Refugee crisis demands European Union-wide surveillance !

The conflicts in the Middle-East and instability in Libya and some parts of Asia and Africa have resulted in a dramatic influx of refugees to the European Union (EU) in recent years. In the first nine months of 2015, more than 600,000 applications for asylum were filed in the EU [1]. With no prospect of change of the international context in the near future, it is likely that the influx of refugees into the EU will continue and may even increase in coming months.

The conflicts in the Middle-East and instability in Libya and some parts of Asia and Africa have resulted in a dramatic influx of refugees to the European Union (EU) in recent years. In the first nine months of 2015, more than 600,000 applications for asylum were filed in the EU [1]. With no prospect of change of the international context in the near future, it is likely that the influx of refugees into the EU will continue and may even increase in coming months.
We have witnessed numerous large displacements of populations in recent years and 'Refugee health' has become an area of concern for national and international, governmental and non-governmental organisations. Much has been learned from responding to these humanitarian crises.
Although refugees are facing a similar spectrum of non-communicable diseases to those experienced by the indigenous population of their countries of origin, trauma and injuries, sexual and reproductive health issues, violence and psychosocial disorders are among the most frequent health problems refugees encounter. Disruption of healthcare delivery systems in their countries of origin and limited access to healthcare during their journey result in the interruption of treatments often required for the control of chronic diseases [2].
Refugee populations entering the EU/European Economic Area (EEA), and particularly children, are at risk of exposure to infectious diseases in the same way as other EU residents, and in some cases may be more vulnerable because of the interruption of public health programmes, notably for immunisation, in their country of origin, as well as through various barriers to access healthcare such as language, culture etc. It is therefore important that they benefit from protection from infectious diseases, including those prevented through routine vaccinations. In addition, these refugees may be at specific risk for certain infectious diseases in relation to their country of origin, countries traversed during their migration, and the conditions they experienced during their mostly difficult journeys.
It is important to note that refugees should not be seen as representing a threat to Europeans regarding infectious diseases, but rather as being themselves vulnerable for such diseases. For example, poor living conditions and close contact in crowded shelters and refugee camps may increase the risk for the spread of lice and/or fleas, which in rare cases can carry diseases such as louse-borne diseases (relapsing fever due to Borrelia recurrentis, trench fever due to Bartonella quintana, epidemic typhus due to Rickettsia prowazekii), murine typhus and mites (scabies). In recent months, sporadic cases of louse-borne relapsing fever (LBRF) have been reported in Belgium, Finland, Germany and the Netherlands among migrants from Eritrea, Somalia and Sudan [3][4][5]. LBRF is a disease transmitted by body lice that caused major epidemics in the first half of the 20 th century in Europe [6,7] and is known to have occurred occasionally among homeless people in recent years, without spreading to the general population [8]. Recent reports from Italy indicate that transmission of LBRF is likely to have occurred in shelters for refugees in the EU, resulting in the risk of cross-border spread as refugees are frequently moving to other countries [9,10]. Media are reporting outbreaks of scabies and diarrhoea, notably in Calais, France, in relation to poor housing and hygiene conditions [11].
Meningococcal disease outbreaks have been associated with overcrowding overall and in refugee settings. Contributing factors include sharing dormitories, poor hygiene, and limited access to medical care [12] and that meningococcal carriage rates have been shown to be higher in individuals in overcrowded settings. Most cases are acquired through exposure to asymptomatic carriers [13]. Meningococcal disease has usually been reported in children, but is still a leading cause of both meningitis and sepsis in adolescents, young adults and adults. In addition, overcrowding has been associated with increased transmission of measles, varicella and influenza.
As we are approaching winter, the travelling and living conditions for refugees in transit to Europe or in reception centres after their arrival is likely to deteriorate, with even more overcrowding in shelters with insufficient hygiene and therefore increased risk of transmission of communicable diseases. With the start of the influenza season, there is obviously a risk of increased influenza transmission.
Given the numbers and mobility of the refugee populations, the infectious disease risk can only be contained through a coordinated response at the EU level. That includes (i) raising awareness of the risks and types of infection that refugees may have been exposed to and may continue to be exposed to in reception centres, (ii) providing appropriate hygienic and medical countermeasures and (iii) ensuring ready access to medical diagnosis and treatment services. However, such a response will require that Europe has good information on the health situation of the refugees on the move in the EU.
Currently, the basic information that would allow a competent assessment of the situation is not available. The exact number of refugees is not known, and its assessment is hampered because refugees may avoid registration in fear of being sent back [14] and because they continue to move through different European countries. No comprehensive surveillance data is currently being gathered and only sporadic reports by organisations and institutions providing care for these populations are available.
Refugees are not currently a threat for Europe with respect to communicable diseases, but they are a priority group for communicable disease prevention and control efforts because they are more vulnerable.
The scale of the current influx of refugees is inevitably putting pressure on public health systems in frontline receiving countries. Protecting the health of this vulnerable group is complicated further by the potential occurrence of communicable diseases that have not been commonly or widely seen within Europe, creating challenges in terms of recognition and case management. It is vital to ensure that public health authorities have the right information to target resources and provide appropriate measures.
Given these challenges, the European Centre for Disease Prevention and Control (ECDC) will continue to work with its partners in Europe, including public health authorities in the Member States and the European Commission, to strengthen the evidence base guiding prevention and control measures and adding to the current evidence which pinpoint adequate hygiene conditions and vaccination services as the most immediate needs. Strengthening and coordinating surveillance will require continuing efforts to improve the quantity and quality of surveillance data collected through a EU-wide surveillance scheme. It will allow to ensure that interventions aimed at improving health of Introduction Food poisoning caused by enterotoxigenic Staphylococcus aureus is one of the most common foodborne diseases [1]. In France, which has a long-established foodborne disease surveillance system able to detect fairly rare events [2], staphylococcal food poisoning (SFP) has ranked in recent years as the first cause of foodborne outbreaks: of 1,288 reported foodborne outbreaks in 2012, 300 (23%) were due to SFP [3]. SFPs are thought to be under-reported for several reasons. First, because of the short duration of symptoms, only an estimated 10% of SFP patients visit a hospital [4]. Even if patients seek medical care, the physician often does not deem a stool analysis necessary. If a stool analysis is performed, the microbiological routine procedures often do not include testing for the presence of enterotoxigenic S. aureus unless specifically requested by the physician [5]. In addition, staphylococcal enterotoxin (SE) is highly stable and heat-resistant. Although the bacteria may have been inactivated by heating the food prior to consumption and can therefore be isolated neither from food nor the stool of the patient, the highly stable enterotoxins performed by S. aureus in the food may still be emetically active [6].
In contrast to most other gastrointestinal infections, the onset of SFP symptoms is very rapid, usually within a few hours after ingestion of the contaminated food. The median incubation period of aetiologically confirmed SFP outbreaks occurring in the United States between 1998 and 2008 was estimated to be four hours (5-95 percentile: two to seven hours) [7]. Symptoms in cases in these outbreaks typically included abdominal cramps (72%), vomiting (87%), and diarrhoea (89%). Fever (9%) was infrequently reported. The median duration of illness was 15 hours (5-95 percentile: 4-60 hours) [7].

Enterotoxin A
A phylogenetic dendrogram (neighbour joining tree) was generated for 39 Staphylococcus aureus isolates based on the allelic profiles of 1,625 available of 1,878 queried MLST+ target genes. The scale bars indicate the number of differing alleles comprising the calculated distance. The colours represent the origin of outbreak-related strains (orange: stool samples from hospitalised patients; blue: food samples; green: throat or nose samples from colonised staff members). The genotype column shows the combined data of multilocus sequence typing (prefix ST), spa-typing (prefix t), and MLVA typing (prefix m).
of food samples, and colonisation by S. aureus of catering employees at the event. In particular, we characterised the S. aureus isolates from patients, food items obtained from the buffet, and food handlers using traditional typing methods (PCR, spa-typing, and multilocus variable-number tandem repeat analysis (MLVA)), as well as whole genome sequencing.

The event
From 12 to 15 June 2014, an equestrian show-jumping event with approximately 140 participating international athletes and 300 horses took place in Luxembourg. Approximately one to three hours after eating a buffet lunch in the tented VIP restaurant on 12 June, 11 persons with symptoms of vomiting, diarrhoea, and prostration were taken by ambulance to the emergency departments of two hospitals where they received parenteral fluids. The official health inspection service was informed immediately of the incident and microbiological analysis of stool samples from hospitalised patients was ordered. Official food safety inspectors proceeded immediately to take samples from remaining buffet items for microbiological analysis. An inspection of the professional caterer's onsite restaurant and offsite kitchen did not reveal any major food safety deficiencies as specified in regulation (EC) 852/2004. The next morning, on 13 June, local newspapers announced salmon tartare as a potential culprit. A few hours after having the buffet lunch in the VIP restaurant on 13 June, a further 20 persons fell ill with the same symptoms and were transferred by ambulance to hospital emergency departments. The event organiser stopped serving any prepared meals for the remainder of the event. On 14 and 15 June, there were no further reports of gastrointestinal illness related to the event. While approximately 150-200 persons were estimated to have consumed the buffet lunch in the VIP restaurant on both days and a total of 31 persons were admitted to hospital emergency departments over the two days, the exact number of affected persons is unknown. There were no reports of illness among those people who ate at the other food-serving premises at the event: a non-VIP lunch buffet operated by the same caterer but with different menus, and a barbecue stall hosted by non-professional club members.

Microbiological examination of stool samples
Culture of stool samples for bacterial pathogens (including Salmonella, Campylobacter and verotoxigenic Escherichia coli) conducted in three hospital laboratories revealed the presence of S. aureus in ten patients and Enterococcus in one patient. Isolates of S. aureus were immediately referred to the National Health Laboratory for further molecular characterisation.

