Study Group News

An outbreak of measles is ongoing in Geneva, Switzerland, since January 2011, in the context of a measles epidemic in neighbouring Rhône-Alpes, France. A total of 41 confirmed cases have been reported, the majority among young adults, many unaware of their non-immune status. There is no large clustering of cases and 14 cases were imported or linked to imported cases. Catch-up vaccination, especially among young adults, may be necessary to prevent further extension of this outbreak.


Background
Between 1 January and 2 March 2011, 58 suspected cases of measles were notified to the Geneva health authorities in Switzerland.Of the 58 suspected cases, 41 (8.9 cases per 100,000 population) were confirmed (by laboratory confirmation or evidence of an epidemiological link) and were living in Geneva.In the previous 18 months (July 2009 to December 2010), only nine confirmed cases had been notified.The last national outbreak of measles in Switzerland lasted from November 2006 to July 2010, with 4,410 reported cases, including 84 in Geneva [1].
A large outbreak of measles is currently being reported in the neighbouring region of Rhône-Alpes, France, where more than 900 cases have been notified from January to mid-February 2011 [2].Geneva canton (population 464,000) is located on the eastern part of Switzerland and shares 103 km of its border with France and 4.5 km with the rest of Switzerland.
In Switzerland, measles notification has been mandatory since 1999.Physicians should report to local health authorities any patient presenting with maculopapular rash associated with fever and any of the following: cough, coryza or conjunctivitis.Notification of confirmed cases is also mandatory for laboratories.Childhood measles vaccination has been recommended since 1975: the current vaccination policy is to vaccinate twice with measles-mumps-rubella (MMR) at the age of 12 months and 15-24 months (the second dose was introduced in 1996) [3].The most recent estimate of national coverage among children aged two years is 89.8% for one dose and 74.6% for two doses (unpublished data).

Case definition
A case was defined as a Geneva canton resident presenting with the above clinical symptoms between 1 January and 2 March 2011, with either a detectable titre of measles virus-specific IgM antibodies, detection of measles virus RNA by PCR or a clinically significant increase in measles virus-specific IgG (laboratory-confirmed cases) or evidence of an epidemiological link with a laboratory confirmed case (epidemiologically linked cases).

Case description
Of the 41 cases included in the analysis, 33 were laboratory confirmed (by IgM or PCR) and eight were epidemiologically linked cases.The epidemic curve is presented in Figure 1.An additional 14 notified cases were not included, even though they were diagnosed in Geneva and reported to the health authorities, because they were French residents (n=11) or living in the canton of Vaud (n=3).All worked, attended school or consulted a physician in Geneva.Three suspected cases were reported and later ruled out due to negative serology results.
The male to female sex ratio of the 41 cases was 1:2 and their median age was 20 years (range: 11 months-58 years).Of 35 cases with known immunisation status, 25 had not been vaccinated with MMR vaccine, eight had received one dose and two at least two doses.Cases are presented by age group and vaccination status in Figure 2.
Six patients presented with at least one complication such as pneumonia (n=4), bronchitis (n=1), respiratory failure (n=1) and otitis (n=1).Eight patients were hospitalised: one was in intensive care with respiratory failure, one had pneumonia, three had a general alteration of heath status (in one case, this was associated with hypoxemia and in one, with fever) and two were kept in for overnight observation.The reason for hospitalisation is still unknown for one patient.No deaths were reported.All but one of the patients with complications or who were hospitalised were adults (median age: 36 years).
Of the 41 cases, 14 were imported (n=8) or epidemiologically linked with an imported case (n=6).These cases came from or were epidemiologically linked to cases from France (n=9), the canton of Vaud (n=4) and, possibly, Argentina (n=1).
Four clusters were identified: in two clusters, there were four cases per cluster, and in two clusters, there were two cases per cluster, giving a total of 12 cases.Transmission occurred in settings such as families (three occurrences), schools (n=2), social contacts (n=4) and the health service (n=1).

Control measures
In Switzerland, notification is mandatory within 24 hours of diagnosis and control measures are implemented as early as possible by local health authorities and school health services without waiting for laboratory confirmation.Cases are isolated at home for four days after the beginning of their rash.Extensive and rapid contact tracing is conducted as an emergency measure so that contacts and relatives of cases can be informed and their vaccination or immunisation status assessed.Control measures are customised and depend on the age, immunisation status of the contact and when the contact took place.They include postexposure vaccination of unvaccinated or non-immune contacts with recent exposure (less than 72 hours), administration of immunoglobulins to unvaccinated or non-immune pregnant women and newborns less than six months old.Unvaccinated or non-immune siblings, close and classmate contacts are quarantined at home for 18 days, with vaccination at the end of the quarantine period.A total of 20 exposed non-immune persons were quarantined.Five developed measles but there were no subsequent cases; seven are still quarantined.
In addition, Geneva health authorities regularly sent epidemiological updates and practical information by email to local physicians.These include reducing the age of first measles vaccination from 12 to 9 months of age.A press release, individual emails to all Geneva university students, and information letters to directors of schools, day-care centres and crèches have been sent out by the university or school health services.The main message has been targeted to young adults, recommending them to check their immunisation status and be vaccinated if necessary.

