Gonococcal infections and emergence of gonococcal decreased susceptibility to cephalosporins in France , 2001 to 2012

G La Ruche (g.laruche@invs.sante.fr)1, A Goubard2, B Berçot3, E Cambau3, C Semaille1, P Sednaoui2 1. French Institute for Public Heath Surveillance, Department of infectious diseases, Saint-Maurice, France 2. Institut Alfred Fournier, National Reference Laboratory for gonorrhoea, Paris, France 3. Laboratory of Bacteriology, Virology and Hygiene, Saint-Louis/Lariboisière/Fernand Widal hospitals, National Reference associated Laboratory for Gonorrhoea, Paris, France


Surveillance and outbreak reports
Gonococcal infections and emergence of gonococcal decreased susceptibility to cephalosporins in France, 2001 to 2012

Introduction
Although gonorrhoea is a common sexually transmitted infection (STI), normally responsible for uncomplicated genital infections, the disease can sometimes lead to severe complications (salpingitis, epididymoorchitis, septicaemia) [1,2].It also increases the risk of transmission by human immunodeficiency virus (HIV) [2].Because of its short incubation time (mainly 2 to 5 days) [3] and characteristic symptoms in men, the incidence of gonorrhoea can be used as a very sensitive indicator of the relaxation of safe sexual behaviours, and as an early warning signal for increased risk of HIV transmission [2].
Following acquired immunodeficiency syndrome (AIDS) prevention campaigns in the 1980s and at the beginning of the 1990s, the number of cases of gonorrhoea in certain European Union (EU) and European Free Trade Association (EFTA) countries [4], including France [5], dropped significantly.This drop was the result of a reduction of at-risk sexual behaviours [6].The advent of antiretroviral multitherapy in the mid-1990s brought with it a resurgence of these behaviours in the general population, especially in men who have sex with men (MSM).It also led to a resurgence in cases of gonorrhoea in some EU/EFTA countries [5,6] as well as in the United States [7].
In France, the epidemiological surveillance of gonorrhoea is based on two voluntary sentinel networks relying on clinicians (the RésIST network) or public and private laboratories (the Rénago network -National Gonorrhoea Network -) [8,9].Since the beginning of the 2000s, an increase in gonococcal infections has been observed by these networks, both in men and women [9].The RésIST network also highlighted that this increase exists irrespective of sexual orientation [8,9].
At the end of the 1990s and the beginning of the 2000s, concomitantly with the resurgence of gonoccocal infections, several countries reported increasing levels of gonococcal resistance to ciprofloxacin [2,10], a fluoroquinolone antibiotic, which was used at the time as a single-dose first-line treatment for uncomplicated urogenital gonorrhoea.Since the mid-2000s, singledose third generation cephalosporins (TGC), specifically oral cefixime and injectable ceftriaxone, became the only remaining recommendable antimicrobial class [11].These treatments were officially recommended in France at the end of 2005 [12].However, the subsequent development of clinical and biological resistance to TGC, especially to cefixime, and, since the end of the 2000s, to ceftriaxone, may lead to a therapeutic dead end in the coming years [2,11].Surveillance of gonococcal susceptibility to these antibiotics is therefore essential.
Given this worrying context, the objective of this study was to describe the evolution of gonococcal infections and the changes in the susceptibility of gonorrhoea strains to antibiotics in France between 2001 and 2012.Factors associated with decreased susceptibility to TGC are also assessed.

Methods
The laboratories in the Rénago network are distributed throughout all of metropolitan France.This network helps monitor the resistance of Neisseria gonorrhoeae strains to antibiotics.Laboratory participation to surveillance is voluntary.
The laboratories receive patients referred by their doctor who prescribed a microbiological diagnosis for gonorrhoea.An anonymous epidemiological file, collected by the network laboratory microbiologist, provided the following information for each patient: sex, age, date of sample, anatomical site of sample, presence of symptoms, co-infection with another STI, probable country of infection, information on partner's infection, the place of consultation and specialisation of treating physician, type of laboratory and geographical area to which it belongs.The sexual orientation of the patient is not reported.Information is consolidated and analysed by the Institut de Veille Sanitaire (InVS).Because of the large proportion of missing data for four variables (presence of symptoms, co-infection with another STI, probable country of infection, and information on patient's partner infection status), the latter were used only for univariate analysis for descriptive purposes.All other variables were included in the multivariate analysis; p<0.05 was used as the level for significance.Only the results of multivariate analysis are presented here, unadjusted and adjusted odds-ratios being very similar.For the analyses, the data concerning the years from 2001 to 2009 were pooled to highlight recent developments of the characteristics of gonococcal infections and of antibiotic susceptibility of strains in the period from 2010 to 2012.
Gonococcal infection is laboratory confirmed by a positive culture or a positive nucleic-acid amplification test (NAAT) for N. gonorrhoeae by the network laboratories.Strains isolated by culture by the network laboratories are sent for antibiotic susceptibility testing to the Alfred Fournier Institute in Paris, which is the national reference laboratory (NRL) for gonorrhoea.
Because of the very high number of strains received by the NRL as well as budget constraints, the InVS and the NRL decided to separate epidemiological and microbiological surveillance.Although the epidemiological data collection continued for all laboratories in the network, the number of laboratories sending their strains to the NRL was reduced by about fifty per cent from the beginning of January 2012 (in 2012, 63 of the 133 laboratories in the network participated to the microbiological surveillance).Nevertheless, a homogeneous geographical distribution of laboratories continuing this microbiological surveillance was ensured (Figure 1).
The NLR determines the minimum inhibitory concentration (MIC) for six antibiotics (penicillin G, tetracycline, ciprofloxacin, spectinomycin, ceftriaxone, and, since 2008, cefixime) using Etest strips (first from AB Biodisk, Solna, Sweden; then from AES Laboratory, Combourg, France, and from May 2011 from i2a, Pérols, France).These antibiotics were or are still recommended for the treatment of gonorrhoea [11].The presence of beta-lactamases is detected using Api NH (bioMérieux, Marcy-l'Etoile, France) identification test kits and, when there is any doubt, using a chromogenic cephalosporin test (cefinase, bioMérieux).The criteria used to define resistance are those used by the European Committee on Antimicrobial Susceptibility Testing -EUCAST 2013 [13].For TGC, no defined threshold exists for resistance; strains with a MIC>0.125 mg/L for cefixime and for ceftriaxone are regarded as having a decreased susceptibility.A strain was considered multi-resistant if it was resistant to penicillin G, tetracycline and ciprofloxacin, and if the MIC for cefixime or for ceftriaxone was >0.125 mg/L.
The factors associated with a reduction in susceptibility of gonorrhoea to TGC in patients aged at least 15 years-old were determined through univariate and multivariate analyses using Stata v.11.0 software, excluding missing values.As the number of strains with MIC>0.125 mg/L for TGC was low, we studied the factors associated with a MIC≥0.094mg/L for cefixime and a MIC≥0.032mg/L for ceftriaxone.These values were chosen arbitrarily to have a sufficient number of strains with MICs above these cut-offs to allow comparison analyses.Taking into account such cut-offs, 6% of strains had decreased susceptibility to these two antibiotics.