Case-control study
Following their recovery from illness and after the food samples had been analysed, eight cases who had been admitted to emergency care were contacted by telephone to get initial information on potential food exposures. All food items and symptoms reported by cases were included in a final questionnaire administered by telephone to 22 cases and 21 controls. Cases were defined as persons with sudden gastrointestinal illness (at least one symptom: vomiting, diarrhoea, abdominal cramps or nausea) who had eaten buffet lunch at the VIP restaurant on 12 or 13 June. Controls were defined as persons who had eaten buffet lunch at the VIP restaurant on 12 or 13 June, without any gastrointestinal symptoms. Non-hospitalised cases and controls were contacted using information provided by the event organiser.

Characterisation of S. aureus isolates and whole genome sequencing
Isolates of S. aureus obtained from patients, food, and catering employees were confirmed by MALDI-TOF mass spectrometry (Bruker, Brussels, Belgium). Confirmed isolates of S. aureus were further characterised for the presence of nuc, mecA, toxic shock syndrome toxin 1 (TSST-1), and Panton-Valentine leukocidine (PVL) [8] as well as genes coding for staphylococcal enterotoxins A (sea), B, C, D E, H, I, and J [9]. Isolates exhibiting sea were further characterised by sequencing the PCR products and compared to strains containing allelic sea variants FRI100, FRI287A, and N315. In addition, isolates were subjected to spa-typing [8] and MLVA typing [10]. Whole genome sequencing of isolates was performed on a Miseq Desktop Sequencer using the Nextera DNA sample preparation kit (Illumina, Eindhoven, The Netherlands) with an average coverage of 59 fold (range 27-140 fold). Antimicrobial resistance genes, virulence factors and multi-locus sequence types (MLST) were determined by submitting the raw read files to public webserver tools hosted by the Center for Genomic Epidemiology in Denmark [11][12][13]. After sequencing, whole genome MLST+ was conducted using the Seqsphere+ v2.3 pipeline (Ridom, Münster, Germany). Briefly, after trimming reads until the average quality was 30 in a window of 30 bases, the trimmed reads were mapped to the reference genome NC_002951.2 and the allelic profiles of 1,878 target genes were determined based on the MLST+ scheme developed previously [14]. Final phylogenetic analysis was based on 10 patient isolates, 6 food isolates, 22  Results from the analytical epidemiological case-control study (Table) implicated consumption of pasta salad with pesto as the most likely vehicle of SFP. Eighteen of 22 cases reported eating this food item compared to 3 of 21 controls (p<0.0001). All 14 interviewed cases who had been hospitalised reported eating the pasta salad with pesto. Unfortunately, there were no leftovers of the pasta salad with pesto when sampling was taking place and so this dish was not available for microbiological testing. Eating cured ham or salmon tartare were not statistically significant risk factors (p=0.45). One interviewed patient reported not having eaten ham at the buffet for religious reasons.

Food samples
Isolates of S. aureus with a genotype identical to patient isolates (MLST ST-8, spa-type t024, MLVA-type 4698) were detected in cured ham samples (range <40-5,200 colony-forming units (CFU)/g and shiitake mushrooms (<40 CFU/g) sampled at the event site and in cured ham samples (enumeration range <40-120 CFU/g) obtained at the offsite catering kitchen where the ham was sliced and stored ( Figure). Non-enterotoxigenic isolates of S. aureus with a different genotype to patient isolates were found in cooked asparagus (<40 CFU/g, MLST ST-398, spa-type t571, MLVA type 1039), the floating island dessert (<40 CFU/g, MLST ST-398, spa-type t1184, MLVA-type 567) and several samples of cooked ham (range 50-320 CFU/g, MLST ST-398, spa-type t571, MLVA-type 4789). Unsliced complete legs of cured and cooked hams obtained from the supplying butcher were negative for S. aureus. All 18 food items sampled from the event buffet were negative for Salmonella and E. coli. One food item (cooked asparagus) was positive for presumptive Bacillus cereus (840 CFU/g).
The pasta salad with pesto could not be sampled during food inspection, as there were no leftovers from this dish. The primary ingredients used to make the pesto sauce for the pasta salad (fresh basil, hard cheese, and pine nuts) were all negative for S. aureus.

Staphylococcal carriage study
Thirty-eight of the 49 catering employees at the event were screened for nasal/throat carriage of S. aureus.
Median age of the screened employees was 32.5 years (range 17-50 years), and 11 were women. Twenty-two employees were found to be colonised by S. aureus: three staff members were colonised by strains identical to those found in patients ( Figure). Another four employees were colonised by S. aureus isolates exhibiting sea, but a different genotype than the outbreak strain. None of the seven employees colonised by isolates exhibiting sea reported wounds or gastrointestinal disease prior to the event. Overall, 17 different genotypes were observed among the 22 colonised employees. None of the isolates in food, patients, or catering employees were meticillin-resistant or exhibited pvl.

Whole genome sequencing
The whole genome phylogeny ( Figure), as determined by 1,625 of 1,878 MLST and MLST+ target genes that were present in all 39 isolates, clearly delineated the outbreak isolates. S. aureus isolates found in 10 patients were identical to those isolated from cured ham, shiitake mushrooms and from three catering employees. Interestingly, the Luxembourg outbreak strain had 347 allele differences with a strain that led to the intoxication of 27 boy scouts in Switzerland in 2010, although both strains share a common spa-type t024 [15]. Two of the three food isolates which differed from the outbreak strain were also observed among catering employees. These belonged to livestock-associated sequence type ST398 with spa-types t571 or t1184.

Discussion
Studies of foodborne outbreaks, in which enterotoxigenic isolates were detected in patients, food, and food handlers, are rare [16][17][18]. Our report shows that, even in the era of whole genome sequencing, public health investigations of foodborne outbreaks remain very dependent on classical case-control investigations for interpretation of events. Whereas initial microbiological typing results suggested cured ham as the main vehicle for the intoxication, the case-control study clearly identified the pasta salad with pesto as the most likely source, which was no longer available for microbiological testing.
In our outbreak, there was good evidence that the pathogen responsible for the outbreak was S. aureus, because identical enterotoxigenic strains of S. aureus with a common spa-type but rare MLVA type were recovered from the stools of 10 hospitalised cases. Because three catering employees were colonised by a strain with the same genotype, it is likely that at least one of them may represent the source of food contamination, either via manual contact or through respiratory secretions [19]. However, because catering employees were screened a week after the outbreak, it cannot also be ruled out that some staff members became colonised only during or after the event [20].
One of the probable factors contributing to the outbreak may have been the unusually hot weather for the season, with maximum temperatures ranging between 25 °C and 32 °C during the week preceding the event, compared with a historical average of 21 °C. The food safety inspection at the catering facility revealed that a fridge had stopped working properly a few days prior to the event, although the catering staff denied using this fridge to store any of the dishes. The pasta salad with pesto was reported to have been pre-cooked and sealed into plastic bags in 2 kg portions, and then cooled down in a fast refrigeration unit. Nevertheless, the fact that S. aureus was detected in several dishes including cured and cooked ham, at concentrations up to 5,200 CFU/g, suggests that the cold chain before or during the event was interrupted to allow sufficient microbial growth during or following food manipulation.
A major limitation of our study is that the food item identified by the case-control study, pasta salad with pesto, was no longer available for testing and thus there is no microbiological evidence that the pasta salad with pesto was contaminated with the outbreak strain. However, matrices with similar biochemical properties like potato salad have been confirmed before as vehicles of SFP in France [21] and Switzerland [15]. In the latter case, a strain with identical spa type t024 and enterotoxin A FRI100 allele led to the intoxication of 27 boy scouts. The sea gene found in our outbreak strain is the dominant sea allele described in S. aureus isolates that are associated with food poisoning outbreaks worldwide [19,[21][22][23] and in enterotoxigenic isolates recovered from food handlers [24].
The epidemiological results from our carriage study are consistent with previous findings in similar studies. Our finding of 58% carriers among food handlers concurs with longitudinal studies showing that approximately 20% of persons are persistent nasal carriers and an additional 30% are intermittent carriers of S. aureus [25]. The high genetic diversity among asymptomatic carriers was also observed in similar studies in Germany [26], Switzerland [27], and Bosnia [28]. Interestingly, we found meticillin-susceptible livestock-associated strains with ST398 spa type t571 and variants thereof in both catering employees and in food. Similar clones have recently emerged causing severe infections in neighbouring France and Belgium [29,30], while remaining rare in Germany [31].
Although WGS has been applied to meticillin-resistant S. aureus in hospital and long-term care settings [32][33][34] and to other foodborne pathogens [35,36], to our knowledge our study is the first to report WGS as a tool in a staphylococcal food poisoning outbreak. While WGS showed virtually identical groupings to MLVA, one major advantage of WGS is that it is a universal method applicable to any bacterial species and that it provides further data on the presence of genes encoding virulence and resistance factors.
JM coordinated the various investigations, collated strains from different sources, constructed phylogenies, conducted the statistical analysis for the case-control study, and wrote the manuscript; FD conducted the classical genotyping including MLVA, spa typing and virulence factor detection by PCR; GM was responsible for the laboratory analysis of food items; CR and CO conducted the whole genome sequencing; CO assisted with bioinformatics and with preparing the figure; SJ provided reference material and assisted with interpretation; MP was responsible for the microbiological analysis of human strains; PH led the food inspection; PW was responsible for the public health response and the case-control data collection.