Discussion
This outbreak occurred in the context of a large measles epidemic in neighbouring Rhône-Alpes region in France.In addition, cases have also been recently diagnosed in the Lausanne and Basel regions, but very few are being reported in other parts of Switzerland.
The epidemiology of infectious diseases in the canton of Geneva is closely related to its neighbouring regions for obvious economic and geographic reasons.As a consequence, about one third of the cases in the outbreak were imported or related to imported cases, mostly from the bordering department of Haute-Savoie.The 14 cases who were not included in this analysis as they did not reside in Geneva were investigated in collaboration with the local French health authorities.
Most cases in this outbreak were adults, many of whom were not aware of their immune status.There was a delay in the diagnosis of several cases as early presentation of measles can be quite similar to influenza (seasonal influenza peaked in Geneva between weeks 1 and 7 of 2011).In some instances, there were multiple consultations before measles was diagnosed.However, only one healthcare-related case has been documented so far.Control measures have been implemented early for all cases including those whose measles diagnosis has not yet been confirmed.Post-exposure vaccination is often ineffective because it is implemented too late.As secondary attack rate in unvaccinated household members is high [4], quarantine of non-immunised relatives and close contacts and classmates is enforced and has proven effective in previous outbreaks in Geneva [5].None of the five quarantined contacts who developed measles transmitted the virus to others.
Although national MMR vaccination coverage remains below the threshold required for measles elimination in Switzerland [6,7], it is somewhat higher in Geneva.
In 2007, 89.7% of 28-month-old infants had received two doses of measles vaccine [7][8][9] and in 2009, the corresponding figure was 91.7%, according to the most recent analysis of the Geneva vaccination database [10], in June 2010.Progressive accumulation of non-or insufficiently immunised persons is therefore inevitable and explains this outbreak, along with multiple introduction of infectious patients into the Geneva community.

Conclusion and recommendations
Fairly high MMR vaccination coverage in children as well as early and effective prevention measures have probably contributed to reducing the magnitude of this outbreak, as indicated by the absence of any large clusters of cases so far.However, the influx of cases from neighbouring regions may continue and the potential for extension of the outbreak is substantial.
Catch-up vaccination is critical to prevent further extension of this outbreak, especially among young adults, particularly students.Careful scrutiny of the situation should continue as well as close collaboration with the neighbouring health authorities.[3].

Rapid communications
In order to assess how many people in central European countries may have been exposed to the virus, including individuals with subclinical disease, we analysed the WNV neutralising antibody titre of intravenous immunoglobulins (IVIG), a blood product containing pooled immunoglobulins from the plasma of more than a thousand donors from Austria, Germany and the Czech Republic.The suitability of this approach for assessing the cumulative number of WNV infections in a large donor population has recently been demonstrated through the use of IVIG to follow the spread of WNV in the United States [35].1B).Plotting the WNV neutralisation titres of the respective IVIG lots against the year of product release, a statistically significant increase over the years 2006 to 2010 was observed (p=0.0269one way ANOVA and p=0.004, post test for linear trend; Figure 1A).

Fully
To specifically discriminate antibodies induced by WNV infections of EU plasma donors from possibly crossreactive antibodies against TBEV, a Flavivirus related to WNV and widely endemic in central Europe, the TBEV neutralisation capacity of the IVIG lots was also determined, after the first lots with higher WNV activity had been detected.This was done for 30 of the 55 IVIG lots (Figure 1B).We assessed a potential contribution of TBEV neutralising antibody titres to the measured WNV antibody titres by correlation analysis and found a significant correlation (r 2 =0.by approximately 6-8 months.The IVIG lots containing significantly higher WNV-neutralising capacity (Figure 1, black diamonds) did not follow a seasonal pattern.
The non-structural flavivirus protein NS1 is only expressed during infection, but not present in the inactivated whole virus vaccines that are used in the countries analysed here.In addition, reactivity of antibodies with the NS1 protein is specific to the flavivirus serotype, and thus infections with either WNV or TBEV can be differentiated [36].We therefore analysed the antibody specificity of six randomly selected IVIG lots, produced in 2007 (n=1) and 2009 (n=5), by Western blot.TBEV-or WNV-infected Vero cells as well as recombinant WNV NS1 antigen were used as a positive and uninfected Vero cells as a negative control [37,38].The blots were incubated with IVIG produced from plasma collected in Austria, Germany and the Czech Republic (Figure 2A), or with a control serum from TBEV-infected mice (Figure 2B).IVIG interacted with the flavivirus structural envelope protein E as well as the WNV-specific NS1 (Figure 2A).In contrast, the control mouse serum reacted strongly only with the E protein of TBEV-infected cells and weakly with WNVinfected cells (Figure 2B).

