Pneumococcal carriage and disease in adults in England 2011-2019: the importance of adults as a reservoir for pneumococcus in communities.

BACKGROUND: Pneumococcal carriage in children has been extensively studied, but carriage in healthy adults and its relationship to invasive pneumococcal disease (IPD) is less understood. METHODS: Nasal wash samples from adults without close contact with young children (Liverpool, UK), 2011-2019, were cultured, and culture-negative samples tested by PCR. Pneumococcal carriage in adults 18-44 years was compared with carriage among PCV-vaccinated children 13-48 months (nasopharyngeal swabs, Thames Valley, UK) and IPD data for England for the same ages for 2014-2019. Age-group specific serotype invasiveness was calculated and used with national IPD data to estimate carriage serotype distributions for adults aged 65+ years. RESULTS: In total 98 isolates (97 carriers) were identified from 1,631 adults aged 18+ years (age and sex standardized carriage prevalence 6.4%), with only three identified solely by PCR. Despite different carriage and IPD serotype distributions between adults and children, serotype invasiveness was highly correlated (R=0.9). Serotypes 3, 37 and 8 represented a higher proportion of adult carriage than expected from direct low-level transmission from children to adults. The predicted carriage serotype distributions for 65+ years aligned more closely with the carriage serotype distribution for young adults than young children. CONCLUSIONS: The nasal wash technique is highly sensitive; additional benefit of PCR is limited. Comparison of carriage serotype distributions suggests some serotypes may be circulating preferentially within these specific young adults. Our data suggest that for some serotypes carried by adults 65+ years, other adults may be an important reservoir for transmission. Age groups such as older children should also be considered.

International links between Streptococcus pneumoniae vaccine serotype 4 sequence type (ST) 801 in Northern European shipyard outbreaks of invasive pneumococcal disease.

BACKGROUND: Pneumococcal disease outbreaks of vaccine preventable serotype 4 sequence type (ST)801 in shipyards have been reported in several countries. We aimed to use genomics to establish any international links between them. METHODS: Sequence data from ST801-related outbreak isolates from Norway (n = 17), Finland (n = 11) and Northern Ireland (n = 2) were combined with invasive pneumococcal disease surveillance from the respective countries, and ST801-related genomes from an international collection (n = 41 of > 40,000), totalling 106 genomes. Raw data were mapped and recombination excluded before phylogenetic dating. RESULTS: Outbreak isolates were relatively diverse, with up to 100 SNPs (single nucleotide polymorphisms) and a common ancestor estimated around the year 2000. However, 19 Norwegian and Finnish isolates were nearly indistinguishable (0-2 SNPs) with the common ancestor dated around 2017. CONCLUSION: The total diversity of ST801 within the outbreaks could not be explained by recent transmission alone, suggesting that harsh environmental and associated living conditions reported in the shipyards may facilitate invasion of colonising pneumococci. However, near identical strains in the Norwegian and Finnish outbreaks does suggest that transmission between international shipyards also contributed to those outbreaks. This indicates the need for improved preventative measures in this working population including pneumococcal vaccination.

A patient with respiratory toxigenic diphtheria in Greece after more than 30 years.

The introduction of treatment and systematic vaccination has significantly reduced diphtheria mortality; however, toxigenic strains continue to circulate worldwide. The emergence of an indigenous diphtheria case with fatal outcome in Greece, after 30 years, raised challenges for laboratory confirmation, clinical and public health management. Toxigenic Corynebacterium diphtheriae was isolated from an incompletely vaccinated 8-year-old boy with underlying conditions. The child passed away due to respiratory distress syndrome, before the administration of diphtheria antitoxin (DAT). All close contacts in family, school and hospital settings were investigated. Pharyngeal swabs were obtained to determine asymptomatic carriage. Chemoprophylaxis was given for 7 days to all close contacts and a booster dose to those incompletely vaccinated. Testing revealed a classmate, belonging to a subpopulation group (Roma), and incompletely vaccinated, as an asymptomatic carrier with an indistinguishable toxigenic strain (same novel multilocus sequence type, designated ST698). This case highlights the role of asymptomatic carriage, as the entry of toxigenic strains into susceptible populations can put individuals and their environment at risk. Maintenance of high-level epidemiological and microbiological surveillance, implementation of systematic vaccination in children and adults with primary and booster doses, availability of a DAT stockpile, and allowing timely administration are the cornerstone to prevent similar incidents in the future.

