Vol. 16 Weekly issue 31 4 August 2011

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1 E u r o p e s j o u r n a l o n i n f e c t i o u s d i s e a s e e p i d e m i o l o g y, p r e v e n t i o n a n d c o n t r o l Vol. 16 Weekly issue 31 4 August 2011 Editorials Enterohaemorrhagic Escherichia coli O104:H4: are we prepared now? 2 by AW Friedrich Rapid communications Ongoing measles outbreak in Romania, by A Stanescu, D Janta, E Lupulescu, G Necula, M Lazar, G Molnar, A Pistol Evidence of enzootic circulation of West Nile virus (Nea Santa-Greece-2010, lineage 2), Greece, May to July by A Chaskopoulou, CI Dovas, SC Chaintoutis, I Bouzalas, G Ara, M Papanastassopoulou Household transmission of haemolytic uraemic syndrome associated with Escherichia coli O104:H4, south-western France, June by B Aldabe, Y Delmas, G Gault, B Vendrely, B Llanas, M Charron, C Castor, N Ong, FX Weill, P Mariani- Kurkdjian, F Terrier, M Desjardin, J Simões, B Le Bihan, C Combe, P Rolland Surveillance and outbreak reports Secondary transmissions during the outbreak of Shiga toxin-producing Escherichia coli O104 in Hesse, Germany, by AM Hauri, U Götsch, I Strotmann, J Krahn, G Bettge-Weller, HJ Westbrock, O Bellinger, H Upphoff Review articles Usutu virus potential risk of human disease in Europe 22 by A Vázquez, MA Jiménez-Clavero, L Franco, O Donoso-Mantke, V Sambri, M Niedrig, H Zeller, A Tenorio

2 Editorials Enterohaemorrhagic Escherichia coli O104:H4: are we prepared now? A W Friedrich (alex.friedrich@umcg.nl) 1 1. Department of Medical Microbiology and Infection Control, University Medical Centre Groningen, University of Groningen, the Netherlands Citation style for this article: Friedrich AW. Enterohaemorrhagic Escherichia coli O104:H4: are we prepared now? Euro Surveill. 2011;16(31):pii= Available online: Article published on 4 August 2011 It is over. The outbreak of the enterohaemorrhagic Escherichia coli (EHEC) O104:H4 infection that had its major focus in Germany [1] and affected people in many other European countries has officially come to an end [2]. While the media coverage has been decreasing, the scientific community has been working to understand the reason why this dramatic outbreak occurred. We have learnt that the pathogen is not a totally new clone, but is a slight variant of a known although rarely described EHEC, called HUSEC-41 [3] with an extended-spectrum beta-lactamase (ESBL) resistance. Furthermore, the strain carries genes typically found in two types of pathogenic E. coli, the enteroaggregative E. coli (EAEC) and EHEC [4,5]. It specifically carries the genes for the classical haemolytic uraemic syndrome (HUS)-associated Shiga toxin 2. Despite the efforts that have been made, major questions currently remain unanswered, such as why women were affected more than men, why the attack rate was so high, what the primary source was and what the reservoir is, how long people are carriers, what the importance of the ESBL resistance is, what the infectious dose is for this outbreak strain and what the role of secondary transmission is via symptomatic or asymptomatic carriers, directly to other persons or indirectly via an index source, such as food. It is known that up to 15% of EHEC cases can be a result of secondary transmission arising from household contact with people who have sporadic EHEC infections [6]. In this issue of Eurosurveillance, two articles, Aldabe et al. [7] and Hauri et al. [8] report on secondary transmission during the EHEC O104:H4 outbreak. The first reports on a symptomatic man who transmitted EHEC to his wife and young daughter during the EHEC O104:H4 infection in France [7]. Interestingly, the EHEC that was isolated from the mother apparently lost its ESBL resistance, confirming the known mobility of plasmids carrying resistance genes. This fact should be taken into consideration in diagnostic laboratories if ESBL resistance of EHEC O104:H4 is used for primary selection of the pathogen from stools without using also non-selective enrichment and detection of Shiga toxin genes. The second article [8] illustrates in detail the history of six possible household transmissions, two possible nosocomial and one possible laboratory transmission in the German State of Hesse, where satellite clusters occurred. These cases throw light on three crucial issues. First, secondary transmission of EHEC O104:H4 was shown not to be more frequent than expected. Second, the importance of microbiological serotyping was highlighted, as EHEC of other HUS-associated serogoups (O157, O91, and O103) were also identified during the outbreak. Serotyping data are rarely available, due to the need for time-consuming techniques usually only carried out in specialised reference labs. This shows the need for the development of rapid seroand pathotyping methods for all HUS-associated E. coli strains. Third, infection control in hospitalised patients with EHEC infection needs specific consideration, as does laboratory safety in the handling of EHEC. It is not without reason that in most countries of the European Union EHEC is classified as a biosafety level (BSL)-3** microorganism (but no high-efficiency particulate air (HEPA) filter is required). Both articles illustrate the importance of personal hygiene in preventing secondary transmission. In general, EHEC does not behave differently to any other organism transmitted via the faecal oral route, but our preventive doors for such organisms seem to stay wide open. We have become used to the fact that hundreds of thousands of Europeans have diarrhoea every year and a certain lack of basic hygiene seems to be acceptable, as usually nothing very severe happens. We often lack time for hand hygiene as we consider it not to be of great importance. However, diarrhoea is not a normal state. We forget that most enteropathogens are less infectious than EHEC or do not lead to such severe disease with such social visibility. This brings us to the biggest challenge. Circulating highly pathogenic and/ or multiresistant microorganisms can be detected at a very early stage, before large outbreaks of disease occur. Preventive microbiology is a basis for preventive 2

