Dr Heli Harvala

Jaśmina Kondracka

Chloe Leanne Mcintyre

E Carol

Mcwilliam Leitch

Kate E Templeton

Peter Simmonds
email: heli.simmonds@hotmail.com

Jaśmina Kondracka

Chloe Mcwilliam Mcintyre

E Leitch

Kate ; Templeton

Nigel Mcleish

Jasmina Kondracka

Chloe L Mcintyre

Kate Templeton

Comparison of human parechovirus and enterovirus detection frequencies in cerebrospinal fluid samples collected over a 5-year period in Edinburgh -HPeV type 3 identified as the most common picornavirus type

Keywords: Sepsis, meningitis, surveillance, serotype, neonatal Sepsis, meningitis, surveillance, neonatal, serotype, molecular diagnostics

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Enteroviruses discovered more recently have not been classified into these subgroups, nor have their antigenic properties been examined in detail; instead they are assigned as enterovirus types numbered chronologically in order of their identification (33 types by the end of 2009). The 93 human EVs fall into 4 genetically distinct species, HEV-A to D [START_REF] Hyypia | Classification of enteroviruses based on molecular and biological properties[END_REF][START_REF] Stanway | Family Picornaviridae[END_REF], with species B variants (including echoviruses, CBVs and CAV9) being the viral cause of CNS-associated infections in Europe identified most frequently.

Along with this reorganisation of enteroviruses was the recognition that two echoviruses, E22 and E23, were different genetically and biologically from other members in the Enterovirus genus. These two serologically distinct viruses were originally isolated and characterised over 50 years ago during a summer diarrhoea outbreak in America [START_REF] Wigand | Properties of ECHO types 22, 23 and 24 viruses[END_REF]. However, they were recognised as genetically distinct from enteroviruses in the early 1990s [START_REF] Hyypia | A distinct picornavirus group identified by sequence analysis[END_REF] and subsequently reassigned to a new picornavirus genus, Parechovirus [START_REF] King | Virus taxonomy. Seventh Report of the International Committee for the Taxonomy of Viruses[END_REF] as human parechovirus (HPeV) types 1 and 2. Since then, a further twelve types (HPeV3 to 14) known to infect human have been discovered [START_REF] Harvala | Parechoviruses in children: understanding a new infection[END_REF]. Although HPeV infections are common and the clinical course is usually mild, they have been recently associated with sepsis-like disease in very young children [START_REF] Boivin | Human parechovirus 3 and neonatal infections[END_REF], Harvala et al., 2009, Wolthers et al., 2008).

There is a high incidence of EV and HPeV infections in early childhood, as indicated by the high seroprevalence by the time children reach school age [START_REF] Joki-Korpela | Parechoviruses, a novel group of human picornaviruses[END_REF][START_REF] Tauriainen | Human parechovirus 1 infections in young children--no association with type 1 diabetes[END_REF]. Although these infections are mostly asymptomatic, newborn infants are considered to be at a higher risk of severe EV (and HPeV) infection because of the immaturity of their immune system. Previous reports suggest that the risk of symptomatic neonatal EV and HPeV disease varies by infecting type, and that CBVs, E11 and HPeV3 are most commonly associated with severe neonatal sepsis-like illness [START_REF] Harvala | Aetiological role of human parechovirus type 3 in neonatal sepsis identified by direct typing assay on cerebrospinal fluid[END_REF], Khetsuriani et al., 2006a, Khetsuriani et al., 2006b). However, fever in young infants can be bacterial in origin and thus it often necessitates a complete set of investigations including blood and urine culture, lumbar puncture as well as the initiation of broad-spectrum antibiotic therapy. This may be avoided in many cases if a rapid diagnosis of EV and HPeV infections could be made.

Traditionally, the diagnosis of EV or HPeV infections had relied on obtaining a positive viral culture from stool, nasopharyngeal swab or cerebrospinal fluid (CSF) samples. This often slow, laborious and insensitive virus detection method has been largely replaced by nucleic acid amplification methods such as reverse-transcriptase polymerase chain reaction (RT-PCR) for detection of viral RNA [START_REF] Arola | Identification of enteroviruses in clinical specimens by competitive PCR followed by genetic typing using sequence analysis[END_REF][START_REF] Chapman | Molecular detection and identification of enteroviruses using enzymatic amplification and nucleic acid hybridization[END_REF], Hyypia et al., 1989[START_REF] Olive | Detection and differentiation of picornaviruses in clinical samples following genomic amplification[END_REF][START_REF] Zoll | General primer-mediated polymerase chain reaction for detection of enteroviruses: application for diagnostic routine and persistent infections[END_REF].

Typically, RT-PCR assays target the 5'untranslated region, highly conserved between all four EV species, and between all known types of HPeV. Although extremely useful for virus screening, amplicon sequences from the 5'NCR provide little or no information on the (sero)type of the infecting virus. Reliable type identification therefore depends on amplification and genetic analysis of structural gene regions such as VP1. Because of sequence variability in these coding regions, implementation of reliable typing assays for both EVs and HPeV requires considerably greater optimization of primer design and amplification conditions to achieve the sensitivity required for direct use on clinical specimens, particularly CSF. However, it is recognized that molecular typing is important as a necessary part of ongoing poliovirus surveillance but also for the molecular epidemiological purposes and for better understanding of symptoms and clinical outcomes. This study describes the large-scale implementation of previously developed highly sensitive typing assays for EVs and HPeV to investigate the (sero)types and epidemiology of these viruses over the past five years in a large Scottish Virology Centre. [START_REF] Welch | Detection of enterovirus viraemia in blood donors[END_REF]. Real-time RT-PCR was introduced into routine use in the beginning of 2009 (modified from [START_REF] Dierssen | Rapid routine detection of enterovirus RNA in cerebrospinal fluid by a one-step real-time RT-PCR assay[END_REF], at the same time screening of CSF samples (total of 2000) for mumps virus by real-time RT-PCR was initiated [START_REF] Krause | Real-time PCR for mumps diagnosis on clinical specimens--comparison with results of conventional methods of virus detection and nested PCR[END_REF].