Evolution of the number of gonococcal infections and analysed strains
Between 2001 and 2012, 13,400 cases of gonorrhoea were reported by the network's laboratories.From these cases, a total of 10,501 strains were sent to the NLR.The NLR succeeded in recultivating 8,649 (82%) of them to determine the susceptibility of N. gonorrhoeae to antibiotics.The proportion of strains sent to the NLR and then recultivated remained quite stable from 2005 to 2012 (84% on average for this period, range 82 to 87%).
Figure 2 shows the increase in the number of reported cases and the number of strains studied by the NLR

Evolution of the susceptibility of gonococcal strains to antibiotics
The analysis is based on 8,649 gonococcal strains tested by the NLR.Proportionally, more strains were tested from men (70%: 7,569/10,815) than women (42%:   4).
In 2010, for the first time two strains of 1,400 (0.14%; 95% CI: 0.02-0.57)showed decreased susceptibility to ceftriaxone, and both of these also showed decreased susceptibility to cefixime.Both cases were in men.One was about twenty years of age (MIC of 0.19 mg/L for ceftriaxone and 0.5 mg/L for cefixime).The other was a MSM in his fifties (MIC of 0.75 mg/L for ceftriaxone and 2 mg/L for cefixime) [14].No decreased susceptibility to ceftriaxone was subsequently found in 2011 or in 2012.The upward trend in MIC values between 2008 and 2010 was not observed in the 2011 to 2012 period (Figure 5).
Restricted analysis of those laboratories which reported throughout the whole study period and continued to participate in microbiological surveillance during 2012 provided similar results in terms of the evolution of the susceptibility of gonococcal strains to antibiotics between 2001 and 2012 (data not shown).
Factors associated with a reduction in the susceptibility of gonococcal strains to third generation cephalosporins in patients 15 years and older Univariate analysis was performed using the variables from Table 1.The proportion of strains with decreased susceptibility to cefixime (MIC≥0.094mg/L) and to ceftriaxone (MIC≥0.032mg/L) (  For each variable, the number of strains with decreased susceptibility (n) is given relative to the total tested for the antibiotic (N) as well as the percentage.The multivariate analysis includes all the variables in the Table .Significant results (p<0.05) are in bold.
a Rénago is the national gonorrhoea network, a sentinel network based on voluntarily participating public and private laboratories.b Cefixime tested from 2008 onwards; MIC≥0.094mg/L for 385 strains of 6,004 (6.4%).c Ceftriaxone: MIC≥0.032mg/L for 554 strains of 8,645 (6.4%).d Specialised facilities for the management of STI: STI clinics, free anonymous counselling and testing services and family planning centres.
geographical area.This proportion was also greater in the absence of gonorrhoea-associated STI, notably in the absence of co-infection with chlamydia.Furthermore the proportion of strains with decreased susceptibility to cefixime was associated with anatomical site of sample, presence of symptoms and place of consultation.
Multivariate analysis (Table 3) was performed using the variables from Table 1 excluding four variables with a large proportion of missing data (as mentioned in the methods section).The proportion of strains with decreased susceptibility to cefixime (MIC≥0.094mg/L) increased in the 2010 to 2012 period.Indeed, this proportion was 2.7 times greater in 2012 than for the period between 2001 and 2009.This reduced susceptibility increased with the patients' age.It was almost four times higher for strains derived from the pharynx than for those derived from the urethra, even when analysis was restricted to male patients (adjusted odds ratio 4.2; 95% CI: 1.5-11.6;p=0.006).Finally, the decrease in susceptibility to cefixime was found to be higher in the north-eastern area of France.
The proportion of strains with decreased susceptibility to ceftriaxone increased in the 2010 to 2012 period, this increase being particularly sharp in 2010.Taking 2010 as the reference year, the proportion of strains with decreased susceptibility to ceftriaxone (MIC≥0.032mg/L) significantly diminished in the 2011 to 2012 period.As was the case for cefixime, decreased susceptibility for ceftriaxone increased with patients' age, was higher for pharyngeal-based strains (even when restricting the analysis to male patients) (adjusted odds ratio: 3.4; 95% CI: 1.4-8.2;p=0.008) and was higher in the north-eastern area of France.
Multivariate analysis of the 2001 to 2012 period, restricted to the laboratories which continued microbiological surveillance in 2012, provided similar results (data not shown).

Discussion
The microbiological surveillance network in France shows that the proportion of resistant strains to antibiotics fluctuated around 13% for penicillin G over the whole study period.It increased throughout the study for tetracycline, reaching 56% in 2012.For ciprofloxacin it increased between 2003 and 2006 after which it remained high at around 42%.These three families of antibiotics have a resistance level which prevents them from being used in current practice for the treatment of gonorrhoea.Indeed, according to the World Health Organization, first-line treatment must cure at least 95% of patients and must not be used if more than 5% of strains are resistant [2].
Gonorrhoea was susceptible to spectinomycin throughout the study period.The marketing of this antibiotic in France stopped in 2008 and then started again since 2011.However, therapeutic failures for pharyngeal-based infections have led it to become a second-line treatment when there are contraindications to beta-lactam antibiotics [15].
The proportion of strains with decreased susceptibility to cefixime (MIC>0.125mg/L) remained moderate, less than 5%, but it quadrupled between 2011 and 2012.Furthermore, the proportion of isolates with high MIC values (MIC≥0.094mg/L) increased consistently in recent years (Figure 4 and Table 3).The only two strains presenting MICs>0.125 mg/L to ceftriaxone were both detected in France in 2010, and one of these was associated with therapeutic failure using cefixime in an infected patient [14].With respect to ceftriaxone, the number or proportion of isolates with high MIC values (MIC≥0.032mg/L) did not increase in 2011 or 2012.
The very rare cases of decreased susceptibility to ceftriaxone [14,[16][17][18][19] and cases of therapeutic failure with cefixime described in Japan, Canada and Europe [14,[20][21][22][23], confirm the recommendations made by the French Agency for Medicine published in 2005 [12], which were reaffirmed in 2008 [15].These guidelines recommend the use of ceftriaxone (500 mg singledose injection) in first-line treatment, and that the use of cefixime (400 mg single-dose oral) be reserved for cases of patient refusal or when parenteral treatment is not possible.
Nevertheless, the increase in gonococcal decreased susceptibility to cefixime observed in France suggests that clinicians may have continued to prescribe this antibiotic as a first-line treatment after the recommendations of 2005 were published (and reaffirmed in 2008).Indeed, a study performed in 2008 on general practitioners in the 'Sentinelles' network showed that only a minority of prescribing doctors were aware of the 2005 recommendation to abandon ciprofloxacin in favour of TGC [24].By analogy, it is possible that in 2012 doctors had not yet sufficiently implemented the recommendation reiterated in 2008 regarding the use of TGCs [15].A new study on doctors' prescriptions could help support this hypothesis.Another hypothesis to explain the recent increase in gonococcal resistance to cefixime is the spread of a resistant clone [25].
The levels of resistance to penicillin G, tetracycline and ciprofloxacin in France were higher than those found in the United States, but of the same order for TGC and multidrug resistance [26].In contrast, the levels of resistance in France were of the same order as European levels for penicillin G and ciprofloxacin, but lower for TGC [27].
We currently do not have data on the susceptibility of gonorrhoea to azithromycin.In France this antibiotic is taken in a single, 1 g dose with ceftriaxone in suspected cases of urethritis and cervicitis [12,15].Elsewhere, notably in the United States, azithromycin is recommended in monotherapy for gonococcal infections, in the case of allergies to TGC [28].Because of resistance to azithromycin described in various countries [26,29] this antibiotic is being tested within the Rénago network as of 2013.
Our study shows factors associated with a reduction in the susceptibility to TGC.We acknowledge that the combination of data from 2001 to 2009 only allowed to highlight the recent epidemiological and microbiological changes.The proportion of strains with decreased susceptibility to TGC increased with patients' age.This has already been observed for other antibiotics (penicillin and tetracycline) in Canada [29].A lower proportion of gonococcal resistant strains in the case of co-infection with chlamydia was found for cefixime in the United Kingdom [30] and for ciprofloxacin at the European level [27], although the reason for this is not clearly established.
The proportion of strains with decreased susceptibility to TGC did not differ significantly according to sex.Anal-based strains did not present higher decreases in susceptibility than urethral-based strains.However, the proportion of pharyngeal-based strains with decreased susceptibility to TGC was three to four times higher than urethral-based strains.This may be explained by the horizontal transfer of gene mutations associated with decreased susceptibility to TGC from oral commensal Neisseria [31].Anal and pharyngeal gonorrhoea in men was probably acquired through homosexual transmission, although the absence of information on sexual behaviours from the Rénago network prevents us from being certain about this.Several studies have shown that MSM are likely to be infected more frequently and more quickly than heterosexuals by resistant gonococcal strains [26,30,32].This fact highlights the need for specific surveillance of antibiotic susceptibility in MSM.We found that the monitoring of anal strains brought information on resistance similar to those of urethral strains.Despite the absence of behavioural data, our study shows, however, that increased monitoring of pharyngealbased strains with respect to decreased susceptibility to TGC seems essential.
Finally, we observed a higher level of decreased susceptibility in the north-eastern area of France.It is hard to interpret the reason for this.Obviously this area should remain under close scrutiny, while still keeping the national distribution of the network's laboratories homogeneous.
In the Rénago network, an increase in the number of declared gonococcal infections occurred over the 2001 to 2012 period.Several interlinked phenomena contributed to this: the real growth in infections, the increase in NAAT diagnoses, the increase in the number of participating laboratories and the growing shift towards the consolidation of laboratories.Despite this complex evolution, the Rénago network continued to help monitor gonococcal resistance to antibiotics.This microbiological surveillance network would appear to be reliable as the changes that have been made to it have (the reduction by half of the number of laboratories submitting strains to the NRL), a priori, had little or no effect on the proportion of resistant strains observed.Restricted analysis to those laboratories which continued to participate in microbiological surveillance in 2012 provides strong evidence for this.The emergence of resistance to TGC is extremely worrying as the latter represent the last line of treatment, with no therapeutic alternatives currently available [31].This fact justifies the continuation of microbiological surveillance of gonorrhoea at a collective level in order to adjust therapeutic recommendations.Meanwhile, surveillance of therapeutic failure with respect to TGC is currently being implemented at the European level [33].Given the increase in NAAT diagnosis, it is essential that medical laboratories continue to perform gonococcal cultures which adapt care to the individual's needs while waiting for molecular tests on susceptibility to antibiotics to be developed and made accessible [2].