3 medical advice and decision-making to protect people from infections. In future, European-wide coordination of preventive microbiology will be crucial for early detection of major health threats caused by infectious diseases. Its success will depend on our international and interdisciplinary efforts to foster protection against infection. This outbreak is over. Let us get prepared! References 1. Frank C, Werber D, Cramer JP, Askar M, Faber M, an der Heiden M, et al. Epidemic profile of Shiga-toxin-producing Escherichia coli O104:H4 Outbreak in Germany - preliminary report. N Engl J Med Jun 22. [Epub ahead of print] 2. Robert Koch Institute (RKI). Informationen zum EHEC/HUS- Ausbruchsgeschehen Ende des Ausbruchs [Information about the EHEC/HUS-outbreak end of the outbreak ]. Berlin: RKI; German. Available from: nn_467482/de/content/infaz/e/ehec/info-hus,templateid=ra w,property=publicationfile.pdf/info-hus.pdf 3. Mellmann A, Bielaszewska M, Köck R, Friedrich AW, Fruth A, Middendorf B, et al. Analysis of collection of hemolytic uremic syndrome-associated enterohemorrhagic Escherichia coli. Emerg Infect Dis. 2008;14(8): Bielaszewska M, Mellmann A, Zhang W, Köck R, Fruth A, Bauwens A, et al. Characterisation of the Escherichia coli strain associated with an outbreak of haemolytic uraemic syndrome in Germany, 2011: a microbiological study. Lancet Infect Dis Jun 22. [Epub ahead of print] 5. Mellmann A, Harmsen D, Cummings CA, Zentz EB, Leopold SR, Rico A, et al. Prospective genomic characterization of the German enterohemorrhagic Escherichia coli O104:H4 outbreak by rapid next generation sequencing technology. PLoS One. 2011;6(7):e Parry SM, Salmon RL. Sporadic STEC O157 infection: secondary household transmission in Wales. Emerg Infect Dis. 1998;4: Aldabe B, Delmas Y, Gault G, Vendrely B, Llanas B, Charron M, et al. Household transmission of haemolytic uraemic syndrome associated with Escherichia coli O104:H4, south-western France, June Euro Surveill. 2011;16(31):pii= Available from: aspx?articleid= Hauri AM, Götsch U, Strotmann I, Krahn J, Bettge-Weller G, Westbrock HJ, et al. Secondary transmissions during the outbreak of Shiga toxin-producing Escherichia coli O104 in Hesse, Germany, Euro Surveill. 2011;16(31):pii= Available from: aspx?articleid=

4 Rapid communications Ongoing measles outbreak in Romania, 2011 A Stanescu (aurora.stanescu@insp.gov.ro) 1,2, D Janta 1,2, E Lupulescu 3, G Necula 3, M Lazar 3, G Molnar 4, A Pistol 1,2 1. National Institute of Public Health, National Centre for Communicable Diseases Surveillance and Control, Bucharest, Romania 2. These authors contributed equally to this work 3. National Institute of Research and Development for Microbiology and Immunology Cantacuzino National Reference Laboratory for Measles and Rubella, Bucharest, Romania 4. Ministry of Health, Bucharest, Romania Citation style for this article: Stanescu A, Janta D, Lupulescu E, Necula G, Lazar M, Molnar G, Pistol A. Ongoing measles outbreak in Romania, Euro Surveill. 2011;16(31):pii= Available online: Euro Surveill. 2011;16(31):pii= Available online: Article published on 4 August 2011 Since January 2011 Romania has been experiencing a measles outbreak with 2,072 cases notified in 29 of the 42 Romanian districts. Most cases occurred in the north-western part of the country among unvaccinated children with the highest number of cases (893 cases) registered in children aged one to four years. This report underlines once more the need for additional measures targeting susceptible populations to achieve high vaccination coverage with two doses of measles-mumps-rubella vaccine. Between January and June 2011, 2,072 measles cases were notified in 29 of the 42 Romanian districts with most cases registered in the north-western part of the country mainly among unvaccinated children. No measles-related deaths have so far been notified in An outbreak of measles was first noticed in late August 2010 in the north-eastern part of Romania [1] and by the end of the year, 193 cases were registered in the whole country. Measles is a statutorily notifiable disease since 1978 in Romania, and medical practitioners have to immediately report all suspected measles cases to the local public health authorities. At national level, the National Centre for Communicable Diseases Surveillance and Control in Bucharest collects and analyses all notifications of measles cases. National case-based notification was initiated in 1999 and the European Union (EU) case definition and case classification have been adopted since 2005 [2]. The monovalent measles-containing vaccine was introduced in 1979 in the Romanian immunisation schedule for children aged 9-11 months. In 1994, the second measles vaccine dose was introduced for children aged between six and seven years (first school grade). The combined measles-mumps rubella (MMR) vaccine replaced the monovalent measles vaccine in 2004 and was recommended as a first dose for children aged months. The second MMR vaccine has been recommended for children aged between six and seven years since October Between 2000 and 2008 the national measles vaccination coverage for children aged between 18 and 24 months with the first dose of measles-containing vaccine was estimated at 97%-98% and for children aged seven years, the vaccination coverage with the second dose of measles-containing vaccine was estimated at 96%-98% [3]. In the last two years a constant decrease could be noticed in the measles vaccination coverage for children aged 12 months. According to the vaccination coverage reports, in 2009, the coverage for the first MMR vaccine dose was 85.1% (95% CI: ) at the age of 12 months and reached the target of 95% (95% CI: ) coverage for children aged 18 months. A high number of children remain unvaccinated not only in the hard-to-reach communities but also in the general population, due to parental refusal and scepticism regarding the benefits of the vaccination. Vaccination coverage for the second dose of MMR vaccine is reported every year by the school medical staff to the local health authorities after the school vaccination campaign. In 2010, the reported coverage for the second dose of measles-containing vaccine, calculated using the number of doses administrated divided by the total number of eligible children aged seven years was 93.4% (95% CI: ). Here we report an ongoing measles outbreak in Romania by analysing measles data available from 1 January to 30 June Descriptive analysis was performed using the national surveillance standardised form sent by the public health authorities of each district to the National Centre for Communicable Diseases Surveillance and Control. Outbreak description From the beginning of 2011 until 30 June, a total number of 2,072 measles cases were notified by the local public health authorities. The highest number of cases was registered among children aged between one and four years (893 cases), followed by the five-nine year-olds (445 cases) and the infants under one year of age (303 cases). Among the year-olds there were 189 cases identified, 150 cases in those aged 20 years and above and 92 cases were registered among adolescents aged 4