F

For HPeV screening and type identification, extracted RNA from 3739 anonymised CSF samples (representing over 90% of all CSF sample obtained during the period) were deposited in the Specialist Virology Centre CSF sample archive and tested for HPeVs by RT-PCR (Harvala et al., 2008, McLeish et al. manuscript in preparation). Samples were anonymised and archived according to protocol approved by the Lothian Regional Ethics Committee (08/S11/02/2). Stored data included age band, recorded clinical information, referral source, month of sample collection and results of routine virological testing of the sample. Amplification of VP1 sequences. Sufficient volumes of CSF were available for typing from 159 of the 201 EV-positive samples. Extracted RNA was amplified by a combined RT and first round PCR using the Superscript III (Invitrogen) followed by a second amplification reaction with nested primers specific for species A and B VP1 sequences as previously described [START_REF] Leitch | Direct identification of human enterovirus serotypes in cerebrospinal fluid by amplification and sequencing of the VP1 region[END_REF]. Positive HPeV samples were amplified in the VP3/VP1 junction region and sequenced between nucleotide positions 2182 and 2437 to identify HPeV types as described previously [START_REF] Harvala | Epidemiology and clinical associations of human parechovirus respiratory infections[END_REF].

Nucleotide sequencing and serotype identification. Amplified DNA was directly sequenced using the BigDye Terminator kit (Applied Biosystems, Warrington, UK).

Sequences were edited and aligned using the Simmonics sequence editor (http://www.virus-evolution.org). Sequences have been assigned GenBank accession numbers xxxxx-yyyyy. remarkably high percentage of all EV infections (94/201; 47%) were found in children under the age of three months (Fig. 2). The highest frequency of HSV1 and VZV infections was found in study subjects over 65 years of age, whereas HSV2 and Mumps virus infections occurred predominantly in the younger adults group (21-36 years and 37-65 years; data not shown). There was no significant over-representation of males among those infected with EV (103 male, 97 female) or HPeV (16 male, 15 female).

Adults infected with EV presented almost exclusively with suspected viral meningitis, whereas young children with HPeV and EV infections were referred with fever and sepsis-like illness (Table S1; Supplementary Material). Overall, HPeV presented with greater disease severity; four of 31 (13%) HPeV-positive subjects were treated on the High-Dependency / Intensive Care Units at time sampling compared to 5 from 198 (2.5%) infected with EV (p = 0.023; Fisher's Exact Test).

EV and HPeV infections in young infants. The remarkable high incidence of HPeV infections in children under the age of three months were largely confined to spring time on even years (22% in March 2006, 25% in April 2008and 22% in March 2010; Fig. 3), whereas EV infections were absent in this age group during these months. The prevalence of EV infections (11.8%) in young infants (<3 months) generally exceeded that of HPeV infections (4.1%). Interestingly, the frequency of EV infections was 12.0% (88/748) in children under the age of 3 months referred from home, while only 6 EV infections were diagnosed in 586 (1%) neonates remaining in hospital after birth in RIE Neonatal Unit (NNU) (p <0.001; Fig. 4). A similar discordance in HPeV incidence was observed between these two groups with a 4% detection frequency EV and HPeV type identification. To identify infecting EV serotypes, amplification of VP1 gene was performed. Regions amplified in VP1 allowed previously assigned EV serotypes to be reliably identified [START_REF] Leitch | Direct identification of human enterovirus serotypes in cerebrospinal fluid by amplification and sequencing of the VP1 region[END_REF]. An extremely high proportion of (uncultured) CSF samples could be directly typed for EVs (145/151; 96%), despite the availability of very limited CSF sample volumes and frequently low viral loads. Six samples failed to be detected by VP1 primers, but their identity as species A/B serotypes was confirmed by sequencing the 5'UTR. For HPeV, all 5'UTR screening positive CSF samples (31/31) could be amplified and typed in the VP3/VP1 region.

A total of 16 different species B and 4 species A EV serotypes were detected in this study. The most frequently detected enterovirus types were E9 (23; 15%), CAV9 (22; 14%), E6 (19; 13%), E30 (17; 11%), E18 (9; 6%) and CBV5 (8; 5%) serotypes, along with occasional detections of species A serotypes CAV2, CAV6, CAV16 and EV71 (Fig. 5). Rapid changes in serotype frequencies were observed. Each serotype infected a wide age range, although E6 and E30 infections were largely or entirely absent in children less than three months of age (data not shown).

Almost all

Similarly to HPeV3 infections (30/30), all E16 infections (5 patients) as well as most E25 (6/7; 86%) and E11 (5/7; 71%) infections were seen in young infants (<3 months of age). Most very young children infected with EV or HPeV presented with sepsislike disease, whereas older children and adults commonly had meningitis (Table S1; Supplementary data). All EV infections were reported to the Health Protection Agency, England. Furthermore, HSV2, VZV and mumps virus were associated with meningitis and HSV1 with encephalitis (data not shown).

Discussion

This study reports the results of comprehensive screening of CSF samples for enteroviruses and parechoviruses over a 5-year period using molecular techniques for detection and virus characterization. Because of the equivalent sensitivity of the screening PCR for HEV-A and HEV-B viruses (as well as for HEV-C and HEV-D), our rates of detection are uninfluenced by variability in ease of in vitro culture, an important drawback of previous enterovirus surveillance reports that relied on prior virus isolation for identification of serotypes. Similarly, PCR offered a truer reflection of the relative importance of parechoviruses in CNS-related disease; HPeVs, particularly type 3, are known to be refractory frequently to in vitro culturing [START_REF] Benschop | Detection of enterovirus and human parechovirus genotypes from clinical stiool samples; PCR and direct molecular typing, culture characteristics and serotyping[END_REF].

Enterovirus diversity.

Over the 5-year study period, the most common enterovirus serotypes identified in Edinburgh were E9, E6, CAV9, E30 and E18. Three of these (E9, E30 and CAV9) were also among the most abundant serotypes isolated from clinical specimens in the UK previously in 1975 to 1994 [START_REF] Maguire | Enterovirus infections in England and Wales: laboratory surveillance data: 1975 to 1994[END_REF]. E6, E9 and E30 were also among the most frequently reported serotypes in the United States in 1970 to 2005 (Khetsuriani et al., 2006a), and similarly E30 and E6 in continental Europe in 2000s [START_REF] Antona | Surveillance of enteroviruses in France, 2000-2004[END_REF][START_REF] Blomqvist | Eight years of experience with molecular identification of human enteroviruses[END_REF]. In contrast to most previous surveillance data based on virus isolation outside South-East Asia, a total of four different species A enterovirus serotypes were detected (CAV2, CAV6, CAV16 and EV71) in association with neonatal sepsis. This higher frequency of species A variants is consistent with a previous study of viraemia frequency in Scottish blood donors, where CAV16 and EV71 were amongst the identified EV types identified most frequently despite never being isolated contemporaneously by the virus diagnostic services serving the same catchment area [START_REF] Welch | Detection of enterovirus viraemia in blood donors[END_REF]. Other evidence for the widespread circulation of EV species A variants is provided by the frequent molecular detection of EV71, CAV6, CAV10 and CAV4 in faecal sample screening of healthy Norwegian infants [START_REF] Witso | High prevalence of human enterovirus a infections in natural circulation of human enteroviruses[END_REF] and in Finnish national surveillance [START_REF] Blomqvist | Eight years of experience with molecular identification of human enteroviruses[END_REF].

