Legionnaires' disease in Europe, 1996.

WHO News and activities/Notes et activites OMS Legionnaires' disease in Europe, 1996 The twelfth meeting of the European Working Group on Legionella Infections (EWGLI), which was co-sponsored by WHO, was held in Lisbon, on 1-3 June 1997. Below is a summary of the major topics discussed. Epidemiology Information on cases of Legionnaires' disease in Europe was obtained through the completed annual reporting forms prepared by the PHLS Communica- ble Disease Surveillance Centre (CDSC). For 1996, the aggregated data show a total of 1563 reports of Legionnaires' disease from 24 European countries, an increase of nearly 300 cases from 1995 and nearly 400 more cases than were reported in 1994. Most of the increase in 1996 was due to the large community outbreak that occurred in Spain. The number of deaths reported in 1996 was 77, an overall case fatality rate of 4.9% compared with 7.7% in 1995. However, the data for 647 cases from five countries contained no information on the num- bers of deaths associated with these cases. In countries where more complete information was re- ported, the case fatality rate varied from 4% to 30%. A total of 22 outbreaks or clusters were de- tected by individual countries in 1996. Of these, two were linked to hospitals, eight to the community, and twelve to travel. Of these outbreaks ten were linked to hot or cold water systems, 6 to wet cooling sys- tems, and one to spa water. The source was unknown in five instances, one of which was the nosocomial outbreak in Spain, while the rest were associated with travel. The total number of travel-associated cases of Legionnaires' disease in 1996 increased compared with 1995 and 1994. In 1996, 31 such cases from nine countries were linked to travel in the same country of residence and 215 cases from 15 countries to travel outside the country of residence. Surveillance has improved in many countries to the extent that incomplete epidemiological informa- tion decreased from 50% in 1995 to 38% in 1996. The overall number of cases reported from Europe has continued to increase since 1994, probably be- cause of improvements in reporting from individual Reprint No. 5844 countries. However, improved surveillance of deaths caused by Legionnaires' disease remains a priority in Europe in order to monitor the continued effective- ness of control measures in community, hospital, and hotel settings. Epidemiological typing markers There is a real willingness to develop a European strategy for typing Legionella spp. For example, at the meeting, one group reported having sent 115 coded isolates of Legionella to 13 laboratories in 11 countries and to have collated all the typing data obtained by the different laboratories using their own respective techniques. Phase II of the study will use a more limited but common set of techniques in all the laboratories. New techniques to type Legionella at the level of the species or the subspecies were also presented. In this respect, the panel of monoclonal antibodies to serotype Legionella pneumophila described at the meeting will be very helpful in limiting the number of strains that cross-react with antisera. Analysis of the intergenic 16S-23S RNA gene by the polymerase chain reaction (PCR) will prob- ably become a technique easily carried out by every laboratory, permitting rapid identification of all spe- cies of Legionella. Other taxonomic techniques that were presented included the analysis of the fatty acid or lipopolysaccharide composition. For these studies it appears important to exchange strains between laboratories to obtain a maximum number of strains for each species and not to limit the evaluation of a new technique to a single reference strain. The use of markers to type strains isolated from outbreaks using the following techniques was de- scribed: arbitrarily primed PCR and monoclonal antibody; pulsed field gel electrophoresis and arbi- trarily primed PCR; pulsed field gel electrophoresis, comparing Finnish and EWGLI collection isolates. The results indicate the considerable diversity exhib- ited by Legionella pneumophila serogroup 1. The limits of pulsed field gel electrophoresis were demonstrated by the results of an analysis of strains of L. pneumophila not belonging to serogroup 1. Identical profiles were obtained for strains of