Toxigenic Corynebacterium ulcerans associated with upper respiratory infections in cats and dogs.

OBJECTIVES: To describe infection in companion animals with the zoonotic pathogen Corynebacterium ulcerans and to determine its prevalence in clinically-affected and healthy animals. MATERIALS AND METHODS: The clinical presentation and treatment of three cases of C. ulcerans infection is described. Two studies to determine C. ulcerans prevalence rates were undertaken: (a) a prospective study of nasal samples from healthy animals, 479 dogs and 72 cats; (b) a retrospective analysis of records of nasal samples collected over a 10-year period from 189 dogs and 64 cats affected by respiratory signs. RESULTS: Toxigenic C. ulcerans was isolated from four cats with nasal discharge while concurrent C. ulcerans and mecC methicillin-resistant S. aureus infection was detected in a dog suffering from chronic nasal discharge. Clinical features were not distinctive and all cases recovered following antimicrobial treatment. Multilocus sequence typing supported a common source for isolates from the shelter cats. Carriage rates of C. ulcerans in healthy animals were 0.42% (2/479) in dogs and 0.00% (0/72) in cats whereas in animals with signs of upper respiratory tract infection prevalence rates were 0.53% (1/189) in dogs and 6.25% (4/64) in cats. CLINICAL SIGNIFICANCE: Clinicians should be aware that dogs and cats can be infected with (or carriers of) toxigenic C. ulcerans Considering the potential zoonotic risk, assistance from medical and public health colleagues should be sought in confirmed cases.

Current epidemiology of tetanus in England, 2001-2014.

Public Health England conducts enhanced national surveillance of tetanus, a potentially life-threatening vaccine-preventable disease. A standardized questionnaire was used to ascertain clinical and demographic details of individuals reported with clinically suspected tetanus. The 96 cases identified between 2001 and 2014 were analysed. The average annual incidence was 0·13/million (95% confidence interval 0·10-0·16) of which 50·0% were male. Where reported, 70·3% of injuries occurred in the home/garden (45/64). Overall, 40·3% (31/77) cases were in people who inject drugs (PWID), including a cluster of 22 cases during 2003-2004. Where known (n = 68), only 8·8% were age-appropriately immunized. The overall case-fatality rate was 11·0% (9/82). All tetanus-associated deaths occurred in adults aged >45 years, none of whom were fully immunized. Due to the success of the childhood immunization programme, tetanus remains a rare disease in England with the majority of cases occurring in older unimmunized or partially immunized adults. Minor injuries in the home/garden were the most commonly reported likely sources of infection, although cases in PWID increased during this period. It is essential that high routine vaccine coverage is maintained and that susceptible individuals, particularly older adults, are protected through vaccination and are offered timely post-exposure management following a tetanus-prone wound.

Analysis of Bordetella pertussis clinical isolates circulating in European countries during the period 1998-2012.

Despite more than 50 years of vaccination, pertussis is still an endemic disease, with regular epidemic outbreaks. With the exception of Poland, European countries have replaced whole-cell vaccines (WCVs) by acellular vaccines (ACVs) in the 1990s. Worldwide, antigenic divergence in vaccine antigens has been found between vaccine strains and circulating strains. In this work, 466 Bordetella pertussis isolates collected in the period 1998-2012 from 13 European countries were characterised by multi-locus antigen sequence typing (MAST) of the pertussis toxin promoter (ptxP) and of the genes coding for proteins used in the ACVs: pertussis toxin (Ptx), pertactin (Prn), type 2 fimbriae (Fim2) and type 3 fimbriae (Fim3). Isolates were further characterised by fimbrial serotyping, multi-locus variable-number tandem repeat analysis (MLVA) and pulsed-field gel electrophoresis (PFGE). The results showed a very similar B. pertussis population for 12 countries using ACVs, while Poland, which uses a WCV, was quite distinct, suggesting that ACVs and WCVs select for different B. pertussis populations. This study forms a baseline for future studies on the effect of vaccination programmes on B. pertussis populations.

