Sequence-based typing (SBT), analogous to multilocus sequence typing (MLST), is the current \"gold standard\" typing method for investigation of legionellosis outbreaks caused by Legionella pneumophila However, as common sequence types (STs) cause many infections, some investigations remain unresolved. In this study, various whole-genome sequencing (WGS)-based methods were evaluated according to published guidelines, including (i) a single nucleotide polymorphism (SNP)-based method, (ii) extended MLST using different numbers of genes, (iii) determination of gene presence or absence, and (iv) a kmer-based method. L. pneumophila serogroup 1 isolates (n = 106) from the standard \"typing panel,\" previously used by the European Society for Clinical Microbiology Study Group on Legionella Infections (ESGLI), were tested together with another 229 isolates. Over 98% of isolates were considered typeable using the SNP- and kmer-based methods. Percentages of isolates with complete extended MLST profiles ranged from 99.1% (50 genes) to 86.8% (1,455 genes), while only 41.5% produced a full profile with the gene presence/absence scheme. Replicates demonstrated that all methods offer 100% reproducibility. Indices of discrimination range from 0.972 (ribosomal MLST) to 0.999 (SNP based), and all values were higher than that achieved with SBT (0.940). Epidemiological concordance is generally inversely related to discriminatory power. We propose that an extended MLST scheme with ∼50 genes provides optimal epidemiological concordance while substantially improving the discrimination offered by SBT and can be used as part of a hierarchical typing scheme that should maintain backwards compatibility and increase discrimination where necessary. This analysis will be useful for the ESGLI to design a scheme that has the potential to become the new gold standard typing method for L. pneumophila.

Sequence-based typing (SBT) is the internationally recognized standard method for genotyping Legionella pneumophila. To date all strains of serogroup 1 (SG1) and some of SGs 2 to 14 yield a seven-allele profile and can be assigned a sequence type (ST). However, for some strains belonging to SGs 2 to 14, the targeted region of the neuA gene could not be amplified using the published standard primers. We determined the DNA sequence of a neuA gene homolog located in the lipopolysaccharide synthesis locus of strain Dallas-1E. By using newly designed degenerate consensus primers based on the neuA homolog in strains Dallas-1E, Philadelphia-1, Paris, Lens, and Corby, we were able to obtain DNA sequences for all 48 non-SG1 strains which were untypeable by the standard method. Our data show that the neuA gene is present in all L. pneumophila strains but differs significantly in some non-SG1 strains at both the DNA and amino acid levels. The new primers can be used to amplify and sequence the neuA gene in all strains and can substitute for the standard primers. This offers the possibility of assigning an ST to all strains of L. pneumophila.

The standard sequence-based method for the typing of Legionella pneumophila serogroup 1 strains was extended by using the gspA and neuA alleles. The use of neuA as a seventh allele for typing significantly increased the index of discrimination calculated for a panel of unrelated strains (from 0.932 to 0.963) and subdivided some known large common complexes (e.g., 1,4,3,1,1,1). This modification to the standard method is proposed as the method of choice in the epidemiological investigation of L. pneumophila infections.

Community-acquired pneumonia (CAP) is associated with considerable morbidity and mortality. The incidence of CAP in the Netherlands is estimated to be 5-10 per 1000 per year. This guideline can be used for the scientifically-based diagnosis and antibiotic treatment of adults with CAP. Streptococcus pneumoniae is the most frequent causative agent In 30-50% of patients, the aetiological pathogen cannot be identified. In the Netherlands, the resistance of S. pneumoniae to penicillin is less than 1%. In addition to patient history and physical examination, chest radiography is indispensable to the diagnosis of CAP. Cultures of sputum, blood, and, if present, pleural effusion are needed to detect the causative agent. Bronchoscopy can be considered if the patient\'s condition deteriorates during antibiotic therapy. Urinary antigen detection is important if signs of legionellosis are present; only Legionella pneumophila serotype can be identified with this technique. The severity of CAP and the risk factors can be measured by the pneumonia severity index, which may be helpful in deciding whether to hospitalise a patient. The choice of antibiotic therapy is based on the intention of providing optimal therapy, the epidemiological features ofvarious microorganisms in the Netherlands, and an inference of the most likely pathogen, based on comorbidity. Empirical antibiotic therapy should target primarily S. pneumoniae because of its high incidence. In both seriously ill patients and those suspected of having legionellosis, antibiotic therapy should also target L. pneumophila. Antibiotic therapy should be adjusted if the pathogen is subsequently identified. Parapneumonic effusion frequently occurs in cases of CAP. Drainage is indicated if the pleural fluid contains bacteria or yields a pH < 7.0. Influenza vaccination is recommended in the elderly to prevent CAP.

A previously described sequence-based epidemiological typing method for clinical and environmental isolates of Legionella pneumophila serogroup 1 was extended by the investigation of three additional gene targets and modification of one of the previous targets. Excellent typeability, reproducibility, and epidemiological concordance were determined for isolates belonging to both serogroup 1 and the other serogroups investigated. Gene fragments were amplified from genomic DNA, and PCR amplicons were sequenced by using forward and reverse primers. Consensus sequences are entered into an online database, which allows the assignment of individual allele numbers. The resulting sequence-based type or allelic profile comprises a string of the individual allele numbers separated by commas, e.g., 1,4,3,1,1,1, in a predetermined order, i.e., flaA, pilE, asd, mip, mompS, and proA. The index of discrimination (D) obtained with these six loci was calculated following analysis of a panel of 79 unrelated clinical isolates. A D value of > 0.94 was obtained, and this value appears to be sufficient for use in the epidemiological investigation of outbreaks caused by L. pneumophila. The D value rose to 0.98 when the results of the analysis were combined with those of monoclonal antibody subgrouping. Sequence-based typing of L. pneumophila is epidemiologically concordant and discriminatory, and the data are easily transportable. This consensus method will assist in the epidemiological investigation of L. pneumophila infections, especially travel-associated cases, by which it will allow a rapid comparison of isolates obtained in more than one country.

Community-acquired pneumonia remains a common and serious condition worldwide. Severe community-acquired pneumonia requiring ICU admission is a distinct entity with different pathogens, outcomes, and management. The mortality rate in severe community-acquired pneumonia can be more than 50%. Over the past decade, some international guidelines for the management of community-acquired pneumonia have been developed in an attempt to optimize patient care. These guidelines have developed prediction tools to direct clinicians in the management of community-acquired pneumonia, including when to admit a patient to the ICU and selecting appropriate investigations and antimicrobial therapy. The individual recommendations of these guidelines and the guidelines as a whole require further studies.

In Italy, 35 clusters of travel associated Legionnaires\' disease were identified from July 2002, when the European Guidelines for Control and Prevention of Travel Associated Legionnaires\' Disease have been adopted by the EWGLINET network, to October 2003. Eight per cent (28.6%) would not have been identified without the network. The clusters detected were small, ranging from 2 cases to a maximum of 6. All clusters involved 5 camping sites and 30 hotels/residences, and an overall of 87 patients. The diagnosis was confirmed in 92.0% of the cases and mainly performed by urinary antigen detection (84.7%). A clinical isolate was available only in one case. Following environmental investigations, samples were collected for all the 35 clusters from the water system, and Legionella pneumophila was found in 23 occasions (65.7%). In 15 resorts out of 35, investigations were already in progress at the time of EWGLI cluster notification, since in Italy full environmental investigation is performed even after notification of a single case. Control measures were implemented in all accommodation sites at risk and one hotel only was closed. In all the 35 clusters, reports were completed and sent on time, highlighting that it is possible to comply with the procedures requested by the European Guidelines.