The clinical utility of Legionella urinary antigen assays for the diagnosis of Legionnaires' disease was assessed by using samples from 317 culture-proven cases. The sensitivities of the Binax enzyme immunoassay (EIA) and Biotest EIA were found to be 93.7 and 94.4% for travel-associated infection and 86.5 and 76.0% for community-acquired infection but only 44.2 and 45.7% for nosocomially acquired infection, respectively.

OBJECTIVES: To compare genotypic methods for epidemiologic typing of Legionella pneumophila serogroup (sg) 1, in order to determine the best available method within Europe for implementation and standardization by members of the European Working Group on Legionella Infections. METHODS: Coded isolates (114) of L. pneumophila sg 1 comprising one epidemiologically 'unrelated' (79) and one 'related' panel of isolates (35) were sent to 12 laboratories in 11 European countries. Analysis was undertaken in each laboratory using one or more of the following methods: ribotyping, restriction fragment length polymorphism analysis, restriction endonuclease analysis, pulsed-field gel electrophoresis (PFGE), PCR using arbitrary/repeat sequence primers (AP-, AP/rep-PCR), and amplified fragment length polymorphism (AFLP) analysis. Results were analyzed visually or using gel analysis software. Each method was assessed for its: index of discrimination (D), epidemiologic concordance (E), speed of application and ease of use. In addition, phenotypic analysis was performed in two laboratories using monoclonal antibodies (mAbs). RESULTS: The D of each of the genotypic methods ranged from 0.840 for ribotyping to 0.990 for PFGE using Sfil: E ranged from 0.06 for AP- and AP/rep-PCR to 1.00 for ribotyping using Pstl/EcoRI and AFLP: in general, E was inversely related to D. Although offering only limited discrimination (D=0.838), mAb typing was both rapid and highly epidemiologically concordant (E=1.00). CONCLUSIONS: Two methods, PFGE using Sfil and AFLP, were selected for further study. AFLP is rapid and highly epidemiologically concordant (E=1.00), but is not highly discriminatory. This method will be developed as a rapid screening tool. PFGE using Sfil is highly discriminatory but, in the present study, yielded low values of E (0.12-0.71). Attempts will be made to rigorously standardize this method for use as the reference method. Primary screening of isolates by mAb subgrouping is recommended.

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The new BinaxNOW Immunochromatographic (ICT) Assay for the detection of Legionella pneumophila antigens was used to test 535 urine specimens from patients with and without Legionnaires' disease. The speci®city, calculated by testing 112 samples from patients with pneumonia of aetiologies other than Legionella infection, and 167 urine specimens from urinary tract infections, was found to be 97.1% if the manufacturer's guidelines were followed. However, it was determined that the`false positive' results characterised by very weak bands could be discounted by reexamination of the results at 60 min, yielding a speci®city of 100%. With this minor modi®cation of the procedure applied to examination of urine samples from 117 patients with legionellosis con®rmed by isolation of L. pneumophila and 70 patients who had seroconverted to L. pneumophila serogroup 1, sensitivity was calculated to be 79.7%. In comparison, the sensitivities of the Binax Urinary Antigen Enzyme Immunoassay (EIA) and Biotest Urin Antigen EIA were estimated to be 79.1 and 83.4%, respectively. Eleven cases (5.9%) were positive by BinaxNOW assay but negative by Binax or Biotest EIA, or both. The sensitivities of all assays increased to c. 94% if only diagnosis of cases con®rmed by isolation of serogroup 1 L. pneumophila was considered, although the sensitivity for infections caused by L. pneumophila serogroup 1 monoclonal antibody (MAb) subgroup Bellingham was signi®cantly lower than for other MAb subgroups. The Biotest EIA recognised 10 (45%) of the 22 cases not caused by L. pneumophila serogroup 1, whereas the two Binax kits detected only three each. The ICT assay BinaxNOW can be recommended as a rapid speci®c test for the diagnosis of Legionnaires' diseases caused by L. pneumophila serogroup 1, although very weak bands should be interpreted cautiously.