Investigations into the emergence of pertactin-deficient Bordetella pertussis isolates in six European countries, 1996 to 2012.

Pathogen adaptation has been proposed to contribute to the resurgence of pertussis. A striking recent example is the emergence of isolates deficient in the vaccine component pertactin (Prn). This study explores the emergence of such Prn-deficient isolates in six European countries. During 2007 to 2009, 0/83 isolates from the Netherlands, 0/18 from the United Kingdom, 0/17 Finland, 0/23 Denmark, 4/99 Sweden and 5/20 from Norway of the isolates collected were Prn-deficient. In the Netherlands and Sweden, respectively 4/146 and 1/8 were observed in a later period (2010­12). The Prn-deficient isolates were genetically diverse and different mutations were found to inactivate the prn gene. These are indications that Prn-deficiency is subject to positive selective pressure. We hypothesise that the switch from whole cell to acellular pertussis vaccines has affected the balance between 'costs and benefits' of Prn production by Bordetella pertussis to the extent that isolates that do not produce Prn are able to expand. The absence of Prn-deficient isolates in some countries may point to ways to prevent or delay the spread of Prn-deficient strains. In order to substantiate this hypothesis, trends in the European B. pertussis population should be monitored continuously.

Extension of the Legionella pneumophila sequence-based typing scheme to include strains carrying a variant of the N-acylneuraminate cytidylyltransferase gene.

Sequence-based typing (SBT) combined with monoclonal antibody subgrouping of Legionella pneumophila isolates is at present considered to be the reference standard during epidemiological investigation of Legionnaires' disease outbreaks. In some isolates of L. pneumophila, the seventh allele of the standard SBT scheme, neuA, is not amplified, because a homologue that is refractory to amplification with the standard neuA primers is present. Consequently, a complete seven-allele profile, and hence a sequence type, cannot be obtained. Subsequently, primers were designed to amplify both neuA and the homologue, but these yielded suboptimal sequencing results. In this study, novel primers specific for the neuA homologue were designed and internationally validated by members of the ESCMID Study Group for Legionella Infections at national and regional Legionella reference laboratories with a modified version of the online L. pneumophila sequence quality tool. To date, the addition of the neuAh target to the SBT protocol has allowed full typing data to be obtained for 108 isolates of 11 different serogroups, namely 1, 2, 3, 4, 5, 6, 7, 8, 10, 13, and 14, which could not previously be typed with the standard SBT neuA primers. Further studies are necessary to determine why it is still not possible to obtain either a neuA or a neuAh allele from three serogroup 11 isolates.

Evaluation of PCR methods for the diagnosis of pertussis by the European surveillance network for vaccine-preventable diseases (EUVAC.NET).

This study aimed to evaluate the performance of polymerase chain reaction (PCR) methods used for the diagnosis of pertussis in laboratories within Europe in 2011. National reference laboratories in 25 European countries were contacted and a total of 24 laboratories from 19 countries agreed to participate in the study. A panel of seven samples of DNA from Bordetella pertussis, Bordetella parapertussis and Bordetella holmesii plus a negative control were distributed and analysed according to the routine PCR methods in each laboratory. The study took place in 2011. Nineteen laboratories used a real-time PCR approach, four laboratories used block-based PCR and one laboratory used a combination of methods. Six different combinations of amplification targets were used, and ten laboratories tested only for the presence of B. pertussis DNA. All laboratories (24/24) correctly identified a sample with high concentration of B. pertussis DNA, while three misidentified the B. parapertussis DNA as B. pertussis and 15 misidentified the B. holmesii DNA as either B. pertussis or B. parapertussis. There was a wide variation in the methods used for PCR-based diagnosis of pertussis among the European laboratories. Several laboratories were not able to discriminate between DNA samples from different Bordetella species.