Discussion and conclusion
We found that IVIG preparations manufactured from plasma collected in Austria, Germany and the Czech Republic, contained neutralising antibodies against WNV at titres which have increased significantly since 2009.As WNV and TBEV are related flaviviruses, albeit distantly, we quantified neutralising antibody titres against TBEV in these IVIG lots, to investigate crossreactivity between these two viruses as a potentially confounding variable.Indeed, very high TBEV NT 50 titres of between 400 and 3,000 were observed in all  investigated years, a result of vaccination and possibly subclinical TBEV infections.Despite a significant correlation of TBEV and WNV neutralisation titres in approximately 60% of the IVIG lots, 11 of the 30 lots contained significantly higher (p<0.0001)neutralisation titres against WNV (mean±standard error of the means (SEM): 6.5±0.6,n=11, compared with 2.8±0.1, n=19) that did not correlate with the TBEV-neutralising capacity.IVIG lots produced from plasma collected in Austria, Germany and the Czech Republic were shown by Western Blot to contain specific antibodies against WNV NS1, the most useful differentiation marker for flavivirus infections in humans [36], which provided further evidence for past WNV infections in plasma donors from the central part of the EU.As the detection of antibodies to the WNV NS1 protein is serotype-specific, a possible contribution of antibodies against dengue virus can be excluded.The theoretical possibility of a contribution of neutralising antibodies to Usutu virus (USUV) [39], a virus that belongs to the same serocomplex as WNV, was not evaluated in vitro, as even the highest USUV activity in Austria as observed during the summer of 2003 [40] had no impact on the WNVspecific neutralisation titres in IVIG lots [41].Moreover, despite a comprehensive surveillance programme for dead birds and mosquitoes in Austria [42], where around 45% of the plasma is collected for production of the IVIG lots tested in this study, no evidence of human exposure to USUV could be found [43], which makes a significant contribution of USUV antibodies to WNV neutralisation titres determined in the present study unlikely.However, additional studies would be relevant to describe this issue more precisely.
These results demonstrate WNV seropositivity in asymptomatic plasma donors from Austria, Germany and the Czech Republic, although some of the seropositivity could be due to travel-related infections.The present epidemiological situation in these countries is thus similar to the one in Greece before the recent epidemic.The causative agent of the outbreak in Greece, WNV lineage 2, has only once before been isolated from humans with severe clinical progression, in an outbreak in Volgograd, Russia in 2007 [18].Before those two outbreaks, this lineage was considered to be less virulent in humans compared to isolates belonging to the lineage 1 [44].It has been suggested that the evolution from low to high human pathogenicity is associated with mutations of only a few amino acids, most likely in the non structural proteins [45].
The WNV neutralisation capacity of IVIG lots produced from Austria, Germany and the Czech Republic has increased from a mean NT 50 of 2.5 in 2006 and 2007 to 4.2 at the beginning of 2010, which accounts for an increase of 1.7 in the WNV neutralising capacity (Figure 1).The TBEV neutralisation titres of IVIG have not changed over time, and thus the increase in WNV neutralisation titres is most likely a true reflection of increased virus circulation.Using the same, fully validated assay, reconvalescent sera from human WNV cases in North America have earlier been shown to have a mean NT 50 of 208 [41], 120-fold higher than the increase now observed.This would indicate past WNV exposure of just under 1% of the population of Austria, Germany and the Czech Republic.The increasing WNV seropositivity in these countries marks this virus as a potential public health concern in this area, and a future epidemic associated with human morbidity and mortality similar to that observed in summer 2010 in Greece cannot be excluded.

Introduction
West Nile virus (WNV) was originally identified in 1937 in northern Uganda [1].Birds, especially those in the Corvidae family are the natural reservoir of the virus, which is mainly transmitted to humans through the bite of infected Culex mosquitoes [2,3].Transmission through receipt of blood products and transplantation has also been documented [4,5].Rare cases of vertical transmission (i.e.transplacentally or through breast milk) and laboratory-acquired infection have also been reported [6].
During the last 20 years, several WNV outbreaks have occurred throughout the world.In addition to the United States [7], where the virus was first identified in 1999 [2,3,6], large WNV outbreaks have also been reported in Europe and in the Mediterranean basin [8,9].The largest European outbreak occurred in 1996 in Bucharest, Romania [10].In the Mediterranean area, the first outbreak of human encephalitis was identified in the Camargue, France, in 1962 [11], followed by other epidemics in the 1990s involving a relatively high number of human cases in Algeria [8], Tunisia [12] and Israel [13,14].In the past few years, cases of WNV encephalitis have also been reported in the Volgograd region in Russia [15], in Hungary [16] and Romania [17], and very recently in Greece [18].
In Italy, the first outbreak of WNV infection was reported in 1998 in horses in Tuscany [19].The virus reemerged in north-eastern Italy in summer 2008, when equine cases of WNV neuroinvasive infection were notified in the regions Veneto and Emilia Romagna [20].An extraordinary WNV surveillance programme was subsequently activated, which led to the notification of nine human cases of West Nile neuroinvasive disease (WNND) in the summer of 2008 [21,22] and a further 16 cases in late summer 2009; all cases occurred in the regions Emilia Romagna, Veneto and Lombardia, in wet areas surrounding the Po river [23][24][25].
Since WNV infection is generally asymptomatic with encephalitis occurring in less than 1% of cases, we conducted a seroepidemiology investigation in the Rovigo province in northern Italy, where a high incidence of WNND was observed, in order to estimate the extent of the epidemic and to better plan intervention strategies.