Clearly, the adoption of PCR-based methods for virus detection provides a better opportunity to assess population enterovirus diversity. Despite this, no individuals were found to be infected with species C or D EVs, an observation that suggests either their much more restricted community incidence or a greatly reduced likelihood of infected individuals developing the severe disease manifestations that would lead to diagnostic virology testing. The recently implemented weekly screening of sewage in the Edinburgh area has indeed revealed the presence of numerous species C serotypes (Calvert et al., unpublished observation) providing evidence in favour of the latter explanation (despite the evident neurotropism of the now eradicated polioviruses).

Detection of HPeV3.

In contrast to the diversity of enteroviruses detected in CSF, all but one of the 31 parechovirus-positive samples identified were type 3. Over the 5year study period, it was the most prevalent picornavirus type in CNS-related infection. Comparison of genetic variants in the current study with those detected in previous surveillance provides evidence for a major change in parechovirus molecular epidemiology; HPeV1 (named as E22 at that time and thus part of enterovirus surveillance) was the second most prevalent serotype in the UK detected by virus isolation between 1975 and 1994 [START_REF] Maguire | Enterovirus infections in England and Wales: laboratory surveillance data: 1975 to 1994[END_REF]. Similarly, HPeV1 was [START_REF] Hovi | Relative abundance of enterovirus serotypes in sewage differs from that in patients: clinical and epidemiological implications[END_REF], Khetsuriani et al., 2006a). Although it could be argued that the lack of HPeV3 detection in these earlier surveys directly resulted from putative insensitivity of virus isolation for this virus type [START_REF] Benschop | Detection of enterovirus and human parechovirus genotypes from clinical stiool samples; PCR and direct molecular typing, culture characteristics and serotyping[END_REF], it is nevertheless clear that a major change in either the community circulation of HPeV1 or its disease outcomes has occurred over the last 3 decades.

HPeV3 infections differed considerably in their seasonality from enteroviruses, being detected exclusively in CSF samples collected in Edinburgh in 2006, 2008and 2010 (Fig. 3). This biennial cycle is consistent with previous reports of much higher frequencies of HPeV3 infections in even-numbered years between 2000 and 2006, and the virtual absence in intervening years [START_REF] Benschop | High prevalence of human parechovirus genotypes in the Amsterdam region and the identification of specific HPeV variants by direct genotyping of stool samples[END_REF][START_REF] Van Der Sanden | Prevalence of human parechovirus in the Netherlands in 2000 to 2007[END_REF]. To an extent, this periodicity resembles the regular increases in reported cases of several of the commonly circulating EV serotypes. For example, E30 shows a 4-5 year periodicity based on both USA and European surveillance data [START_REF] Antona | Surveillance of enteroviruses in France, 2000-2004[END_REF], Khetsuriani et al., 2006a, Trallero et al., 2010), while E9 shows a 2-3 year cycle. The re-appearance of E30 and E9 variants after an eclipse phase is associated with the emergence of novel recombinant genetic lineages of the viruses [START_REF] Leitch | Transmission networks and population turnover of echovirus 30[END_REF], McWilliam Leitch et al., 2010). This is however not the case in HPeV3; the variants circulating in 2006 and 2008 were almost identical in the VP3/VP1 region and sequence analysis of the 3Dpol region did not provide any evidence of the emergence of novel recombinant forms over the ellipse period [START_REF] Calvert | The recombination dynamics of human parechoviruses; investigation of typespecific differences in frequency and epidemiological correlates[END_REF]. The phenomena underlying the biennial cycle of HPeV3 infections and its much greater frequency in spring thus remains to be determined. CNS-associated infections in young children. There were several differences in the age distribution between different EV serotypes and HPeVs. A remarkably high proportion of all EV infections (47%) were observed in infants younger than three months of age. This is higher than reported previously; enterovirus infection in the neonatal period accounted for about 10% of the total number of reported cases of enterovirus infections in the United States, whereas children <1 year of age accounted for 44% of reports (Khetsuriani et al., 2006a). National surveillance data from France and the UK has shown that the vast majority of EV infections occur in young children, with infants below the age of one year accounting for about a third of all cases [START_REF] Antona | Surveillance of enteroviruses in France, 2000-2004[END_REF][START_REF] Maguire | Enterovirus infections in England and Wales: laboratory surveillance data: 1975 to 1994[END_REF].

As found in the current study, it has also been shown that the relative frequency and predominant serotypes of enteroviruses affecting neonates differ from the pattern observed in the general population. Indeed, the remarkable variability in the proportion of young infants infected with enteroviruses could be partly accounted by seasonal changes in the circulation of serotypes associated with CNS-related disease in young infants. These include CBVs, E11, E16 and E25, each of which typically target neonates [START_REF] Verboon-Maciolek | Diagnosis of enterovirus infection in the first 2 months of life by real-time polymerase chain reaction[END_REF], and E9 and E30 which infect neonates much less frequently than older persons (Khetsuriani et al., 2006a, Khetsuriani et al., 2006b). In this study, the lowest proportions of EV infections in young infants (<3 months of age; 36%) were observed in 2007, the year when E9 and E30 were the two viruses detected most frequently, accounting for 57% of all EV infections. Conversely, the highest proportion of young infants (<3 months of age) infected with EV (57%) occurred in 2009, when CBVs, and E11 were amongst the two most prevalent viruses. Furthermore, E16 was positively associated with infections in very young children (<3 months; all infections in this age group). Although marked differences in the age distribution of different EV serotypes were observed, an even more polarised age distribution was observed in HPeV infections. These were detected exclusively in CSF samples obtained from young infants (<3 months), and entirely absent from older children and adults. These findings are consistent with the previous analysis of HPeV infection [START_REF] Harvala | Aetiological role of human parechovirus type 3 in neonatal sepsis identified by direct typing assay on cerebrospinal fluid[END_REF]; the parallel observation that HPeV3 detection in respiratory samples was similarly age-restricted providing evidence that the virus circulation is limited to children in this very young age group. This association is therefore not the result of biased sampling and greater likelihood of sampling because of disease severity, or a clinical need to identify viral from bacterial causes of sepsis.

