Seventh International Meeting of the European Laboratory Working Group on Diphtheria-Vienna , June 2002

The Seventh International Meeting of the European Laboratory Working Group on Diphtheria (ELWGD) was held in Vienna, Austria in June 2002 and brought together the microbiologists and epidemiologists responsible for diphtheria in many countries throughout the world. The ELWGD was formed in 1993 in response to the epidemics of diphtheria in Russia and the Newly Independent States (NIS) of the former Soviet Union (1,2). These epidemics, and others elsewhere, highlighted the importance of strengthening and maintaining both epidemiological surveillance and reliable laboratory screening. Since December 2001, these areas have been progressed and strengthened by European Commission (EC) funding to undertake a feasibility study for diphtheria surveillance amongst European Union (EU) member states and accession countries, and to establish a definitive and official European diphtheria surveillance network called DIPNET (EC DG SANCO Agreement No. S12.324473 (2001 CVG4-012)). This was the first international meeting of the ELWGD to be organised and funded under the auspice of the DIPNET (European Diphtheria Surveillance Network).

The Seventh International Meeting of the European Laboratory Working Group on Diphtheria (ELWGD) was held in Vienna, Austria in June 2002 and brought together the microbiologists and epidemiologists responsible for diphtheria in many countries throughout the world.The ELWGD was formed in 1993 in response to the epidemics of diphtheria in Russia and the Newly Independent States (NIS) of the former Soviet Union (1,2).These epidemics, and others elsewhere, highlighted the importance of strengthening and maintaining both epidemiological surveillance and reliable laboratory screening.Since December 2001, these areas have been progressed and strengthened by European Commission (EC) funding to undertake a feasibility study for diphtheria surveillance amongst European Union (EU) member states and accession countries, and to establish a definitive and official European diphtheria surveillance network called DIPNET (EC DG SANCO Agreement No. S12.324473 (2001 CVG4-012)).This was the first international meeting of the ELWGD to be organised and funded under the auspice of the DIPNET (European Diphtheria Surveillance Network).D IPNET will continue a collaborative and coordinated approach to support countries to improve diphtheria surveillance for early detection of cases and contacts by accurate microbiological and epidemiological surveillance (3,4,5).The network of national and international laboratories includes, ➤ D IPNET continue son approche collaborative et coordonnée pour aider les pays à améliorer notamment la détection précoce des cas et des contacts grâce à une surveillance microbiologique et épidémiologique (3,4,5).Le réseau des laboratoires nationaux et internationaux comprend des microbiologistes et ➤ ➤ des épidémiologistes de la plupart des pays membres et des pays candidats.La communication entre laboratoires et autorités de santé publique améliore la capacité des états membres à répondre rapidement à cette menace pour la santé publique (6).
A partir de 1998, la région de Saint-Pétersbourg a également connu une augmentation annuelle de l'incidence de la diphtérie (2,6 pour 100 000), touchant essentiellement les patients adultes.Depuis 2000, la plupart des cas de diphtérie sont liés à la souche C. diphtheriae de variété mitis, ribotype Otchakov.Récemment, un dépistage et des analyses sérologiques chez différentes catégories sociales et professionnelles à risque ont été réalisés dans cette région.Sur les 472 prélèvements de sérum provenant de sujets âgés de 20 à 69 ans analysés, trente-cinq pour cent étaient sen-➤ not only microbiologists, but also epidemiologists from most of the EU member states and accession countries.Communication between laboratories and public health authorities within and between countries enhances the capacity of an EU member state to respond rapidly to this public health threat (6).
Participants in this meeting came from 39 different countries including 12 EU member states, six accession countries, 12 NIS countries, and Brazil, Canada, India, Israel, Norway, Switzerland, Turkey, and the United States (US).This report summarises the presentations given at this meeting which provided an interesting overview of the surveillance of diphtheria within the Russian Federation and NIS as well as the EU member states and accession countries, a review of the clinical and microbiological aspects of infection caused by corynebacteria, and an update on recent studies on the molecular and genetic characteristics of Corynebacterium diphtheriae and C. ulcerans.