Setting
The study area was the province of Rovigo, Veneto region, north-east Italy.This province, which has around 250,000 inhabitants, borders with the Po river and the Emilia-Romagna region.All territory is a level land characterised by extraordinary biodiversity, mainly because of the presence of freshwater and brackish water wetlands, including flooded deciduous woodlands, open lagoons of shallow water and river mouths.The high humidity level makes this area particularly attractive for mosquitoes, in particular during summer.It is located at the crossroads of bird migration routes connecting Europe, the Mediterranean basin and Africa, hosting a high number and wide range of migrating birds throughout the year.Thus, the high concentration of mosquitoes and migrating birds creates opportunities for vector-borne viruses such as WNV [26].

Study design
The study involved the three blood donation centres of Rovigo province which collected about

Laboratory testing
WNV NAAT screening was performed using Cobas Taq Screen West Nile Virus test on a Cobas s201 system (Roche Molecular Systems) on pools of aliquots from six individual plasma specimens.Specimens included in WNV RNA-positive pools were re-tested individually with the same WNV NAAT kit.
WNV IgM and IgG testing was done using the WNV IgM capture DxSelect ELISA and IgG DxSelect ELISA kits (Focus Diagnostics), respectively, as reported [18].All serum samples which tested positive in the ELISA were further analysed by anti-West Nile virus IIFT IgG and IgM immunofluorescence assays (IFA) (Euroimmun AG), and, to rule out cross-reactivity with other flaviviruses and confirm the result, also with the PRNT, according to the previously described protocol [18].

Statistical analysis
Prevalence of antibodies to WNV was calculated as the ratio between sera confirmed positive by PRNT and all tested sera.The 95% confidence intervals (CI) of the prevalence were calculated using the binomial distribution.Prevalence estimates were also stratified by blood donation centre and by month of donation.Chisquare test was used to evaluate if the prevalence by blood donation centre and by month of donation was statistically significant.
Sensitivity, specificity, positive predictive (PPV) and negative predictive value (NPV) of IgG and IgM with respect to PRNT were also calculated, assuming that all sera not evaluated by PRNT were negative.This assumption favours highest estimates for specificity and NPV.
Odds ratios (OR) were calculated to evaluate the association between age, sex and open-air/indoors job of the donor with confirmed positivity by PRNT compared   a WNV IFA is evaluated as a second line test for WNV ELISA-positive samples.Note: It was assumed that all sera tested negative by ELISA would also have been negative in IFA and PRNT even when these tests were not performed.
to matched sera not tested/not confirmed by PRNT.The OR was calculated taking into account matching by day and blood donation centre.A conditional logistic model was used to simultaneously adjust the OR for the effect of each evaluated factor with respect to the others.