Application of Legionella pneumophila-specific quantitative real-time PCR combined with direct amplification and sequence-based typing in the diagnosis and epidemiological investigation of Legionnaires' disease.

The detection of Legionella pneumophila DNA in clinical specimens using quantitative real-time polymerase chain reaction (qPCR) combined with direct sequence-based typing (SBT) offers rapid confirmation and timely intervention in the investigation of cases of Legionnaires' disease (LD). We assessed the utility of a specific L. pneumophila qPCR assay targeting the macrophage infectivity potentiator (mip) gene and internal process control with three clinical specimen types from confirmed LD cases. The assay was completely specific for L. pneumophila, as demonstrated by positive results for 39/39 strains from all subspecies and 16 serogroups. No cross-reaction was observed with any of the 54 Legionella non-pneumophila (0/69 strains) or 21 non-Legionella (0/58 strains). All L. pneumophila culture-positive respiratory samples (81/81) were qPCR-positive. Of 80 culture-negative samples tested, 47 (58.8%) were qPCR-positive and none were inhibitory. PCR was significantly more sensitive than culture for samples taken ≤ 2 days of hospitalisation (94.7% vs. 79.6%), with the difference being even more marked for samples taken between 3 and 14 days (79.3% vs. 47.8%). Overall, the sensitivity of the qPCR was ∼30% greater than that of culture and direct typing on culture-negative PCR-positive samples resulted in full 7-allele profiles from 23/46, 5 to 6 alleles from 8/46 and ≥ 1 allele from 43/46 strains.

What to do and what not to do in serological diagnosis of pertussis: recommendations from EU reference laboratories.

Bordetella pertussis-specific antibodies can be detected by enzyme-linked immunosorbent assays (ELISAs) or multiplex immunoassays. Assays use purified or mixed antigens, and only pertussis toxin (PT) is specific for B. pertussis. The interpretation of results can be based on dual-sample or single-sample serology using one or two cut-offs. The EU Pertstrain group recommends that: (i) ELISAs and multiplex immunoassays should use purified non-detoxified PT as an antigen, that they should have a broad linear range and that they should express results quantitatively in International Units per millilitre (IU/ml); (ii) a single or dual diagnostic cut-off for single-serum serology using IgG-anti-PT between 50 and 120 IU/ml should be used, and diagnostic serology cannot be validly interpreted for one year after vaccination with acellular pertussis (aP) vaccines; (iii) IgA-anti-PT should only be used with indeterminate IgG-anti-PT levels or when a second sample cannot be obtained. This group discourages using: (i) other antigens in routine diagnostics, as they are not specific; (ii) micro-agglutination, due to its lack of sensitivity; (iii) immunoblots for pertussis serodiagnosis, as results cannot be quantified; (iv) other methods, such as complement fixation or indirect immunofluorescence, due to their low sensitivity and/or specificity.

Antimicrobial susceptibility testing of historical and recent clinical isolates of Bordetella pertussis in the United Kingdom using the Etest method.

Reports of the development of antimicrobial resistance by Bordetella pertussis to macrolides in the United States and Taiwan, together with a recent increase in pertussis notifications and laboratory-confirmed cases in England and Wales in 2008, prompted the examination of historical and recent clinical isolates from patients for evidence of such resistance in our collection. Isolates submitted to our laboratory as part of the enhanced surveillance scheme for pertussis, from 2001 to 2009, were tested against three agents, erythromycin, clarithromycin and azithromycin, by the Etest (bioMérieux) method. All isolates (n = 583) were fully susceptible to all three agents tested (minimum inhibitory concentrations [MICs] 256 microg/ml. Although no evidence of resistance was found in the strains tested from the United Kingdom, screening for antimicrobial resistance of B. pertussis may be warranted in cases that are unresponsive to macrolide treatment and to provide early warning of such emergence in the future.