The observation that both EV or HPeV infections were restricted almost entirely to infants who had been admitted to hospital from home (Fig. 4) and were almost entirely absent from children in the same age range who had remained hospitalised after birth, provides clear evidence for community sources of infection that are largely curtailed in a hospital setting. These observations reassure that nosocomial transmission of EVs and HPeV in the Edinburgh neonatal unit was extremely uncommon over the five-year study period. The findings also indicate how diagnostic screening for EVs and HPeV may be targeted more effectively as there would be a case to exclude those who have remained in hospital after birth from testing.

Why has the incidence of neonatal EV and HPeV infections increased? One of the major findings in the current study was the remarkable frequency of EV and HPeV infections in children less than three months old, presenting with sepsis-like illnesses.

The observed frequencies are much higher than reported previously in this age range for both virus groups [START_REF] Figueroa | An outbreak of acute flaccid paralysis in Jamaica associated with echovirus type 22[END_REF], Khetsuriani et al., 2006a, Legay et al., 2002a), and cannot be entirely attributed to improvement in the effectiveness of screening methodologies (although the greater ability of PCR to detect HPeV3 and species A EVs compared to virus culture undoubtedly contributed partly to the increased detection frequency in CSF).

In seeking to explain this change, it might be proposed that HPeV3 and several of EVs specifically found in neonates may have changed in their biological properties (associated with a greater propensity to infect systemically), in their epidemiology and transmission, or that there have been systematic changes in host susceptibility over the past decades. One factor supporting the latter hypothesis is the evidence for a progressive lowering of maternal seroprevalence and lack of passively transferred protective neutralizing antibodies transferred passively in an increasing proportion of neonate and infants. The changing epidemiology of enterovirus infections has indeed been demonstrated clearly in Finland [START_REF] Viskari | Can enterovirus infections explain the increasing incidence of type 1 diabetes?[END_REF]: the proportion of pregnant women lacking antibodies to CBV4 increased from 6% in 1983 to 17% in 1995 (p<0.001). Increasing number of young infants will therefore lack the protective maternal antibodies to viruses circulating in the community, and increase the likelihood of developing severe CNS-associated (sepsis-like) EV disease.

Whether such changes in the viral circulation account for the increasing incidence of HPeV3-associated sepsis-like illness in neonates is unclear, as are the underlying reasons for the major clinical and epidemiological differences from HPeV1, which is well documented to cause asymptomatic or mild infections and shows a strikingly high incidence in the first two years of life [START_REF] Joki-Korpela | Diagnosis and epidemiology of echovirus 22 infections[END_REF][START_REF] Tapia | Longitudinal observation of parechovirus in stool samples from Norwegian infants[END_REF][START_REF] Tauriainen | Human parechovirus 1 infections in young children--no association with type 1 diabetes[END_REF]. The close to universal adult seropositivity for HPeV1 contrasts with that of HPeV3; over 30% of child-bearing aged women has been shown to lack antibodies to HPeV3 [START_REF] Ito | Isolation and identification of a novel human parechovirus[END_REF]. However, unlike the situation with EVs, the lower adult seroprevalence of HPeV3 may have resulted directly from its relatively recent emergence compared to other HPeV types.

Through analysis of the genetic diversity of circulating currently and historical isolates of HPeV3, it has been estimated that a common ancestor for this type appeared in the human population as recently as 1987 [START_REF] Calvert | The recombination dynamics of human parechoviruses; investigation of typespecific differences in frequency and epidemiological correlates[END_REF], approximately 10 years before it was first isolated in Japan and Canada (Abed & Boivin, 2005[START_REF] Ito | Isolation and identification of a novel human parechovirus[END_REF]. HPeV3 may therefore have not spread sufficiently far into adult human populations to provide the essential levels of maternal protection required to prevent neonatal disease. This proposed scenario contrasts with HPeV1, where close to universal maternal seropositivity may account for the current almost complete lack of neonatal disease associated with this type. Sewage surveillance in Edinburgh indeed documents the predominance of HPeV1 over other HPeV types circulating in the local community (Calvert et al., unpublished observations). Interestingly, diagnosed HPeV1 infections were substantially more frequent and more severe in the 1980s [START_REF] Figueroa | An outbreak of acute flaccid paralysis in Jamaica associated with echovirus type 22[END_REF], Koskiniemi et al., 1989, Legay et al., 2002b), where it accounted for close to 8% of all HPeV/EV infections in national surveillance systems [START_REF] Antona | Surveillance of enteroviruses in France, 2000-2004[END_REF], Khetsuriani et al., 2006a, Khetsuriani et al., 2006b[START_REF] Maguire | Enterovirus infections in England and Wales: laboratory surveillance data: 1975 to 1994[END_REF]. We might speculate that the emergence of HPeV1 simply preceded that of HPeV3 and these disease presentations originated similarly from a lack of universal maternal exposure at that time.

Combined, these findings emphasises the importance of ongoing surveillance of EVs and HPeVs in our understanding of human picornavirus-related disease. In particular, the evidence for relatively recent emergence and marked changes in the Similarly, the frequency of HPeV infections varied from 4.1% (31/748) to 0.2%

(1/586). Both differences were statistically significant (p<0.0001, Chi-squared test). A total number of CSF sample tested in this age group (< 3 months) was 1334.

  EV) and parechoviruses (HPeV), members of the virus family Picornaviridae, are important human pathogens responsible for a wide spectrum of illnesses varying from the common cold to severe infections of the central nervous system (CNS) and myocardium. Enteroviruses have been divided traditionally into polioviruses (PVs, 3 serotypes), coxsackie A viruses (CAVs, 23 serotypes), coxsackie B viruses (CBVs, 6 serotypes) and echoviruses (E, 28 serotypes), mainly on the basis of their pathogenicity in laboratory animals.

