Establishment of the European meningococcal strain collection genome library (EMSC-GL) for the 2011 to 2012 epidemiological year.

Invasive meningococcal disease surveillance in Europe combines isolate characterisation and epidemiological data to support public health intervention. A representative European Meningococcal Strain Collection (EMSC) of IMD isolates was obtained, and whole genome sequenced to characterise 799 EMSC isolates from the epidemiological year July 2011-June 2012. To establish a genome library (GL), the isolate information was deposited in the pubMLST.org/neisseria database. Genomes were curated and annotated at 2,429 meningococcal loci, including those defining clonal complex, capsule, antigens, and antimicrobial resistance. Most genomes contained genes encoding B (n = 525; 65.7%) or C (n = 163; 20.4%) capsules; isolates were genetically highly diverse, with >20 genomic lineages, five of which comprising 60.7% (n = 485) of isolates. There were >350 antigenic fine-types: 307 were present once, the most frequent (P1.7-2,4:F5-1) comprised 8% (n = 64) of isolates. Each genome was characterised for Bexsero Antigen Sequence Typing (BAST): 25.5% (n = 204) of isolates contained alleles encoding the fHbp and/or the PorA VR1 vaccine component, but most genomes (n = 513; 64.2%) did not contain the NadA component. EMSC-GL will support an integrated surveillance of disease-associated genotypes in Europe, enabling the monitoring of hyperinvasive lineages, outbreak identification, and supporting vaccine programme implementation.

European public health policies for managing contacts of invasive meningococcal disease cases better harmonised in 2013 than in 2007.

In 2007, a European survey identified variation in country policies on public health management of invasive meningococcal disease (IMD). In 2009-10, the European Centre for Disease Prevention and Control (ECDC) published evidence-based guidance on IMD. We therefore surveyed again European countries to describe policies for managing IMD cases and contacts in 2013. We asked national IMD public health experts from 32 European countries to complete a questionnaire focusing on post-exposure prophylaxis (PEP) for IMD contacts and meningococcal vaccination. Proportions in 2007 and 2013 were compared using the chi-squared test. All 32 countries responded, with responses from two regions for Belgium and Italy; half stated having used ECDC guidance to update national recommendations. PEP was recommended to close contacts in 33 of 34 countries/regions, mainly ciprofloxacin for adults (29/32 countries) and rifampicin for children (29/32 countries). ECDC guidance for managing IMD contacts in airplanes was strictly followed by five countries/regions. Twenty-three countries/regions participated in both surveys. Compared with 2007, in 2013, more countries/regions recommended i) ceftriaxone for children (15/23 vs 6/20; p = 0.03), ii) PEP for all children in the same preschool group (8/23 vs 17/23; p = 0.02). More countries/regions recommended evidence-based measures for IMD public health management in 2013 than 2007. However, some discrepancies remain and they call for further harmonisation.

Molecular typing methods for outbreak detection and surveillance of invasive disease caused by Neisseria meningitidis, Haemophilus influenzae and Streptococcus pneumoniae, a review.

Invasive disease caused by the encapsulated bacteria Neisseria meningitidis, Haemophilus influenzae and Streptococcus pneumoniae remains an important cause of morbidity and mortality worldwide, despite the introduction of successful conjugate polysaccharide vaccines that target disease-associated strains. In addition, resistance, or more accurately reduced susceptibility, to therapeutic antibiotics is spreading in populations of these organisms. There is therefore a continuing requirement for the surveillance of vaccine and non-vaccine antigens and antibiotic susceptibilities among isolates from invasive disease, which is only partially met by conventional methods. This need can be met with molecular and especially nucleotide sequence-based typing methods, which are fully developed in the case of N. meningitidis and which could be more widely deployed in clinical laboratories for S. pneumoniae and H. influenzae.

Multicenter study for defining the breakpoint for rifampin resistance in Neisseria meningitidis by rpoB sequencing.