Distribution of Legionella pneumophila serogroups, monoclonal antibody subgroups and DNA sequence types in recent clinical and environmental isolates from England and Wales (2000-2008).

Clinical isolates of Legionella pneumophila, obtained from 167 patients, who acquired their illness in the community in England and Wales between January 2000 and March 2008, were compared with 276 environmental isolates of L. pneumophila obtained over the same period as part of the routine sampling of 'managed' water systems. The 443 isolates were typed by monoclonal antibody (mAb) subgrouping and the internationally standardised, seven-gene loci, sequence-based typing (SBT) scheme of the European Working Group for Legionella Infections (EWGLI). Of the clinical isolates, 97.6% were L. pneumophila serogroup (sgp) 1, compared with only 55.8% of environmental isolates (P = 0.0002); 91.6% were subgrouped as mAb3/1+ve, compared with only 8.3% of environmental isolates (P < 0.0001). The isolates were very diverse, with SBT identifying 111 sequence types (STs) (index of diversity [IOD] 0.954). Among the clinical isolates, 42 ST were seen, with one (ST47) accounting for 25.7% and three (ST47, ST37 and ST62) accounting for 46.1% of all isolates. Eighty-two STs were identified among the environmental isolates, with two (ST1 and ST79) accounting for 34.1% of these. Comparison of the STs seen among clinical and environmental isolates showed that there was very little overlap between the two populations (P < 0.0001), with common clinical strains found in the environment very infrequently: 0.4, 0.7 and 0% (ST47, ST37 and ST62, respectively), and common environmental strains rarely causing disease: 4.8 and 1.2% (ST1 and ST79, respectively). Combining phenotypic and genotypic data identified 144 phenons (IOD 0.970); 52 among clinical isolates and 101 among environmental isolates. The most abundant clinical strain, mAb 'Allentown' ST47, accounted for 22.8% of cases, but was only found once in the environment. Conversely, mAb 'Oxford/OLDA' ST1 was the most common environmental strain (17.0%), but only caused two infections. A review of the published data shows that mAb 'Allentown' ST47 is also an important cause of infection in France and possibly in the Netherlands. However, it was not found in a large study of German clinical isolates. This study confirms previous work showing that just a few strains of L. pneumophila cause the majority of community-acquired Legionella infection in England and Wales, and that these clinically significant strains are only rarely found in managed water systems. These data suggest that knowing which particular strain is present in an environment might be at least as important as knowing the quantity in which legionellae are present.

Pertussis outbreak on a neonatal unit: identification of a healthcare worker as the likely source.

We describe the investigation and containment of an outbreak of pertussis on a neonatal unit. Bacterial culture, polymerase chain reaction (PCR) and serology were used to confirm suspected cases. Two infants with pertussis were identified and a nurse with prolonged cough was traced as the likely source. Control interventions included mass chemoprophylaxis of healthcare workers and patients and exclusion from work of healthcare workers with cough. The use of PCR allowed rapid assessment of the extent of the outbreak. This outbreak highlights the risk to hospitalised infants posed by circulation of Bordetella pertussis in young adults and illustrates the utility of PCR in rapidly assessing the extent of outbreaks. Prevention strategies such as universal vaccination of adolescents, or selective vaccination of healthcare workers, should be considered in the UK.

Guidelines for the validation and application of typing methods for use in bacterial epidemiology.