  Fig.1). In comparison, EV was detected throughout the 5-year study period in a total

  748) in the former group compared to 0.2% (1/485; p <0.001; Fisher's Exact Test) among neonates remaining in the NNU).

  EVs were typed as E9 in early 2007, and were replaced entirely by E30 and E6 in the latter half of the year. No virus predominated in 2005, 2006, 2008 and 2009, where 7, 6, 9 and 11 different serotypes circulated respectively. Most recently in 2010, CAV9 became the predominant serotype. All except one of the HPeV types identified were HPeV3 (30/31), whereas the remaining one was HPeV type 1B. Over the 5-year study period, HPeV3 was the most prevalent picornavirus in CNS-related infections.

  other parts of Europe and in the United States in the 1980's

Fig. 1 .

 1 Figure legends

Fig. 2 .

 2 Fig. 2. The positivity rate of HEV and HPeV infections in each age category

Fig. 4 .

 4 Fig. 4. The frequency of EV infection was 11.8% (87/748) in children under the age

Fig. 5 .

 5 Fig. 5. The number of individual HEV serotypes and HPeV types detected over the 5

Journal of Medical Virology


Elisa Martro

Ana Valero

Elena Jordana-Lluch

Verónica Saludes

Ramón Planas

Fernando Gonzalez

Vicente Ausina

Maria Alma Bracho

Elisa Martró

Fernando González-Candelas

Hepatitis C virus sequences from different patients confirm the existence and transmissibility of subtype 2q, a rare subtype circulating in the metropolitan area of Barcelona, Spain

Keywords: HCV, genotype 2, subtype 2q, complete genome

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

for both structural and nonstructural (NS) proteins [START_REF] Choo | Genetic organization and diversity of the hepatitis C virus[END_REF]. HCV is characterized by a high level of genetic variability, which has allowed to classify the virus into 6 major genotypes (differing from each other by 31-33% at the nucleotide level), and a number of subtypes within each genotype (differing from each other by 20-25% at the nucleotide level) [START_REF] Kuiken | Nomenclature and numbering of the hepatitis C virus[END_REF]. On the basis of a single genomic sequence from a Canadian emigrant from the Democratic Republic of Congo, genotype 7a has been assigned provisionally [START_REF] Murphy | Use of sequence analysis of the NS5B region for routine genotyping of hepatitis C virus with reference to C/E1 and 5' untranslated region sequences 10[END_REF]]. Different genotypes show specific geographical locations, whilst certain subtypes (1a, 1b, 2a and 3a) have become widely distributed as a result of transmission through intravenous drug use, blood products, and health-care-related procedures [START_REF] Kuiken | Nomenclature and numbering of the hepatitis C virus[END_REF].

HCV genotype is a major predictor of a sustained virological response to antiviral therapy (pegylated alpha-interferon plus ribavirin); HCV genotyping is required to tailor dose and duration of treatment [START_REF] Ghany | Diagnosis, management, and treatment of hepatitis C: an update[END_REF]]. The target of choice for commercialized molecular diagnostics and most genotyping assays is the 5'NC region, which is highly conserved. However, subtype identification based on this region is not always reliable. Although subtyping is not clinically relevant in the context of the current therapy, accurate subtype identification is useful for understanding the epidemiology and virological features of HCV. Besides, it might be clinically important in the coming era of specifically targeted antiviral therapy for HCV, since subtypespecific resistance profiles have been described for subtypes 1a and 1b both in infected patients [START_REF] Kieffer | Telaprevir and pegylated interferon-alpha-2a inhibit wild-type and resistant genotype 1 hepatitis C virus replication in patients[END_REF], Sarrazin et al. 2007] and in vitro [START_REF] Mccown | GT-1a or GT-1b subtype-specific resistance profiles for hepatitis C virus inhibitors telaprevir and HCV-796[END_REF]].

Consensus criteria have been established for HCV classification and nomenclature, and confirmed designation status of new subtypes with epidemiological value requires rigorous phylogenetic analysis of one or more complete genome sequences of the candidate subtype with no epidemiological connection [START_REF] Kuiken | Nomenclature and numbering of the hepatitis C virus[END_REF].

Eighteen subtypes within genotype 2 have been assigned so far (2a to 2r), but only four have been confirmed (2a, 2b, 2c, and 2k) and a fifth one proposed (2i) on the basis of their complete genome sequences.

In a previous study comparing three commercial kits for HCV-genotyping based on the 5'NC region [START_REF] Martró | Evaluation of a new assay in comparison with reverse hybridization and sequencing methods for hepatitis C virus genotyping targeting both 5' noncoding and nonstructural 5b genomic regions[END_REF], three specimens that showed discordant results were subjected to NS5B sequencing. Upon phylogenetic analysis, they did not cluster with any of the previously confirmed genotype 2 subtypes, but formed a monophyletic group with the only representative sequence of the provisionally assigned 2q subtype [START_REF] Tokita | The entire nucleotide sequences of three hepatitis C virus isolates in genetic groups 7-9 and comparison with those in the other eight genetic groups[END_REF]]. This study reports the HCV genomic sequence for two of those isolates and a partial NS5B sequence for the third one, and suggests changing the status of subtype 2q from proposed to confirmed designation.

METHODS

Patients and specimens.

Three patients with a discordant genotyping result based on the 5'NC region were selected retrospectively from a previous study [START_REF] Martró | Evaluation of a new assay in comparison with reverse hybridization and sequencing methods for hepatitis C virus genotyping targeting both 5' noncoding and nonstructural 5b genomic regions[END_REF].

Isolates from patients 963 and 852 were genotyped as 2a/2c by reverse hybridization (Inno-LiPA HCV II Assay, Innogenetics, Gent, Belgium) and were indeterminate by Once the rest of components were added, the reaction was incubated at 50ºC for 60 min (for random hexadeoxynucleotides) or 55ºC for 60 min (for primer), followed by 15 min at 70ºC. 1) with ClustalW [START_REF] Thompson | CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positionspecific gap penalties and weight matrix choice[END_REF]] implemented in MEGA v4.0 [START_REF] Tamura | MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0[END_REF]]. Alignments corresponding to partial genomic regions (Core, E1 and NS5B) were also obtained. The alignments included the most similar sequences found in a BLAST search against HCV sequences, including all 2k deposited sequences and sequences for all genotype 2 subtypes available, as well as reference sequences for the other genotypes. The sequence from patient 985 was also considered for the analysis of the NS5B region.

