistently bactericidal.

The in-vitro activities of the topical agents azelaic acid, nitrofurazone, silver sulphadiazine and mupirocin have been determined against 80 strains of MRSA collected from worldwide sources. MICs were determined by agar dilution (with an inoculum of approximately 5.0 x 10(5) cfu) in Iso-Sensitest agar, and MBCs were measured by replica-plating from MIC plates using velvet pads. The agents tested were uniformly active against MRSA, mupirocin being the most active (MIC50 0.15 mg/L) followed by nitrofurazone (MIC50 19 mg/L), silver sulphadiazine (MIC50 85 mg/L) and azelaic acid (MIC50 850 mg/L). Concentrations of azelaic acid, nitrofurazone and silver sulphadiazine close to the MIC were bactericidal, but mupirocin was only bactericidal at concentrations substantially greater than the MIC. In time-kill experiments, azelaic acid and nitrofurazone were gradually bactericidal, silver sulphadiazine was rapidly bactericidal and mupirocin was not bactericidal. Silver sulphadiazine killed sulphonamide-sensitive and sulphonamide-resistant strains equally rapidly. No resistant mutants were found to azelaic acid, nitrofurazone or silver sulphadiazine in an inoculum of 10(9) cfu, but two strains yielded (frequency: 1.0 x 10(-9)) mutants resistant to mupirocin. Our in-vitro results suggest azelaic acid, nitrofurazone and silver sulphadiazine could be of use for clearing staphylococcal carriage.

Rokitamycin was more active than erythromycin against erythromycin-sensitive strains of Staphylococcus aureus and enterococci, but somewhat less active against coagulase-negative staphylococci. Strains with inducible resistance to erythromycin were uniformly resistant to erythromycin, while rokitamycin was active against such strains. Strains with constitutive resistance to erythromycin were also uniformly resistant to erythromycin, and most were also resistant to rokitamycin. However, 5 of 21 coagulase-negative staphylococci and 2 of 20 enterococci remained sensitive to rokitamycin. This is a novel finding, perhaps suggesting a new mechanism of macrolide resistance.

Cook-chill food prepared by the Catering Department of the Royal Free Hospital was examined over an 8-month period commencing October 1989. Total aerobic viable counts (TAVCs) were performed on 200 food specimens which were also examined for Listeria using selective enrichment culture. Ten of 200 food samples tested had TAVCs greater than 10(5) colony forming units (cfu) g-1, and nine of these were from non-vegetarian (meat) dishes. Although most of the food prepared by cook-chill had microbial loads within the limits recommended by current Department of Health guidelines (i.e. less than 10(5) cfu g-1), our findings for the non-vegetarian dishes suggest that extra caution is required when preparing such food. Listeria were not isolated from any food sample. In a survey of listeria faecal carriage, Listeria monocytogenes was isolated from only two of 100 faeces specimens obtained from patients.

The in vitro activities of nine quinolones (seven fluoroquinolones, nalidixic acid, and acrosoxacin) against methicillin-resistant Staphylococcus aureus (MRSA) were compared with those of the glycopeptides teicoplanin and vancomycin. MICs against 160 strains of ciprofloxacin-susceptible (MIC, less than 2.0 micrograms/ml) MRSA and 40 strains of ciprofloxacin-resistant (MIC, greater than or equal to 2.0 micrograms/ml) MRSA were determined. The following MICs for 50% of the strains tested (in micrograms per milliliter) were obtained for ciprofloxacin-susceptible and -resistant strains, respectively: tosufloxacin, 0.06 and 2.0; ofloxacin, 0.25 and 16; ciprofloxacin, 0.5 and 16; pefloxacin, 0.5 and 32; acrosoxacin, 1.0 and greater than 256; enoxacin, 1.0 and 64; fleroxacin, 1.0 and 32; norfloxacin, 2.0 and 64; nalidixic acid, 64 and 512; teicoplanin, 1.0 and 1.0; vancomycin, 2.0 and 2.0. In mutation rate studies using a range of antibiotic concentrations to reflect those achievable in vivo, resistant mutants grew only on plates containing nalidixic acid (rate of mutation to resistance, 10(-7) to 10(-8) and on plates containing low concentrations of ciprofloxacin, enoxacin, and norfloxacin (rate of mutation to resistance, 10(-8) to 10(-9). In time-kill studies, 99.9% killing was found within 8 h for all of the quinolones tested (norfloxacin and nalidixic acid were not tested). Teicoplanin and vancomycin were less rapidly bactericidal. For the clinical isolates of ciprofloxacin-resistant MRSA, different levels and patterns of quinolone resistance were found. Generally, cross-resistance among the fluoroquinolones was complete; however, incomplete cross-resistance did occur with the nonfluorinated quinolone acrosoxacin.