Introduction
Since the 1990s, some significant changes in serogroup Y Neisseria meningitidis (MenY) epidemiology have been reported worldwide. During the beginning of this period, an increase of MenY cases was observed in the United States (US) [1], as well as in Latin American countries [2]. In Colombia the proportion of MenY cases peaked at 50% in 2006 [3]. MenY incidence increased also in Europe [4,5]. In France, MenY accounted for only 5.5% of all cases of invasive meningococcal disease (IMD) in 2010 but for 10% in 2013 [6]. In Norway and in Finland, MenY represented respectively 31% and 38% of all cases reported in 2010 [7,8]. MenY emergence was observed also in Sweden, with an increase of the incidence from < 0.05 cases per 100,000 inhabitants in 2000 to 0.23 in 2010 [9].
In Italy, although the incidence of IMD remained stable since 2007 (around 0.3 cases/100,000 inhabitants; data from National Surveillance System http:// www.iss.it/mabi/), some changes were noted in the frequency distribution of specific meningococcal serogroups. In our country, similarly to other European countries, serogroup B and C are responsible for the majority of IMD cases, however, an increase in the proportion of MenY has been observed, from 4% before 2005 to 7% in 2006 [10]. Some changes in the distribution of serogroups may be due to the introduction of the meningococcal serogroup C conjugate (MCC) vaccination (between 2005 and 2007), which has been included in the 2012 to 2014 national immunisation plan (NIP), in accordance with regional policies; the vaccine is recommended to all children between 13 and 15 months of age, and to 11 to 18 year-old individuals, if not previously vaccinated, and to those belonging to risk categories [11].
The aims of present study were: (i) to describe the trend of MenY IMD cases from 2007 to 2013 in Italy and (ii) to investigate the clinical and epidemiological features and the molecular characteristics of MenY cases.

Bacterial isolates
In Italy, notification of all cases of IMD is mandatory. Clinical and epidemiological information and meningococcal isolates are collected in the frame of the National Surveillance System coordinated by the National Reference Laboratory (NRL) of the Istituto Superiore di Sanità.
Every year, the NRL receives an average of 75% of the meningococci isolated by local hospital laboratories throughout the country. Epidemiological and microbiological data for each IMD case are managed using a dedicated database. Local laboratories send the isolates to the NRL, where they are stored at -80 °C before complete microbiological characterisation.

Microbiological analyses
Serogroup is confirmed by slide agglutination with commercial antisera (Remel Europe, Ltd, United Kingdom) or by multiplex polymerase chain reaction (PCR) [12]. Susceptibility to ceftriaxone, ciprofloxacin, penicillin G and rifampicin is determined by E-test method (bioMérieux SA -France) on Mueller-Hinton agar (Oxoid) supplemented with 5% of sheep blood. The breakpoints are those recommended by the European Committee on Antimicrobial Susceptibility Testing -EUCAST version 5.0, 1 January 2015 (http://www.eucast.org/).
eBurst MLST data were analysed by eBURST, version 3, (http:// eburst.mlst.net) [14]. eBurst analysis was set up referring to the most stringent setting of identity of alleles in six of the seven housekeeping genes.

Statistical analysis
The data were analysed using EpiInfo (version 3.4.5. July 30, 2013). Odds ratios (OR), 95% confidence intervals (CI) and p values, were obtained to measure the strength of the association between serogroup Y and other variables. Statistical differences were tested using standard tests (i.e. chi-squared and chi-squared for trend); the level of statistical significance is set at p value < 0.05.

Results
From 2007 to 2013, a total of 1,157 IMD cases were detected in Italy, with an average annual incidence of 0.27 cases per 100,000 inhabitants. The annual proportions of IMD cases attributable to the principal serogroups (B, C, Y, W) by year are shown in Figure 1.  with all the other age groups (OR: 3.3; 95% CI: 1.94-5.59), (data not shown).
Differences with regard to the risk of being infected with MenY according to sex were not statistically significant.
As expected, meningitis and septicaemia represented the main clinical pictures among IMD cases. There was no significant difference in MenY infection among cases with different clinical presentation. However, among patients with MenY, an increase of septicaemia, from 19% (3/16 cases) in 2011 to 42% (8/19 cases) in 2013 was observed. The respective proportion of serogroup Y in the south and the islands was higher than in northern and central Italy, with an OR of 2.18 (Table). The outcome, available for 52 of 81 cases, was fatal for three patients: two women (67 and 45 years-old) and a six year-old child with sepsis, corresponding to a case fatality ratio of six per cent.
A total of 59 samples from the 81 serogroup Y IMD cases, were received by the NRL, allowing further typing. Moreover bacterial isolates derived from 50 patients respectively, were also obtained, and could be used for antibiotic susceptibility testing. All MenY isolates retrieved from cases were susceptible to ceftriaxone, ciprofloxacin and rifampicin. Moreover, 21 of 50 isolates showed a decreased susceptibility to penicillin G (minimum inhibitory concentration (MIC) 50 and MIC 90 were 0.047 and 0.125mg/L, respectively).  All cc23 samples analysed (n=48) harbour a mutation in the lpxL1 gene. In particular, 38 were lpxL1 type XVII, seven type VI, two type V and one type XVI. However, no associations between a specific lpxL1 type, ST and age group were identified. Moreover, no differential association with the clinical picture of meningitis and septicaemia was found. Isolates belonging to cc167 showed a lpxL1 sequence identical to the reference (GenBank accession number: AE002098.2).

Molecular analyses
The lpxL1 genotype was analysed in a subsample of non-serogroup Y meningococci. Among 20 serogroup C strains only one showed the mutation type III in lpxL1 gene.

Discussion
As already reported, a stable increase of MenY cases has been observed in Italy since 2004 [10]. Noteworthy, the proportion of MenY among IMD cases increased almost eight times between 2007 (2%) and 2013 (17%).
Previous studies reported that, relative to other N. meningitidis serogroups, MenY is usually found in older patients [15][16][17] Of note, since 2011, an increase of septicaemia cases attributed to serogroup Y was observed. Overall, the case fatality ratio among IMD cases caused by serogroup Y was six per cent. As already reported [10], a high proportion (42%) of MenY isolates with decreased sensitivity to penicillin was found.
Several reports indicated the cc23 as one of the most frequently detected in invasive MenY cases: in particular, it was responsible for an increase of IMD incidence in the 1990s in the US [1] and was associated with 94% of serogroup Y meningococci isolated between 2000 and 2005 [19]. From 1999 to 2003, in Canada 65.7% of invasive MenY strains were cc23 [20], whereas in Taiwan this cc characterised 11 of 13 MenY causing disease between 2001 and 2002 [21]. In South Africa, during the years 2003 to 2007, 11% of invasive MenY belonged to cc23 [18]. In Europe, during the 1990s, the cc23 was isolated more frequently from healthy carriers than from invasive meningococcal cases [22,23]. Nevertheless, in Sweden, from 2000 to 2010, the cc23 was identified in the three major clones responsible for the increased number of IMD cases [9] and in England it was found in the 56% of MenY causing IMD during the years from 2007 to 2009 [13]. In Italy, cc23 was the main cc among invasive MenY: it was detected in 89% of MenY samples from 1998 to 2006 [10], and in 54 of 56 (96%) of samples from 2007 to 2013. However, the identification in this study of 25 different finetypes suggests that more than a single strain is responsible for the MenY increase in Italy.
All the cc23 isolates analysed in this work harboured a mutation in the lpxL1 gene, and in particular, the mutation XVII was the most frequently found (79%  studies have demonstrated the presence of lpxL1 mutations in N. meningitidis carrier strains cc23 and in meningococci isolated from cases of chronic meningococcaemia and meningitis [13,[24][25][26][27]. In contrast, as shown from the results reported here, MenY cc23 with a mutated lpxL1 was associated indifferently with meningitis or septicaemia. In conclusion, in Italy, IMD due to serogroup Y is steadily increasing, especially among five to 14 year-old patients, with predominance of isolates belonging to cc23 and harbouring lpxL1 mutation. Overall, these results have significant public health implications. They support the potential utility of vaccination with the quadrivalent-meningococcal vaccine (ACWY) and/ or the opportunity of a booster dose with this vaccine among children and young adolescents previously immunised with the MCC vaccine. More than 10 years since the beginning of vaccination with the MCC vaccine, there is evidence of a different epidemiology of IMD in Italy. The results are consistent with those of other studies that reported an increase of MenY and, more recently, of MenW infections [6,28] and provide further information which can be used to decide if and when the quadrivalent vaccination should be introduced. The quadrivalent meningococcal vaccine (ACWY) is safe and immunogenic; however, the costeffectiveness of a booster with MCC vs the latter vaccine is still debated. In Italy, the use of quadrivalent vaccine is currently recommended for people at risk and for people who live in or travel to countries where meningococcal disease is hyperendemic or epidemic; this policy is likely to change. In fact, the dynamic nature of IMD epidemiology is well known [29]. In this respect, monitoring changes in the trend of the different serogroups and the microbiological features of meningococci is key to generate scientific evidence which is essential for producing appropriate vaccine recommendations.

Conflict of interest
None declared.