Results
During the period from 1 August to 31 October 2009, 5,726 blood donations were collected in Rovigo province and tested by WNV NAAT.One blood donation, not included in the seroprevalence study, was WNV RNApositive (17.5 per 100,000 donations; 95% CI: 0.4-97.3).Details on this positive case have been reported previously [25].
Among the 2,507 serum samples evaluated for IgG and IgM, 94 (3.7%) were positive in the WNV ELISA IgG and/ or IgM screening and further tested by IFA and PRNT (Table 1).Of the 94 ELISA-positive samples, 70 (75%) were also WNV IFA-positive and 17 (18%) were confirmed by PRNT.The PRNT-confirmed cases included eight (47%) cases with both WNV IgM and IgG and nine (53%) with only WNV IgG detected by ELISA and/or IFA.
The estimated overall prevalence of WNV antibodies confirmed by PRNT was 6.8 per 1,000 tested sera (95% CI: 4.0-10.9).Stratifying by month of donation no particular fluctuations were observed, with prevalences that varied from 6.2 per 1,000 in September to 7.6 in October (p=0.99,Chi-square test, data not shown).
The prevalence differed significantly by blood donation centre, with the highest prevalence in Trecenta (17.9 per 1,000 donations) and the lowest in Adria and Rovigo (4.0 per 1,000 donations) (p<0.01).
Table 2 shows the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of WNV ELISA alone and WNV ELISA followed by WNV IFA as a second-line test compared with PRNT results as the gold standard, and assuming that all ELISA-negative samples that were not also evaluated by PRNT, would have been negative in the PRNT.The sensitivity of WNV IgM detection for predicting WNV infection was low, whether by ELISA alone or by a combination of ELISA and IFA; however, the possibility of false negative results of PRNT at early phases of infection was not excluded in this study.The sensitivity of WNV IgG detection, either alone or in combination with WNV IgM detection by ELISA was about 50%.The addition of WNV IFA as a second-line test for ELISA-positive samples did not improve test sensitivity and PPV.
Table 3 shows the association of the blood donors' characteristics with being WNV positive in PRNT in a matched case-control analysis.While age and sex were not found to be significantly associated with being WNV-positive in PRNT, working outdoors was associated with a statistically significant higher risk (>5 times) of WNV infection than working indoors (p=0.05).
Finally, a map of the province was produced, plotting the geographical coordinates of the PRNT-positive donors and the cases of WNND notified in 2009 and resident in the Rovigo province.Most subjects with a PRNT-confirmed WNV infection lived close to rivers in western areas of the Rovigo province, without any apparent clustering or association with the place of residence of subjects diagnosed with WNND (data not shown).

Discussion
In accordance with national guidelines [27] that recommend screening of blood donations in affected areas where at least one human case with WNND has been detected, all blood donations performed in Rovigo province during summer 2009 were individually tested by NAAT.Only one of 5,726 blood donations resulted positive by NAAT, which corresponds to an estimated risk of WNV transmission of about 17.5 per 100,000 blood donations.With the limitation of the small samples size in our study, this risk appears to be lower than that reported in the United States during the peak of the WNV epidemic in 2002 [28] and in Canada in the period from 2006 to 2007 [29].
In order to estimate the extent of WNV infection among humans, we also conducted a serosurvey in a selection of blood donations in the study area; the estimated overall prevalence of anti-WNV antibodies was 6.8 per 1,000 sera (95% CI: 4.0-10.9).This prevalence estimate was lower than that found in a previous survey (15.6 per 1,000), that was conducted in the same province but was restricted to a population considered at risk of environmental exposure represented by farm employees who worked in areas where WNVseropositive horses had been identified [22].
A retrospective screening of solid organ donors in several Italian regions found a higher prevalence of anti-WNV antibodies than the present study on blood donors [30].This discrepancy could be accounted for by differences in target populations and sampling strategies as well as laboratory methods used for testing and for confirmation of positive results.We considered as positive only those samples which were confirmed by neutralisation assays.In fact, less than 20% of WNV ELISA-positive samples were confirmed by PRNT.After combining ELISA and IFA, the specificity did not increase significantly for those samples which were positive in both tests.Since the PPV of a test tends to decrease in low prevalence areas, our results suggest that neutralisation assays should be used to confirm positive results, especially in areas with a low risk of infection.
The prevalence in our study varied widely among geographical areas, ranging from about 18 per 1,000 blood donations in Trecenta to 4.0 per 1,000 in Adria and Rovigo.Moreover, the geographical distribution of infected individuals suggested that most cases were resident in areas near rivers and other water sources.This is consistent with other studies on WNV and other mosquito-borne infections, such as La Crosse encephalitis [3,31].However, we could not evaluate the association between WNV seroprevalence and proximity to water sources in our samples because of the lack of available geo-reference data on WNV-negative blood donations.For the same reason, any factor explaining the geographical variation could not be evaluated due to limited environmental data.
With regard to risk factors for infection, no statistically significant difference was found for age or sex, while a statistically significant association was found with working outdoors compared with other jobs.These findings, which suggest a higher risk of exposure to mosquito bites, contradict studies conducted in Romania, where having spent more than six hours outdoors during the day was not found associated with West Nile virus infection [32].
In conclusion, this study estimated the seroprevalence of WNV among blood donors of an affected area of Italy as 6.8 per 1,000 sera and indicates that WNV seroprevalence can vary widely even between different geographical areas within the same province.Although WNV NAT-positive samples were rare in our population, blood screening is needed in order to reduce the risk of WNV transmission to vulnerable recipients.