Identification of clinical isolates of Neisseria meningitidis that are resistant to rifampin is important to avoid prophylaxis failure in contacts of patients, but it is hindered by the absence of a breakpoint for resistance, despite many efforts toward standardization. We examined a large number (n = 392) of clinical meningococcal isolates, spanning 25 years (1984 to 2009), that were collected in 11 European countries, Argentina, and the Central African Republic. The collection comprises all clinical isolates with MICs of > or = 0.25 mg/liter (n = 161) received by the national reference laboratories for meningococci in the participating countries. Representative isolates displaying rifampin MICs of < 0.25 mg/liter were also examined (n = 231). Typing of isolates was performed, and a 660-bp DNA fragment of the rpoB gene was sequenced. Sequences differing by at least one nucleotide were defined as unique rpoB alleles. The geometric mean of the MICs was calculated for isolates displaying the same allele. The clinical isolates displaying rifampin MICs of > 1 mg/liter possessed rpoB alleles with nonsynonymous mutations at four critical amino acid residues, D542, H552, S548, and S557, that were absent in the alleles found in all isolates with MICs of < or = 1 mg/liter. Rifampin-susceptible isolates could be defined as those with MICs of < or = 1 mg/liter. The rpoB allele sequence and isolate data have been incorporated into the PubMLST Neisseria database (http://pubmlst.org/neisseria/). The rifampin-resistant isolates belonged to diverse genetic lineages and were associated with lower levels of bacteremia and inflammatory cytokines in mice. This biological cost may explain the lack of clonal expansion of these isolates.

A surveillance network for meningococcal disease in Europe.

Between 1999 and 2004, the European Union Invasive Bacterial Infections Surveillance Network (EU-IBIS) received c. 50,000 reports of meningococcal disease from 27 participating countries. Analysis has demonstrated a major decline in the incidence of invasive disease in those countries that have introduced routine vaccination against serogroup C infection. The establishment of rapid reporting of W135 and B2a/B2b strains has been able to provide early reassurance that these strains are not emerging as major public health problems in Europe. Between September 2001 and February 2005, the EU-MenNet project offered further opportunities for enhancing this data resource. Collaborative projects included: improving the EU-IBIS website; reviewing case ascertainment in Europe; reviewing cost-effectiveness studies for meningococcal serogroup C conjugate (MCC) vaccination; international comparisons of MCC vaccine efficacy; and mathematical modelling studies. In addition, linking of data from the European Meningococcal Multi-locus Sequence Type Centre to epidemiological data was performed. Particular clonal complexes were found to be preferentially associated with certain serogroups. Case fatality was also found to vary with clonal complex, suggesting that genotype can be a marker for hypervirulence. The importance of close collaboration between networks of epidemiologists, microbiologists, and the wider scientific and public health community is demonstrated.

Invasive meningococcal disease in Austria 2002: assessment of completeness of notification by comparison of two independent data sources.

AIM OF THE STUDY: The notified incidence of meningococcal disease in European countries varies from <1 case per 100,000 inhabitants to approximately 7 cases per 100,000. Assessing the true burden of disease is important for setting priorities in health services and for estimating the benefit of interventions such as vaccination. Completeness and timeliness of reporting is also essential for the early recognition of outbreaks. The objective of this study was to assess the completeness of surveillance data on invasive meningococcal disease in Austria at the National Reference Center for Meningococci for the year 2002. METHODS: The data stored at the reference center was compared with an independent database containing the main diagnosis documented in the obligatory hospital discharge dataset of all Austrian hospitals (coded in ICD-10 since 2001). All mismatches were reviewed in order to exclude possible errors and identify true cases of meningococcal disease that had not been reported to the reference center. The number of cases not recorded by either data source was estimated using the capture-recapture method. RESULTS: The first comparison of the two data sources identified 50 cases not registered at the national reference center. Screening of the ICD codes from these 50 patients through the hospitals reduced the number of under-reported cases to 10, of which 6 showed symptoms compatible with meningococcal disease, although microbiological confirmation was missing. Re-evaluation of the case histories of these 6 patients by a clinical expert for meningococcal disease identified them as probable cases. The main reason for correction of the diagnosis in 27 cases was an obvious coding error: these patients had been treated in hospitals for illnesses not related to meningococcal disease. In 72 cases, the two databases were in agreement. Eleven cases of meningococcal disease were notified solely to the national reference center. Addition of the newly recognized cases of invasive meningococcal disease increased the total number of cases from 83 (incidence, 1.03/100,000) to 93 (incidence, 1.16/100,000). Estimation of the "true" number of cases of meningococcal disease, using the capture-recapture method, gave a final total of 95 cases (95% CI, 93-98) and an incidence of 1.18/100,000. The completeness (sensitivity) of the original notification at the national reference center was therefore 87.4% (83 of 95 cases). CONCLUSION: All probable cases of meningococcal disease, even those (still) lacking microbiological confirmation, should be reported to the public health authorities as soon as possible, in order to ensure the necessary prompt prophylactic action (e.g., chemoprophylaxis).