Introduction
The global rise of carbapenemase-producing Enterobacteriaceae (CPE) is alarming and represents an increasing threat to healthcare delivery and patient safety in Europe and beyond.
In 2012, the European Centre for Disease Prevention and Control (ECDC) launched the 'European survey of carbapenemase-producing Enterobacteriaceae (EuSCAPE)' project to improve the understanding of the occurrence and epidemiology of CPE, to increase awareness of the spread of CPE and to build laboratory capacity for diagnosis and surveillance in Europe. In February 2013, a self-assessment questionnaire was sent to one national expert (NE) from each of the EuSCAPE participating countries (i.e. 28 European Union (EU) Member States, Iceland, Norway, the seven EU enlargement countries (Albania, Bosnia and Herzegovina, Kosovo*, Montenegro, the former Yugoslav Republic of Macedonia, Serbia and Turkey) and Israel, to gather information on the current awareness of and knowledge about the spread of CPE, the public health responses and the available national guidelines on detection, surveillance, prevention and control, as well as on the capacity for laboratory diagnosis and surveillance. NEs were chosen based on their national and international laboratory and/ or epidemiological experience in CPE among experts from national reference or expert laboratories, from the European Antimicrobial Resistance Surveillance Network (EARS-Net), and from the ECDC Coordinating Competent Bodies (National Focal Points for antimicrobial resistance and National Focal Points for microbiology) and ECDC National Correspondents for EU enlargement countries. The answers collected from the NEs showed that the epidemiological situation for CPE had worsened since 2010 and CPE continued to spread in European hospitals [1][2][3]. Answers also indicated that the knowledge and awareness of the spread of CPE and the laboratory capacity for diagnosis and surveillance were heterogeneous among countries [1,2]. These findings highlighted the urgent need for a coordinated European effort towards early diagnosis, active surveillance and guidance on infection control measures [1,2].
In September and October 2013, the EuSCAPE project supported laboratory capacity building for diagnosis and surveillance by hosting a 'train-the-trainer' workshop at the European level for national laboratory experts on the identification and confirmation of CPE, and by carrying out an external quality assessment (EQA) of national reference/expert laboratories. The workshop and the EQA aimed at ensuring performance quality, consistency and comparability of data between participating countries and laboratories. Between November 2013 and April 2014, 36 European countries participated in the first European-wide structured survey of CPE (data not shown). The participating reference/expert laboratories were asked to collect CPE isolates of Klebsiella pneumoniae and Escherichia coli together with clinical data on these CPE-related infections to gain an understanding on the prevalence and epidemiology of CPE, as well as the risk factors associated with CPE infections in Europe.
In March 2015, after the completion of the EuSCAPE project, a post-EuSCAPE feedback questionnaire was sent to the participating countries to document whether (i) knowledge and awareness regarding the occurrence and spread of CPE had increased, and (ii) national capacity for containment of CPE had changed in terms of surveillance, laboratory reference services, and availability of guidance on infection prevention and control measures for these bacteria, since February 2013.
In this report, we present the analysis of the NEs' answers on behalf of their countries to the post-EuS-CAPE feedback questionnaire and provide summaries of the current epidemiological situation of the spread of CPE in each country.

Methods
The post-EuSCAPE feedback questionnaire was derived from the self-assessment questionnaire issued in February 2013 [1,2]. The questionnaire was divided in five sections. The first two sections explored awareness and knowledge about the occurrence of CPE in each country and collected information on the current national capacity for containment of CPE. The third and fourth sections collected the participants' feedback on the EuSCAPE activities, e.g. laboratory capacity building workshop, EQA exercise and on the impact of the EuSCAPE project on collaborations and networking capacity, respectively. The fifth section investigated Luxembourg Malta desired areas for future ECDC activities on carbapenemresistant Gram-negative bacteria. The questionnaire was sent to the same NEs who participated in a similar survey in February 2013, with the exception of France and the Netherlands. They were invited to coordinate their replies with colleagues in their countries i.e. the ECDC National Focal Points for antimicrobial resistance and the ECDC National Correspondents for EU enlargement countries) to reflect the national situation and to complete the questionnaire online between 3 March and 30 April 2015 (questionnaire available upon request from the corresponding author). They were also asked to provide a description of the emergence and spread of CPE in their country beyond K. pneumoniae and E. coli isolates collected during the EuSCAPE structured survey. The answers were based on their knowledge of national clinical and microbiological data and/or their personal judgement. When necessary, the respondents were contacted for clarification, and corrections were made accordingly. The latest data from For the presentation, countries were arbitrarily grouped in geographic entities independently of the epidemiological stages of CPE spread, geopolitical or economic considerations.
Using the same epidemiological staging system as in 2010 and 2013 (Table 1), all participating countries self-assessed their epidemiological situation of CPE, thereby documenting the progression of CPE within countries and dissemination in Europe between 2013 and 2015. All countries provided a self-assessment of the current national situation.

Overall occurrence of carbapenemaseproducing Enterobacteriaceae
Three countries reported not having identified one single case of CPE, whereas 13 reported regional and inter-regional spread, and four reported an endemic situation. Nine countries reported sporadic occurrence, five reported single hospital outbreak and four reported sporadic hospital outbreaks ( Figure 1, Table  2). Table 2  Occurrence of carbapenemase-producing Enterobacteriaceae by type of carbapenemase All countries were able to rate the occurrence and spread of CPE by type of carbapenemase. As of May 2015, K. pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae still had the widest dissemination in Europe, but carbapenem-hydrolysing oxacillinase-48 (OXA-48)-producing Enterobacteriaceae had almost reached the same spread, a change compared with February 2013, with eight countries reporting regional or inter-regional spread and another two countries reporting an endemic situation ( Figure 2). The distribution of KPC-and OXA-48-producing Enterobacteriaceae varies and does not necessarily overlap, for example, Greece seeing predominantly KPC-producing Enterobacteriaceae and rarely OXA-48-producing Enterobacteriaceae, and Malta seeing almost exclusively OXA-48-producing Enterobacteriaceae.
The European epidemiology for CPE also changed between 2013 and 2015 for New Delhi metallo-beta-lactamase (NDM)-producing Enterobacteriaceae; five countries reported sporadic hospital outbreaks, and seven countries regional or inter-regional spread. No country reported an endemic situation. The epidemiological situation for Verona integron-encoded metallo-beta-lactamase (VIM)-producing Enterobacteriaceae remained stable with some minor country-specific changes. Imipenemase (IMP)-producing Enterobacteriaceae remained rare in Europe ( Table 3).

Description of the emergence and spread of carbapenemase-producing Enterobacteriaceae
The NEs participating in the EuSCAPE provided a description of the emergence and spread of CPE in their country beyond K. pneumoniae and E. coli isolates collected during the EuSCAPE structured survey.

Denmark, Iceland, Finland, Norway, Sweden and the Netherlands
In Denmark, only sporadic occurrence of CPE, mostly related to foreign travel, was observed until 2012 when the situation for CPE changed to sporadic hospital outbreaks with the spread of VIM-4 producing E. Single hospital outbreak Outbreak defined as two or more epidemiologically-associated cases with indistinguishable geno-or phenotype in a single institution. 2a

Sporadic hospital outbreaks
Unrelated hospital outbreaks with independent, i.e. epidemiologically-unrelated introduction or different strains; no autochthonous inter-institutional transmission reported. 2b Regional spread More than one epidemiologically-related hospital outbreak confined to hospitals that are part of the same region or health district, suggestive of regional autochthonous inter-institutional transmission. 3 Inter-regional spread Multiple epidemiologically-related outbreaks occurring in different health districts, suggesting interregional autochthonous inter-institutional transmission. 4 Endemic situation Most hospitals in a country are repeatedly seeing cases admitted from autochthonous sources. 5 Table 1 Epidemiological stages of carbapenemase-producing Enterobacteriaceae spread [1,3]    coli, the identification of NDM-4 producing E. coli and an outbreak of NDM-1 producing Citrobacter freundii [4,5]. Since 2013, the number of CPE cases in Denmark has further increased with multiple epidemiologicallyrelated hospital outbreaks of OXA-48-and NDMproducing Enterobacteriaceae in different regions of the country [6]. In 2014, most of the CPE cases had no history of recent travel aboard. Denmark is now facing an inter-regional spread of CPE.
The situation in Iceland has remained unchanged since 2010 despite active screening. Iceland is one of the few countries in Europe that has not reported any case of CPE.
In In Sweden, most identified cases had a history of foreign travel. In 2014, there was a slight increase in the number of CPE cases due to an outbreak that was only detected through identification of a secondary colonised case. Until 2013, the predominant CPE in Sweden were NDM-producing Enterobacteriaceae closely followed by OXA-48-producing Enterobacteriaceae [9]. Since 2014, OXA-48-producing Enterobacteriaceae became predominant over NDM-producing Enterobacteriaceae, however both are still at low level.
In the Netherlands, KPC-, OXA-48-and NDM-producing Enterobacteriaceae have so far only been responsible for single hospital outbreaks, although a recent interinstitutional outbreak of KPC-producing K. pneumoniae occurred following the transfer of a patient from a nursing home to a hospital [10,11].

Estonia, Latvia and Lithuania
The Baltic countries only recently started to report CPE cases [12,13].
In Latvia, only three cases of CPE have been identified so far, of which the first two VIM-producing isolates were identified during the EuSCAPE structured survey (data not shown).
In Lithuania, surveillance of CPE became mandatory in 2014. Between 1 January and 31 December 2014, 13 CPE cases were reported, consisting of two cases of OXA-48-producing K. pneumoniae, nine cases of NDMproducing Enterobacter cloacae, one case of NDMproducing E. aerogenes and one case of VIM-producing E. cloacae.

Ireland and United Kingdom
In Ireland, sporadic occurrence of CPE, i.e. KPC-, VIMand NDM-1-producing Enterobacteriaceae, had been reported until 2011, with the majority of cases being related to travel abroad [14][15][16]. In 2011, an outbreak of epidemiologically-related KPC-producing K. pneumoniae in two hospitals from two different regions resulted in epidemiological stage 4 of CPE spread in the country [17]. This was concomitant with the first hospital outbreak of OXA-48-producing K. pneumoniae [18]. Since 2013, although the spread of CPE was limited to regional spread in some regions, the overall national situation is considered to have worsened due to an increase in the overall number of reported CPE cases. Furthermore, increasing numbers of hospitals and regions where CPE had not been encountered before 2013, have since reported sporadic cases or outbreaks of CPE. Prior to 2013, KPC-producing Enterobacteriaceae were the main type of CPE responsible for hospital outbreaks, but from 2013 onwards, OXA-48-and NDM-producing Enterobacteriaceae were also responsible for outbreaks.
The United Kingdom (UK), reported the emergence and the spread of NDM-1-producing CPE soon after its first isolation in 2008 from a patient repatriated to Sweden from a hospital in India, and this led to a National Resistance Alert 3 notice by the Department of Health [19,20]. To date, the UK has reported the largest number of NDM-producing CPE cases among European countries and has seen multiple NDM variants. The number of CPE isolates received by the national reference laboratory has increased continuously since 2008. In 2014, an increasing number of NDM-or OXA-48-producing isolates was reported compared with previous years with a marked increase in carbapenemase-producing E. coli.