Introduction
Echinococcus multilocularis, causative agent of alveolar echinococcosis in humans, has become the target of intensive research at the beginning of the 1980s [1].In the past decades, the known range of the parasite in Europe has expanded.The formerly known geographical range of the parasite included regions of (by date of detection) Austria, Switzerland, France, Germany, Liechtenstein, Luxembourg, Denmark, the Baltic states, Belgium, the Netherlands, northern Italy, Poland, the Czech Republic, Slovakia, northern Hungary and Slovenia [2,3].Recently, the occurrence of E. multilocularis was for the first time confirmed also in Sweden [4,5], and in southern Denmark, a new highendemic focus was detected [6].The importance of the issue is highlighted by a recent regulation issued by the European Commission in 2011 [7].The aim of the measures outlined in the regulation is to minimise the risk of introducing the parasite into territories where it has not yet been detected, namely Finland, the United Kingdom, Ireland and Malta [7].
In Slovakia, E. multilocularis was detected for the first time in red foxes in 1999 [8] and intensive epidemiological studies have been carried out since.During the surveillance of E. multilocularis between 2000 and 2010, more than 4,700 red foxes (Vulpes vulpes) from all regions of the country have been examined.The total average prevalence of the parasite was 30.3% and the existence of highly endemic areas in northern regions of Slovakia was revealed [9].
The survey presents a comprehensive picture of the epidemiological situation of E. multilocularis in highly endemic regions of northern Slovakia from its first detection in 1999 until 2012 and includes results from the territory of the High Tatras National Park.Moreover, we report here the first list of confirmed human cases of this serious parasitic disease.[10] with modification of using 0.09 mm mesh analytic sieves for washed fraction filtration [9] was applied for E. multilocularis detection.

Climatic data collection
Influence of climatic factors (mean annual air temperature and mean annual precipitation) on the prevalence rate of E. multilocularis in red foxes in individual years was assessed.The data were obtained from datasets of Statistical Office of the Slovak Republic [11].Data measured at three weather observatories in northern Slovakia (Oravská Lesná -Žilina Region, Poprad and Stropkov -Prešov Region) were used for this purpose.

Collection and examination of dog samples for Echinococcus multilocularis
To determine the role of dogs in the spread of the disease in highly endemic areas, 138 animals originating from the northern areas of Slovakia (Prešov and Žilina Region) were collected between 2002 and 2005.Faecal samples came from stray and owned dogs that were not dewormed regularly with the last deworming treatment at least four months before examination.In order to reduce the risk of infection, all samples were deep-frozen at -80 °C for at least seven days prior to examination.
Data concerning the age, sex, locality and usage of dogs were gathered with the help of a questionnaire.Other questions were related to how often the dogs could move freely in rural areas, were fed with raw viscera or caught rodents.Animals aged up to 10 months were classified as young, older animals were identified as adults.
Nested PCR was used to detect the presence of E. multilocularis DNA in faecal samples [12].

Statistical analyses
The prevalence of E. multilocularis in the examined dog and red fox population was calculated.The seroprevalence values were given with 95% confidence intervals (CI), and odds ratios (OR) were calculated to estimate the association between variables included in the study (age, sex, utilisation, etc.) and the risk of echinococcosis.All statistical analyses were conducted using STATISTICA 6 Base (StatSoft, Inc, 2001).

Surveillance of Echinococcus multilocularis in red foxes in northern Slovakia during 2000 to 2012
Since 2000, a total of 1,875 red foxes originating from the northern Slovak regions have been examined for E. multilocularis presence.The tapeworm was detected in the small intestines of 779 foxes (41.6 %), with a mean prevalence rate in the Žilina and Prešov Region of 47.7% and 38.5%, respectively (Figure 1).

Echinococcus multilocularis in dogs
Of 138 dog faeces examined, E. multilocularis DNA was confirmed in four (2.9 %) samples.The majority, 119 samples, came from the Prešov Region with three infected animals (2.5 %).Among the 19 animals from the Žilina Region, one sample (5.2 %) was found to be positive.
The positive animals were used as guard and shepherd dogs, but neither usage of the dogs nor their age or sex influenced the occurrence of parasites significantly (p>0.05).
Data about free movement in rural areas, feeding and catching rodents were known for 82 cases.Positivity for E. multilocularis was closely correlated with the possibility to catch the rodents (p=0.04;OR=9.4; 95% CI: 0.9-95.9)and uncontrolled movement in rural areas (p=0.02;OR=14.5;95% CI: 1.4-145.5).Feeding with raw viscera was not identified as a risk factor for infection (Table 2).