Introduction
Clostridium difficile infection is a common nosocomial problem in elderly patients following antibiotic treatment [1].C. difficile is typically isolated from faeces of 1-5% of healthy individuals, but colonisation increases upon admission to hospitals.Hospital-based studies performed in 1999 to 2001 showed that nosocomial spread of C. difficile was rare in Swedish hospitals in which infection control routines were implemented, although there were small outbreaks associated with multiresistant PCR-ribotype 012/SE17 [2][3][4]

Screening of C. difficile isolates with decreased susceptibility to moxifloxacin (2008)
An invitation to participate in the study, including instructions, methods and study protocol, was sent to all 28 regional clinical laboratories in Sweden.Faecal specimens sent by the clinicians for routine C. difficile detection were tested in the regional laboratories according to their standard methodology (ELISA/ EIA was the most commonly used method, in 24 of the 28 laboratories, followed by cell cytotoxicity assay).Sample material that was positive for C. difficile was inoculated anaerobically on taurocholate-cefoxitincycloserine-fructose agar (TCCFA) for 48 hours and toxigenic C. difficile colonies were subcultured on blood agar.Susceptibility to moxifloxacin was tested using disc diffusion (5 µg) on IsoSensitest agar (Oxoid, United Kingdom) supplemented with 5% defibrinated horse blood and 20 µg/ml beta-NAD incubated at 36 °C for 48 hours.C. difficile strains with an inhibition zone diameter >16 mm (ATCC9689) and 6 mm (Ö07-1702) were used as controls.All C. difficile isolates with zone diameter <16 mm were sent to the Swedish Institute for Infectious Disease Control for typing.A protocol with strain number, diameter of the inhibition zone, and, if possible, the total number of screened isolates were included each shipment.Upon arrival, samples were inoculated on both TCCFA and blood agar and incubated anaerobically at 36 °C for 48 hours.Isolated strains were verified for susceptibility to moxifloxacin using the disc diffusion method described above and Etest (AB Biodisk, Sweden).Of 641 sent samples, 37 were excluded because C. difficile could not be isolated and/ or the sample was contaminated; a further 19 isolates were omitted due to an inhibition zone diameter >16 mm, giving a final 585 C. difficile isolates.The study period was from 7 December 2007 to 1 September 2008.Since no patient data were included, we could not judge how many patients or how many cases of C. difficile infection there were during the study period.

National surveillance typing (2009)
An invitation to participate in the surveillance programme, including methods and study protocol, was sent to all 28 regional Swedish laboratories.All consecutive strains isolated from C. difficile-positive samples obtained through routine detection during 9-15 April and 21-27 September 2009 were sent to the Swedish Institute for Infectious Disease Control for typing and susceptibility testing for moxifloxacin, erythromycin, clindamycin, metronidazole and vancomycin.Of 432 samples sent, 40 were excluded because they were contaminated or not viable.Of the remaining 392 isolates, 28 were not analysed because their dates of isolation did not comply with the inclusion criteria above, giving a final total of 364 isolates.
Susceptibility testing was performed using Etest on Mueller-Hinton agar medium (BD Diagnostics) at 35°C for 48 hours.A bacterial density of McFarland 1 was used to obtain a next-to-confluent layer of C. difficile bacteria.There were no significant differences in minimum inhibitory concentration (MIC) values using Mueller-Hinton or IsoSensitest agar (data a The three-digit international nomenclature is used for typing, or when reference isolates are missing, SE-type is used.b Ribotypes represented by fewer than three isolates per type.

PCR ribotyping
PCR ribotyping was performed according to the method of Stubbs et al. [8], with modifications described in Svenungsson et al. [4].Reference isolates were obtained from the Cardiff-European Centre for Disease Prevention and Control (ECDC) collection of C. difficile strains (obtained from Ed J. Kuijper, Leiden University Center, Leiden, the Netherlands).Bionumerics software version 6.1 (Applied Maths NV, Sint-Martens-Latem, Belgium) was used for the analysis.The three-digit international nomenclature was used for typing or, when reference isolates were missing, an SE-type was given.

Prescription of antibiotics
Sales data of antibiotics were obtained from Apotekens Service AB, which is responsible for compilation of all drug statistics, including sales, in Sweden.The data include antibiotics prescribed to patients as well as sales to hospitals and other healthcare providers.

Statistical methods
Fisher's exact test and chi-square tests were performed to examine differences in geographical distribution of ribotypes using R v. 2.10.1.Kaleidagraph v. 4.02 (Synergy software) was used for data management and graphics.To avoid type-I error due to multiple statistical tests, a significance threshold of p=0.005 was applied.

C. difficile isolates with decreased susceptibility against moxifloxacin (2008)
A total of 20 of the 28 Swedish regional laboratories covering 15 of the 21 of the counties supplied a total of 585 isolates with reduced susceptibility to moxifloxacin.Of the 15 counties, 10 supplied data on the number of screened isolates: 546 of 2,702 isolates (20%; range: 9-34%) had decreased susceptibility to moxifloxacin.Of the 585 isolates (from the 15 laboratories), 454 (78%) belonged to four PCR ribotypes: 012, SE37, 017 and 046 (Figure 1).Types 012 and 046 were found mainly in the central and southern part of Sweden, while types 017 and SE37 were predominant in the central-east counties of Gävleborg and Stockholm, respectively (Figure 2, shown in purple).The distributions for each of the types 012, 017, 046, 078 and SE37, compared with that of other types, were highly skewed between counties: a chi-square test among counties having more than 10 isolates available for typing gave a p value of <0.001 for each of the types 012, 017, 046, 078 and SE37 (for 9 of the 21 counties).Only two type 027 isolates were found, both from the same patient.