Austria, Czech Republic, Germany, Luxembourg and Slovenia
In Austria, the epidemiological situation worsened between 2010 and 2013, but has since remained unchanged with a low occurrence of CPE and sporadic hospital outbreaks [21][22][23][24]. Between 2010 and 2015, the most frequently confirmed carbapenemase genes by the reference laboratory were bla VIM and bla KPC , but also bla OXA-48 and bla NDM were also found in low numbers. In April 2015, Austria initiated the Austrian surveillance project 'Carba-Net Austria' and organised four laboratory capacity building workshops on the identification of CPE and characterisation of carbapenemases based on the EuSCAPE protocols and training curriculum.
In the Czech Republic, the occurrence of CPE was rare until 2011 with only sporadic cases, and a total of three cases detected between 2009 and 2010. In 2011, however, the number of CPE increased due to the repatriation of patients from hospitals in Italy and Greece and an outbreak following the transfer of a patient from Italy [25]. To contain this increase, the national surveillance included CPE isolates from active screening samples as part of its surveillance scheme and the Ministry of Health issued, in 2012, official national guidelines for the control of CPE covering both infected and colonised cases. No further increase in the occurrence of CPE was observed in 2012 and 2013, and only one outbreak restricted to five patients and four sporadic cases was reported until mid-2013 [26]. During the EuSCAPE survey, the Czech Republic reported only two confirmed CPE cases, of which one involved NDM-1-producing K. pneumoniae from a patient transferred from Ukraine [27].
In Germany, there has been an increasing number of CPE referred to the German National Reference Laboratory for CPE and the German national antibiotic resistance surveillance has showed an increase of resistance to meropenem in K. pneumoniae from 0.1% in 2010 to 0.5% in 2014. Both observations possibly indicated an increase in the prevalence of CPE in Germany albeit on a low level. Several outbreaks with KPC-2-, KPC-3-, NDM-1-and OXA-48-producing Enterobacteriaceae have been documented; notably a protracted KPC-2 outbreak involving over 100 patients and a polyclonal KPC-2 outbreak involving other species besides K. pneumoniae [28]. The most prevalent CPE are in order of importance OXA-48-, KPC-2-, VIM-1-, NDM-1-and KPC-3-producing Enterobacteriaceae. Despite the dominance of OXA-48-producing Enterobacteriaceae, mostly KPC-producing K. pneumoniae outbreaks have been reported in Germany.
Luxembourg has only experienced sporadic cases of VIM-producing CPE [29].
In Slovenia, only sporadic cases of CPE were detected until 2013, with a large proportion of the cases being related to patient transfers from foreign hospitals [30]. The situation changed in October 2014 with the first outbreak of both OXA-48-and NDM-producing E. coli and K. pneumoniae affecting several wards in a single hospital. While one of the first identified patients had been transferred from a foreign hospital, other patients had no history of travel abroad. Some CPE-positive patients belonging to this outbreak were transferred to other hospitals across the country, but no further spread occurred in these hospitals.

Hungary, Poland, Romania and Slovakia
In Hungary, ca 600 VIM-4-producing Enterobacteriaceae isolates -the predominant type of CPE in Hungary -have been collected since 2008. The first KPC-2producing K. pneumoniae isolates were reported from 2008 to 2009 during a local outbreak in the north-eastern part of Hungary and the index case was a patient previously hospitalised in Greece [31]. About 20 KPCproducing isolates, from sporadic cases and mostly associated with medical treatment abroad, have since been collected, with an average of 1 to 2 isolates per year. These were KPC-producing K. pneumoniae until 2015 when the first KPC-producing E. coli was isolated. Only two small outbreaks caused by OXA-48like-producing K. pneumoniae were reported, in 2012 and 2014, and both were linked to patient transfers from Romania and Ukraine, respectively [32]. In total, 20 OXA-48-producing Enterobacteriaceae have been identified so far in Hungary. Since 2013, only sporadic cases of NDM-producing CPE, primarily E. cloacae, have been identified, of which some but not all were linked to Romania.
In Poland, KPC-producing K. pneumoniae were predominant between 2008 and 2012 [33,34]. Since 2012, the epidemiology of CPE has changed with a decreasing number of KPC-producing K. pneumoniae and an increasing number of NDM-1-producing K. pneumoniae. The former primarily occurred in the regions that had experienced outbreaks of KPC-producing K. pneumoniae in 2008-2012. The latter was a consequence of a large inter-regional outbreak of NDM-producing K. pneumoniae that started at the end of 2012 [35], just a few months after the first case of NDM-1-producing K. pneumoniae was found in a patient with previous travel history to Africa [36].
Prior to 2013, Slovakia experienced only one small local epidemic in two hospitals, following an imported case of NDM-1-producing K. pneumoniae [40]. However, the situation changed in December 2013 after the identification of the first case of KPC-2-producing K. pneumoniae in a patient who had been hospitalised in Greece and the subsequent spread of CPE to more than    Bosnia and Herzegovina did not report any CPE, but NDM-1-producing K. pneumoniae had previously been reported in Croatia from a patient transferred from Bosnia and Herzegovina [42,43].
In Bulgaria, the occurrence of CPE has increased since 2012. KPC-2-and VIM-1-producing K. pneumoniae were isolated from a hospitalised patient in Varna and an outbreak caused by NDM-1-producing E. coli was reported from the Military Medical Academy Hospital of Sofia [44,45].
In Croatia, the first reported case of CPE was a NDM-1-producing K. pneumoniae isolated in 2009 in the University Hospital Centre of Zagreb from a patient repatriated from Bosnia and Herzegovina [42]. In February 2011, the first KPC-producing K. pneumoniae was isolated from a patient at the same hospital [46]. A multicentre study performed from 2011 to 2012 in four large hospital centres in Croatia identified a higher prevalence of VIM-1-producing Enterobacteriaceae than of NDM-and KPC-producing Enterobacteriaceae [47]. Since 2014, the epidemiology of CPE in Croatia has changed with the rapid spread of OXA-48-producing Enterobacteriaceae whereas incidence of KPC isolates declined.
Kosovo* is one of the few countries that have not reported any cases of CPE isolated from normally sterile body fluids such as blood cultures and cerebrospinal fluid (CSF), although NDM-1-producing K. pneumoniae were previously reported in Austria, Belgium and in Germany from patients being transferred from hospitals in Kosovo* [43,48,49].
In Montenegro, a laboratory capacity building workshop was organised and phenotypic methods of detection of CPE were implemented in the participating laboratories, leading to the identification of NDM-1producing K. pneumoniae during the EuSCAPE structured survey (data not shown).
In Serbia, NDM-and OXA-48-producing Enterobacteriaceae, as well as NDM-and OXA-48-co-producing Enterobacteriaceae have been isolated during the EuSCAPE structured survey (data not shown). The latter type of CPE was also identified in a patient transferred from Serbia to Switzerland in December 2013 [50].
In the former Yugoslav Republic of Macedonia, only KPCproducing K. pneumoniae have been isolated so far through the EuSCAPE structured survey (data not shown).

Belgium, France, Portugal and Spain
In Belgium, the situation of CPE has seriously worsened with a rapid spread of CPE since 2012, i.e. a doubling in prevalence and incidence in acute care hospitals between 2012 and 2015 and more than 80% of the reported cases being confirmed as autochthonous acquisition, i.e. not travel-related. In addition, there has been an increase in the number of documented regional and inter-regional transmissions of epidemiologically related clusters and/or outbreaks, especially for OXA-48-producing Enterobacteriaceae and to a lesser extent for KPC-producing Enterobacteriaceae. There has also been an increase in the number of outbreaks with one third of the country's hospitals reporting outbreaks of CPE. Another major change in Belgium in 2015 was the marked increase, compared with 2013, in the number of non-travel-related NDM cases with inter-institution regional spread and multiple large difficult-to-control outbreaks occurring in several hospitals.
In France, the number of cases and outbreaks of CPE has steadily increased since 2009 with the sharpest increase during the last quarter of 2014. KPC-producing Enterobacteriaceae however, have been declining since 2012. Most cases were acquired abroad, i.e. through hospitalisation or travel. However, there has been an increase in the number of autochthonous cases, usually OXA-48-producing Enterobacteriaceae. In 2014, the most frequent CPE are OXA-48-producing K. pneumoniae and E. coli, followed by NDM-, VIM-and KPCproducing Enterobacteriaceae.
In Spain, the situation of CPE has worsened in the last few years with an increasing trend in the number of CPE cases and a wide geographic spread [51][52][53][54]. The spread of CPE has currently affected 34/50 Spanish provinces, resulting in a potential inter-regional spread of CPE [47,49, unpublished data]. The most predominant CPE are OXA-48-and VIM-producing K. pneumoniae [51][52][53]. In general, the prevalence of KPC-and NDM-producing Enterobacteriaceae in Spain is low but increasing [51,54]. Recently, an inter-hospital spread of NDM-7-producing K. pneumoniae that belonged to MLST type 437 was described in Madrid [51]. Although not frequent, detection of the polyclonal dissemination of OXA-48-producing E. coli is worrying.
In Portugal, only sporadic isolates or single hospital cases have been described. The most predominant CPE   were KPC-producing Enterobacteriaceae, but OXA-48producing Enterobacteriaceae have also been recently reported [55,56].