L. pneumophila serogroup 6 and for L. pneumophila strains with identical serological cross- reactions. Interest was expressed in finding easier and more rapid typing techniques for subtyping L. pnelumophila serogroup 1 by linking adaptors Bulletin of the World Health Organization, 1998, 76 (2): 213-215 . World Health Organization 1998 213 WHO News and activities/Notes et activites OMS to chromosomal restriction fragments and by per- forming PCR under stringent conditions. Atypical pneumonias: clinical cases Case reports were presented on isolates of L. feelei from patients with pneumonia, on legionellosis in immunosuppressed patients, and on repeated isolations of L. pneumophila serogroup 5 in in- stances where it remained unclear whether it was a long-standing colonization or a chronic infection. Azithromycin had been used successfully to treat legionellosis in immunocompromised patients. It is important that unusual features and observations on the clinical presentation and of the epidemiology of legionella infections are reported in order to in- crease understanding of this disease. Laboratory diagnosis Interest in methods to diagnose Legionnaires' dis- ease by detection of antigen in urine has increased steadily since the early 1980s. At the 1997 meeting three presentations addressed this subject. The first, described the development of an enzyme immunosorbent assay (EIA) from an in-house refer- ence test to a commercial kit. The second, was a multicentred evaluation of this kit and the results indicated that the assay was simple to use, specific, and gave reproducible results. In the third presen- tation, a comparison was described of another com- mercially available kit with a well characterized in-house assay that has been used widely in the United Kingdom. This study found that there was generally good agreement and indicated that the commercial kit gave good results. However, it was recommended that all samples found to be positive should be retested after boiling to eliminate the false-positive results that were occasionally encountered. The results of these studies indicate that detection of L. pneumophila urinary antigen is one of the most efficient ways of diagnosing Legion- naires' disease. Furthermore, now that several commercially produced kits are available, this ap- proach to the diagnosis is no longer confined to a few reference laboratories able to develop and main- tain in-house assays. The continued development of the EWGLI pilot quality assurance scheme for urinary antigen detection is essential if confidence in the results obtained using these assays is to be maintained. Two presentations examined the problem of cross-reactions that might confound the serological diagnosis of Legionnaires' disease. The final two papers addressed the develop- ment of novel methods for the diagnosis of Legion- naires' disease. The first reported results obtained using an enzyme-linked oligonucleotide-sorbent assay (a semi-quantitative PCR-based method). The second described the preparation of a direct- fluorescent reagent using phage display technology. Both methods appeared promising, but it will be some years before their diagnostic use can be properly assessed. Environmental problems Shortcomings in the control of Legionella spp. in cooling towers, hot and cold water systems, and shower boilers, despite recommended control meas- ures, underline the importance of a sustainable con- trol strategy. A novel Legionella amplifier a home-based whirlpool bath with recirculated water has appeared on the market in Japan. Recycling has a certain cachet and the re-use of water is becom- ing commoner even in areas with no shortage of fresh water; therefore the potential risk presented by equipment that enhance the growth of Legionella spp. has to be realized. There is a need for the au- thorities to follow the situation and to update regula- tions and recommendations accordingly. Virulence In contrast to the laboratory diagnosis of Legion- naires' disease, where great strides have been made in the last few years, progress towards understanding the virulence mechanisms of Legionella spp. has been slow. Four papers were presented at the meeting, each exemplifying a very different approach to this problem. The first looked specifically at a case of Legionnaires' disease caused by L. dumoffii