Strong Public Health Recommendations from Weak Evidence? Lessons Learned in Developing Guidance on the Public Health Management of Meningococcal Disease.

The evidence underpinning public health policy is often of low quality, leading to inconsistencies in recommended interventions. One example is the divergence in national policies across Europe for managing contacts of invasive meningococcal disease. Aiming to develop consistent guidance at the European level, a group of experts reviewed the literature and formulated recommendations. The group defined eight priority research questions, searched the literature, and formulated recommendations using GRADE methodology. Five of the research questions are discussed in this paper. After taking into account quality of evidence, benefit, harm, value, preference, burden on patient of the intervention, and resource implications, we made four strong recommendations and five weak recommendations for intervention. Strong recommendations related not only to one question with very low quality of evidence as well as to two questions with moderate to high quality of evidence. The weak recommendations related to two questions with low and very low quality of evidence but also to one question with moderate quality of evidence. GRADE methodology ensures a transparent process and explicit recognition of additional factors that should be considered when making recommendations for policy. This approach can be usefully applied to many areas of public health policy where evidence quality is often low.

Implications of differential age distribution of disease-associated meningococcal lineages for vaccine development.

New vaccines targeting meningococci expressing serogroup B polysaccharide have been developed, with some being licensed in Europe. Coverage depends on the distribution of disease-associated genotypes, which may vary by age. It is well established that a small number of hyperinvasive lineages account for most disease, and these lineages are associated with particular antigens, including vaccine candidates. A collection of 4,048 representative meningococcal disease isolates from 18 European countries, collected over a 3-year period, were characterized by multilocus sequence typing (MLST). Age data were available for 3,147 isolates. The proportions of hyperinvasive lineages, identified as particular clonal complexes (ccs) by MLST, differed among age groups. Subjects <1 year of age experienced lower risk of sequence type 11 (ST-11) cc, ST-32 cc, and ST-269 cc disease and higher risk of disease due to unassigned STs, 1- to 4-year-olds experienced lower risk of ST-11 cc and ST-32 cc disease, 5- to 14-year-olds were less likely to experience ST-11 cc and ST-269 cc disease, and ≥25-year-olds were more likely to experience disease due to less common ccs and unassigned STs. Younger and older subjects were vulnerable to a more diverse set of genotypes, indicating the more clonal nature of genotypes affecting adolescents and young adults. Knowledge of temporal and spatial diversity and the dynamics of meningococcal populations is essential for disease control by vaccines, as coverage is lineage specific. The nonrandom age distribution of hyperinvasive lineages has consequences for the design and implementation of vaccines, as different variants, or perhaps targets, may be required for different age groups.

Epidemiology of invasive meningococcal disease in Austria 2010.

INTRODUCTION: Neisseria meningitidis is a leading cause of meningitis and sepsis worldwide. Thus, monitoring the epidemiology of invasive meningococcal disease (IMD) is an important public health measure. The National Reference Centre for Meningococci at the Austrian Agency for Health and Food Safety is operating the laboratory-based surveillance database. METHODS: IMD isolates from all over Austria are collected at the National Reference Centre for Meningococci, where the microorganism is characterised by serological and molecular methods, as well as by antimicrobial susceptibility testing. These laboratory-based surveillance data from 1995 to 2010 were analysed by time, place and person. Long-term and seasonal trends were analysed by log-linear regression, yielding an annual average percentage change (AAPC), and by cyclic regression models, applying either a simple linear or a negative binomial regression, including one sine and one cosine term. RESULTS: Over the past 16 years, IMD incidence ranged between 0.73 and 1.41/100,000 persons, with a decreasing trend in Serogroup B IMD incidence (AAPC: -2.1%; p = 0.0396) and an increasing trend in Serogroup C IMD incidence (AAPC: +4.3%; p = 0.043). In 2010, 80 cases of IMD were recorded, corresponding to an incidence of 0.95/100,000 population. Ten deaths were registered, which results in a case-fatality of 12.5% and a mortality incidence of 0.12/100,000 population. The predominant serogroup in Austria was serogroup B, followed by serogroups C, Y and W-135. DISCUSSION: Although the incidence of IMD in 2010 is below the average incidence rate over the last 15 years, serogroup C IMD in Austria is increasing. Within the previous 10 years two provinces initiated vaccination campaigns for a monovalent meningococcal conjugate vaccine, following the increasing trend of Sg C IMD incidence. An ongoing study on the impact of these vaccination campaigns on the IMD burden will yield further scientific evidence for supporting the introduction of this vaccine into the publicly funded childhood immunisation program.