For bacterial typing to be useful, the development, validation and appropriate application of typing methods must follow unified criteria. Over a decade ago, ESGEM, the ESCMID (Europen Society for Clinical Microbiology and Infectious Diseases) Study Group on Epidemiological Markers, produced guidelines for optimal use and quality assessment of the then most frequently used typing procedures. We present here an update of these guidelines, taking into account the spectacular increase in the number and quality of typing methods made available over the past decade. Newer and older, phenotypic and genotypic methods for typing of all clinically relevant bacterial species are described according to their principles, advantages and disadvantages. Criteria for their evaluation and application and the interpretation of their results are proposed. Finally, the issues of reporting, standardisation, quality assessment and international networks are discussed. It must be emphasised that typing results can never stand alone and need to be interpreted in the context of all available epidemiological, clinical and demographical data relating to the infectious disease under investigation. A strategic effort on the part of all workers in the field is thus mandatory to combat emerging infectious diseases, as is financial support from national and international granting bodies and health authorities.

Identification of Legionella spp. by 19 European reference laboratories: results of the European Working Group for Legionella Infections External Quality Assessment Scheme using DNA sequencing of the macrophage infectivity potentiator gene and dedicated online tools.

Identification of Legionella spp. can be achieved by DNA sequencing of the macrophage infectivity potentiator (mip) gene. The External Quality Assurance (EQA) scheme described in this report is the first to assess the proficiency of laboratories using this methodology. The results obtained from two EQA distributions sent to European reference laboratories involved in Legionella outbreak control and environmental monitoring are presented. Each distribution contained a panel of ten coded Legionella strains. All strains were from clinical and environmental sources and were considered to be wild-type strains. Participants used dedicated online tools to compare sequence text files against a database of known Legionella spp. The majority of centres (seven of ten, and 11 of 12) correctly identified all strains tested, in the first and second distributions, respectively. Typically, sequence similarity values of 98-100% were obtained when the test strains were compared with sequences contained in the database. In all but one case, lower values indicated a poor quality sequence. The exception was associated with the identification of a putative new species in the first panel. Genotypic identification of Legionella can be achieved by the use of standard protocols, dedicated identification libraries, and online tools. EQA schemes provide an independent measure of performance, and it is recommended that laboratories performing these techniques participate in such schemes, thereby allowing optimisation of and improvements in their performance.

Comparison of clinical and environmental isolates of Legionella pneumophila obtained in the UK over 19 years.

Between January 1980 and December 1998, 3458 cases of Legionnaires' disease were reported to the national surveillance scheme in England and Wales. Of these, 463 (13.4%) were reported as proven by culture and isolation of Legionella spp., with 96.3% being Legionella pneumophila. Serogroup (Sgp), monoclonal antibody (mAb) subgrouping and restriction fragment length polymorphism (RFLP) analysis data were obtained for 321 (69.3%) of these, of which 284 were classified as being unrelated to any other isolate in the study. Typing data were also available for 117 unrelated environmental isolates of L. pneumophila obtained from England and Wales, giving a total of 401 unrelated isolates in the study. Of the clinical isolates, 88.0% were Sgp1, compared with only 42.7% of environmental isolates (p <0.001); 79.6% of clinical isolates were subgrouped as mAb2+, compared with only 12.8% of environmental isolates (p <0.001). RFLP typing identified 67 types among the 401 isolates, with clinical isolates showing significantly less diversity than environmental isolates (index of diversity (IOD) 0.944 vs. 0.958; p <0.05), with three RFLP types (1, 5 and 14) accounting for 40.0% of all clinical isolates. Combining the phenotypic and genotypic data resulted in 173 distinct phenons, with clinical isolates showing significantly less diversity than environmental isolates (IOD 0.964 vs. 0.996; p <0.01). Three phenons accounted for 30% of all clinical isolates. These data strongly suggest that some strains of L. pneumophila are more likely to cause human infection than would be expected from their distribution in the environment.

Assessment of fluorescent amplified fragment length polymorphism analysis for epidemiological genotyping of Legionella pneumophila serogroup 1.