We have used the polymerase chain reaction to detect Mycoplasma genitalium in artificially seeded human throat swab samples as well as in clinical material. On the basis of the published nucleotide sequence of the M. genitalium 140-kDa adhesin gene, primers were chosen to produce an amplified fragment of 281 bp. Five different previously isolated strains, including the type strain of M. genitalium, could all be detected by the polymerase chain reaction, and DNAs from other mycoplasmal and bacterial species yielded negative results. The detection limits were estimated to be approximately 50 organisms by inspection of ethidium bromidestained agarose gels and 4 organisms when a biotinylated oligonucleotide probe was used in filter hybridization. The amplified DNA fragments were subjected to restriction enzyme analysis. DNAs from the five different isolates all possessed EcoRI, SspI, AluI, Sau3AI, and DdeI restriction sites, as predicted from the published sequence. A total of 150 urogenital swabs collected from 100 patients for culturing of Chiamydia trachomatis were tested for the presence of M. genitalium DNA. Ten samples from eight patients were found positive. The amplified DNA fragments from all of our clinical samples possessed the AluI, Sau3AI, and DdeI restriction sites, but three samples from two patients did not contain the SspI site and none of the samples contained the EcoRI site.

This pan-European study included unrelated strains of Legionella pneumophila obtained from 1335 cases of Legionnaires' disease. The isolates were serotyped into the serogroups 1 to 15 by monoclonal antibodies (MAb) and/or rabbit antisera. Additionally, MAb subgrouping was undertaken for isolates belonging to serogroups 1, 4, and 5. Monoclonal types of serogroup 1 were subdivided as having, or not having, the virulence-associated epitope recognized by the MAb 3/1 (Dresden Panel). This epitope is not present on strains belonging to any other serogroups. Taking all Legionella incidents together, MAb 3/1-positive cases were most frequent (66.8%); 11.7% of the isolates belonged to MAb 3/1-negative serogroup 1 subgroups and 21.5% to other serogroups, with serogroups 3 and 6 predominating. Among all serotypes discriminated in this study, monoclonal subtype Philadelphia was the most frequent. If categories of infection were considered, the proportion of MAb 3/1-negative strains differed significantly ( P<0.0005) between community-acquired cases (139/510; 27.3%) and travel-associated (42/295; 14.2%) or hospital-acquired infections (176/329; 53.5%). Moreover, taking distribution in different European areas into account, the proportion of MAb 3/1-negative strains was significantly higher in the Scandinavian region than in the Mediterranean countries or the UK for both community-acquired (48.7% vs. 18.6% or 12.0%; P<0.0005) and nosocomial cases (87.7% vs. 32.6% or 52.6%; P

The aims of this work were to assess (i) the intercentre reproducibility and epidemiological concordance of amplified fragment length polymorphism analysis for epidemiological typing of Legionella pneumophila serogroup 1, and (ii) the suitability of the method for standardisation and implementation by members of the European Working Group on Legionella Infections. Fifty coded isolates comprising two panels of well-characterised strains, a "reproducibility" panel (n=20) and an "epidemiologically related" panel (n=30), were sent to 13 centres in 12 European countries. Analysis was undertaken in each centre following a previously determined standard protocol. Results were analysed by the participants, using gel analysis software where available, and submitted to the coordinating centre. The coordinating centre reanalysed all results visually and selected data-sets with gel analysis software. Data analysis by participants yielded reproducibility (R) values of 0.20-1.00 and epidemiological concordance (E) values of 0.11-1.00, with 6 to 34 types. Following visual analysis by the coordinating centre, R=0.78-1.00, and E=0.67-1.00, with 10-20 types. Analysis of three data-sets by the coordinating centre using gel analysis software yielded R=1.00 and E=1.00, with 12, 13 or 14 types. This method can be used as a simple, rapid screening tool for epidemiological typing of isolates of Legionella pneumophila serogroup 1. Results demonstrate that the method can be highly reproducible (R=1.00) and epidemiologically concordant (E=1.00), with good discrimination. The electropherograms generated are amenable to computer-aided analysis, but strict adherence to a previously defined laboratory protocol is required. Following designation of representative type strains and patterns, this method will be adopted by the European Working Group on Legionella Infections as the first internationally standardised typing method for use in the investigation of travel-associated Legionella infections.

Denmark experienced two waves of Mycoplasma pneumoniae infection during autumn and early winter in 2010 and 2011, respectively. Both affected the whole country. The proportion of positive results was almost the same for both, indicating that the two waves were probably of equal size. High macrolide consumption during the epidemics did not seem to affect levels of macrolide resistance in M. pneumoniae, which remain low in Demark (1% to 3%).