Cyprus, Greece, Israel, Italy, Malta and Turkey
During the EuSCAPE structured survey, Cyprus collected only three CPE isolates (data not shown). In line with this, the latest data from the European Antimicrobial Resistance Surveillance Network (EARS-Net) showed a decreasing trend in the percentage of carbapenem-resistant isolates among invasive, i.e. blood and cerebrospinal fluid (CSF), K. pneumoniae isolates from 15.7% to 5% during 2011 to 2014 [12,57].
Since the early 2000s, Greece has been first facing a nationwide epidemic of polyclonal VIM-producing K. pneumoniae followed by a nation-wide occurrence of  In Italy, it was not until 2010 that CPE became a major issue when KPC-producing K. pneumoniae became endemic, due to a rapid countrywide diffusion mostly caused by strains of clonal complex 258 [64]. This increase in percentages of carbapenem resistance in invasive K. pneumoniae isolates has been documented by EARS-Net since 2010 and the latest data from EARS-Net reported that 32.9% of K. pneumoniae invasive isolates were carbapenem-resistant [12]. NDM-1-and OXA-48-producing Enterobacteriaceae have been reported but their dissemination was still limited and cases were mostly acquired abroad [65][66][67]. In an effort to control and prevent the further spread of CPE, the Ministry of Health issued a circular letter in 2013 asking the public health offices across the country to report all cases of bacteraemia caused by CPE to the regional and national authorities. Although there is still underreporting of CPE, more than 2,000 CPE bacteraemia cases have been reported since publication of the circular letter. One worrisome recent development is the rapid and country-wide dissemination of resistance to colistin in KPC-producing K. pneumoniae [68] and the presence of pandrug-resistant (PDR) strains (data not shown).
In Malta, dissemination of OXA-48-producing Enterobacteriaceae had changed the country's epidemiological level from rare sporadic occurrence before 2010 to an endemic situation by 2013 [1,2]. It is thought that the influx of injured Libyan war victims to the intensive treatment unit of the country's only tertiary care hospital in 2011 contributed to the first outbreak and spread of OXA-48-producing Enterobacteriaceae in the country [69]. Despite initial control of the outbreak, the situation rapidly became endemic in this hospital and OXA-48-producing Enterobacteriaceae spread to other health and residential care entities on the Maltese islands. Until 2014, no KPC-or NDM-producing Enterobacteriaceae were reported while during the same period more than 400 new cases of OXA-48producing Enterobacteriaceae were identified. Since then, the number of new cases of OXA-48-producing Enterobacteriaceae has continued to increase. In addition, sporadic cases of VIM-and NDM-producing Enterobacteriaceae were recently identified, mainly acquired outside the country. EARS-Net data for Malta showed an increase in the percentage of invasive carbapenem-resistant K. pneumoniae, OXA-48 -producing K. pneumoniae, from 3.8% to 9.9% during the period 2011 to 2014 [12].
In Turkey, OXA-48-producing Enterobacteriaceae are endemic, and since 2013, an increasing number of reports have demonstrated the emergence of other types of CPE (e.g. NDM-1-and KPC-producing Enterobacteriaceae) [70]. This was confirmed by the results of the EuSCAPE structured survey (data not shown). Reports of NDM-1-producing Enterobacteriaceae cases have been increasing, especially in hospitals from cities close to the Syrian border. The latter development is in accordance with recent reports on both autochthonous and imported NDM-1-producing Enterobacteriaceae cases in Turkish hospitals [71,72]. In 2015, the first K. pneumoniae co-producing OXA-48 and NDM-1 was isolated from a patient treated in the hospital of Sanliurfa, a city close to the border with Syria [73].
National capacity for surveillance and containment of carbapenamase-resistant Enterobacteriaceae Table 4 summarises the existing surveillance and reference laboratory systems in place as well as the available national guidance documents for the containment of CPE in the participating countries at the time of the survey.

Surveillance of carbapenamase-resistant Enterobacteriaceae
Twenty-five EU Member States, Norway and Iceland had a dedicated national system for surveillance of CPE. Three EU Member States did not have a dedicated national surveillance system but reported carbapenemresistant K. pneumoniae and E. coli from blood and CSF to EARS-Net. Slovenia, one of these, reported that at the time it was developing a dedicated national system for surveillance of CPE for implementation by the end of 2015. The Netherlands, which has a system in place, reported that enhanced surveillance of CPE will take place from 2016 onwards. In order to increase laboratory participation and coverage as well as to improve data quality, the enhanced surveillance should further optimise diagnostic testing and integrate clinical, molecular and epidemiological data for all CPE cases to determine relevant risk factors to target interventions and control potential spread.
All EU enlargement countries and Israel reported participating in the Central Asian and Eastern European Surveillance on Antimicrobial Resistance (CAESAR) network, a joint initiative of the European Society of Clinical Microbiology and Infectious Diseases (ESCMID), the Dutch National Institute for Public Health and the Environment (RIVM) and the World Health Organization Regional Office for Europe (WHO/Europe). However, only Serbia, the former Yugoslav Republic of Macedonia and Turkey have so far reported data to CAESAR using the EARS-Net methodology [74]. Israel, Serbia, the former Yugoslav Republic of Macedonia and Turkey had dedicated national systems for the surveillance of CPE, while Albania, Bosnia and Herzegovina, Kosovo* and Montenegro were developing their surveillance system to be able to report data to CAESAR by 2015 or2016.
Of 31 countries with a dedicated national surveillance system for CPE, 20 countries reported that surveillance of CPE was mandatory for all laboratories, nine countries reported that surveillance of CPE was voluntary and two countries did not specify. In Romania and Serbia, surveillance was voluntary and in form of a sentinel system of individual laboratories. In Ireland, laboratory participation was only mandatory for invasive disease caused by CPE, i.e. isolation from blood and CSF, but remained voluntary for CPE isolated from other body sites (Table 4).

Laboratory capacity for carbapenamase-resistant Enterobacteriaceae
Thirty-four countries reported having an officially nominated national reference laboratory for CPE or a national expert laboratory that fulfilled a similar role. Both Albania and Montenegro reported that the national reference laboratory was in development for implementation by 2015-2016 (Table 4).

Notification to health authorities for carbapenamaseresistant Enterobacteriaceae
Twenty-six countries reported having a national recommendation for reporting to health authorities CPEpositive patients identified by diagnostic laboratories.
In most countries there is mandatory notification for all private and hospital laboratories and for all infections; only seven countries notified CPE cases on a voluntary basis. In two of the latter, notification of CPE cases was voluntary but notification of CPE outbreaks was mandatory. Slovenia and Germany reported that national recommendations or obligations for reporting were going to be implemented by the end of 2015, and for Bosnia and Herzegovina this is planned by 2016-2017 (Table 4).

National plan for containment of and infection control measures for carbapenamase-resistant Enterobacteriaceae
Eleven countries had implemented a national plan for the containment or for preparedness to contain CPE, and another nine countries were developing national containment plans. Spain had no national but regional specific plans.
Twenty-four countries reported having national recommendations or guidelines for infection prevention and control measures to be applied for patients confirmed as being infected or colonised with CPE: for six countries this applied to single CPE cases, for 15 to single CPE cases and outbreaks, for Greece this only applied to outbreaks and two countries did not specify the scope of their recommendations. Twelve of the national recommendations or guidelines were specific guidance documents for prevention and control of CPE, while nine were included as part of a general guidance document for multidrug-resistant organisms (MDROs) that specifically referred to CPE and three included a general guidance document for MDROs not specifically referring to prevention and control of CPE. Kosovo* and Portugal indicated that such recommendations or guidelines are in preparation for implementation by the end of 2015.
The most cited measures in such national recommendations or guidelines were isolation e.g. in single rooms, of suspected/colonised/infected patients and increased hand hygiene compliance (21 countries each), followed by active screening for early detection at admission of patients having been hospitalised abroad, implementation of contact precautions, for visitors and medical staff, and implementation of environmental hygiene procedures e.g. decontamination of equipment and disposal of waste (20 countries each), active screening for early detection of transferred patients from other wards/hospitals at admission (19 countries), cohorting of suspected/colonised/infected patients (18 countries), active screening for early detection of colonised patients at admission (16 countries), dedicated infection control teams (14 countries), separate cohort nursing care e.g. nurses, doctors (13 countries), specialised training for nursing staff in infection control (12 countries), implementation of an antibiotic stewardship programme (11 countries), and audit and feedback to local, regional or national health authorities (10 countries).