Microbiological surveillance of diphtheria in the NIS of the former Soviet Union
The recent epidemic of diphtheria in the NIS began in the Russian Federation in 1990 and affected all 12 NIS by the end of 1994 (7).In 1990, the number of reported diphtheria cases in the NIS was 1 436.However, in 1993 the number of cases dramatically increased to 19 604, then to 47 869 in 1994, and finally reached a peak of 50 434 in 1995.The prominent strains during this epidemic were C. diphtheriae var gravis ribotypes Sankt-Petersburg and Rossija.At the beginning of the epidemic, the case fatality rate was very high and was probably due to inadequate supplies of antitoxin, improper case management, and delayed treatment.Diphtheria control efforts, coordinated by the World Health Organization (WHO), began in the Russian Federation in 1992 and included mass immunisation campaigns which covered most of the adult population (8).Further control efforts and mass immunization campaigns in the other NIS followed.In recent years, the number of cases reported to the European Region of WHO have fallen to approximately 1 500 in 1999 and 2000.
Excellent progress has been achieved in the control of diphtheria in Armenia, Azerbaijan, Belarus, Estonia, Kazakhstan, Lithuania, Republic of Moldova, Turkmenistan, and Uzbekistan, where the incidence of the disease has reached low levels.However, the number of cases are higher in Georgia, Kyrghyzstan, Latvia, the Russian Federation, Tajikistan and the Ukraine (9,10).Some regions appear to have higher incidence rates than others: during recent years, for example, there has been an increase in the number of diphtheria cases reported in the Smolensk region of the Russian Federation.In 2001, 30 cases were reported in Smolensk, giving an incidence of 2.66 per 100 000 population, while the incidence in the Russian Federation as a whole was only 0.57 per 100 000 population.Seventy three per cent of the cases were adults, and two adult patients died.A dramatic increase in carriage rates of toxigenic C. diphtheriae strains was also observed.When there is an increase in the incidence of diphtheria, additional anti-epidemic measures may be needed to increase the coverage rates in all age groups.
Beginning in 1998, an annual increase of diphtheria incidence was also observed in the St Petersburg region, with an incidence of 2.6 per 100 000, mainly in adult patients.Since 2000, most of the diphtheria cases have been caused by toxigenic C. diphtheriae var mitis ribotype Otchakov.Screening and serological investigations amongst different social and professional risk groups have recently been performed in this region.A total of 472 sera from individuals aged between 20 sibles à la diphtérie.Cinquante deux pour cent des sujets entre 40 et 59 ans n'avaient reçu qu'une dose de rappel, et parmi ceux qui en avaient reçu deux, le nombre de personnes sensibles n'était que de 25 %.Pour un contrôle efficace de l'épidémie dans la région, la vaccination des adultes à l'aide de deux doses de rappel peut être nécessaire.L'immunisation avec une dose de rappel a débuté dans la région chez la population adulte, mais il semble que le ribotype Otchakov représente une nouvelle menace pour la santé publique.
Actuellement, la Lettonie présente le taux d'incidence de la diphtérie le plus élevé de tous les Etats membres ou des pays candidats.Entre septembre 1993 et 2001, 1 288 cas de diphtérie, dont 96 décès, ont été déclarés.Deux pics d'incidence ont été ➤ to 69 years were studied, and 35% were susceptible to diphtheria.Fifty two per cent of individuals aged between 40 and 59 years had only ever been given one booster vaccination.Among those who had been given two boosters, the proportion of susceptibles was only 25%.For effective control of the epidemic in this district, immunisation of adults with two boosters may be required.Immunisation of the booster vaccine to the adult population has begun in this region, but it seems that ribotype Otchakov may present a new public health threat in the future.

Epidemiology of diphtheria in EU member states and accession countries
Although the risk of resurgence of diphtheria in EU member state countries is relatively low, the capacity to control and prevent diphtheria must be maintained due to close links with other higher incidence countries.The ability to recognise an imported case of diphtheria rapidly and to control any potential spread of the disease within the community is a public health priority (3).Laboratory facilities, clinical expertise, and surveillance all need to be maintained.Additionally, travellers to countries where the disease is endemic need to be protected, so adequate supplies of antitoxin and toxoid vaccine must be available when required.
Many of the EU member states and accession countries see either no cases or very few cases of diphtheria each year.However, the WHO Collaborating Centre for Diphtheria at the Respiratory and Systemic Infection Laboratory of the Health Protection Agency in the United Kingdom (UK) identified 36 isolates of toxigenic corynebacteria between the beginning of 1997 and the end of 2002 from throat swabs and other samples types referred for toxigenicity testing, 58% (21/36) of which were identified as C. ulcerans.Infection with C. ulcerans in humans is 'traditionally' associated with consumption of raw dairy products or contact with farm animals; however, no such risk factors were identified for about half of the cases.