5 between 15 and 19 years. Among the total number of cases, approximately half occurred in hard-to-reach communities. The monthly incidence increased from 131 cases registered in January to a peak of 515 cases in May, and decreased in June when the number of notified cases was 437 (Figure 1). The laboratory confirmation was performed by detecting measles IgM antibodies in serum samples. Due to many local outbreaks, the laboratory confirmation was performed only in some of the first cases identified in a particular area until D4 genotype was confirmed. For those cases with clear epidemiological link with the outbreak, the epidemiological confirmation criteria were used. Of the 2,072 notified measles cases, 898 were laboratory-confirmed, 1,161 were probable cases with Figure 1 Monthly incidence of notified measles cases, Romania, 1 January 30 June 2011 (n=2,072 cases) Incidence (per 100,000 population) Jan Feb Mar Apr May Jun 2011 documented epidemiological link and 13 were clinical measles cases for whom sera could not be obtained due to parental refusal. RT-PCR techniques to detect measles virus nucleic acid were also used to confirm the first cases from some affected districts. Twelve viruses were genotyped by a nested RT-PCR reaction which targeted a 450 nt region at the C-terminus of the N protein (Nc region). All of them belonged to D4 genotype currently circulating in Europe [4]. Measles spread in 29 districts (including Bucharest) of a total of 42 and the geographical distribution shows a concentration of measles cases in the north-western part of the country (Figure 2). Of the total number of 2,072 measles cases, 800 (38.6%) presented complications: 582 (72.8%) cases developed pneumonia, 203 (25.4%) diarrhoea, eight (1%) malnutrition, five (0.62%) convulsions and two (0.25%) encephalitis. The median age was three years (range: three weeks 43 years). The highest incidence (138.4 per 100,000 population) was in infants not eligible for vaccination (under one year of age), followed by the one to four year-olds (103.4 per 100,000 population) and the five to nine year-olds (42.3 per 100,000 population) (Figure 3). For the older age groups the incidence ranged between 17.1 per 100,000 population and 1.8 per 100,000 population. Figure 2 Distribution of notified measles cases, Romania, 1 January 30 June 2011 (n=2,072 cases) One dot represents one case Satu Mare Maramures Suceava Botosani Arad Timis Bihor Salaj Cluj Alba Hunedoara Bistrita-Nasaud Iasi Neamt Mures Sibiu Harghita Bacau Covasna Brasov Vrancea Vaslui Galati Caras-Severin Gorj Mehedinti Dolj Valcea Olt Buzau Prahova Arges Braila Dambovita Ialomita Bucuresti Ilfov Calarasi Giurgiu Teleorman Tulcea Constanta 5

6 Most cases occurred among unvaccinated children representing 72.8% from the total number of cases registered during period mentioned above. Of these, only 19.8% were not eligible for MMR vaccination due to their age (under 12 months) (Figure 4). Control measures Several control measures have been implemented by the local health authorities in their efforts to stop this outbreak. An additional MMR vaccination campaign started in the affected areas targeting all children aged between seven months and seven years, irrespective of their measles vaccination status. Nevertheless, no change has yet been foreseen in the national immunisation schedule regarding the administration of the first dose of MMR vaccine. The MMR vaccine is supplied by the Ministry of Health and is offered free of charge through the routine immunisation services (family doctors) and special outreach teams. As of 30 June 2011, 4,500 children have been vaccinated with Figure 3 Measles incidence by age groups, Romania, 1 January 30 June 2011 (n=1,922 cases a ) Incidence (per 100,000 population) < 1 year 1-4 years 5-9 years years years Age groups a Cases aged 20 years and above (n=150) are not included in the figure. Figure 4 Percentage of measles cases by age group and vaccination status, 1 January 30 June 2011 (n=2,072 cases) Percentage (%) <1 year a 1-4 years 5-9 years years years 20 years Age groups 0 doses 1 dose 2 doses a 2.6% were vaccinated during the incubation period in the course of the additional vaccination campaign targeted at children aged seven months seven years. measles-containing vaccine, following this additional vaccination campaign. Active case finding was initiated by general practitioners in the areas most affected by the outbreak, as well as contact-tracing in hospitals and in the community. Other activities such as meetings with local public health representatives were undertaken by the national public health authorities in order to increase awareness on the ongoing outbreak not only among physicians but also in the general population. Discussion and conclusions In Romania, the measles incidence dropped from 16.3 and 1.6 per 100,000 population in respectively, to less than 0.1 per 100,000 population in [5-7]. Despite the high national immunisation coverage with MMR vaccine reported during the last 10 years, this outbreak highlights the presence of pockets of individuals vulnerable to measles and particularly those members of hard-to-reach communities but not only. We observed that more parents, even among highly educated persons, lost their confidence in vaccination benefits for their children and this became an important problem that needs to be addressed. The current measles outbreak in Romania and in other European countries reveal the need for increased awareness on the declining confidence that people have in vaccination benefits for their children and for public health intervention focused in hard-to-reach communities. In addition, after the pandemic influenza A(H1N1)2009, a constant scepticism and refusal regarding vaccination in general could be noticed, not only in hard-to-reach communities, but also in the general population. In areas and communities where vaccine coverage remains sub-optimum, large cohorts of susceptible people accumulate and represent a potential for large outbreaks. The large proportion of cases observed in infants suggests an intensely circulating measles virus [7]. Acknowledgements We wish to thank all the colleagues from the local public health authorities for their assistance and involvement in this investigation. References 1. Stanescu A, Muscat M, Romaniuc A, Pipirigeanu R, Lupulescu E, Necula G, et al. Spotlight on measles 2010: An ongoing measles outbreak in the district of Neamt, Romania, August September Euro Surveill. 