This study assessed the reproducibility and epidemiological concordance of double-enzyme fluorescent amplified fragment length polymorphism (fAFLP) analysis for genotyping of Legionella pneumophila serogroup (sg) 1. fAFLP fragment analysis was performed on three different sequencing platforms (one gel- and two capillary-based) in different laboratories with a well-characterised set of 50 strains of L. pneumophila sg 1. fAFLP data were analysed with the Pearson correlation similarity coefficient, using a range of parameters, and dendrogram outputs were converted to arbitrary types after selection of a specified percentage similarity threshold. The results obtained were compared with those obtained by the standard non-fluorescent AFLP method and were found to be broadly concordant. Using optimised settings for each fAFLP method to analyse the panel of 50 strains, epidemiological concordance (E) and reproducibility (R) values of 1.00 were obtained, and the number of types ranged from nine to 15, compared with E=1.00 and R=1.00, with 16 types, for the non-fluorescent AFLP protocol. The study demonstrated the potential of fAFLP for typing strains of L. pneumophila sg 1 on all three platforms; however, inter-platform comparison of fAFLP data was not achieved. fAFLP analysis may have a role in the fingerprinting of multiple isolates during Legionella outbreak investigations, but further work is required before type designations and identification libraries can be developed.

External quality assessment for molecular detection of Bordetella pertussis in European laboratories.

Although the PCR for the detection of Bordetella pertussis is routinely performed in diagnostic laboratories, no quality assessment program has so far been described. We report on the results obtained with two external quality assessment proficiency panels sent to European laboratories. The first proficiency panel contained a series of dilutions of three previously characterized B. pertussis clinical isolates and two negative controls. No false-positive results were reported by six laboratories providing seven data sets. The reported limits of detection of the three B. pertussis strains varied between 4 and 4,000, 9 and 9,000, and 3 and 30,000 CFU/ml, respectively. The second proficiency panel, composed of a series of dilutions of reference strains of B. pertussis, B. holmesii, B. hinzii, and B. bronchiseptica, as well as negative controls, was sent to nine laboratories. One laboratory reported a negative result for a sample and reported a B. parapertussis-positive sample to be positive for B. pertussis. By using the B. pertussis-specific target gene pertactin, one laboratory detected B. pertussis with 100% specificity. All other laboratories, which used IS481-based assays, reported positive results for the samples containing B. holmesii and B. bronchiseptica, species that have occasionally been recovered from human respiratory samples. These data show that the choice of the target gene is particularly critical for the species specificity of B. pertussis PCR assays.

Designation of the European Working Group on Legionella Infection (EWGLI) amplified fragment length polymorphism types of Legionella pneumophila serogroup 1 and results of intercentre proficiency testing Using a standard protocol.

The utility of amplified fragment length polymorphism (AFLP) analysis as a genotyping method for the epidemiological typing of Legionella pneumophila serogroup 1 has been previously demonstrated. This study (i). reports recommendations for the designation of the European Working Group on Legionella Infections (EWGLI) AFLP types, (ii). describes the EWGLI AFLP types identified for the 130 strains in the EWGLI culture collection, and (iii). reports the results of a newly introduced international programme of proficiency testing. Following preliminary analysis of 20 epidemiologically unrelated isolates, 16 major AFLP types were identified. A coded proficiency panel, comprising 12 additional isolates representing 9 of these 16 AFLP types, was sent to 17 centres in 14 European countries where it was analysed following a previously determined standard protocol. The identity of each coded strain (recorded as AFLP type 001-016 or untypeable) was determined by participants with reference to these 16 AFLP types, either visually or using gel analysis software where available, and reported to the coordinating centre. Nine of the 12 strains, including an epidemiologically related pair and two pairs of unrelated isolates of the same type, were correctly identified to the correct AFLP type by all or all but one of the participants. Seven laboratories correctly identified all 12 isolates, and a further seven laboratories correctly identified 11. Type identification scores ranged from 75% (1 centre), 83% (2 centres), and 92% (7 centres) to 100% (7 centres). The AFLP method as described is robust and rapid and allows the genotypic comparison of isolates of Legionella pneumophila between different testing centres without the need for exchange of the strains studied.