In April 1993, the first imported case of diphtheria for nearly thirty years was identified in Finland and was found to be caused by toxigenic C. diphtheriae var gravis.Since then, a total of fifteen isolations of C. diphtheriae have been reported, the majority produced toxin (13/15) and were var gravis (10/15).Twelve of the cases had epidemiological links to Russia and were caused by toxigenic C. diphtheriae.Based on ribotype analysis, most of the var gravis isolates were indistinguishable from the the Russian epidemic clone group Sankt-Peterburg/Rossija (11,12).Despite extensive traffic across the border, the small number of diphtheria cases identified in Finland in recent years suggests that the overall immunity of the population is relatively good (13).
During 2002, the first case of diphtheria since 1989 was identified in France in a female Chinese immigrant who presented with dysphasia and fever.She had lived in France for the last two years and was probably unimmunised.Toxigenic C. diphtheriae var mitis was isolated from a throat swab taken from this patient which revealed a new ribotype pattern Lariboisière.Also in 2002, the first case of diphtheria in Italy since 1998 was seen in a 14 year old boy from Friuli, northern Italy, who presented with fever, sore throat, lymphadenopathy and pseudomembrane.Toxigenic C. ulcerans was isolated from a throat swab, but the source of the infection was not ascertained.

Caractéristiques moléculaires et génétiques de corynebacteria
Le ribotypage, méthode universelle de typage moléculaire des bactéries basé sur la détermination du profil de restriction de l'ARN ribosomial est reconnu comme étant le meilleur marqueur pour le typage moléculaire des isolats de C. diphtheriae.Les endonucléases utilisées en standard étaient BstEII (premier choix) et PvuII (second choix).Les ribotypes obtenus avec ces enzymes ont souvent ➤1994 and 1996 and then between 1999 and the present time.During the first peak, more than 71% diphtheria cases were reported amongst adults, most of whom were unvaccinated, and especially high morbidity levels was seen in those aged between 30 and 59 years (14).This is consistent with the usual epidemiology of the disease in a population that has high levels of childhood vaccination, but incomplete adult coverage.During the last increase in incidence, however, the proportion aged between 30 and 59 years decreased by 19%, and 97% had been vaccinated against diphtheria in childhood.The reasons for this last resurgence are still not completely understood.

Clinical and microbiological aspects of infection caused by potentially toxigenic and other corynebacteria
A large seroepidemiological study of 558 injured patients aged between 18 and 70 years was conducted at an outpatient clinic of a trauma department in Vienna, Austria.Twenty seven per cent of the subjects were susceptible to diphtheria, 27% had basic protection, and 46% were fully protected (15).Multiple linear regression analysis revealed that age and gender both had a significant independent influence on diphtheria immunity level.In another study carried out in northern Greece on sera from 429 healthy children and adults aged between 0 and 80 years, immunity rates against diphtheria were found to be protective in high proportions of individuals up to 20 years of age, after which time immunity levels declined sharply.Another seroepidemiological study in Slovenia also showed a high level of protective immunity in children and young adults and, again, there was evidence of a fall in the level of protective antibodies in adults.
Between 1998 and 2000, antibiotic susceptibility tests were carried out on 148 isolates of C. diphtheriae from diphtheria patients and carriers in the Moscow, Murmansk, Omsk, Kaliningrad, Vladimir and Krasnodar regions of Russia.Sixteen of these isolates (11%), all of which had been characterised as the epidemic clonal group of toxigenic C. diphtheriae var gravis ribotype Sankt-Peterburg/Rossija, were found to be resistant to macrolide and lincosamide antibiotics such as erythromycin, azythromycin, lincomycin, and clindamycin (11).No antibiotic-resistant strains were found among isolates that circulated in Russia between the early 1980s and 1998.
The completion of the genome sequence of C. diphtheriae offers new strategies for studying host-pathogen interactions.An investigation was described that was recently undertaken in Brazil to examine the interactions leading up to and involving the adherence process of C. diphtheriae to human epithelial cells, the primary step made in the colonisation and invasion of the host (16,17).Thirteen strains were tested and displayed varying degrees of attachment to HEp-2 cell monolayers with two distinct adherence patterns: the predominant type called localised (LA), and diffuse (DA) (18).The LA pattern was mainly observed among the glass adherent/sucrose fermenting strains (19).Bacterial adherence to HEp-2 cells was inhibited in varied degrees by carbohydrate moieties.Initial indications point to these carbohydrate components as putative adhesins of C. diphtheriae to human epithelial cells.