and some subsquent work using a guinea-pig model. It was reported that in both the human case and in the animal model the organisms were located not only within alveolar macrophages but also within alveolar epithelial cells and it was proposed that this charac- teristic may explain the rapid and fulminant progress of L. dumoffii pneumonia observed in the patient. As L. dumoffii infections are very rare, and animal models far from ideal, this interesting conjecture may not be easy to substantiate. The second presentation described an attempt to identify putative virulence-associated genes by constructing a genomic library of the Corby isolate of L. pneumophila. Identified was a gene that codes for a 16kDa protein, which appears to be specific to L. pneumophila; however, although of considerable interest, possibly as a diagnostic or identification rea- WHO Bulletin OMS. Vol 76 1998214 WHO News and activities/Notes et activites OMS gent, no evidence has been found that it is associated with virulence. A study to investigate the influence of temperature (22°C or 37°C) and growth condi- tions (intra- versus extraamoebal) on the transcrip- tion of icm and dot, both of which are required for intracellular multiplication of legionellae, was pre- sented. There was no evidence that clear tempera- ture influenced expression of these genes, but intracellular growth might affect the transcripton of icm. The final paper described a study of the involve- ment of tissue necrosis factor-alfa (TNF-c) in the death of susceptible A/J mice inoculated intra- peritoneally with L. pneumophila. The study showed that death provoked by a virulent strain was associated with TNF-ct levels that were higher than those produced by mice infected with an avirulent strain. Of course, as with any such animal models, it is not clear how, if at all, this relates to naturally acquired infection. La legionellose en Europe, 1996 La douzieme r6union du Groupe de travail europeen sur la legionellose (EWGLI), coparrainee par I'OMS, s'est tenue a Lisbonne du ler au 3 juin 1997. On trouvera ci-dessous un r6sum6 des principales questions trait6es. Epidemiologie Les informations concernant les cas de l6gionellose survenus en Europe ont 6t6 obtenues au moyen des formulaires de d6claration pr6par6s par le Com- municable Disease Surveillance Centre (CDSC) du Public Health Laboratory Service (PHLS). En 1996, les donn6es globales font apparaitre un total de 1563 d6clarations de legionellose dans 24 pays europeens, une augmentation de pres de 300 cas depuis 1995, avec pres de 400 cas d6clares de plus qu'en 1994. L'augmentation de 1996 est pour l'essentiel due a la flamb6e communautaire importante survenue en Espagne. Le nombre de deces declar6s en 1996 etait de 77, avec un taux de l6talit6 global de 4,9%, contre 7,7% en 1995. Les donn6es concernant 647 cas survenus dans 5 pays ne comportaient toutefois aucune infor- mation sur le nombre de deces associ6s a ces cas. Dans les pays qui ont fourni des informations plus completes, le taux de letalite s'etageait de 4% a 30%. Les pays ont d6clar6 22 flambees ou agregats spacio-temporels en 1996, dont 2 6taient associ6es a des 6tablissements hospitaliers, 8 a la communaut6, et 12 a des voyages; 10 6taient associees au r6seau de distribution de l'eau, chaude ou froide, 6 a des systemes de climatisation humide, et 1 a une source thermale. L'origine etait inconnue dans 5 cas, dont l'un d'eux 6tait la flambee nosocomiale survenue en Espagne; les autres 6taient associes a des voyages. Le nombre total de cas de l6gionellose associ6s a des voyages en 1996 a augmente compare a 1995 et 1994. En 1996, 31 de ces cas survenus dans 9 pays 6taient liUs a un voyage dans le pays de r6sidence, et 215 cas survenus dans 15 pays a un voyage hors du pays de r6sidence. La surveillance s'est amelioree dans de nombreux pays, au point que le pourcentage d'informations 6pidemiologiques incompletes est passe de 50% en 1995 a 38% en 1996. Le nombre total de cas declares en Europe ne cesse d'augmenter depuis 1994, en raison probablement de l'amelioration de la d6claration par les pays. Cependant, l'amelioration de la surveillance de la mortalit6 par legionellose reste une priorit6 en Europe, de facon a pouvoir surveiller l'efficacite des mesures de lutte dans la communaut6 ainsi qu'en milieu hospitalier et hotelier. Marqueurs