2010;15(40):pii= Available from: aspx?articleid= European Commission. Commission Decision of 19 March 2002 laying down case definitions for reporting communicable diseases to the Community network under Decision No 2119/98/EC of the European Parliament and of the Council (2002/253/EC), Official Journal of the European Union. Luxembourg: Publications Office of the European Union. 3 Apr Available from: LexUriServ.do?uri=OJ:L:2002:086:0044:0062:EN:PDF 6

7 3. World Health Organization (WHO). Immunization coverage. WHO/UNICEF estimates of national immunization coverage. Geneva: WHO. Accessed Jul Available from: who.int/immunization_monitoring/routine/immunization_ coverage/en/index4.html 4. Mankertz A, Mihneva Z, Gold H, Baumgarte S, Baillot A, Helble R, et al. Spread of measles virus D4-Hamburg, Europe, Emerg Infect Dis. 2011;17(8): EUVAC.NET. Measles surveillance annual report EUVAC. NET. 12 Nov Available from: graphics/euvac/pdf/annual_2008.pdf 6. EUVAC.NET. Measles surveillance annual report EUVAC. NET. 25 May Available from: graphics/euvac/pdf/annual_2009.pdf 7. Muscat M, Bang H, Wohlfahrt J, Glismann S, Mølbak K, EUVAC. NET group. Measles in Europe: an epidemiological assessment. Lancet;2009;373(9661):

8 Rapid communications Evidence of enzootic circulation of West Nile virus (Nea Santa-Greece-2010, lineage 2), Greece, May to July 2011 A Chaskopoulou (andahask@ufl.edu) 1, C I Dovas 2, S C Chaintoutis 2, I Bouzalas 2, G Ara 3, M Papanastassopoulou 2 1. United States Department of Agriculture-Agricultural Research Service (USDA-ARS), European Biological Control Laboratory, Thessaloniki, Greece 2. Aristotle University of Thessaloniki, Faculty of Veterinary Medicine, Laboratory of Microbiology and Infectious Diseases, Thessaloniki, Greece 3. American Farm School, Thessaloniki, Greece Citation style for this article: Chaskopoulou A, Dovas CI, Chaintoutis SC, Bouzalas I, Ara G, Papanastassopoulou M. Evidence of enzootic circulation of West Nile virus (Nea Santa-Greece-2010, lineage 2), Greece, May to July Euro Surveill. 2011;16(31):pii= Available online: Article published on 4 August 2011 A West Nile virus (WNV) surveillance network including sentinel chickens was deployed in Thessaloniki county, Greece, from May to July For the first time in summer 2011, a chicken WNV isolate from 6 July was molecularly identified. The partial NS3 sequence was identical to that of the Nea Santa-Greece-2010 WNV lineage 2, detected in central Macedonia in This suggests that WNV is actively circulating in central Macedonia and that it may have overwintered in northern Greece. During 2010, Greece underwent the second largest West Nile virus (WNV) epidemic in Europe in the last two decades with 262 clinical human cases and 35 fatalities [1]. WNV lineage 2 was identified in two pools of Culex mosquitoes (Nea Santa-Greece-2010 virus) [2] and in wild birds [3] that were sampled during the epidemic season of 2010 from areas in close proximity to human cases. No active vector and arbovirus surveillance system was in place in Greece before the epidemic in We initiated a monitoring programme in 2011, from May to November, in order to understand subsequent transmission, to document virus activity, and to better assess the relative importance of vector species. A small scale mosquito and animal surveillance network was established in the county of Thessaloniki, one of the areas with the highest number of human cases during the epidemic of 2010 [1]. The long term objective of this project is to design within the following years an optimum, large scale arbovirus surveillance programme for Thessaloniki. We report here preliminary findings of the study that will have interest for public health authorities. Methods Sentinel chickens Six chicken flocks (six chickens per flock) were placed in stationary cages along the western and eastern edges of Thessaloniki (three flocks on each side) to monitor WNV activity in areas suitable for potential enzootic transmission [4] (Figure 1). These areas combine abundance of mosquito larval sites (e.g. rice fields) and potential habitat for migratory birds (e.g. Axios River delta) that may serve as reservoir populations for WNV. The flocks were placed within or in close proximity to residential communities that experienced abundant mosquito activity. All chickens were confirmed WNV antibody negative prior to placement in the field. For each flock, the chicken cage was divided in six compartments so that each chicken would be kept separate from the others. Chickens were bled through the ulnar vein weekly (about 1ml of blood sample per chicken). Mosquito population monitoring Carbon-dioxide (dry ice) baited Centers for Disease Control (CDC) light traps (John W. Hock, Gainesville, United States (USA)) were deployed once a week at 28 sites in the Thessaloniki area beginning 20 May 2011 (Figure 1). Traps were located at approximately equal intervals in order to provide a geographically representative sampling. Laboratory analysis Chicken plasma (0.5 ml) and sera (0.25 ml) were collected for virus detection and serology, respectively. Serum samples were tested by ELISA for the detection of WNV-specific antibodies using a commercial ELISA kit (ID Screen West Nile Competition, IDVET, France). After the detection of seroconversion, RNA was extracted from selected plasma samples previously taken from the seroconverted birds. RNA extracts were examined using a one tube RT-PCR screening protocol employing a primer pair (WNPolUp: 5 -TTTTGGGAGATGGTGGATGARGA-3 and WNPolDo2: 5 -CCACATGAACCAWATGGCTCTGC-3 ) designed for the specific detection of WNV and targeting a 144 bp part of the nonstructural protein 5 (NS5) gene. Samples found positive by the RT-PCR screening protocol, were additionally subjected to RNA reverse transcription using random hexamers, followed by two PCR assays employing a primer pair (WN-NS3up1: 5 -GCTGGCTTCGAACCTGAAATGTTG-3 8