In 2002, a toxigenic strain of C. diphtheriae var mitis was isolated from a throat swab of a boy aged 11 years from a religious community in Salford, north west England (23).A toxigenic strain of C. diphtheriae var mitis was also isolated from a family member aged 3 years in Jerusalem and the two isolates were found to be indistinguishable from their ribotype patterns.This is an excellent example of how ribotyping can be used in 'tracking' the international spread of this disease.Disease transmission does not necessarily occur as an isolated event in one country but may have implications for spread to other countries (3).
In Belarus, the majority of toxigenic strains in the last few years have been biotype var gravis and their ribotypes were mainly Sankt-Petersburg, Rossija, and Lyon ribotypes.Many of the strains circulating in Belarus, however, have been nontoxigenic tox-bearing (NTTB) strains of C. diphtheriae var mitis that are phenotypically non-toxigenic but carry the tox-gene, and these have been mainly represented by the ribotype Moskva, while the other non-toxigenic strains were mainly ribotype Cluj (11,21).Ribotyping has successfully been used for molecular subtyping and the identification and monitoring of clonal groups (24).
As previously mentioned, human infection with C. ulcerans is usually acquired through contact with animals or by ingestion of unpasteurised dairy products, although risk factors are often undetermined (3).In the UK, the frequency and severity of C. ulcerans infections appear to be increasing.Between 1986 and 2001 a total of 51 C. ulcerans isolates, 84% of which were toxigenic strains, were identified.Ribotyping results of toxigenic C. ulcerans isolated from three cats with bilateral nasal discharge have shown that the profiles produced are so far indistinguishable from human isolates (25).The isolation of toxigenic C. ulcerans from domestic cats, in addition to the possibility of person to person spread, highlights the importance of further research into the role of C. ulcerans in the epidemiology of diphtheria (15).
The complete genome of the C. diphtheriae NCTC 13129 strain, an isolate of the epidemic clone (ribotype Sankt-Petersburg), circulating within eastern Europe during the 1990s, has been sequenced at the Pathogen Sequencing Unit at the Sanger Institute.A whole-genome shotgun technique was used and the final sequence was assembled from 66 099 sequencing reads.Analysis of the sequence was carried out using the software tool Artemis and the Artemis Comparison Tool (ACT) was also used to compare this genome with other bacterial genomes, both at the DNA or protein level.The genome sequence data is available at http://www.sanger.ac.uk/Projects/C_diphtheriae/. ➤ ➤ Le séquençage de ce génome a permis de mener d'autres études sur les caractéristiques moléculaires et génétiques des corynebacteria.Une étude complète des souches NTTB, toutes de biotype C. diphtheriae var mitis, a été réalisée récemment à Moscou (26 27,28).L'absence de production de toxine diphtérique par ces souches s'est avérée liée à une mutation -délétion d'une base au niveau du nucléotide 52-55 -se traduisant par un décalage de lecture entraînant la perte de la séquence de début de lecture du gène tox conduisant à la production d'une protéine tox tronquée.Cependant, des chercheurs ont découvert des propriétés toxigéniques à ces souches NTTB.Ils émettent l'hypothèse que la production de la toxine peut facilement être restaurée après passage à travers une dialyse en cellophane sur un milieu Elek déficient en fer.
➤ The completion of this genome sequence has enabled the development of further studies into the molecular and genetic characteristics of corynebacteria.A comprehensive study of NTTB-strains, all of biotype C. diphtheriae var mitis, was recently carried out in Moscow (26,27,28).The lack of diphtheria toxin production by these strains was explained by a mutation a single base deletion at nucleotides 52-55 -that was the result of a frameshift that subsequently produced a loss of the tox-gene open reading frame and the truncation of the tox protein.However, some researchers have discovered toxigenic properties in these NTTB strains and argued that toxin production can be easily restored after passage through dialysis cellophane on iron-deficient Elek media.
At the University of Colorado in the US, an impressive molecular analysis was carried out on the regulation and function of the diphtheria toxin repressor (DtxR) of C. diphtheriae (27).The conclusion to this study was that DtxR represses siderophore biosynthesis more stringently than diphtheria toxin production, and that that DtxR was not essential for the viability of C. diphtheriae, although those strains with mutations within the dtxR gene did exhibit enhanced susceptibility to oxidative stress (29).
Further research and exploration into the genome sequence of C. diphtheriae are providing sources of inspiration and new understanding of the pathogenicity of toxigenic, NTTB, and non-toxigenic strains.The numbers of participants in this international conference highlighted the expansion of this specialised area of microbiology, with attendees several continents.An international surveillance network within Europe is essential for the control of a resurging, potentially fatal, vaccine-preventable infectious disease such as diphtheria.Diphtheria is an excellent example of a situation where public health services are expected to deliver a timely and coordinated response to a public health threat such as an imported case that may originate outside Europe, but which has the potential to spread within that country.