Human cases of alveolar echinococcosis in Slovakia
The first case of alveolar echinococcosis in Slovakia was confirmed in 2000 in a woman in her 60s from a village in the Žilina Region (north-western Slovakia).Although the first clinical symptoms of the disease had appeared already in 1994, the lack of knowledge about the presence of E. multilocularis on the territory of Slovakia probably resulted in misdiagnosis.In the patient's anamnesis, a history of keeping a dog and collecting forest fruits were of epidemiological importance [18].
In the same year, a second case of autochthonous alveolar echinococcosis was detected.The patient was a woman in her 70s, from a village in mountain area of north-western Slovakia (Žilina Region), who spent a lot of time at her mountain cottage where she grew vegetables.Except weight loss, the patient had no clinical signs, and the disease was noticed only after observing the high erythrocyte sedimentation rate during a routine medical examination.Subsequent USG and CT examination of the abdomen revealed an irregularly shaped focus 12x11x10 cm in the right liver lobe.Serological (ELISA, Western blot) and histological examinations confirmed the diagnosis of alveolar echinococcosis [19].
Three years later, in 2004, two further cases were verified.The patients were men from the Prešov and Žilina Region.Since then, new cases of alveolar echinococcosis have been detected every year (Figure 3).The incidence of the disease has followed an increasing trend.As of April 2013, the total number of alveolar echinococcosis in Slovakia has reached 26 cases (Figure 1).Only three of these patients did not come from the highly endemic Prešov or Žilina Regions.One woman came from the Trenčín Region, situated in the west of Slovakia and neighbouring the Žilina Region, and two persons were from the Košice Region neighbouring the Prešov Region (Figure 1).The mean age of the patients was 52.3±20.7 years, ranging between eight and 78 years.Only negligible differences were recorded between the positivity of women (14 cases) and men (12 cases).
In addition to the 26 confirmed cases of alveolar echinococcosis, at least 10 other cases were detected between 2007 and 2012 in whom the aetiological agent of echinococcosis was not determined.In these patients, the diagnosis of echinococcosis was made after serological and USG examination.However, material for histological or PCR examination that could facilitate the confirmation of diagnosis could not be obtained because of the good response to anthelmintic treatment that made surgical intervention unnecessary, or because the patients neglected to appear for their control examination.
Three of the 26 confirmed patients were younger than 20, one of them even younger than 10 years ( a forest.The patient suffered from headache, fatigue, cough, fever and abdominal enlargement.USG examination confirmed tumour-like infiltrations in the right and left liver lobes and the diagnosis of echinococcosis was confirmed by ELISA, Western blot and histological examination [20].In 2012, two more cases of alveolar echinococcosis in young patients were detected.One was a teenager from the Žilina Region with multiple cysts in the liver and four cysts in the lungs, found by USG examination; the diagnosis was confirmed by PCR after the resection of suspicious parasitic material from the liver.The other case was younger and came from the Prešov Region, with a primary infection site in the right femur; no other lesions were confirmed using USG and PET-CT examinations.This case was living near the forest at the end of a village, in close vicinity to an area often visited by red foxes.The family also owned a dog.Although the first symptoms, pain in the right leg and knee, were not indicative of alveolar echinococcosis, serological examination confirmed the presence of antibodies to Echinococcus spp.Following histological investigation of material obtained during surgical intervention, Western blot of the serum sample and PCR analyses of DNA from the cyst secretion confirmed the diagnosis of alveolar echinococcosis.

Discussion and conclusion
Our monitoring confirms the presence of highly endemic areas of E. multilocularis in the northern part of Slovakia, with a mean prevalence rate of almost 42.0% in its main definitive hosts, red foxes.This 12-year examination of red foxes has unfolded great fluctuation of E. multilocularis prevalence between years, probably depending on climatic conditions.Prevalence rates were highest during the cold and wet years of 2001, 2005 and 2010.In those years precipitation totals were considerably above normal.In contrast, prevalence significantly decreased in the warmer years of 2003, 2007 and 2012, which were the first, third and fourth warmest years since 1871 in Slovakia [17].
Given that E. multilocularis is considered a parasite adapted to cold climate, environmental conditions of northern Slovakia are very suitable for the spread of echinococcosis.The area is characterised by mountain climate with average annual temperatures between -3 and +4 °C.Annually, rainfall varies between 800 and 2000 mm.Main recreational areas of Slovakia are situated in northern Slovakia, which is of relevance from a public health point of view.Žilina Region is dominated by national parks, and 51.0% of its territory is under some form of protection, accounting for 30.0% of Slovakia's total protected area.In the territory of the Prešov Region are situated the only truly alpine mountains in Eastern Europe, the Tatra National Park [11].In the past decades, touristic activities have increased, not only in the National Parks but all over the country.The estimated number of tourists visiting Tatra National Park exceeds 3,500,000 per year [21].In addition, the risk of human infection is also influenced by high population density and urbanisation of red foxes (and other free-living carnivores) in these areas.   of the disease [22,23].Some of the dogs (2.9%) in our study were infected with E. multilocularis and could be a source of human infection.Catching rodents and unattended movement in rural areas were correlated with infection of dogs.Of course, a larger number of samples and further epidemiological analyses would be more valuable, but due to limited staff we did not continue the examination of dogs after 2005.Budke et al. and Ziadinov et al. also recognised a higher probability of infection in dogs that hunt rodents, in hunting dogs and in non-restrained dogs [24,25].Although E. multilocularis is not common in dogs in Slovakia, contact with animals with risk behaviour increases the probability of infection, especially in highly endemic regions of the country.
The list of diagnosed human cases of alveolar echinococcosis confirms northern Slovakia as a risk area.Of 26 infected humans, only three lived outside the Žilina or Prešov Region.However, the prevalence of E. multilocularis in red foxes also reaches 18.4 % in the Košice Region and as much as 40.3 % in the Trenčín Region [9].Thus, the risk of infection in areas in regions other than Žilina and Prešov is not negligible and a rise in the number of new cases is possible in the future.Moreover, as northern Slovakia is a popular tourist destination, we cannot exclude the infection of the three patients in highly endemic localities.
Humans are considered to be unsuitable hosts for E. multilocularis [26].Therefore, it is assumed that repeated or long-term exposure to contaminated environment is needed to establish the infection [27].In addition, the long incubation period (five to 15  The short interval between the first detection of E. multilocularis in foxes and the first human case (with severe organ damage) suggests that the parasite was present in Slovakia before 1999.Since then, increased awareness of public health authorities and physicians the possibility of serological examination of serum samples and new diagnostic methods, together with a constant high prevalence of the parasite in red foxes and increased density of the fox population, has contributed to an increase in the number of human cases detected, especially since 2010.
We can assume that the real number of human alveolar echinococcosis may be several time higher than that shown by official data.A similar situation was recorded  also in other countries; Jorgensen et al., for example, estimate that the incidence of the infection in Germany is three times higher than national surveillance data [31].In Slovakia, confirmed cases of echinococcosis are reportable to the Regional Authorities of Public Health (RAPH) operated under Ministry of Health, and thereafter, the data are sent to the Epidemiological Information System (EPIS) or reported directly to the EPIS.Such a reporting system has some limitations since reports to EPIS come only from physicians and pathologists registered in the system.On the other hand, reports to the RAPH come from microbiological or parasitological laboratories.Due to the fact that each party relies on the other to report diagnosed cases, many cases can be neglected and the incidence can thus be seriously underestimated.Moreover, in many cases the diagnosis is established only as echinococcosis, and the aetiological agent of the disease, E. multilocularis or E. granulosus, is not determined.Similarly, the European Food Safety Authority (EFSA) supposes that current data about the occurrence of human echinococcosis in Member States of the European Union do not provide an accurate picture of the epidemiological situation.
In 2010, approximately 22% of human cases remained undetermined [32].Distinction between infections with E. granulosus and E. multilocularis would be beneficial because the two diseases require different management of prevention and treatment [32].
Despite the fact that alveolar echinococcosis was discovered in Slovakia already 13 years ago and despite (or maybe because of) the popularisation of this medical problem, public knowledge on its epidemiology is still greatly misleading.In Slovakia, as in other European countries, biased information about eating berries and mushrooms as the most important infection risk factor is often promoted in media, while little consideration is given to ownership of dogs and contact with wild carnivores.Nevertheless, several case-control studies aimed at investigating the epidemiology of alveolar echinococcosis have affirmed ownership of dogs and contact with carnivores as the most important risk factors [22,23,33].Therefore, regular deworming treatment of dogs could be an effective preventive measure.In contrast, two studies from 2004 and 2005 did not find an association of alveolar echinococcosis with picking and eating berries and mushrooms from fields and forests [23,34].
The above facts draw attention to the need to revise the existing reporting system, improve the national surveillance system and engage specialists outside the medical community (public health professionals, parasitologists, veterinarians, zoologists and ecologists) for effective management of prevention and control strategies.Knowledge on how to minimise the risk of E. multilocularis transmission is not yet established in new endemic areas.Eradication of the parasite from the environment by means of long-term baiting campaigns in the fox population appears to be an effective tool, but has never been completely successful [35,36].
The baiting programmes depend strongly on several factors, mainly on available financial resources.Therefore the prevention of human alveolar echinococcosis should be based on integrated control measures such as increasing public awareness of hygienic measures, regular anthelmintic treatment of domestic carnivores or vole habitat management [36].