Discussion
In 2013, at the beginning of the EuSCAPE project, knowledge about the spread and occurrence of CPE was heterogeneous among European countries [1]. Moreover, some NEs expressed concerns that underdetection affected the epidemiological self-assessment of their country. Following EuSCAPE activities including a capacity building workshop and an EQA exercise to improve the detection of CPE and the identification of the different carbapenemases circulating in Europe, the results of this follow-up survey provide evidence that the activities contributed to the desired improvement and increased awareness and knowledge of the epidemiology of CPE in many participating countries. After participation in the EuSCAPE project, all countries were able to self-assess their current situation, whereas only 26 countries could do so in 2013. In addition, all participating countries were able to rate the occurrence and spread of CPE according to the type of carbapenemase, while such data were only partially or not available in several European countries in 2013 [1,2].
In 2015, 13/38 countries reported inter-regional spread of or an endemic situation for CPE, compared with 6/38 countries in 2013. In addition, the survey documented the more frequent reporting of OXA-48-and NDMproducing Enterobacteriaceae compared with 2013. For OXA-48-producing Enterobacteriaceae, four countries had reported regional spread and only one country had reported an endemic situation in 2013, while in 2015, three countries reported regional spread, four reported inter-regional spread and two reported an endemic situation. Similarly for NDM-producing Enterobacteriaceae, only Italy and the UK had reported sporadic hospital outbreaks in 2013, while in 2015 six countries reported sporadic hospital outbreaks and seven countries reported regional and inter-regional spread. For the countries that were uncertain about their epidemiological stage in the 2013 survey, the results of this survey reflect an improved ability to detect CPE and identify the different carbapenemases.
For the other countries, the changes in epidemiological stages observed between 2013 and 2015 likely reflect an increasing spread of CPE, as confirmed by the NEs. At the same time, increased awareness of CPE spread and surveillance might also contribute to increased detection and reporting of more advanced epidemiological stages. Indeed, countries with strict screening policies are more likely to report such advanced epidemiological stages.
The establishment of a surveillance system for CPE, based on the notification of CPE cases to health authorities, supported by reference laboratory confirmation and identification as well as, molecular typing services are the cornerstones of efficient monitoring and controlling of the spread of CPE. Many countries have developed dedicated surveillance systems and designated reference laboratories over the last two years, as well as implemented mandatory laboratory participation in CPE surveillance, or mandatory reporting of all cases of infections. However, despite the increased awareness and the worsening of the epidemiological situation in 2015, only 25 of the 38 countries that participated in the EuSCAPE project had enacted mandatory notification of CPE cases to health authorities. Active reporting of CPE cases should be encouraged by making all clinical cases notifiable to public health authorities.
Twenty countries had either implemented a national or regional plan for the containment of, or preparedness to contain, CPE or were developing national containment plans. However, national guidance documents on infection prevention and control of CPE were not available in 14 countries. In an effort to support healthcare professionals, hospital administrators and public health professionals, ECDC published an online directory of guidance documents on prevention and control of carbapenem-resistant Enterobacteriaceae by EU/EEA Member States, ECDC, international and national agencies and professional societies [75,76].
A major impending threat to public health as a consequence of the expanding CPE epidemic in Europe is the emergence of PDR strains causing untreatable infections. Polymyxins, and particularly colistin, represent a last-line option for the treatment of patients infected with CPE. The latest data available from the European Surveillance of Antimicrobial Consumption Network (ESAC-Net) show that consumption of polymyxins, mainly colistin, in Europe, almost doubled between 2009 and 2013 [77]. In parallel to this increasing colistin consumption, colistin resistance is increasing in carbapenem-resistant Enterobacteriaceae [68,77,78]. In Italy, 43% of KPC-producing K. pneumoniae isolates collected during the EuSCAPE structured survey in 2013 to 2014 [68] and 13% of carbapenem-resistant K. pneumoniae isolates from blood cultures reported to EARS-Net in 2014 were resistant to colistin [12]. Approximately 20% of carbapenem-resistant K. pneumoniae isolates from blood cultures reported to EARS-Net in 2014 were resistant to colistin in Romania and Greece [12]. In February 2015, a Greek hospital reported an outbreak of PDR Enterobacteriaceae, via the acquisition of bla VIM by the naturally colistin-resistant Providencia stuartii, in an intensive care unit occurring in September to November 2011 [79].
The accumulation of other resistance markers in CPE strains in addition to colistin resistance makes it likely that Europe will soon witness an increasing number of outbreaks of extensively drug-resistant (XDR) or PDR Enterobacteriaceae [57], for which few or even no treatment options are available. The Unites States Food and Drug Administration (FDA) recently approved the use of a combination of a well-established β-lactam antibiotic, ceftazidime, with a novel β-lactamase inhibitor, avibactam, for treatment of serious infections caused by resistant Gram-negative pathogens. Ceftazidimeavibactam is active against OXA-48-and KPC-producing Enterobacteriaceae but not NDM-or VIM-producing Enterobacteriaceae and would offer a partial solution to treat infections due to XDR or PDR Gram-negative bacteria.
In conclusion, the EuSCAPE project and this followup survey confirm the urgent need for a coordinated European effort for surveillance, control and prevention of CPE in Europe. The project contributed to the improvement of the capacity and ability to detect CPE The national experts, the ECDC National Focal Points for antimicrobial resistance and the ECDC National Correspondents for EU enlargement countries: answered the survey providing country specific data, provided country specific profile, approved the final data and the analysis, and reviewed and provided feedback the manuscript.
The EuSCAPE scientific advisory board and the external consulted experts: reviewed and provided feedback on the manuscript.

Introduction
Neuraminidase inhibitors (NAIs) are a class of influenza antivirals that target the highly conserved enzymatic site of the neuraminidase (NA) glycoprotein on the surface of influenza A and B viruses [1]. The NAIs have become the most widely used antivirals for the treatment or prophylaxis of influenza, particularly since the development of widespread resistance to the adamantanes, the older class of antivirals that block the M2 ion channel protein [2]. Two NAIs, oseltamivir and zanamivir, have been available in many countries since 1999, and two new NAIs, peramivir and laninamivir, have recently been approved for human use in Japan and a small number of other countries. Each of the NAIs is structurally different and therefore binds slightly differently within the NA active site [1]. This difference in binding is advantageous for treatment, as a virus that develops resistance against one NAI, may retain sensitivity to others. For example, the H275Y NA mutation in NA subtype N1-containing viruses confers resistance to oseltamivir but not to zanamivir [3].
Resistance to the NAIs commonly occurs as a result of amino acid mutations within the NA active site, either in the catalytic residues (those that interact directly with the NAIs), or in the framework residues (those that provide structural support for the catalytic residues) [4]. However, not all viruses with resistance to NAIs will pose a public health risk, as mutations that reduce binding can also impact the ability of the NA to interact with the natural substrate during replication [5]. However, in some cases, the mutation can affect NAI sensitivity but not compromise viral 'fitness'. The H275Y mutation that was present in seasonal influenza A(H1N1) viruses between 2007 and 2009 is such an example, as it conferred oseltamivir resistance but did not appear to affect the ability of the virus to replicate and transmit [6,7].
Oseltamivir resistant influenza viruses have been detected at a considerably higher frequency than zanamivir resistant viruses. During human clinical trials, oseltamivir resistance was detected in < 1-4% of adults [8,9] and 5-6% of treated children [10] undergoing oseltamivir treatment, although in observational studies the frequency of resistance in oseltamivir treated children has been as high as 27% [11]. Most significantly, oseltamivir-resistant seasonal A(H1N1) viruses with an H275Y mutation became widespread during 2008, spreading globally even in regions of low drug usage [12,13]. In comparison, there have only been a few reports of zanamivir resistance. The first was in an immunocompromised child undergoing zanamivir treatment where an influenza B virus with a R152K NA mutation was detected that caused a 40-150-fold reduction in zanamivir sensitivity [14,15]. More recently a small number of A(H1N1)pdm09 viruses with an I223R NA mutation have been detected in immunocompromised patients exposed to oseltamivir and/or zanamivir [16,17] and in a patient without previous exposure to NAIs [18], but the change in zanamivir sensitivity as a result of this mutation is relatively minor (10-fold), compared with the larger 45-fold shift in oseltamivir sensitivity [16].
Previously, our group and others reported the detection of former seasonal A(H1N1) virus isolates with a Q136K mutation that conferred a 250-fold reduction in zanamivir sensitivity [19,20]. The Q136K isolates were particularly unusual because the mutation could not be detected in the clinical specimens from which they were derived. This suggested that either the variant virus was being generated in cell culture or it was present in very low levels in the clinical specimen and then selectively amplified during cell culture. The former seasonal A(H1N1) virus stopped circulating soon after the emergence of the A(H1N1)pdm09 virus in 2009, and for the first two years after the A(H1N1) pdm09 viruses started circulating, no Q136K variants were reported. However, here we report the detection of both Q136K and Q136R substitutions in A(H1N1) pdm09 and A(H3N2) viruses between 2011 and 2014, investigate their selection in different cell lines and determine the effect that these and other amino substitutions of the Q136 residue have on NAI susceptibility and NA enzymatic function.

Virus strains, Madin-Darby canine kidney epithelial cells and virus culture
The influenza viruses used in this study were received at the World Health Organization (WHO) Collaborating Centre for Reference and Research on Influenza (WHOCC), Melbourne, Australia through the WHO Global Influenza Surveillance and Response System (GISRS) from countries in the Asia Pacific region. The Q136K or Q136R isolates had initially been isolated and then passaged in MDCK cells in external laboratories before being received and repassaged one to two further times at the WHOCC. WHOCC MDCK cells were originally received from ATCC (CCL-34) and used at passage level 63 to 83 and grown in Dulbecco's modified

Neuraminidase activity and neuraminidase inhibition assays
To determine NA activity, viruses were standardised to an equivalent haemagglutinin (HA) titre using turkey red blood cells, serially diluted (twofold) in assay buffer (  Single nucleotide (nt) mutations were introduced into the NA plasmids to alter the Q136 codon to residues H, K, L, or R using the QuikChange Multi Site Directed  Mutagenesis Kit (Agilent Technologies) and using mutagenesis primers designed according to manufacturer's guidelines and synthesised by GeneWorks (Adelaide, Australia). The NA segment was sequenced directly from the plasmid as described previously to confirm that the desired mutation had been introduced and that no additional mutations were present.
Recombinant viruses composed of the NA gene from one of the viruses described above, and the remaining seven segments from A/Puerto Rico/8/34 (A/PR/8/34 plasmids kindly provided by Dr Robert Webster, St. Jude Children's Research Hospital, Memphis) were generated by reverse genetics. All eight plasmids were transfected into a co-culture of 293T and MDCK cells as previously described [21]. Rescued viruses were subsequently cultured in MDCK cells in maintenance media described above.   Figure 1). This positive selection for the Q136K variant was also observed following passage of the A/ Brisbane/345/2011 isolate in the WHOCC MDCK cells, but was not seen with the other two isolates, where passage in WHOCC MDCK cells resulted in the gradual loss of the Q136K or Q136R variant. The MDCK-SIAT1 cells consistently selected against the Q136K or Q136R variants, with the proportion of each variant gradually decreasing after serial passage (Figure 1). The largest change in mixture proportion was seen following egg passage, which showed that growth of the variants were not well supported in embryonated eggs and were rapidly selected against, such that after a single passage in eggs the Q136K and Q136R viruses were undetectable in two of the isolates, and accounted for < 10% of the viral population in the third isolate ( Figure 1).