The authors gratefully acknowledge the contribution of all participants of the Seventh International Meeting of the ELWGD, the European Commission (EU DG SANCO Agreement No. S12.324473 (2001 CVG4-012)), the Public Health Laboratory Service, the Department of Traumatology at the University of Vienna, the Federal Ministry of Social Security and Generations in Vienna, the Vienna Medical Academy, the WHO Regional Office for Europe, and the Wellcome Trust.
The Programme and Abstracts Book for the meeting are accessible in pdf format on the Eurosurveillance website.■

C. Berghold 1 ,11
C. Kornschober 1 , S. Weber 2 National Reference Centre for Salmonella at the Austrian Agency for Health and Food Safety, Graz, Autriche 2 Austrian Poultry Health Service, Tulln, Autriche Au cours du printemps et de l'été 2002, le Nazionale Referenzzentrale für Salmonellen (Centre National de Référence autrichien pour les salmonelles -NRCS) a observé un foyer d'infections humaines à Salmonella enterica subsp.enterica ser.Enteritidis lysotype 5 (S.Enteritidis PT5) dans deux districts proches l'un de l'autre en Autriche.En 1999, une épidémie également liée à S. Enteritidis PT5 était survenue dans cette région chez un producteur avicole (6 000 poules environ).L'attention a donc été à nouveau portée sur cet élevage.Les analyses bactériologiques des échantillons prélevés dans cette ferme et les investigations épidémiologiques ont montré que l'épidémie de 2002 était liée au même producteur que l'épidémie de 1999.Les 70 isolats humains de S. Enteritidis PT5 confirmée en 2002 ne représentaient qu'une minorité de toutes les infections.Il est probable que plusieurs centaines d'infections soient survenues au cours de l'épidémie de 2002.Depuis août 2001, les nouvelles vagues d'élevage n'étaient plus vaccinées contre Salmonella.Il est probable que ce changement ait causé la réapparition des infections à S. Enteritidis PT5.Dès la fin du mois de septembre 2002, plus aucun oeuf provenant de poules non traitées n'était mis en vente.En octobre 2002, seul un isolat de S. Enteritidis PT5 a été confirmé dans la région.Historique A u milieu de l'année 2002, le Nazionale Referenzzentrale für Salmonellen (Centre National de Référence autrichien des salmonelles -NRCS) a observé un foyer régional d'infections humaines à Salmonella enterica subsp.enterica ser.Enteritidis lysotype 5 (S.Enteritidis PT5) (1).La zone affectée consistait en deux ➤ A regional outbreak of S. Enteritidis phage type 5, traced back to the flocks of an egg producer, Austria C. Berghold 1 , C. Kornschober 1 , S. Weber 2 National Reference Centre for Salmonella at the Austrian Agency for Health and Food Safety, Graz, Austria 2 Austrian Poultry Health Service, Tulln, Austria In the spring and summer of 2002, the Nationale Referenzzentrale für Salmonellen (National Reference Centre for Salmonella -NRCS) in Austria noticed a cluster of human Salmonella enterica subsp.enterica ser.Enteritidis phage type 5 (S.Enteritidis PT5) infections in two neighbouring districts of Austria.Another small outbreak of S. Enteritidis PT5 infections that occurred in the same region in 1999 had been traced back to the flocks of a local egg producer (approximately 6 000 hens).Attention was therefore again directed at this farm.The results of voluntary bacteriological examinations from the farm and further epidemiological investigations identified the same egg producer as the source of the second outbreak.The 70 human isolates of S. Enteritidis PT5 ascertained in 2002 represented a minority of all infections.It is realistic to estimate that several hundred infections occurred in the course of the 2002 outbreak.The farmer had not vaccinated new flocks against Salmonella since August 2001.It is likely that the change in vaccination policy resulted in the reappearance of the S. Enteritidis PT5 infections.By the end of September 2002 the farmer had stopped selling untreated table eggs.In October 2002 only one isolate of S. Enteritidis PT5 was ascertained in the region.Background I n mid-2002, the Nationale Referenzzentrale für Salmonel- len (NRCS) noticed a regional cluster of human Salmonella enterica subsp.enterica ser.Enteritidis phage type 5 (S.Enteritidis PT5) infections (1) in a region consisting of two neighbouring districts (combined population of the two ➤ RAPPORT D'INVESTIGATION OUTBREAK REPORT