Introduction
Lyme borreliosis (LB) is the most common vector-borne disease in the northern hemisphere [1][2][3].The economic and social costs of managing the disease represent an important burden on both health services and society [4,5].Several epidemiological studies describe a 2 to 3.6-fold increase in the incidence of this disease over the last decade, in Europe as well as in the United States (US) [6][7][8][9].
The American surveillance system, based on notifications of observed cases, has been active since 1991.In Europe, most countries do not have national monitoring data at their disposal.The Czech Republic and Slovenia are among the few exceptions [1].In France, the only existing nationwide study is a prospective study conducted by the Sentinelles network with general practitioners (GPs) from 1999 to 2000.It estimated the incidence of the disease at 9.4 per 100,000, with important inter-regional variations [10].Several regional studies were also conducted, some on highrisk populations (forest workers), others on the general population in high-risk regions.In eastern France, disease incidence was estimated at 200 per 100,000 inhabitants, and at over 500 per 100,000 in certain areas [11,12].
Estimates of the epidemiological characteristics of LB are useful to orient control and prevention measures, as well as to assess their effectiveness.These data are also necessary to elaborate factual risk communication messages provided to the lay public or the media.Since 1984, the Sentinelles network collects, processes, forecasts and dispatches epidemiological data on the activity of GPs in France [13], in real time.In 2009, LB was added to the list of the monitored health indicators.
We analysed the data collected by the Sentinelles network over the first four years of LB surveillance in France (2009)(2010)(2011)(2012).We also analysed the national hospitalisation databases from 2004 to 2009 (the latest available data at the time of writing this paper).

Incidence and characteristics of Lyme borreliosis cases (2009-2012)
The Sentinelles network GPs notify weekly the cases of LB they identify during consultations to the network's electronic information system.Notifications are made online throughout the year, in a standardised way.Sentinelles GPs make up a representative sample of the national GPs, both in terms of practice area and age distribution of their patients [14].To be reported by GPs, cases had to meet the following definitions as stated in the European Concerted Action on Lyme Borreliosis criteria: (i) presence of an erythema migrans (clinical diagnosis); or (ii) appearance of neurological, articular (arthritis only), cutaneous or cardiac symptoms evocative of Lyme disease, in a patient with positive serology [15].
For each notified case, a standardised questionnaire provided information on the patient's age and sex, the date of diagnosis, a history of tick bite before the consultation (date of bite), presence of asthenia, myalgia, cutaneous manifestations (erythema migrans with a single or numerous lesion(s), lymphocytoma, acrodermatitis (ACA)), neurological manifestations (meningoradiculitis, clinical signs of meningitis, meningoencephalitis, radiculitis, facial paralysis, events related to another cranial nerve), the presence of arthritis (articulation(s) concerned) or cardiac events (atrioventricular block, pericarditis, myocarditis, other) and, when available, the results of cerebrospinal fluid (CSF) analysis and serological tests.Three of the authors (AV, CA, TH) validated the reported cases when they met the adopted case definition.Validation of cases is an ongoing regular activity of the surveillance procedure and involves checking the consistency of data reported in the standardised questionnaire for each reported cases.When needed, GPs are contacted for more information.Cases of meningoradiculitis or unilateral facial paralysis were validated even if CSF fluid analysis had not been done, when they were clinically very suggestive (consensus agreement between three authors, AV, CA and TH) in patients who reported a history of erythema migrans less than two months before the onset of neurological manifestations [16].
Cases reported from 1 January 2009 through 31 December 2012 were analysed.The annual incidence rate was calculated as follows: the average number of cases notified by Sentinelles GPs (adjusted for participation and geographic distribution) was multiplied by the total number of private GPs practicing in France [17].This product was then divided by the French population [18].Confidence intervals were estimated under the assumption that the number of reported cases followed a Poisson distribution.

Lyme borreliosis hospitalisations (2004-2009)
Hospitalisation data were collected by reviewing all hospital discharge reports containing an LB code from 1 January 2004 through 31 December 2009 (the latest available data at the time of writing this paper), all obtained through the Programme de Médicalisation des Systèmes d'Information (PMSI) data processing centre [19].This database is a national register of all discharges from all short-stay/acute-care hospitals.It collects data described by the physicians who took care of the patients during their hospitalisation, using the International Classification of Diseases, 10th revision (ICD-10) [20].We identified all the hospitalisation reports for which a LB code was reported, i.e.M01.Also, seasonality and the mean duration of hospitalisation were presented.To estimate the average annual incidence rate of hospitalisations (national and regional), the number of hospital stays in a given geographical area was divided by the average population in the study period, and then multiplied by 100,000 [18].

Incidence and characteristics of Lyme borreliosis cases (2009-2012)
From 2009 through 2012, GPs of the Sentinelles network reported 441 cases of Lyme borreliosis, 110 of which were not chosen for validation because they did not meet the criteria.These were mostly late manifestations without serological confirmation (n=42), tick bites without any clinical event (n=25), insufficiently completed questionnaires (n=19), positive serology without any associated clinical event (n=8), diagnosis errors later confirmed by the GP (n=7), data capture errors (n=4), prevalent but non incident cases (n=5).The remaining 331 cases were validated and analysed.
Women represented 52% of these 331 cases (p=0.37).The age distribution revealed two peaks, between 5 and 10 years and between 50 and 70 years (Figure 3).A tick bite was reported by 74% of patients.The average delay between the bite and the date of diagnosis was 28 days, with a median of 10 days.Most reported cases had erythema migrans (93.6%), with a single lesion in 91% of cases and multiple lesions in 9% of cases.Other clinical forms of the disease were reported in a limited number of cases: cutaneous lymphocytoma (n=4), ACA (n=3), arthritis (n=9), neurological disorders (n=5), and one patient presented both arthritis and neurological disorders.For one patient only presenting a neurological form of LB, a lumbar puncture was performed to confirm the diagnosis.

Lyme borreliosis hospitalisations (2004-2009)
Among the 14,348 files extracted in France from the PMSI from 2004 through 2009, 5,727 were considered compatible with a diagnosis of Lyme disease, i.e. 40% of all hospitalisation records with an LB code.Therefore, the estimated average incidence of hospitalisations was 954 per year (Figure 4), which represents an estimated annual average hospitalisation rate over the period of 1.55 per 100,000 inhabitants (95% CI: 1.42-1.70),ranging from 1.38 to 1.87.Regional variations of hospitalisation rates were remarkable, ranging from 6.72 per 100,000 inhabitants per year in Alsace, to 0.30 in Corsica (Figure 1B).