National surveillance typing programme (2009)
Of 364 C. difficile isolates supplied by all 28 regional laboratories, type SE21 was found in highest number followed by types 001, 020, 078, 012 and 023 (Figure  a The three-digit international nomenclature is used for typing, or when reference isolates are missing, SE-type is used.b Ribotypes represented by fewer than three isolates per type.

Prescription of moxifloxacin in Sweden in 2008 and 2009
The mean prescription rate of moxifloxacin per county was 0.020 defined daily dose (DDD) per 1,000 inhabitants per day for 2008 and 2009.The prescription rate differed markedly between counties (range: 0-0.088DDD per 1,000 inhabitants per day) and was highest in the south-east of Sweden (Figure 6).Some of the counties in the south-east also had a high proportion of PCR ribotypes 012 and 046 (Figure 5).However, the prescription rate of moxifloxacin was comparatively low in the counties where types 017 and SE37 dominated.[23]).From previous geographically localised studies in Sweden in 1999 to 2001, the six most common types were 014/SE21, 012, 001, 020, 002 and 005 [2][3][4].All these were still among the most common types in 2009.There were thus only small changes in the distribution of major types over a period of 10 years, a result quite different from the shift in strain types that has occurred in England [7].One exception might be type 078, which was the fourth most common type in 2009.Type 078 may be an emerging type in Europe and is commonly found in domestic animals, suggesting transmission through the food chain [24][25][26].

Discussion and conclusion
The studies presented here have several weaknesses.All laboratories did not report a full set of data, particularly from the first screening study.This was mostly due to the fact that several laboratories were in the process of setting up culturing methods and were thus unable to supply isolates from the first day of the study period.Also, the two studies were not of identical design: the first aimed to collect consecutive moxifloxacin-resistant isolates during a longer time period and the second collected all isolates during two shorter time periods.In addition, the short study periods in 2009 implies a poor detection of seasonal variability.We also did not ask for detailed information about the number of patients or the number of cases during the study periods -thus the true prevalence could not be calculated.Despite these limitations, the regional clustering of certain types observed in 2008 was confirmed in 2009.Some of the remaining questions will be answered by the voluntary reporting system, but since all laboratories are not yet participating, it is too early draw any conclusions from this system.
The proportion of C. difficile isolates with reduced susceptibility to moxifloxacin was 16% in Sweden 2009 and thus less than half of the 37.5% reported in Europe in 2005 [5].Decreased susceptibility to moxifloxacin has been found in several C. difficile types isolated in Europe and worldwide, and appears to be common in dominant types including 001, 018, 027 and 106 in, for example, England [7], Scotland [27], Germany [28] and Italy [5].We found decreased susceptibility mainly in types 012, 017, 046 and 231/SE37.Type 012 is related to serogroup C [29] and has been reported as a common type in Sweden, Greece and Hungary [2,19] while toxin A-negative, toxin B-positive type 017 has been found to be associated with outbreaks in, for example, Poland, Canada and the Netherlands [6,30].Regarding type 231/SE37, the proportion of this type was 0.8 % at the Karolinska University Hospital, Huddinge, Sweden, in 1999 [4], but there is no information on whether this type has caused any outbreaks in other countries.
Despite reports of increased prevalence of moxifloxacin resistance in C. difficile [5,[31][32][33], including outbreaks of C. difficile 027 occurring shortly after replacing levofloxacin with moxifloxacin/gatifloxacin [34], it is currently unclear whether moxifloxacin use implies higher risk for C. difficile infection than other commonly used antibiotics [35][36][37][38].Thus, we can neither confirm nor exclude the possibility that moxifloxacin has contributed to the selection of types 046 and 012 in certain Swedish counties.In this context, it should be pointed out that types 012, 017, 046 and 231/SE37 had decreased susceptibility also to clindamycin and erythromycin.Moreover, type 012 was shown to be resistant to many other antibiotics that were not tested in this report [32].Thus, their success as pathogens may just reflect general selection due to the overall antibiotic use in healthcare settings [7].Nevertheless, the cause for the variable prescription rates of moxifloxacin in Sweden needs to be investigated further. In

Figure 2
Figure 2Reactivity of intravenous immunoglobulin lots produced from plasma collected in Austria, Germany and the Czech Republic with viral proteins

Figure 1
Figure 1West Nile virus and tick-borne encephalitis virus neutralisation by intravenous immunoglobulin lots produced from plasma collected in Austria, Germany and the Czech Republic (N=55)