Maximum likelihood phylogenetic trees were constructed from the corresponding alignments with the evolutionary model implemented in PHYML that best fitted the data according to jModeltest [START_REF] Posada | jModelTest: phylogenetic model averaging[END_REF]]. The robustness of the tree topologies was assessed by bootstrap analysis implemented in PHYML [START_REF] Guindon | A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood[END_REF] with 1000 pseudorandom resamplings of the sequences. GenBank accession numbers of the reference sequences used in each phylogenetic reconstruction are shown in Figure 1.

Pairwise nucleotide identities between genotype 2 subtypes including the newly described 2q sequences were calculated using MEGA v4.0, using an alignment encompassing nucleotide positions 31 (5'NC region) to 9336 (end of the 2i sequence DQ155561) of the H77 reference sequence (GenBank accession number AF009606).

Reference sequences for the following HCV subtypes were used in this analysis: Recombination analysis. Recombination analysis was performed using the recombination detection program RDP v3.44 [START_REF] Martin | RDP: detection of recombination amongst aligned sequences[END_REF] with an alignment that included the two genomic sequences generated in this study along with the 27 representative complete sequences used in the phylogenetic analyses [START_REF] Heath | Recombination patterns in aphthoviruses mirror those found in other picornaviruses[END_REF]]. This program implements several methods for the identification of recombinant sequences and recombination breakpoints. Recombination analysis was performed using six of them: two phylogenetic methods that detect recombination when different regions of the genome lead to discordant topologies (RDP, and Bootscan/Recscan [Martin et al. 2005]), and four nucleotide substitution methods that search either for a significant clustering of substitutions or for a fit to an expected statistical distribution (GeneConv [START_REF] Padidam | Possible emergence of new geminiviruses by frequent recombination[END_REF], Chimaera [START_REF] Posada | Evaluation of methods for detecting recombination from DNA sequences: computer simulations[END_REF], MaxChi [START_REF] Smith | Analyzing the mosaic structure of genes[END_REF], and SiScan [START_REF] Gibbs | Sister-scanning: a Monte Carlo procedure for assessing signals in recombinant sequences[END_REF]). Default RDP v3.44 settings were used throughout, except that sequences were considered to be linear. Only putative recombination events inferred by more than two methods were taken into account, as performance evaluation of these recombination detection methods indicated that the results of a single method are not completely reliable [START_REF] Posada | Evaluation of methods for detecting recombination from DNA sequences: empirical data[END_REF]].

RESULTS

The HCV genomic sequences obtained were 9398 and 9566 nt in length for patients 852 and 963, respectively. Both sequences were partial at the 5' end of the 5'NC region; the first one ended 18 nt before the end of the polyprotein coding region (the NS5B region is partial at the 3' end), and the later included part of the 3'NC region. Figure 1a shows the unrooted ML phylogenetic tree corresponding to the polyprotein-encoding nucleotide sequence. For genotype 2, only the five fully sequenced subtypes could be included in the analysis (2a, 2b, 2c, 2i and 2k), since complete genome sequences are still lacking for the other 13 assigned subtypes. This tree shows a highly supported grouping of sequences from patients 852 and 963, which would constitute a highly supported clade along with subtype 2k. Similar results were obtained in the phylogenetic analysis of the full Core region (573 nt) in which a few additional subtype 2 sequences were included (Figure 1b).

Phylogenetic analysis of partial E1 and NS5B regions provided additional information. The phylogenetic tree of a NS5B subregion (309 nt) included the three patients considered in this study (852, 963 and 985, for which only this partial NS5B sequence could be obtained). As shown in Figure 1d, these three sequences cluster together with the only representative sequence of the provisionally assigned 2q subtype (GenBank accession number D86529) with a high bootstrap support. Interestingly, the latter corresponds to isolate BA045, which was obtained in the 90's from a patient residing in Barcelona, Spain, who had not travelled abroad, and had acquired HCV infection through transfusion (M. A. Barrera, personal communication). Sequence AF037240 from a patient from Burkina Faso (West Africa) and described as a putative new genotype 2 subtype [Jeannel et al. 1998], was the sequence related most closely to the 2q cluster. Similarly, the phylogenetic tree of the E1 region (336 nt) shows that the two 2q sequences clustered with maximum bootstrap support (Figure 1c), and nontypable sequences EF115812 and EF115840 from patients in Quebec, Canada [START_REF] Murphy | Use of sequence analysis of the NS5B region for routine genotyping of hepatitis C virus with reference to C/E1 and 5' untranslated region sequences 10[END_REF]], were the most closely related sequences. no evidence that the two newly described 2q genomes had been involved in recombination events involving other genotypes or genotype 2 subtypes.

Finally, when considering the coding region, the two subtype 2q genomes sequenced showed a 90% identity to each other at the nucleotide level, while both sequences differed by 17.5 to 22.6% from the other confirmed genotype 2 subtypes (Table II). The highest identity was observed between subtypes 2q and 2c (82.23%) while the identity between 2q and 2k was slightly lower (80.54%). However, genetic distances estimated by maximum likelihood (which attempt to estimate the true amount of change occurred between two subtypes) were 0.40 and 0.28 respectively, which reveals the closest relationship between 2q and 2k subtypes. When the length of the full coding region as well as the ten protein regions were compared between genotype 2 subtypes, all subtype 2q protein regions were identical in length to those of subtypes 2a, 2b and 2k (Table III).

DISCUSSION

Genetic diversity is greatest within HCV genotypes 6, 3 and 2, which contains more divergent sequences than other genotypes [START_REF] Kuiken | Nomenclature and numbering of the hepatitis C virus[END_REF].

Genotype 2 is highly prevalent and genetically diverse in sub-Saharan Africa and in Martinique [START_REF] Candotti | Frequent recovery and broad genotype 2 diversity characterize hepatitis C virus infection in Ghana, West Africa[END_REF], Jeannel et al. 1998[START_REF] Martial | Hepatitis C virus (HCV) genotypes in the Caribbean island of Martinique: evidence for a large radiation of HCV-2 and for a recent introduction from Europe of HCV-4[END_REF][START_REF] Ruggieri | Heterogeneity of hepatitis C virus genotype 2 variants in West Central Africa (Guinea Conakry)[END_REF]]. In Europe, previous studies found a prevalence of 11% in France, with a considerable diversity [START_REF] Thomas | Genetic diversity of HCV genotype 2 strains in south western France[END_REF]], and a 3% in Spain [START_REF] Echevarria | Follow-up of the prevalence of hepatitis C virus genotypes in Spain during a nine-year period (1996-2004)[END_REF]].