Discussion
Data from the Sentinelles GP network and administrative hospitalisation database allowed for an updated and ongoing description of the epidemiology of LB in France.Although they covered different time periods, there was consistency between the data sources in regard to age distribution, seasonality and spatial distribution.The study confirmed that the incidence was greater in the eastern and central regions of France, increased in summer, and was higher among young children and older adults.It also confirmed that GPs play a major role in diagnosing and treating the early forms of the infection.Even though they were unusual, severe forms made hospitalisation necessary.
On the basis of the 331 cases collected and validated by the Sentinelles network, the LB incidence was estimated at 42 (95% CI: 37-48) for 100,000 inhabitants.The actual LB incidence could in fact be higher than this estimate, since patients who did not consult a GP were not covered by this study.There is no published or unpublished information that could help assess the magnitude of this effect.However, nowadays in France, people cannot go directly to a specialist without first consulting a GP.It is therefore likely that the vast majority of patients had been seen by a GP.The incidence rates estimated here can be compared with incidence rates in other European countries, although such comparisons must be interpreted with caution.Indeed, there are differences between countries in the process of data collection.Only some countries use a continuous surveillance system, and case definitions and laboratory confirmation methods vary [1,9].In 2005, the highest incidence rates of LB in Europe were reported in Slovenia with 206 cases per 100,000 inhabitants, Austria with 135 cases per 100,000 inhabitants and the Netherlands with 103 cases per 100,000 inhabitants [9].A high yearly incidence rate of LB (131 per 100,000 inhabitants) was also reported in Switzerland between 2008 and 2011 [21].Incidence measured in Germany in 2006 amounted to 37.3 per 100,000 inhabitants in six of the 16 federal states [8].In the Czech Republic in 2006, it was 42.6 per 100,000, close to the national incidence in France.In Belgium, it was 16 per 100,000 in 2005 [9], whereas the Belgian network of sentinel GP estimated the incidence rate of erythema migrans at 90.2 per 100,000 in 2009 [22].The incidence rate was below 1 per 100,000 in Italy and Portugal in 2005 [9].
The only earlier available data on French national incidence of LB were provided by a study conducted by the Sentinelles network in 1999 and 2000.It estimated the national incidence rate at 9.4 per 100,000 inhabitants [10].The higher incidence reported in the present study could suggest that the LB incidence rate has increased in France.However, these incidences must be compared with caution, since different artifacts may have contributed to the higher numbers.Firstly, it has been shown Incidence rates per 100,000 inhabitants.that the incidence of LB is highly sensitive to changes in surveillance methods [23].Indeed, the methodology used by Letrilliart et al. in 1999-2000 was different from the one we applied, and probably led to an underestimation of the incidence rate in their study.At the time, data were not collected through a routine systematic and standardised surveillance, as they have been since 2009.The routine surveillance of the Sentinelles network is based on periodic reports from sentinel physicians (usually weekly reports).Thus, if a physician does not provide report for several weeks, they will not be counted as a participating during those weeks.In the 1999 survey, it was assumed that physicians reported cases actively during the entire study period.Thus, compared to routine surveillance, the number of cases declared in the 1999 study was attributed to an overestimated number of participating GPs, resulting in lower incidence estimates.Secondly, LB today is better known to the public and the GPs than it was in 1999, which may have contributed to the strong increase in estimated LB incidence we report here [6].Finally, the number of hospitalised cases was stable over the period 2004-2009 and seems incompatible with a four-fold increase in incidence suggested by a comparison of incidence estimates derived from the 1999-2000 study and the 2009-12 surveillance data.The hospital discharge database being a stable system, surveillance bias is unlikely, and the database can therefore provide useful data for trend analyses.
Nevertheless, numerous studies and surveillance data from other European countries and the US showed Incidence rates per 100,000 inhabitants.that the LB incidence may indeed have increased over the past few decades.In the US, a continued surveillance system was created in 1991, and the incidence has increased by 101% since then [6].In Europe, an increase in LB was observed in several countries [9].Between 1990 and 2001, the incidence rate in the Czech Republic doubled [1]).In Germany, Fulop et al. describe a 110% increase between 2001 and 2006 [8].
In the United Kingdom, case numbers have increased 3.6-fold between 2001 and 2011 [24].In addition, some factors not related to surveillance artifacts may have contributed to an actual incidence increase, such as climate changes [25,26], increases in the population of wild animals hosting ticks, modifications in agricultural and forest landscapes that can lead to a higher density of tick populations [1], or reduction in biodiversity that can increase the prevalence of ticks carrying Borellia [27].
Regional incidence rates varied considerably in France, from 0 per 100,000 inhabitants to 184 per 100,000.These variations could be explained by participation biases.However, they were the same as those observed in the earlier Sentinelles network study [10] and, more importantly, the regional hospitalisation incidences corroborated the GP surveillance data, as shown by the similar distribution of GP and hospitalisation data in Figure 1.Other published studies confirmed the regional distribution reported here [11].
In the US, incidence also varied among states, from less than 0.01 per 100,000 inhabitants in Montana or Colorado, to 73 per 100,000 in Connecticut [6].The confirmation of interregional variations is an asset to guide future studies and develop more efficient public health actions.
In 94% of the cases in France, the infection was diagnosed at the erythema migrans stage.In the US, the proportion of erythema migrans was lower, estimated at 69% [6].However, the data collection process in the US included cases reported through laboratorybased surveillance that are more likely to have late manifestations of LB [23].An earlier study conducted in France with GPs, specialists and hospital practitioners reported a proportion of erythema migrans of 68% [11].In Europe, published data varied from 65% to 95% [11,28,29].These observations suggest that either the incidence of late Lyme disease forms in general practice in France is underestimated or the incidence of erythema migrans is overdiagnosed.Indeed, the lesion can be confused with other dermatological disorders, and the positive predictive value of an erythema migrans diagnosis made by GPs in France has been estimated at 72% [30].
There was a marked difference in the sex ratio between outpatients (predominantly female) and inpatients (predominantly male), for which we have no explanation.Sex differences in the risk of contracting tick bites, incidence rates, and clinical picture of erythema migrans have been reported, although the biological, immunological, and sociological mechanisms causing these differences have not been determined [31].
There are limits to the use of data coming from the national hospitalisation statistics, including diagnosis and coding mistakes, unintentional omissions, partial reporting of pre-existing conditions, lack of information about treatments (whether or not patients received an LB-effective antibiotic could help validate the diagnosis), the frequent unavailability of medical history forms to the certifying practitioner or difficulties in determining the initial cause of hospitalisation when several associated pathologies are involved.Indeed, it is possible that the recorded diagnoses were given by physicians who did not base their decision on accepted case definitions.Also, the diseases defined by the ICD-10 codes that we used are not specific for LB, their aetiologies may be numerous and in many cases idiopathic.It has been previously shown that the positive predictive value of the LB code in the PMSI database was 65% [32].We therefore considered it necessary to set up a selection process for the reported hospitalisations.In the end, we only kept 40% of the registered reports.Nevertheless, the predictive value of our case definition could remain low due to the lack of specificity of the retained hospitalisation definitions (related to the complexity of databases such as the PMSI hospitalisation database).Therefore, our study could also overestimate the hospitalisation rates for LB in France.However, the PMSI database proved a useful tool to monitor trends over time, determine the seasonality, determine high-risk regions, and provide details on characteristics of hospitalised patients.

Conclusions
In conclusion, a countrywide sentinel network and hospitalisation statistics produced epidemiological data that were sustainable and consistent over time and space.They were suitable for following trends and estimating the burden of this disease for which there is significant public concern.Determination of the healthcare burden associated with LB can inform public health policy and enable valid analysis of the efficiency of LB control measures.Furthermore, we believe that this study could help elaborate factual risk communication messages provided to the lay public or the media.Healthcare providers should continue to invest attention into prompt recognition and early, appropriate therapy for LB.Also, public health strategies should keep promoting the use of repellent, daily checks for ticks and their prompt removal, mainly in the age groups, in the regions and during the months with the highest incidences.

Introduction
Influenza infection causes a major burden of illness in adults and children [1,2].Seasonal trivalent influenza vaccines (TIVs) are effective in preventing a range of laboratory-confirmed outcomes [3], but effectiveness varies by severity and season, the presence of comorbidities and age [4,5].
The SHIVERS (Southern Hemisphere Influenza Vaccine Effectiveness, Research and Surveillance) study has allowed estimation of vaccine effectiveness (VE) against influenza illness requiring hospitalisation since 2012 and against influenza illness requiring primary care (general practice) since 2013.
In New Zealand, seasonal non-adjuvanted inactivated trivalent influenza vaccine is available annually free of charge to all adults aged 65 years and over, pregnant women and all those over six months of age with chronic medical conditions that are likely to increase the severity of the infection.Influenza vaccines are also available on the private market for all others over six months of age.