9 RT-PCR screening protocol targeting NS5. All RNA samples were negative except in the case of the sentinel chicken in the city of Agios Athanasios (40⁰ N, 22⁰ E) which seroconverted on 13 July. More specifically, a band of expected size was obtained from one PCR product derived from a sample taken from that respective chicken, one week before seroconversion (6 July). The specific RNA extract was subjected to nested PCR, targeting the partial NS3 gene sequence, which was subsequently determined. The sequence was deposited in GenBank database under accession number JN and according to BLAST algorithm, it presented highest nucleotide sequence identity (99.73%) to that from Nea Santa-Greece-2010 virus derived from a Culex mosquito pool tested during the 2010 epidemic in Central Macedonia [7]. The inferred partial NS3 amino acid sequence was 100% identical to that of the Nea Santa-Greece-2010 WNV lineage 2. As in the Nea Santa-Greece-2010 virus NS3 sequence, the inferred NS3 residue 249 was determined to be proline, similar to several neuroinvasive lineage 1 WNV strains [6]. In contrast, all other investigated lineage 2 viruses have a NS3 protein with a histidine at this position [7]. and WN-NS3do1: 5 -CAATGATGGTGGGTTTCACGCT-3 ) targeting a 778 bp part of the nonstructural protein 3 (NS3) gene, and a nested primer pair (WN-NS3up2: 5 -GCAAGATACTTCCCCAAATCATCAAGG-3 and WN-NS3do2: 5 -TGTCTGGGATCTCTGTTTGCATGTC-3 ) targeting a respective 423 bp part. The nested PCR products were bidirectionally sequenced. The NS3 gene was selected for molecular characterisation because it is phylogenetically informative [5] and it encodes a protein residue (NS3-249) subject to adaptive evolution leading to increased viremia potential and virulence [6]. Results Seroconversion of the first sentinel chicken was detected in the agricultural area of west Thessaloniki in the city of Chalastra (40⁰ N, 22⁰ E) (Figure 1) on 29 June. On 13 July a second chicken seroconversion was detected in the city of Agios Athanasios (40⁰ N, 22⁰ E), followed by a third chicken seroconversion on 20 July in the same area. All prior samples collected from the three seroconverted chickens were tested using the one tube Figure 1 Location of mosquito traps (n=28) and sentinel chicken flocks (n=6) for West Nile virus surveillance, Thessaloniki county, Greece, 2011 Agios Athanasios Chalastra Thessaloniki Axios River Delta 10 kilometres Mosquito trap location Chicken flock location (collocated with mosquito trap) Agios Athanasios and Chlalastra are the two cities where enzootic circulation of the virus was detected. 9

10 The cumulative number of Culex mosquitoes trapped weekly in the agricultural area of Thessaloniki (Figure 2) was low (n=142) during the last week of May and the first two weeks of June. The population rapidly increased during the second half of June, with a peak (n=23,867) at the end of the month. During the following two weeks, the population decreased and then started building up again during the third week of July. So far, the most prevalent mosquito species in both residential and agricultural areas (rice-fields) were Culex pipiens followed by Culex modestus Ficalbi. It should be noted that C. modestus populations started building up significantly in early July. Testing for WNV in mosquitoes is in progress but no results are available at this stage of the surveillance programme. Discussion This is the first report of enzootic circulation of WNV Nea Santa-Greece-2010 in Greece during 2011, one year after the WNV epidemic in Greece. The virus in 2010 was detected in Nea Santa from a Culex mosquito pool [2], and in 2011 we detected an identical isolate (molecular characterization based on NS3 gene) in the agricultural area of west Thessaloniki in the city of Agios Athanasios, approximately 21 km southwest of Nea Santa. The 2011 Greek WNV isolate shows close genetic relationship to the lineage 2 goshhawk-hungary-2004 strain that emerged in Hungary in 2004 but differs from the latter in that it maintains the amino acid substitution H249P found in the Nea Santa-Greece-2010 isolate, which may be associated with increased virulence [7]. WNV lineage 1 strains are distributed in north Africa, Europe, America, Asia and Australia, whereas lineage 2 are mostly distributed in south Africa and Madagascar. Due to increased illness and death caused by WNV lineage 1 compared to lineage 2 in the past, lineage 2 strains were previously considered to be less virulent. However, recent evidence from Africa and Hungary demonstrated that lineage 2 strains may also result in severe disease [8,9]. Transmission in the sentinel chickens occurred immediately after the first significant Culex population peak, as has been observed in WNV outbreaks [11,12]. Two of the principal WNV vectors in Europe, C. pipiens and C. modestus [13], are highly abundant in the agricultural area of Thessaloniki and both species may be associated with the transmission of the virus. In Greece, so far, the virus has been isolated from two pools of C. pipiens mosquitoes during the epidemic of 2010 [2]. More studies are needed to increase our knowledge on the role of the aforementioned species in the enzootic, epizootic and tangential (e.g. to humans) transmission of WNV in Greece. Monitoring disease activity by using sentinel animals can provide critical information regarding periods of increased transmission. Surveillance networks involving sentinel animals and mosquitoes have been used in many parts of the world as an early warning system aiming to identify periods and locations of elevated risk of WNV disease transmission [14,15,16]. The rationale behind these surveillance networks is (i) to increase our understanding of the epidemiology of arboviruses, (ii) to identify the circumstances favourable to the appearance of the disease in humans before this occurs, and (iii) to guide mosquito control efforts in time and space to reduce the impact or likelihood of an epidemic. Arbovirus surveillance systems can be expensive and labour intensive, with weekly monitoring of chickens. This is nevertheless feasible and these systems have been successfully