The authors have investigated the activity of ramoplanin against 162 isolates of MRSA from some twenty-six countries around the world. MICs were determined by the plate dilution method in isosensitest agar with an inoculum of 10(6) cfu. MBCs were measured by replication, using velvet pads, from MIC plates after 24 h incubation at 37 degrees C. Time-kill curves were determined from viable counts of cultures in Isosensitest broth (inoculum ca. 5.0 x 10(6) cfu/ml) taken at intervals during shaking culture at 37 degrees C for up to 24 h. Ciprofloxacin, mupirocin, rifampicin, teicoplanin and vancomycin were used as comparison compounds. The following MIC90 (MBC90) values (mg/l) were obtained against a selection of 60 strains: ciprofloxacin 0.8 (1.8), mupirocin 0.27 (19.0), ramoplanin 0.5 (1.0), rifampicin 0.007 (0.01), teicoplanin 1.2 (greater than 32) and vancomycin 2.2 (greater than 32.0). In time-kill experiments, ramoplanin at 20 mg/l and ciprofloxacin at 3.0 mg/l produced 99.9% killing in less than 4h. Mupirocin at 4.0 mg/l was only slowly bactericidal. No resistance was found to mupirocin, ramoplanin, teicoplanin or vancomycin in the 162 isolates tested, whereas ca. 20% resistance was found to ciprofloxacin and rifampicin. The absence of resistance, the high intrinsic activity and the rapid bactericidal effect of ramoplanin against this diverse group of MRSA are very encouraging, and suggest that clinical trials are indicated.

The in-vitro activities of five anti-staphylococcal agents, teicoplanin, ramoplanin, paldimycin, DuP 721 and DuP 105 have been compared to vancomycin. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) have been determined for a collection of methicillin and gentamicin resistant Staphylococcus aureus (MGRSA), comprising 75 strains obtained from 22 centres. In terms of geometric mean MICs (inoculum size 10(5) cfu) paldimycin was the most active agent (0.4 mg/l) followed by ramoplanin (0.75 mg/l), teicoplanin (1.0 mg/l), DuP 721 and vancomycin (2.0 mg/l) and DuP 105 (6.8 mg/l). Ramoplanin was bactericidal within six hours to all strains at a concentration of 1.0 mg/l. The MBC90s for vancomycin and teicoplanin were greater than 32 mg/l after 22 h exposure to antibiotic and 2.5 and 4.0 mg/l respectively after 26 h exposure. Paldimycin was bactericidal against only some strains, while DuP 721 and DuP 105 were not bactericidal. Ramoplanin is the most interesting of the new antibiotics, on account of its rapid and consistent bactericidal activity.

Antibiotic resistance patterns were determined for 106 strains of methicillin-resistant Staphylococcus aureus (MRSA) from 21 countries. Resistance to gentamicin, tobramycin, netilmicin, amikacin, streptomycin, or erthromycin was recorded in more than 90% of strains. Resistance to the other compounds tested was as follows: tetracycline 86%, minocycline 76%, trimethoprim 69%, clindamycin 66%, neomycin 59%, chloramphenicol 39%, rifampicin 26%, fosfomycin 22%, ciprofloxacin 17%, fusidic acid 12%, bacitracin 2%, and novobiocin 1%. All the stains were sensitive to mupirocin, pristinamycin, ramoplanin, teicoplanin, and vancomycin. There were geographical patterns of resistance: MRSA from the UK and Australia were predominantly resistant to trimethoprim, whereas many strains from centres in Europe and the USA were sensitive. MRSA that were resistant to ciprofloxacin were of French and German origin. 15 strains, 12 of which came from France, Turkey, or Brazil, were resistant either to thirteen or to fourteen agents.