Genotype 2 represents 4.3% of the patients treated in the Hospital Universitari Germans Trias i Pujol (period 2003-2009, N=493), where a variety of subtypes have been identified, including 2a, 2b, 2c, 2i, 2j and 2q [START_REF] Martró | Evaluation of a new assay in comparison with reverse hybridization and sequencing methods for hepatitis C virus genotyping targeting both 5' noncoding and nonstructural 5b genomic regions[END_REF]. New genotype 2 subtypes with limited distribution have been proposed on the basis of a few partial genome sequences in certain geographical regions not only in Africa [Jeannel et al. 1998[START_REF] Ruggieri | Heterogeneity of hepatitis C virus genotype 2 variants in West Central Africa (Guinea Conakry)[END_REF]], but also in Europe [START_REF] Thomas | Genetic diversity of HCV genotype 2 strains in south western France[END_REF][START_REF] Van Doorn | Sequence analysis of hepatitis C virus genotypes 1 to 5 reveals multiple novel subtypes in the Benelux countries[END_REF]]. Among HCV-2 subtypes, 2a, 2b, 2c and 2k are worldwide distributed while subtypes 2d to 2p, 2r and five other tentative subtypes [START_REF] Cantaloube | Molecular characterization of genotype 2 and 4 hepatitis C virus isolates in French blood donors[END_REF]] have been detected in only one or a few countries. HCV 2q, which had been proposed previously on the basis of a single partial NS5B sequence from a patient from Barcelona [START_REF] Tokita | The entire nucleotide sequences of three hepatitis C virus isolates in genetic groups 7-9 and comparison with those in the other eight genetic groups[END_REF]] is another example of a geographically restricted subtype.

In this study, another three patients in the Barcelona metropolitan area were identified as bearing this subtype, and most of its genome has been sequenced in two of the three cases. Patients' ages and clinical records agree with the hypothesis that HCV-2q could have been emerged in Spain as a result of blood transfusion, probably before HCV screening was implemented in blood banks in 1990. In fact, the most common transmission route among patients infected by minority genotype 2 subtypes in the close area of Midi-Pyrenées in France was through blood transfusion, while subtype 2a was transmitted via intravenous drug use [START_REF] Thomas | Genetic diversity of HCV genotype 2 strains in south western France[END_REF]].

Three commercial genotyping assays based on the 5'NC region either failed to assign a genotype or to subtype correctly the three 2q isolates. Nucleotide sequencing and phylogenetic analysis of more variable genomic regions has been recommended for HCV genotyping in consensus proposals [START_REF] Simmonds | Consensus proposals for a unified system of nomenclature of hepatitis C virus genotypes[END_REF] 2002, [START_REF] Martró | Evaluation of a new assay in comparison with reverse hybridization and sequencing methods for hepatitis C virus genotyping targeting both 5' noncoding and nonstructural 5b genomic regions[END_REF][START_REF] Tamalet | Genomic and phylogenetic analysis of hepatitis C virus isolates: a survey of 535 strains circulating in southern France[END_REF][START_REF] Zeuzem | Evaluation of a reverse hybridization assay for genotyping of hepatitis C virus[END_REF]. Furthermore, only sequencing and phylogenetic analysis of coding regions, such as NS5B or Core, provides the chance to identify variants not yet classified.

A level of nucleotide differences of at least 15% is expected between subtypes within each genotype, including newly described variants [START_REF] Kuiken | Nomenclature and numbering of the hepatitis C virus[END_REF]. Accordingly, subtype 2q shows a nucleotide difference of 17.5-22.6% from the other confirmed genotype 2 subtypes. Phylogenetic analysis of the coding region showed that, among confirmed genotype 2 subtypes, subtype 2q was most closely related to subtype 2k. Furthermore, in the phylogenetic analysis of the NS5B and E1 regions, non-typable genotype 2 sequences in patients form Burkina Faso and Canada were identified as the most closely related sequences to subtype 2q. Several recombinants of different genotypes including genotype 2 have been described in several countries: 2k/1b [START_REF] Kalinina | Full-length open reading frame of a recombinant hepatitis C virus strain from St. Petersburg: proposed mechanism for its formation[END_REF]], 2i/6p [START_REF] Noppornpanth | Identification of a naturally occurring recombinant genotype 2/6 hepatitis C virus[END_REF]], and 2b/1b [START_REF] Kageyama | A natural inter-genotypic (2b/1b) recombinant of hepatitis C virus in the Philippines[END_REF]], and 2k/5 [START_REF] Legrand-Abravanel | New natural intergenotypic (2/5) recombinant of hepatitis C virus[END_REF]]. Nevertheless, the two newly described 2q sequences showed no evidence of recombination.

In conclusion, phylogenetic, identity, and recombination analyses indicate that subtype 2q now meets the requirements for confirmed designation status according to consensus criteria for HCV classification and nomenclature [Kuiken andSimmonds 2009, Simmonds et al. 2005

Figure 1 .

 1 Figure 1. Maximum likelihood phylogenetic trees. (a) Unrooted phylogenetic tree of the coding region (positions 342 to 9336), nucleotide substitution model GTR + I + G (proportion of invariable sites: 0.301, gamma shape parameter: 0.841); (b) genotype 2 subtree of the Core region (positions 342 to 914), nucleotide substitution model GTR + I + G (proportion of invariable sites: 0.436, gamma shape parameter: 0.671); (c) genotype 2 subtree of the E1 region (positions 927 to 1262), nucleotide substitution model GTR + I + G (proportion of invariable sites: 0.436, gamma shape parameter: 0.671); (d) genotype 2 subtree of the NS5B region (positions 8283 to 8591), nucleotide substitution model GTR + I + G (proportion of invariable sites: 0.279, gamma shape parameter: 0.833). Positions are given according to the H77 reference sequence. Sequences are identified by genotype, subtype and GenBank accession number. Sequences that have not been assigned to any subtype are denoted as "2_". Sequences newly described in this study are underlined. Nodes supported by a bootstrap support value >70% are indicated. The scale bar represents number of substitutions per nucleotide position. 47x35mm (600 x 600 DPI)