established in some regions [16]. In urban centres of increased vulnerability to mosquito Figure 2 Culex female mosquito abundance in the agricultural area of west Thessaloniki, Greece, 2011 (n=74,909) Up to now, no WNV genomic sequences have been published from the human cases during the 2010 epidemic and there is no direct evidence to incriminate the WNV Nea Santa-Greece-2010 strain as the cause of the 2010 human epidemic. The discovery of the same strain in sentinel chickens in 2011 suggests that the virus was able to overwinter in this region, consistent with current opinion on the endemicity of WNV in Europe [10]. Specifically, the reoccurrence of WNV in continuous years in the same places in Romania and Italy, involving humans and equines, is likely linked to the endemicity of the infection in the areas rather than to a new introduction of the virus [10]. This situation appears to parallel that experienced in California, USA, which has a similar climate (warm temperate, seasonal winter rainfall) [11], where WNV was introduced in 2003, quickly spread throughout the state, and became endemic with the ability to overwinter in a cycle between winter mosquitoes and birds. Number of trapped female a Culex mosquitoes per week 30,000 Culex mosquitoes 25,000 20,000 15,000 10,000 5, May Mean Temperature ⁰C 01 Jun 09 Jun 16 Jun 23 Jun 1 st seropositive chicken 30 Jun Sampling date, Jul 2 nd seropositive chicken 14 Jul 3 rd seropositive chicken a Female mosquitoes were targeted by mosquito traps and accidental male mosquito collection was negligible (<0.1%). 21 Jul Temperature ⁰C 10

11 borne epidemics, such as Thessaloniki (approximately 1 million inhabitants, in close proximity to prolific mosquito breeding environments), there is a need and demand for such systems and the benefits associated with their successful deployment outweigh the associated costs. This is the first active arbovirus surveillance system in place in Thessaloniki and in order to optimise its use, extensive data are required in the following years. These data could help create a useful disease surveillance tool that may increase our understanding of the disease transmission cycle and help the local authorities to design a local WNV response plan based on the disease transmission levels. It is encouraging, that through a small scale surveillance system, like the one described in this paper, we were able to detect WNV enzootic circulation in Greece before the onset of any human cases for the year of Acknowledgements This project is funded by the Mosquito Control Programme Contract of Thessaloniki County. The Development Agency of Thessaloniki (ANETH) is the implementation body of the programme and the funding is provided by the Greek government, the Regional Authority of Central Macedonia, the Local Union of Communities and Municipalities of Thessaloniki County (TEDK) and the eight municipalities of Thessaloniki County. We also thank all the residents of the Thessaloniki region who kindly accepted to host the sentinel chickens for the duration of the study. Most importantly, we thank the field technical team Nikos Kampragkos and Aristeidis Kampragkos for their excellent cooperation. 11. Reisen W, Lothrop H, Chiles R, Madon M, Cossen C, Woods L, et al. West Nile virus in California. Emerg Infect Dis. 2004; 10(8): Sacramento-Yolo Mosquito and Vector Control District. Annual Report, [Accessed 26 Jul 2011]. Available from: AnnualReport2006.pdf 13. Hubalek Z, Halouzka J. West Nile fever a reemerging mosquito-borne viral disease in Europe. Emerg Infect Dis. 1999; 5(5): Cernescu C, Nedelcu N, Tardei G, Ruta S, Tsai TF. Continued transmission of West Nile virus to humans in southeastern Romania, Infect Dis. 2000; 181(2): Gubler DJ, Petersen LR, Roehrig JT, Campbell GL, M.D., Ph.D.Komar N, Nasci RS et al. Epidemic/Epizootic West Nile Virus in the United States: Guidelines for Surveillance, Prevention, and Control. Centers of Disease Control and Prevention; [Accessed 26 Jul 2011]. Available from: Connelly CR, Carlson DB, editors Florida Coordinating Council on mosquito control. Florida mosquito control: The state of the mission as defined by mosquito controllers, regulators, and environmental managers. Vero Beach, Florida: University of Florida, Institute of Food and Agricultural Sciences, Florida Medical Entomology Laboratory. [Accessed 25 Jul 2011]. Available from: Documents/Florida_Mosquito_Control_White_Paper.pdf References 1. Hellenic Center for Disease Control and Prevention (HCDC). Report on West Nile Virus Epidemic, [Accessed 27 Jul 2011]. Greek. Available from: stories/keelpno/ios_ditikou_neilou/ekthesi_epidimias_2010_ revised.pdf 2. Papa A, Xanthopoulou K, Gewehr S, Mourelatos S. Detection of West Nile virus lineage 2 in mosquitoes during a human outbreak in Greece. Clin Microbiol Infect. 2011; 17(8): Valiakos G, Touloudi A, Iacovakis C, Athanasiou L, Bitsas P, Spyrou V, et al. Molecular detection and phylogenetic analysis of West Nile virus lineage 2 in sedentary wild birds (Eurasian magpie), Greece, Euro Surveill. 2011; 16(18):pii= Available from: aspx?articleid= Komar N. West Nile virus surveillance using sentinel birds. Ann N Y Acad Sci. 2001; 951: Gray RR, Veras NMC, Santos LA, Salemi M. Evolutionary characterization of the West Nile Virus complete genome. Mol Phylogenet Evol. 2010; 56(1): Brault AC, Huang CY, Langevin SA, Kinney RM, Bowen RA, Ramey WN, et al. A single positively selected West Nile viral mutation confers increased virogenesis in American crows. Nat Genet. 2007; 39(9): Papa A, Bakonyi T, Xanthopoulou K, Vasquez A, Tenorio A, Nowotny N. Genetic characterization of West Nile virus lineage 2, Greece, Emerg Infect Dis. 2011; 17(5): Murgue B, Zeller H, Deubel V. The ecology and epidemiology of West Nile virus in Africa, Europe and Asia. Curr Top Microbiol Immunol. 2002; 267: Erdelyi K, Ursu K, Ferenczi E, Szeredi L, Ratz F, Skare J, et al. Clinical and pathologic features of lineage 2 West Nile virus infections in birds of prey in Hungary. Vector Borne Zoonotic Dis. 2007; 7(2): Calistri P, Giovannini A, Hubalek Z, Ionescu A, Monaco F, Savini G, et al. Epidemiology of west nile in europe and in the mediterranean basin. Open Virol J. 2010; 4:

12 Rapid communications Household transmission of haemolytic uraemic syndrome associated with Escherichia coli O104:H4, south-western France, June 2011 B Aldabe 1,2, Y Delmas 2,3, G Gault 1, B Vendrely 3, B Llanas 3, M Charron 1, C Castor 1, N Ong 1, F X Weill 4, P Mariani-Kurkdjian 5, F Terrier 6, M Desjardin 6, J Simões 7, B Le Bihan 7, C Combe 3,8, P Rolland (Patrick.ROLLAND@ars.sante.fr) 1,8 1. French Institute for Public Health Surveillance (Institut de Veille Sanitaire; InVS), Regional office Cire Aquitaine, Bordeaux, France 2. These authors contributed equally to this work 3. Bordeaux University Hospital, Bordeaux, France 4. Institut Pasteur, National Reference Centre for Escherichia coli and Shigella, Paris, France 5. Robert Debré Hospital, Associated Laboratory to the National Reference Centre for Escherichia coli and Shigella, Paris, France 6. Robert Picqué Military Hospital, Villenave-D Ornon, France 7. Regional Health Agency of Aquitaine, Bordeaux, France 8. Both authors contributed equally to this work Citation style for this article: Aldabe B, Delmas Y, Gault G, Vendrely B, Llanas B, Charron M, Castor C, Ong N, Weill FX, Mariani-Kurkdjian P, Terrier F, Desjardin M, Simões J, Le Bihan B, Combe C, Rolland P. Household transmission of haemolytic uraemic syndrome associated with Escherichia coli O104:H4, south-western France, June Euro Surveill. 2011;16(31):pii= Available online: Article published on 4 August 2011 Following the outbreak of haemolytic uraemic syndrome (HUS) on June 2011 in south-western France, household transmission due to Escherichia coli O104:H4 was suspected for two cases who developed symptoms 9 and 10 days after onset of symptoms of the index case. The analysis of exposures and of the incubation period is in favour of a secondary transmission within the family. Recommendations should be reinforced to prevent person-to-person transmission within households. Introduction On 30 June 2011, an outbreak of haemolytic uraemic syndrome (HUS) and bloody diarrhoea was reported among attendees of an open day event at a children s community centre that took place on 8 June in a town near Bordeaux, south-western France [1]. The identified strain was Shiga toxin 2-producing Escherichia coli O104:H4, with the same characteristics as the strain that caused the recent outbreak in Germany [2,3,4]. As of 26 July 2011, 15 cases of bloody diarrhoea have been observed in relation with this event, nine of whom have developed HUS. An investigation was conducted to identify the vehicles of infection and to guide control measures. Preliminary results of interviews and trawling questionnaires suggested sprouts as the vehicle of transmission. Here we describe the two cases of HUS for whom household transmission of E. coli O104:H4 was suspected. The possibility of person-to-person transmission of E. coli O104:H4 has also been reported in the Netherlands [5]. Case descriptions Patient A On 18 June, a man in his 40s was admitted in a hospital near Bordeaux, with abdominal pain and bloody diarrhoea of two days. Stool samples were sent to the National Reference Centre for E. coli and Shigella in Paris. Four days after admission, the patient left the hospital and returned home. On 27 June, he was hospitalised again in the nephrology department of Bordeaux University Hospital with a diagnosis of HUS. Test results from stool samples showed the presence of E. coli O104:H4 possessing the stx2 gene, encoding Shiga toxin 2. The strain was negative for the gene coding for intimin (eae) but positive for aggr which regulates the expression of aggregative adherence fimbriae. The antimicrobial resistance pattern of the strain was similar to than seen in the outbreak strain in recent E. coli O104:H4 outbreak in Germany: ampicillin-resistant (R), cefotaxime-r, ceftazidime-r, imipenem-sensitive (S), streptomycin-r, kanamycin-s, gentamicin-s, sulfamethoxazole-r, trimethoprim-r, cotrimoxazole-r, tetracycline-r, chloramphenicol-s, nalidixic acid-r, and ciprofloxacin-r. The PCR analysis indicated the presence of the bla CTX-M-15 gene, encoding an extended-spectrum beta-lactamase (ESBL), and the presence of the bla TEM gene, encoding a penicillinase. After treatment, the patient gradually recovered from HUS. He returned home on 6 July having received specific hygiene guidelines from the hospital, notably recommendations about hand-washing. Patient A had participated in the open day event on 8 June, accompanied by his three-year-old child (Patient B). His wife and his second five-year-old child did not attend. Patient A s interview revealed that he had consumed three kinds of sprouts (fenugreek, mustard, and rocket) during the event. Patient B The three-year-old child of Patient A presented first symptoms of illness on 26 June, i.e. 18 days after the 12

13 open day and nine days after the onset of symptoms in Patient A. At onset, the child had abdominal pain followed after three days by bloody diarrhoea. Shiga toxin 2-producing E. coli O104:H4 was isolated from stool samples. Some bacterial colonies produced the ESBL, whereas others only the penicillinase. However, both types of colonies were also resistant to streptomycin, sulfamethoxazole, trimethoprim, cotrimoxazole, tetracycline, and nalidixic acid, as observed for other outbreak isolates [1]. On 3 July, the child developed anaemia, haemolysis and high urine protein-tocreatinine ratio, compatible with HUS and was admitted to the paediatric department of Bordeaux University Hospital. On 4 July, thrombocytopenia developed. The child gradually recovered after treatment and was discharged from hospital on 8 July. Patient B had participated in the open day event but, according to Patient A, did not consume sprouts. As the children had no access to the buffet unless accompanied by an adult, it is unlikely that the child has eaten any sprouts without the father s knowledge. Patient C On 2 July, a woman in her 30s, wife of Patient A and mother of Patient B, was admitted to Bordeaux University Hospital with bloody diarrhoea of six days, i.e. she had had the first signs of disease on 27 June, respectively 10 and 1 days after the onset of illness in Patient A and Patient B. Shiga toxin 2-producing E. coli O104:H4 was isolated from her stool samples. It was resistant to ampicillin, streptomycin, sulfamethoxazole, trimethoprim, cotrimoxazole, tetracycline, and nalidixic acid, but susceptible to extended-spectrum cephalosporins (i.e. only production of the penicillinase). The absence of the CTX-M-15 ESBL compared to the isolates from Patient A and some