Surveillance of invasive pneumococcal disease in 30 EU countries: Towards a European system?

In this era of new pneumococcal conjugate vaccines (PCV), we described and compared surveillance of invasive pneumococcal disease (IPD) and PCV policies in 30 European countries to provide guidance for Europe-wide surveillance. We confirmed the heterogeneity of surveillance systems and case definitions across countries but identified elements common to all countries, such as the availability of serotyping and the surveillance of pneumococcal meningitis. PCV impact was monitored in 11/15 countries using it. We propose steps for the monitoring of incidence rates and serotype distribution at EU level, to assess the need to introduce PCV and monitor its impact once introduced.

CD14 C-159T and toll-like receptor 4 Asp299Gly polymorphisms in surviving meningococcal disease patients.

BACKGROUND: Carriage of Neisseria meningitidis occurs approximately in 10% of the population, onset of invasive meningococcal disease (IMD) cannot be predicted and differs between ages. It remains unclear, which host factors determine invasion of the bloodstream by the bacteria. Innate immunity has a very important role in the first recognition of invading pathogens. The functional single nucleotide polymorphisms (SNPs) CD14 C-159T and toll-like receptor 4 (TLR4) Asp299Gly have been associated with the risk of gram-negative infections. However, their role in development of IMD still remains unclear. Our aim was to investigate the influence of CD14 C-159T and TLR4 Asp299Gly polymorphisms on the risk of IMD. METHODOLOGY/PRINCIPAL FINDINGS: It was a retrospective case control study. Surviving Austrian meningococcal disease patients were enrolled by sending buccal swabs for DNA analysis. 185 cases with a proven meningococcal infection and 770 healthy controls were enrolled. In surviving meningococcal disease patients DNA analysis of CD14 C-159T and TLR 4 Asp299Gly polymorphisms was performed, as they are part of the innate immune response to bacterial determinants. CD14 C-159T and TLR4 Asp299Gly SNPs were not significantly associated with the presence of IMD when compared to healthy controls. The odds ratio for CD14 C-159T SNP was 1.14 (95% confidence interval (CI) 0.91-1.43; p = 0.266). In TLR4 Asp 299 Gly SNP the odds ratio was 0.78 (CI 0.47-1.43; p = 0.359). CONCLUSION/SIGNIFICANCE: We could not observe a significant influence of CD14 C-159T and TLR4 Asp299Gly polymorphisms on the risk of developing IMD in surviving meningococcal disease patients. To our knowledge, this is the first study investigating the influence of the CD14 C-159T SNP on the susceptibility to IMD.

Target gene sequencing to characterize the penicillin G susceptibility of Neisseria meningitidis.

Clinical isolates of Neisseria meningitidis with reduced susceptibility to penicillin G (intermediate isolates, Pen(I)) harbor alterations in the penA gene encoding the penicillin binding protein 2 (PBP2). A 402-bp DNA fragment in the 3' half of penA was sequenced from a collection of 1,670 meningococcal clinical isolates from 22 countries that spanned 60 years. Phenotyping, genotyping, and the determination of MICs of penicillin G were also performed. A total of 139 different penA alleles were detected with 38 alleles that were highly related, clustered together in maximum-likelihood analysis and corresponded to the penicillin G-susceptible isolates. The remaining 101 penA alleles were highly diverse, corresponded to different genotypes or phenotypes, and accounted for 38% of isolates, but no clonal expansion was detected. Analysis of the altered alleles that were represented by at least five isolates showed high correlation with the Pen(I) phenotype. The deduced amino acid sequence of the corresponding PBP2 comprised five amino acid residues that were always altered. This correlation was not complete for rare alleles, suggesting that other mechanisms may also be involved in conferring reduced susceptibility to penicillin. Evidence of mosaic structures through events of interspecies recombination was also detected in altered alleles. A new website was created based on the data from this work (http://neisseria.org/nm/typing/penA). These data argue for the use of penA sequencing to identify isolates with reduced susceptibility to penicillin G and as a tool to improve typing of meningococcal isolates, as well as to analyze DNA exchange among Neisseria species.