Methods
Ethics approval for the study was obtained from the Northern A Health and Disability Ethics Committee (NTX/11/11/102 AM02).

Study design
In both hospital and community settings, we conducted a study using a standard case test-negative design [6], drawing on an urban population of approximately 838,000 people in Central, South and East Auckland [7].
For community cases, we recruited 18 sentinel general practices with 103,884 enrolled patients.Patients in these sentinel practices were broadly representative of the ethnically diverse urban population of Auckland by age and sex distribution, but with more Pacific people (27% in the practices compared with 15% in the source population) and slightly fewer people of Asian descent (14% versus 19%, respectively) [7].
The practices recruited individuals aged six months and older who presented to a general practitioner or practice nurse with ILI, defined as a history of fever or measured fever of ≥38 °C and cough, with onset during the preceding 10 days [8,9].
All patients presenting to one of the sentinel general practices with suspected respiratory infections were screened by the general practitioner or nurse for ILI.All identified ILI cases were entered on an electronic form in the practice management system and a nasopharyngeal or throat swab was collected for influenza virus testing from all consenting patients.
For hospitalised patients, we enrolled individuals aged six months and older who were admitted with SARI to one of the four public hospitals covering the whole population in this catchment area.On the basis of the World Health Organization (WHO) definition, SARI was defined as hospitalisation with a patient-reported history of a fever or a measured temperature of ≥38 °C and cough with onset within the past 10 days [10] the first three days, then declines steadily until days 7-9 [9].We also excluded patients with incomplete data for vaccination status or age, all infants under 6 months of age, children aged under 9 years who were only given one dose of trivalent inactivated influenza vaccine, patients who were vaccinated less than 14 days before admission to hospital or presentation to general practice.For patients with multiple episodes, the first influenza virus-positive episode was used for the analysis, or the first illness episode if there was no influenza virus-positive episode.
The influenza season was defined as starting when there were two consecutive weeks with two or more cases and ending when there were no consecutive weeks with two or more cases.Analysis was thus undertaken from 3 June to 10 November 2013 for SARI cases and from 13 May to 27 October 2013 for ILI cases (Figures 1 and 2).

Participant information
For all ILI patients, variables extracted from the electronic form and patient management system included age, sex, ethnicity, chronic medical conditions and current smoking status, socio-economic status as identified by the New Zealand deprivation status (a meshblock measure reflecting eight dimensions of deprivation distributed into deciles) [11] and a subjective assessment of obesity by the clinician as body mass index measurements were not consistently available.
Similar information was collected for all SARI patients, but for this group we also collected the following: a patient-or caregiver-reported measure of dependence (which assessed requirement for assistance with normal activity or full dependency on nursing care); a measure based on long-term use of oxygen that we labelled 'frailty'; a history of chronic medical conditions; and a self-defined, standardised functional wellbeing health status score from a national survey [12], combining fair or poor well-being versus all other more positive well-being scores.
In New Zealand, almost all influenza vaccinations are administered in general practices.For ILI cases, vaccination status was taken from the general practice record.SARI vaccination status for the 12 months before hospitalisation was determined by self-report.

Laboratory methods
Nasopharyngeal swabs, aspirates and other respiratory samples were collected according to hospital or general practice standard procedures.Samples were tested using the United States Centers for Disease Control and Prevention (CDC) real time RT-PCR protocol [13] or the AusDiagnostic PCR protocol [14].The AusDiagnostic assay had a sensitivity of 100% and  specificity of 96.6% when the United States CDC method was used as the comparator [15].RT-PCR assays detected influenza virus types A and B and subtyping was performed for type A. All influenza virus PCR-positive samples were forwarded to the National Influenza Centre and characterised antigenically using established methods [14].

Statistical analysis
Univariate chi-squared tests were used to compare characteristics of patients who were influenza virus positive (cases) and negative (controls).A multivariate logistic model restricted to the test-negative controls was used to calculate an adjusted odds ratio for the propensity to be vaccinated for a range of patient characteristics possibly associated with vaccination (Table 1) and used in a previous study [16].The results from this propensity model are presented as odds ratios.This propensity model was then applied to the complete dataset to generate individual propensity scores for vaccination.In order to ensure these propensity scores were linear with respect to influenza virus positive status, we used the cubic spline for these scores as an adjustment variable for estimating VE.For both SARI and ILI, we calculated the crude VE, adjusting only for the timing of presentation relative to the influenza season (defined as weeks from the peak), and the adjusted VE, which included the timing of presentation and the cubic spline of the fitted values of the propensity model.VE estimates were calculated against both SARI and ILI, by influenza virus type and subtype and by age group (6 months-17 years, 18-64 years and ≥65 years).
For all patient characteristics, other than age and vaccination status, each missing data point was imputed as the baseline (referent) value for the corresponding variable.The baseline values were: non-Māori, non-Pacific ethnicity, female, not in New Zealand deprivation groups 8,9 or 10 (the three lowest deprivation deciles), not pregnant, current non-smoker, without chronic disease, not obese, with self-rated health average or better, not using long-term oxygen and living without assistance).All male patients and all female patients not aged 13-55 years were assumed to be not pregnant.Overall, 53 SARI (5%) cases and 1 ILI (0.1%) case had any imputed covariates.Sensitivity analyses were performed using only those individuals with complete data.
As a further sensitivity analysis, we also compared the overall VE estimate from the propensity-adjusted model with an epidemiological model that included covariates that were assessed as potential confounders and/ or effect modifiers (Table 1) and a statistical model, derived from the epidemiological model, where only covariates that were significant at the 0.05 level were included in the final model.

Results
Characteristics and vaccination status of participants ILI and SARI patients in this study by influenza virus status are shown in Figures 1 and 2.

Figure 3
Flowchart of all selected, recruited and tested patients with influenza-like illness and severe acute respiratory infection for influenza vaccine effectiveness analysis, New Zealand, 2013 influenza season Of the 482 ILI patients who tested influenza virus positive, 44 (9%) were vaccinated, compared with 177/1,013 (17%) who tested negative (Figure 3).The proportion vaccinated did not change throughout the season.In those excluded because of incomplete laboratory tests, self-reported vaccination in the previous 12 months among SARI cases was 49/135 (36%), slightly less than the proportion of included SARI cases, 454/1,042 (44%).In the ILI cases, the proportion vaccinated was much higher, at 8/21 (38%), in those excluded because of incomplete laboratory tests compared with the ILI cases included, of whom 9% (44/482) were vaccinated.
Influenza-positive cases and influenza-negative controls were compared across a range of patient characteristics.SARI and ILI patients who were aged 6 months to 5 years or over 80 years, and those presenting outside the influenza season were less likely to test positive.In comparison with the community patients, the hospitalised patients were more likely to be vaccinated, to be older, to live in a deprived area, to be of Māori or Pacific ethnicity, to be a current smoker and to be obese (Table 1).Details on pregnancy were poorly recorded but less than 3% (30/1,042) of other data fields were missing for both SARI and ILI patients.
Although vaccination was more common in SARI patients, the same factors affected the propensity to be vaccinated in persons with ILI or SARI.The adjusted odds ratios for the association of various patient characteristics with the likelihood of vaccination showed that older age groups and those with chronic diseases were most likely to be vaccinated (Table 3).In contrast, there was no statistically significant difference in the likelihood of vaccination by ethnicity, sex, deprivation score, pregnancy, obesity, self-rated health, smoking, assisted living or the timing of the admission relative to the influenza season (Table 3).
For both SARI and ILI influenza-positive cases, the vaccination rate was constant over time.