isolates from Patient B might be due to the mobilisation of insertion sequences usually present in the vicinity of the bla CTX-M-15 gene. This might be in relation to the absence of selective pressure by this class of antimicrobials. Patient C developed symptoms of anaemia, haemolysis, thrombocytopenia and proteinuria on 8 July. After treatment, she recovered gradually and she left the hospital on 12 July. Patient C had not attended the open day event and not consumed any type of sprouts during the two months previously. Family members The five-year-old child spent the whole period at home. It did not develop any symptoms but was admitted to the paediatric department of Bordeaux University Hospital for observation from 3 to 4 July. Two other relatives of Patients A, B and C shared meals in the family s house on 26 June. One of them stayed in the family s house between 29 June and 3 July and complained of severe fatigue. The other relative developed mild diarrhoea on 28 June. A rectal swab was performed for both with an O104 serology for the relative with severe fatigue. Both relatives stool samples were negative for the presence of E. coli O104:H4. Hypothesis of transmission We hypothesised that Patients B and C both probably acquired HUS by secondary transmission from Patient A because they developed illness 9 and 10 days, respectively, after Patient A s symptom onset. Figure Two cases of probable household transmission of haemolytic uraemic syndrome due to Escherichia coli O104:H4, southwestern France, June 2011 home not at home home DSO home H 10 days home not at home home DSO home H 18 days 9 days DSO H home H 9 days June 2011 July 2011 Open day (community event) Meals in the family s house Patient C Patient B Patient A (index case) Time period between open day and DSO of Patients A and B (who attented the open day). Time period between DSO of Patient A (index case) and Patients B and C DSO: date of symptom onset; H: hospitalised (the day of discharge was not counted as day of hospitalisation). 13

14 Although Patient B attended the open day event, food-borne transmission from the sprouts to patient B is unlikely because she reportedly did not eat any sprouts. Moreover, an incubation period of 18 days would be unusually long. Both Patients B and C had spent time with Patient A on the day when he first experienced symptoms and after his return from hospital. During his first hospitalisation, his family did not stay in Bordeaux and had no contact with him. A recent study by the German outbreak investigation team showed that the median incubation period of E. coli O104:H4 in that outbreak was eight days (interquartile range: 7 9 days) [3]. Therefore, two scenarios can be considered for the household described here: transmission on 17 June when Patient A first presented symptoms at home, or transmission between 22 and 26 June after his return from hospital. Between 22 and 26 June, patient A had symptoms of diarrhoea and severe fatigue; during this period, he had prepared some meals. Recommendations for hygiene and infection control Following the detection of the outbreak on 22 June 2011, recommendations for hygiene and infection control were disseminated, starting on 24 June, to the general population and to the participants of the open day through several press releases, the website of the Ministry of Health, and local physicians. Furthermore, following the probable household transmission described here, a letter was sent on 5 July to the participants of the open day. This letter stressed the importance of personal hygiene measures, and safe food preparation practices, to reduce the risk of transmission. No other secondary cases in connection with the community event have been reported to date. References 1. Gault G, Weill FX, Mariani-Kurkdjian P, Jourdan-da Silva N, King L, Aldabe B, et al. Outbreak of haemolytic uraemic syndrome and bloody diarrhoea due to Escherichia coli O104:H4, southwest France, June Euro Surveill. 2011;16(26):pii= Available from: aspx?articleid= Frank C, Faber MS, Askar M, Bernard H, Fruth A, Gilsdorf A, et al. Large and ongoing outbreak of haemolytic uraemic syndrome, Germany, May Euro Surveill. 2011;16(21):pii= Available from: eurosurveillance.org/viewarticle.aspx?articleid= Frank C, Werber D, Cramer JP, Askar M, Faber M, Heiden MA, et al. Epidemic Profile of Shiga-Toxin-Producing Escherichia coli O104:H4 Outbreak in Germany Preliminary Report. N Engl J Med. 22 Jun 2011 [Epub ahead of print]. doi: / nejmoa Bielaszewska M, Mellmann A, Zhang W, Köck R, Fruth A, Bauwens A, et al. Characterisation of the Escherichia coli strain associated with an outbreak of haemolytic uraemic syndrome in Germany, 2011: a microbiological study. Lancet Infect Dis. 23 Jun 2011 [Epub ahead of print]. doi: / S (11) Kuijper E J, Soonawala D, Vermont, van Dissel J T. Household transmission of haemolytic uraemic syndrome associated with Escherichia coli O104:H4 in the Netherlands, May Euro Surveill. 2011;16(25): Available from: eurosurveillance.org/viewarticle.aspx?articleid= Snedeker KG, Shaw DJ, Locking ME, Prescott RJ. Primary and secondary cases in Escherichia coli 0157 outbreaks: a statistical analysis. BMC Infect Dis. 2009;9: Mellmann A, Harmsen D, Cummings CA, Zentz EB, Leopold SR, Rico A, et al. Prospective genomic characterization of the German enterohemorrhagic Escherichia coli O104:H4 outbreak by rapid next generation sequencing technology. Plos One. 2011;6(7):e Here, as in the household transmission of E. coli O104:H4 reported in the Netherlands [5], the index case was an adult. In our episode, one of the two secondary cases was also an adult. A review of 90 confirmed outbreaks caused by classical E. coli O157, showed that a lower median age of the index case was associated with a higher rate of secondary cases and that young children were most likely to become infected [6]. The unusual transmission from adult to adult observed in our episode is in line with the preponderance of cases in adults reported in the German outbreak [3]. This unusual pattern could be attributable to the specific properties of this strain [4,7]. Measures to prevent secondary transmission among adults should be strictly implemented. Acknowledgements We thank N Jourdan, V Vaillant and H de Valk for comments on the manuscript. Agreement of patients Written consent of patients to this report has been obtained. 14