Effectiveness of antibiotics given before admission in reducing mortality from meningococcal disease: systematic review.

OBJECTIVE: To review the evidence for effectiveness of treatment with antibiotics before admission in reducing case fatality from meningococcal disease. DESIGN: Systematic review. DATA SOURCES: Cochrane register of trials and systematic reviews, database of abstracts of reviews of effectiveness, health technology assessment, and national research register in England and Wales, Medline, Embase, and CAB Health. INCLUDED STUDIES: Studies describing vital outcome of at least 10 cases of meningococcal disease classified by whether or not antibiotics were given before admission to hospital. RESULTS: 14 observational studies met the review criteria. Oral antibiotic treatment given before admission was associated with reduced mortality among cases (combined risk ratio 0.17, 95% confidence interval 0.07 to 0.44). In seven studies in which all included patients were seen in primary care, the association between parenteral antibiotics before admission and outcome was inconsistent (chi2 for heterogeneity 11.02, P = 0.09). After adjustment for the proportion given parenteral antibiotics before admission, there was no residual heterogeneity. A higher proportion of patients given parenteral antibiotics before admission was associated with reduced mortality after such treatment and vice versa (P = 0.04). CONCLUSION: Confounding by severity is the most likely explanation both for the beneficial effect of oral antibiotics and the harmful effect observed in some studies of parenteral antibiotics. We cannot conclude whether or not antibiotics given before admission have an effect on case fatality. The data are consistent with benefit when a substantial proportion of cases are treated.

Bacterial protein microarrays for identification of new potential diagnostic markers for Neisseria meningitidis infections.

Neisseria meningitidis is the most common cause of meningitis and causes epidemic outbreaks. One trait of N. meningitidis, which is associated with most of the currently recognized virulence determinants, is the presence of phase-variable genes that are suspected to enhance its ability to cause an invasive disease. To detect the immune responses to phase-variable expressed proteins, we applied protein microarray technology for the screening of meningitis patient sera. We amplified all 102 known phase-variable genes from N. meningitidis serogroup B strain MC58 by polymerase chain reaction and subcloned them for expression in Escherichia coli. With this approach, we were able to express and purify 67 recombinant proteins representing 66% of the annotated genes. These were spotted robotically onto coated glass slides to generate protein microarrays, which were screened using 20 sera of patients suffering from meningitis, as well as healthy controls. From these screening experiments, 47 proteins emerged as immunogenic, exhibiting a variable degree of seroreactivity with some of the patient sera. Nine proteins elicited an immune response in more than three patients, with one of them, the phase-variable opacity protein OpaV (NMB0442), showing responses in 11 patient sera. This is the first time that protein microarray technology has been applied for the investigation of genetic phase variation in pathogens. The identification of disease-specific proteins is a significant target in biomedical research, as such proteins may have medical, diagnostic, and commercial potential as disease markers.

Vaccine strategies of meningococcal disease: results of a 10-year population-based study.

UNLABELLED: Invasive meningococcal disease (IMD) is an important cause of morbidity and mortality in children and adults. This study was conducted to determine a possible increase in IMD in recent years with special interest focused on serogroup C disease. From January 1st 1993 to December 31st 2002, IMD was studied in one million residents of Austria. We used active, population-based surveillance data from the Office of Public Health. A total of 126 patients with positive blood and/or cerebrospinal fluid culture or positive swabs for Neisseria meningitidis were studied. The median age of all patients was 9.5 years (range 1 month to 63 years). The average incidence of all IMD subgroups was 1.05 cases per 100,000 person years and was highest in children 0-4 years old (7.08 cases per 100,000 person years) followed by young adults aged 15 to 19 years (4.35 cases per 100,000 person years). Serogroup C IMD occurred in 1.30 cases/100,000 person years in patients aged 0 to 4 years and in 1.92 cases/100,000 person years in patients aged 15 to 19 years. Overall mortality was 11.1%. There was a significant increase (P =0.001) in IMD due to serogroup B disease within the last 10 years. In contrast, serogroup C disease did not increase during the last decade. CONCLUSION: Currently, we do not recommend mass vaccination against serogroup C disease in Austria, but young adults aged 15 to 19 years display a high incidence of meningococcal C disease. In this age group, vaccination against serogroup C disease should be considered.

Interlaboratory comparison of PCR-based identification and genogrouping of Neisseria meningitidis.