Figure 1 a
Figure 1Geographical distribution of the 133 laboratories in the Rénago a network according to participation in microbiological surveillance, France, 2012

Figure 2
Figure 2Annual number of reported gonococcal infections and strains tested by the national reference laboratory for gonorrhoea, Rénago network a , France, 2001-2012

Figure 3
Figure 3Evolution of the resistance of gonococcal strains to penicillin G, tetracycline and ciprofloxacin, Rénago a network, 2001-2012 (n=8,649 strains)

Figure 1
Figure 1Occurrence of alveolar echinococcosis in humans (n=26) and mean prevalence of Echinococcus multilocularis in red foxes in the Žilina and Prešov Regions, Slovakia 2000-2012 Due to close contact with humans and contamination of soil around houses and in gardens, dogs present a considerable risk factor in the spread of echinococcosis to humans.Stehr-Green et al. and Kern et al. identified ownership of a dog as associated with acquisition

Figure 2
Figure 2Positive correlation between Echinococcus multilocularis prevalence in red foxes and mean annual precipitation a , northern Slovakia, 2000-2010

Figure 3
Figure 3Annual incidence rates of Lyme borreliosis by age group as estimated by the general practitioners' Sentinelles  network, France, 2009-2012

Figure 2
Figure 2Study participants with severe acute respiratory infections who were influenza virus positive and negative by week, New Zealand, 2013 influenza season

Table 1
Characteristics of cases of gonorrhoea, Rénago a network,France 2001France  -2012 (n=13,400)    (n=13,400) STI: sexually transmitted infection.a Rénago is the national gonorrhoea network, a sentinel network based on voluntarily participating public and private laboratories.b Specialised facilities for the management of STI: STI clinics, free anonymous counselling and testing services and family planning centres.

Table 3
Factors associated with decreased susceptibility of gonococcal strains to third generation cephalosporins in patients 15 years-old and over, Rénago network a , 2001-2012
found dead, road-killed or legally hunted by workers of the Tatra National Park Research Station.Small intestines of the animals were deepfrozen at -80°C prior to necropsy and parasitological examination.Sedimentation and counting method out twice a year, in spring/summer and autumn/winter campaigns.The number of samples and the time schedule of campaigns were set by the State Veterinary and Food Administration of the Slovak Republic.From each of 79 Slovak districts, a representative sample of at least six foxes per campaign was shot.Animals originating from the protected area of the High Tatras National Park were cystic lesions found during imaging examination (ultrasonography (USG), radiography, computed tomography (CT)) were sent to the Institute of Parasitology SAS (IP SAS) for serological examination.At the beginning, IP SAS was the only laboratory where antibodies to Echinococcus could be examined.After a few years, private laboratories started with serological diagnostics and it could not be estimated how many samples were sent elsewhere.Currently, IP SAS is the only laboratory performing Western blot and PCR tests.Specific antibodies to E. multilocularis were detected by modified ELISA[13,14]or commercial ELISA test kits (NovaLisa Echinococcus IgG, NovaTec Immunodiagnostica, Germany), and positive sera of patients who had not undergone surgery, i.e. for whom PCR samples or histology were not available, were examined also by Western blot (Echinococcus WB IgG, LDBIO Diagnostics, France).Imaging techniques (USG, CT, magnetic resonance imaging (MRI)) were performed to detect the presence of parasitic lesions in the liver and other organs, and material obtained during surgery was submitted to histological and/or PCR examination[15].Some clinical cases were detected accidentally, during surgical intervention for another disease.
Human samples and data about the patients were collected in cooperation with medical doctors and hospitals, especially with infectious disease and surgical clinics from different regions in Slovakia.Sera from patients suspected for echinococcosis, e.g.patients with clinical symptoms of echinococcosis or patients with The diagnosis was confirmed if at least two of following four parameters were verified[16]: (i) typical organ lesions detected by imaging techniques, (ii) presence of antibodies to Echinococcus multilocularis (ELISA, Western Blot), (iii) histological findings compatible with E. multilocularis metacestode, or (iv) detection of E. multilocularis DNA by PCR methods.

Table 3
).In 2006, alveolar echinococcosis was diagnosed in a teenager from the Prešov Region, living in close vicinity of

Table 1
Occurrence of Echinococcus multilocularis in red foxes, northern Slovakia, 2000-2012 NA: data become available in full-scale form with a delay of two years.a Measured at three weather observatories in northern Slovakia (Oravská Lesná, Poprad, Stropkov).

Table 1
ICD-10 codes of clinical disorders that may be related to Lyme borreliosis ICD-10 chapters and group of conditions concerned a ICD-10 code concernedChapter VI: Diseases of the nervous system Study participants with influenza-like illness who were influenza positive or negative by week, New Zealand, 2013 influenza season Patients who were identified at seven days post onset of symptoms were excluded from the ILI and SARI analysis, based on the pattern of shedding, which peaks in Figure 1 ILI: influenza-like illness.Weeks 18 to 52 are shown (29 April to 31 December 2013).

Table 1
Characteristics of study participants with influenza-like illness and severe acute respiratory infection, New Zealand, 2013 influenza season a Unless otherwise indicated.b A meshblock measure reflecting eight dimensions of deprivation distributed into deciles.c Subjective assessment of obesity by the clinician.d A self-defined, standardised functional well-being health status score.e Defined as currently on long-term oxygen use.f Requirement for assistance with normal activity or full dependency on nursing care, as reported by the patient or caregiver.
ILI: influenza-like illness; SARI: severe acute respiratory infection.total of 1,042 SARI admissions and 1,495 ILI patients were included in the analysis, of whom 224 (21%) and 482 (32%) were influenza virus positive, respectively.Of the 224 SARI admissions who tested influenza virus positive, 82 (37%) were vaccinated, compared with 372/818 (45%) who tested negative.Despite our attempts, we were unable to verify self-reported vaccination status of SARI patients with the vaccine providers. A The influenza viruses isolated from all of New Zealand during February to September 2013 were forwarded to the WHO Collaborating Centre for Research and Surveillance of Influenza in Melbourne, Australia, for further antigenic characterisation.Most of the New Zealand influenza A(H1N1) viruses reacted well with ferret antisera raised against A/California/7/2009 virus.Almost all of the A(H3N2) viruses reacted well with ferret antisera raised against cell-propagated A/Victoria/361/2011 or A/Texas/50/2012 viruses.B/ Yamagata lineage viruses were the predominant B viruses in New Zealand in 2013.Although this lineage was included in the 2013 southern hemisphere vaccine formulation, antigenic drift was observed in these viruses, as they reacted better with ferret sera raised against B/Massachusetts/2/2012-like virus (selected for the southern hemisphere 2014 vaccine) than B/ Wisconsin/1/2010 virus (included in the southern hemisphere 2013 vaccine).

Table 3
Characteristics of non-influenza virus-positive study patients with severe acute respiratory infection and influenza-like illness (controls) and their association with influenza vaccination status, New Zealand, 2013 influenza season OR: adjusted odds ratio compared with referent group: female, aged 46-64 years, non-Māori, non-Pacific ethnicity, not in the New Zealand deprivation measure of the three lowest deciles (8,9 or 10), not pregnant, current non-smoker, without chronic disease, not obese, with self-rated health average or better, not on long-term oxygen use, living without assistance and admitted to hospital for severe acute respiratory infection during the peak influenza season.
a A meshblock measure reflecting eight dimensions of deprivation distributed into deciles.b Subjective assessment of obesity by the clinician.c A self-defined, standardised functional well-being health status score.d Defined as currently on long-term oxygen use.e Requirement for assistance with normal activity or full dependency on nursing care, as reported by the patient or caregiver.f Admission or presentation before 1 June 2013.