de typage epidemiologique Il existe une r6elle volonte de mettre au point une strategie europeenne de typage de Legionella spp. Lors de la reunion, un groupe a signale qu'il a envoy6 115 isolements cod6s de Legionella a 13 laboratoires de 11 pays, et recueilli les r6sultats du typage par les diff6rents laboratoires qui ont utilise leurs propres techniques de typage. La phase II de l'etude consistera a utiliser des techniques moins nombreuses mais identiques dans tous les laboratoires. Des nouvelles techniques de typage de Legionella au niveau de l'espece et de la sous- esp&ce ont 6galement ete exposees. La batterie d'anticorps monoclonaux pour le serotypage de Legionella pneumophila decrite au cours de la r6union permettra de limiter le nombre de souches qui donnent des reactions croisees avec les antiserums. L'analyse du gene situ6 entre les genes codant les ARN 16S et 23S par la PCR (amplification genique) devrait devenir une technique facile a r6aliser par tous les laboratoires, permettant l'identification rapide de toutes les especes de Legionella. D'autres techniques taxonomiques ont ete present6es, et notamment l'analyse de la compo- sition en acides gras et en lipopolyosides. Dans ces 6tudes, il semble important que les laboratoires 6changent des souches pour avoir le nombre maxi- Bulletin de l'Organisation mondiale de la Sante, 1998, 76 (2): 215-217 . Organisation mondiale de la Santd 1998 215 WHO News and activities/Notes et activites OMS mum de souches par espece et ne pas limiter l'evaluation d'une nouvelle technique a une seule souche de reference. L'utilisation de marqueurs de typage des souches isolees au cours des flambees a ete decrite pour les techniques suivantes: PCR par amorce arbitraire et anticorps monoclonaux; electrophorese en champ pulse et PCR par amorce arbitraire; electrophorese en champ pulse comparant les collections d'isolements du Groupe de travail europeen sur la legionellose et celles de la Finlande. Les resultats sont une illustration de la diversite considerable de Legionella pneumophila serogroupe 1. Les limites de l'electrophorese en champ pulse ont ete mises en evidence par les resultats de l'analyse de souches de L. pneumophila n'appartenant pas au serogroupe 1. Des profils identiques ont ete obtenus pour les souches de L. pneumophila serogroupe 6 et les souches de L. pneumophila donnant des reactions croisees identiques. L'interet a 6te manifeste pour des techniques plus faciles et plus rapides pour le sous-typage de L. pneumophila serogroupe 1 en associant des adaptateurs aux fragments de restriction des chro- mosomes et en realisant la PCR dans des conditions rigoureuses. Pneumopathies atypiques: cas cliniques Divers cas ont et6 examines concernant des souches de L. feelei isolees chez des patients atteints de pneumopathie, des iegionelloses chez des patients immunodeprimes et l'isolement repete de L. pneumophila serogroupe 5 alors qu'on ignore s'il s'agit d'une colonisation de longue duree ou d'une infection chronique. L'azithromycine a ete utilis6e avec succes chez des patients immunodeprimes. Il est important de signaler les manifestations et les obser- vations inhabituelles, tant au niveau clinique que de l'epidemiologie, pour mieux comprendre la maladie. Diagnostic au laboratoire L'int6ret pour les methodes de diagnostic par recherche de l'antigene dans l'urine ne cesse de croitre depuis les annees 80. Trois communications faites a la reunion de 1997 traitent de la question. La premiere decrit la mise au point pour le com- merce d'une methode immunoenzymatique utilisant un immunoadsorbant (EIA) a partir d'une methode de reference elaboree par un laboratoire. La deuxieme est une evaluation multicentrique de cette methode, montrant qu'elle est simple, specifique, et donne des resultats reproductibles. La troisieme communication porte sur la comparaison entre la methode d'un laboratoire, bien caractdrisee et tres utilisee au Royaume-Uni, et un autre test du com- merce. L'etude montre que l'accord est en general bon, et que le test du commerce donne de bons resultats. Il a cependant ete recommande de tester de nouveau apres ebullition tous les pr6levements trouves positifs pour eliminer les faux positifs quelquefois observes. Les etudes indiquent que la recherche de l'antigene urinaire de L. pneumophila est l'un des outils