Twenty clinical samples (18 cerebrospinal fluid samples and 2 articular fluid samples) were sent to 11 meningococcus reference centers located in 11 different countries. Ten of these laboratories are participating in the EU-MenNet program (a European Union-funded program) and are members of the European Monitoring Group on Meningococci. The remaining laboratory was located in Burkina Faso. Neisseria meningitidis was sought by detecting several meningococcus-specific genes (crgA, ctrA, 16S rRNA, and porA). The PCR-based nonculture method for the detection of N. meningitidis gave similar results between participants with a mean sensitivity and specificity of 89.7 and 92.7%, respectively. Most of the laboratories also performed genogrouping assays (siaD and mynB/sacC). The performance of genogrouping was more variable between laboratories, with a mean sensitivity of 72.7%. Genogroup B gave the best correlation between participants, as all laboratories routinely perform this PCR. The results for genogroups A and W135 were less similar between the eight participating laboratories that performed these PCRs.

Effectiveness of antibiotics in preventing meningococcal disease after a case: systematic review.

OBJECTIVE: To summarise the evidence for the role of antibiotics in preventing further cases of meningococcal disease through chemoprophylaxis given to the index patient, household contacts, and children in day care settings after a single case. DESIGN: Systematic review. METHODS: Studies were identified by searching Embase (1983-2003), Medline (1965-2003), and CAB Health (1973-2003) and by contacting the World Health Organization and the European meningococcal disease surveillance network and examining references of identified papers. The review included all studies with at least 10 cases in which outcomes were compared between treated and untreated groups. MAIN OUTCOME MEASURE: Subsequent cases of meningococcal disease 1-30 days after onset of disease in the index patient. RESULTS: Four observational studies and one small trial met the inclusion criteria. Meta-analysis of studies on chemoprophylaxis given to household contacts showed a significant reduction in risk (risk ratio 0.11, 95% confidence interval 0.02 to 0.58). The number needed to treat to prevent a case was estimated as 218 (121 to 1135). Primary outcome data were not available in studies of chemoprophylaxis given to the index patient: when prophylaxis had not been given, rate of carriage after discharge from hospital was estimated as 3% (0 to 6), probably an underestimate of the true rate. No studies of chemoprophylaxis in day care settings were identified that met the inclusion criteria. CONCLUSION: There have been no high quality experimental trials looking at control policies for meningococcal disease. The best available evidence is from retrospective studies. The risk of meningococcal disease in household contacts of a patient can be reduced by an estimated 89% if they take antibiotics known to eradicate meningococcal carriage. Chemoprophylaxis should be recommended for the index patient and all household contacts.

Interlaboratory comparison of agar dilution and Etest methods for determining the MICs of antibiotics used in management of Neisseria meningitidis infections.

Previous studies have shown that there is considerable variation in the methods and media used to determine the susceptibility of Neisseria meningitidis to antimicrobial agents in different countries. In this study, national and regional reference laboratories used a standardized methodology to determine the MICs of antibiotics used in the management of meningococcal infection. Fourteen laboratories participated in the study, determining the susceptibility to penicillin G, rifampin, cefotaxime, ceftriaxone, ciprofloxacin, and ofloxacin of a collection of 17 meningococci, of which 11 strains were previously defined as having intermediate resistance to penicillin (Pen(I)) by sequencing and restriction fragment length polymorphism analysis of the penA gene. The MIC was determined by agar dilution and Etest with Mueller-Hinton agar (MH), MH supplemented with sheep blood (MH+B), and MH supplemented with heated (chocolated) blood. Several laboratories encountered problems obtaining confluent growth with unsupplemented MH. MH+B was considered to give the most congruent and reproducible results among the study laboratories. The modal MIC for MH+B for each antibiotic and method was calculated to define the MIC consensus, allowing assessment of each individual laboratory's data in relation to the others. The agreement in each antibiotic/method/medium combination was defined as the percentage of laboratories with a result within one dilution of the modal result. For the whole study, an agreement of 90.6% was observed between agar dilution and Etest methods. The agreement in each laboratory/antibiotic/method combination ranged from 98.2% to 69.7%, with six laboratories demonstrating agreement higher than 90% and 11 more than 80%. The ability of the laboratories to detect the Pen(I) isolates ranged from 18.2% to 100%. The apparent difficulty in interpreting susceptibility to rifampin, particularly with the Etest method, is very interesting.