Figure 6
Figure 6Extent of prescription of moxifloxacin by county,Sweden,  2008 and2009 Increasing West Nile virus antibody titres in central European plasma donors from 2006 to 2010 IntroductionClinical cases of West Nile virus (WNV) infection in the European Union (EU) have so far been limited to

Table Cases of
West Nile virus infection and seropositivity in EU countries, Russia and Israel, by year of occurrence, 1962-2010 CFR: case fatality rate; NI: no information available; WNND: case with West Nile neuroinvasive disease; WNV: West Nile virus.
18,500 blood donations in 2009.During the period from 1 August to 31 October 2009, all blood donations were routinely evaluated by nucleic acid amplification test (NAAT) for WNV to identify potentially viraemic donations.In order to study the prevalence of WNV in the area, we tested, during the period from 20 July to 15 November 2009, serum samples from 25 blood donations per day for IgG and IgM antibodies to WNV.Any IgM-or IgGpositive sample was further evaluated by immunofluorescence and by plaque-reduction neutralisation test (PRNT) for confirmation.The number of serum samples collected at each centre was proportional to the volume of donations performed in the year 2008.Thus, each day, five donations were sampled from Adria (Centre 1), five from Trecenta (Centre 2), and 15 from Rovigo (Centre 3), choosing serum samples from the first consecutive daily donors who gave their consent to the study.All samples were handled anonymously by technicians and researchers involved in this study.To evaluate potential risk factors associated with WNV infection, a case-control study matched by day and donation centre was performed.More specifically, for all donors that had IgG and/or IgM to WNV confirmed by PRNT, specific information about age, sex, address and type of job was retrospectively collected.For each positive donor, we collected the same information from four negative cases who were randomly chosen among the donors seen on the same day in the same centre.Although details on the type of job were collected, we decided after preliminary analysis to create a dummy variable classifying the job as done predominantly in the open air or not, e.g. a builder was classified as an open-air worker while a bank clerk's work was classified as indoors.

Table 3
Adjusted odds ratios of being PRNT-positive for West Nile virus, associated with blood donor characteristics, conditional logistic model, Rovigo province, Italy, 20 July-15 November 2009 (n=17) AOR: adjusted odds ratios; CI: confidence interval; PRNT: plaque reduction neutralisation test.a PRNT-negative blood donors were matched to positive ones by day and centre of donation.
The frequency is calculated as the number of isolates of the indicated type divided by the total number of screened isolates for each county multiplied by 100. a

Table Proportion of
Clostridium difficile PCR ribotypes with decreased susceptibility to moxifloxacin, clindamycin and erythromycin,Sweden, 2009 (n=364) a Breakpoints were chosen according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST ) tentative ecological cut-off for detection of isolates with resistance mechanisms, as suggested by the European Study Group for C. difficile.erythromycin(Table).No isolate was resistant to metronidazole or vancomycin.
PCR ribotyping has been used for studying C. difficile epidemiology for several years, and details of the first large C. difficile PCR ribotype library were published byStubbs et al. in 1999 [8].When the European Centre for Disease Prevention and Control (ECDC) and the Centre for Infectious Disease Control Netherlands initiated a pan-European C. difficile infection survey (ECDIS) study in 2008[19], a C. difficile reference strain collection with defined PCR ribotypes was formed (the Cardiff-ECDC collection).One purpose of the collection was to support national laboratories in setting up local typing systems and to aid surveillance studies and international comparisons.We found that a majority of the C. difficile strains in Sweden was represented in this reference strain collection.Exceptions were type SE37, for example, which was confirmed in the ECDIS study as one of six novel types (PCR ribotype 231)[19].Another unknown type was SE21, which closely resembled types SE21a and SE21b as well as reference strains of PCR-ribotypes 014 and 020[4].Type SE21 was verified as a member of the 014/020 group: in total this group comprised 19% of all C. difficile isolates in our study in Sweden in 2009.The proportion of the 014/020 group among all C. difficile isolates was 16% in Europe in 2008[19].Much interest in recent years has been focused on the worldwide spread of the hypervirulent C. difficile type 027[20,21].Outbreaks in northern Europe in 2007 to 2009[10][11]13,22]prompted us to screen for type 027 isolates with decreased susceptibility to moxifloxacin.Of the 585 isolates analysed during 2008, only two type 027 isolates were detected (from the same patient).Furthermore, since no type 027 isolate with reduced susceptibility to moxifloxacin was identified in 2009, such isolates are probably rare in Sweden (compare with conclusion, in our two nationwide C. difficile surveillance studies in Sweden in 2008 and 2009, there was a geographical clustering of PCR-ribotypes 012, 017, 046 and a novel type (231/SE37), suggesting transmission and local outbreaks.Our results support the need for a central European or international C. difficile database with capability to continuously report and characterise emerging types, as well as a national surveillance system to monitor and control C. difficile incidence, type distribution, resistance pattern and related antibiotic use.