diagnostiques de la legionellose les plus efficaces. De plus, maintenant que plusieurs tests existent dans le commerce, cette methode de diag- nostic n'est plus reservee a quelques laboratoires de reference capables de mettre au point leurs propres test et de les utiliser. Il faut absolument poursuivre le developpement du protocole pilote du Groupe de travail europeen (EWGLI) sur l'assurance de la qualite du test de recherche de l'antigene urinaire, si l'on veut pouvoir continuer a se fier aux resultats obtenus avec ces methodes. Deux communications ont examine le probleme des reactions croisees susceptibles de perturber le diagnostic serologique de la legionellose. Les deux dernieres communications ont traite de la mise au point de nouvelles methodes de diagnostic de la maladie. La premiere fait etat des resultats obtenus avec un titrage utilisant un adsorbant oligonucleotidique lie a une enzyme (methode semi-quantitative par PCR). La seconde decrit la preparation d'un reactif pour fluorescence directe par mise en evidence de bacteriophages. Ces deux methodes semblent prometteuses, mais il faudra quelques annees avant de pouvoir evaluer correctement leur interet diagnostique. Problemes environnementaux L'echec de la lutte contre Legionella spp. observe, malgrd les mesures recommandees, avec les tours de refroidissement, les reseaux de distribution d'eau chaude et froide, et les chauffe-eau des douches, souligne l'importance d'une strategie de lutte durable. Un nouveau systeme de bain bouillonnant domestique avec remise en circulation de l'eau apparu dans le commerce au Japon entraine la proliferation des legionelles. Le recyclage est bien vu, et la reutilisation de l'eau est de plus en plus frequente, meme Ia oiu il n'y a pas de penurie. Il convient donc de prendre la mesure des risques presentes par des appareils qui favorisent la proliferation de Legionella spp. Il est indispensable que les autorites suivent la situation de pres et adaptent la reglementation et les recommandations en consequence. WHO Bulletin OMS. Vol 76 1998216 WHO News and activities/Notes et activites OMS Virulence Si le diagnostic de la legionellose au laboratoire a fait des progres considerables ces dernieres annees, il n'en est pas de meme pour la compr6hension de la virulence. Quatre communications presentees lors de la reunion manifestent des approches tres diff6rentes de la question. La premiere s'est interessee 'a un cas de l6gionellose du 'a L. dumoffi et aux travaux ulterieurs sur un modele cobaye. Chez le cas humain comme chez le modele animal les germes n'etaient pas localis6s que dans les macrophages alveolaires mais aussi dans les cellules epitheliales alveolaires, ce qui, selon les auteurs, pourrait expliquer le caractere foudroyant de la pneumopathie 'a L. dumoffi observee chez ce patient. Les infections 'a L. dumoffi etant tres rares et les modeles animaux loin d'etre parfaits, cette hypothese int6ressante ne sera peut-etre pas facile a verifier. La deuxieme communication a decrit une tenta- tive pour identifier des genes qui seraient associes a la virulence en construisant une banque genomique de l'isolement Corby de L. pneumophila. On a identifi6 un gene codant une prot6ine de 16kDa qui semble specifique de L. pneumophila; si son int6ret est consid6rable, peut-etre comme reactif de diag- nostic ou d'identification, rien n'indique qu'elle est associ6e a la virulence. Une etude de l'influence de la temperature (22°C ou 37 °C) et des conditions de developpement (intra- ou extra-amibien) sur la tran- scription des genes icm et dot, tous deux necessaires a la multiplication intracellulaire des l6gionelles a 6te presentee. Rien n'indique clairement que la temperature influe sur l'expression de ces genes, mais le developpement intracellulaire pourrait modi- fier la transcription du gene icm. La derniere communication a detcrit une etude sur l'implication du facteur alpha de necrose des tumeurs (TNF-a) dans la mort des souris A/J sensibles inoculees avec L. pneumophila par voie intraperitoneale. Elle montre que la mort due a une souche virulente est associee a des taux de TNF-a plus elev6s que ceux observes chez la souris contaminee par une souche avirulente. Comme avec tous les modeles animaux, on ignore quel est le lien avec l'infection naturelle, ni meme s'il y en a un. WHO Bulletin OMS. Vol 76 1998 217