Neuraminidase activity and neuraminidase inhibitor susceptibility of reverse genetics derived Q136K, R, L and H variants
Examination (in 2014) of human and avian N1 and N2 sequences from the public sequence databases Global Initiative on Sharing Avian Influenza Data (GISAID) and GenBank revealed not only the Q136R and Q136K substitutions, but also Q136L and Q136H, present in a small number (less than 1%) of sequences from A(H3N2), A(H1N1) and A(H5N1) virus isolates. Site directed mutagenesis and reverse genetics were used to better investigate the phenotypic effect of the Q136K, Q136R, Q136L and Q136H substitutions in the NAs from A(H1N1)pdm09 and A(H3N2) viruses. 7:1 reassortants containing either the N1 or N2 WT NA (no mutations) or variant NA (Q136K, R, L or H) on a PR/8 backbone were successfully generated by reverse genetics.
For N1 reassortants, the Q136H mutant retained full NA activity, while the Q136R, Q136K and Q136L mutants had between 33 and 54% of the WT NA activity ( Table  2). For the same mutations in the N2 NA, the Q136H substitution also had no effect on NA activity, whereas the Q136R substitution caused a minor reduction in NA activity (82% activity of WT), and the Q136K and Q136L substitutions caused large reductions in NA activity (40% activity of WT) ( Table 2).
Analysis of the N1 reassortants for NAI susceptibility showed that the Q136H substitution had no effect, whereas the Q136L mutant demonstrated a moderate 32-fold increase in zanamivir IC 50 and a minor 4-to 12-fold increase in oseltamivir, peramivir and laninamivir IC 50 compared with the respective WT. In comparison, the Q136R substitution caused a 659-to 810-fold increase in zanamivir, peramivir and laninamivir IC 50 compared with WT, while the Q136K substitution caused a 126-to 589-fold increase against the same NAIs (Table 2). Both the Q136K and Q136R substitutions had no effect on oseltamivir susceptibility.
The large effect of the Q136R and Q136K substitutions observed in the N1 NA was not observed in the N2 NA. Q136R caused only a minor (two-to threefold) change in oseltamivir, peramivir and zanamivir IC 50 . A Q136L or a Q136K substitution caused a moderate 10-to 13-fold increase in zanamivir and oseltamivir IC 50 and a threefold increase in peramivir IC 50 but no change in laninamivir sensitivity compared with the WT. Q136H in N2 had no effect on NAI sensitivity, similar to that observed in the N1 NAs (Table 2).

Viral function of reverse genetics derived Q136K, R, L and H variants
The thermostability and HA/NA balance of the reassortant variant viruses were determined. The Q136L and Q136H N1 variants retained high NA activity across the 38 °C to 54 °C temperature range, equivalent to that of the WT virus. However the N1 reassortants with Q136K and Q136R mutations showed a substantial loss of activity at 54 °C (67% and 22% remaining activity compared to 37 °C respectively) (Figure 2A). The N2 reassortant WT had some loss of activity at 54 °C (30% remaining activity), as did the NAs with Q136R and Q136H mutations (8-21% remaining activity), while the NAs with Q136K and Q136L mutations maintained high activity (> 80%) across the entire temperature range ( Figure 2B).
All N1 and N2 reassortant viruses contained the PR/8 HA and all showed good cell binding at 4 °C, as indicated by low viral titres in the supernatant. After incubation at 37 °C, the N1 WT reassortant and the Q136L variant showed full restoration of HA titre, demonstrating an active NA enzyme, whereas the Q136K and Q136R variants had only partial restoration of HA titre, suggesting that the NA activity was insufficient to cleave all bound virus from the cells ( Figure 3A). All of the N2 reassortants, with the exception of Q136K, showed full restoration of HA titre following incubation at 37 °C ( Figure 3B).

Discussion
In this study we describe the detection of A(H1N1) pdm09 influenza virus isolates, and to a lesser extent A(H3N2) viruses, with amino acid substitutions at the Q136 NA residue that reduce zanamivir, peramivir and laninamivir susceptibility. Surveillance data show that the Q136K and Q136R substitutions occurred sporadically in A(H1N1)pdm09 cultured isolates, with periods such as 2011 where a relatively high detection rate was observed, compared with other years where they were absent. Importantly, in all cases, the residue substitution that was present in the isolate, was not detected in the virus from the clinical specimen demonstrating that the mutation was either arising, or being selected for, during MDCK cell culture passage. Because the 'gold-standard' for laboratory assessment of NAI susceptibility is the phenotypic NA inhibition assay, which requires a cell culture isolate for testing, there is concern that viruses such as these can be reported as being 'resistant' when in fact the virus that came from the patient was sensitive. In addition, the process of cell culture may also select against a resistant virus, meaning that a variant virus is not detected when it was present in the clinical specimen. Misdiagnosis can have an impact on the therapies being used in patient management and may unnecessarily result in therapy being stopped, modified or inappropriately continued. In addition to the Q136K/R variants described here, there are many other NA mutations that alter NAI susceptibility and also appear to be selected during MDCK cell culture [19,23]. Interestingly these seem to be increasingly reported for influenza B viruses [23][24][25].
Therefore sequence analysis of the influenza viruses in the original specimen remains important when laboratories detect mutations in cultured isolates.
While conventional MDCK cells selected for the Q136K and Q136R A(H1N1)pdm09 variants, their growth was not supported in eggs, with a single passage resulting in the near complete loss of the variant virus. MDCK-SIAT1 cells also did not appear to give selective growth advantage to the Q136 variants. MDCK-SIAT1 cells have enhanced binding due to an increased concentration of α2,6-linked sialic acids on the MDCK cell surface [26], which may mean that viruses with reduced NA activity, such as the Q136K/R variants, have reduced replication in this cell line, possibly explaining the difference with the conventional MDCK cell lines. If clinical samples are available in the future that have been shown to result in MDCK isolates with Q136K/R mutations, it would be useful to test whether primary isolation into MDCK-SIAT1 or human bronchial epithelial cells prevents this initial selection of the variant virus.
Although the Q136K and Q136R variants detected here were all cell culture derived variants, other studies have reported mutations at Q136 that were detected in viruses from clinical specimens. The Q136K mutation was detected in an A(H3N2) virus, together with an E119V NA mutation, in a patient who had previously undergone a bone marrow transplant, following a treatment course of both inhaled and intravenous zanamivir and oseltamivir [27]. Mutations at Q136 have also been detected in ferrets infected with influenza A(H5N1). The Q136L variant was detected in the nasal wash of a zanamivir treated ferret infected with an A(H5N1) virus [28], while a Q136H mutation was detected in an A(H5N1) virus from a ferret not being treated with an antiviral [29]. These reports demonstrate that viruses with these mutations have the potential to infect or replicate in vivo both in the presence or absence of zanamivir pressure.
The zanamivir concentration in sputum 12 hours postinhalation has been reported to be between 159 and 4,315 nM [30]. Therefore, while the correlation between the drug concentration in sputum and the drug concentration at the site of viral replication is not clear, it is anticipated that only the Q136K and Q136R mutations in the A(H1N1)pdm09 virus may potentially impact the clinical effectiveness of zanamivir. The Q136L mutation in both N1 and N2 NAs and Q136K in N2 NA caused mild increases in zanamivir and oseltamivir IC 50 , which are expected to be below the concentrations present at the sites of replication in treated individuals [30,31].
An evaluation of the ability of the Q136K and Q136R A(H1N1)pdm09 variants to replicate and transmit in animal models will provide useful insights into the potential risk that these viruses may pose to public health. Where possible these future studies would benefit from using Q136K and Q136R variants that were naturally occurring, rather than cell culture derived or generated by reverse genetics. One limitation of this study is that the N1 and N2 NAs with Q136 substitutions were assessed in 1:7 reassortant viruses generated by reverse genetics on a PR/8 backbone, therefore there is potential that the HA/NA balance between the variant NA molecules and the HA from PR/8 may be different from that seen in the 'natural' isolate. The in vitro assays showed that as a result of the Q136K mutation the N1 reassortant had a moderate loss in NA activity and thermostability. A reduction in NA enzyme activity and surface expression due to the Q136K NA mutation has been previously reported [32,33]. Pizzorno et al. [33] also demonstrated that an A(H1N1)pdm09 Q136K variant had compromised replication compared with a WT virus in mice, while in a ferret model the variant was able to transmit between contact ferrets, but at slower rate than for the WT virus. However, a study in guinea pigs found that the Q136K variant did not transmit between animals [32]. Taken together, these studies indicate that the replication and transmissibility of the Q136K variant in the A(H1N1)pdm09 virus appears to be compromised and therefore is unlikely to circulate through the human population. However, compensatory mutations in the NA or other genes that may occur in the A(H1N1)pdm09 virus in the future may buffer the compromising effect of the Q136K mutation and improve overall replication and transmissibility of the variant, in a manner similar to that seen for the H275Y mutation [7,34]. To date there has been no evaluation of the in vivo fitness of the Q136R A(H1N1) pdm09 variant.
In this study we have highlighted the challenges that cell culture derived mutations, such as Q136K and Q136R, can pose to the analysis and interpretation of viruses for NAI susceptibility. This further reaffirms the need to sequence viruses from the clinical specimens of any isolate that shows reduced susceptibility in a phenotypic NA inhibition assay to avoid misdiagnosis and any unnecessary change in patient management with respect to the use of antivirals. Our findings highlight the effect of mutations at the Q136 residue of N1 viruses on laninamivir, peramivir or zanamivir susceptibility, and therefore close monitoring of viruses for these mutations in patients being treated with these antivirals is important.