  real-time PCR (HCV genotyping analyte specific reagent [ASR] assay; Abbott Molecular Inc., Des Plaines, IL), while isolate 985 was genotyped as 2a/2c by reverse hybridization, 2c by sequencing (TruGene HCV 5'NC genotyping kit, Bayer HealthCare, Berkeley, CA), and 2a by the real-time PCR assay. Only by means of NS5B sequencing and phylogenetic analysis evidence could be obtained showing that the three isolates belonged probably to the previously proposed subtype 2q. The clinical and epidemiological characteristics of the three patients are summarized in Table I. Sera obtained at the date of HCV diagnosis had been conserved at -80ºC. Patient 985 had insufficient serum volume and only HCV isolates from patients 963 and 852 could be subjected to full-length genome sequencing. This study was approved by the Clinical Research Ethics Committee ("Comité Ético de Investigación Clínica", CEIC) at Hospital Universitari Germans Trias i Pujol (HUGTIP). RNA extraction, RT-PCR and sequencing. HCV RNA was extracted from 200 µl of serum using the High Pure Viral RNA Kit (Roche) or QIAamp ® viral RNA Mini kit (QIAGEN ® ) following the manufacturer's protocol. Reverse transcription (RT) was performed with 10 µl of extracted RNA in a total reaction volume of 20 µl containing 4 µl of 5× First-Strand buffer, 5 mM DTT, 200 U of SuperScript ® III Reverse Transcriptase (Invitrogen), 40 U of rRNasin ® Ribonuclease Inhibitor (Promega), 0.5 mM of each dNTP, and either 0.75 µg of random hexadeoxynucleotides (Amersham-Pharmacia Biotech) or 0.5 µM of antigenomic primer. The mix containing dNTP, random hexadeoxynucleotides or primer, and the RNA was incubated at 65ºC for 5 min.

  Recombination signals were investigated by means of phylogenetic and nucleotide substitution methods. These analyses based on the complete genome alignment showed

  ]. Accordingly, commercialized assays based on the Core (Versant HCV genotype 2.0 assay, Siemens Medical Solutions Diagnostics) or the NS5B regions (Abbott RealTime HCV Genotype II, Abbott Molecular Diagnostics) have been developed over the last years for a better discrimination of genotype 1 subtypes. However, subtype assignment for the other genotypes still relies on the 5'NC region and often leads to errors[Chen and Weck

  TableIin supplementary material lists the primers used to obtain overlapping RT-PCR products covering most of the viral genome. Primer design was initially based on primers used in the sequencing of the 2i/6p recombinant (GenBank accession number DQ155560)[START_REF] Noppornpanth | Identification of a naturally occurring recombinant genotype 2/6 hepatitis C virus[END_REF]]. First round and hemi-nested PCR amplifications were carried out in a final reaction volume of 50 µl containing either 5 µl of the RT product (in the case of first round PCR) or 1 µl of the first round PCR product (in the case of hemi-nested PCR), 5 µl of 10× PCR buffer with 15 mM MgCl 2 ), 200 µM

	The obtained sequences were deposited in the EMBL Nucleotide Sequence Database
	with accession numbers FN666428 (patient 963), FN666429 (patient 852), and
	FN666430 (patient 985).
	Phylogenetic reconstructions and identity analysis. The nucleotide sequences
	corresponding to the complete polyprotein from patients 963 and 852 were aligned with
	27 homologous sequences representative of the main HCV genotypes and subtypes (see
	GenBank accession numbers, genotypes and subtypes in Figure
	of each dNTP, 400 nM each of both genomic sense and antigenomic sense primers, and 1 U of TaKaRa Taq™ (Takara Bio Europe SAS). Amplification was performed on 2700 or 9700 GeneAmp ® PCR systems (Applied Biosystems) under the following conditions: F o r
	94°C for 2 min; 5 cycles, 94°C for 30 s, 50-65°C (depending on the primers used) for P
	30 s, 72°C for 3 min; 35 cycles, 94°C for 30 s, 52°C for 30 s and 72°C for 3 min; 72°C e
	for 10 min. For the 3'NC region, the following conditions were used: 94°C for 2 min; 5 e r
	cycles, 94°C for 30 s, 55°C for 1 min, 72°C for 2 min; 35 cycles, 94°C for 30 s, 52°C for 1 min, 72°C for 2 min; 72°C for 10 min. Amplified products were checked by R
	e electrophoresis on 1.8% agarose gels stained with ethidium bromide, and purified with either High Pure PCR Products Purification Kit (Roche) or QIAquick PCR purification v i e kit (QIAGEN ® ). Direct sequencing of PCR products was performed for both strands using the w
	BigDyeTM Terminator v3.1 Ready Reaction Cycle Sequencing Kit on ABI Prism 3730
	or 3100-Avant Genetic Analyzers (Applied Biosystems) and the primers listed in Table
	II in supplementary material. The obtained chromatogram files were assembled,
	verified, and edited using the Staden Package [Staden et al. 2000].

Table I . Characteristics of the four patients identified so far as being infected with HCV subtype 2q.

 I ]. Furthermore, subtype 2q is of epidemiological value, since it has spread among several patients in the Barcelona metropolitan area.

	de Investigaciones Sanitarias" (ISCIII-FIS), and grants CD05/00258 ("Contratos FIGURE LEGENDS TABLES
	Postdoctorales de Perfeccionamiento") and "Miguel Servet" from "Ministerio de
	Ciencia e Innovacion" (MICINN), within the "Plan Nacional de Investigación científica, Figure 1. Maximum likelihood phylogenetic trees. (a) Unrooted phylogenetic tree of
	Desarrollo e Innovación Tecnológica (I+D+I)" (EM). the coding region (positions 342 to 9336), nucleotide substitution model GTR + I + G
	Patient ID
	(proportion of invariable sites: 0.301, gamma shape parameter: 0.841); (b) genotype 2 Characteristic 852 963 985 BA045
	Gender subtree of the Core region (positions 342 to 914), nucleotide substitution model GTR + Male Female Female Male HCV risk factor Surgery, several Multiparous, two Cervical carcinoma Transfusion
	transfusions (1959) I + G (proportion of invariable sites: 0.436, gamma shape parameter: 0.671); (c) abortions (<1990), surgery (1990), curettage (1992) possible transfusion
	F o r F o r F o r F o r F o r F o r F o r genotype 2 subtree of the E1 region (positions 927 to 1262), nucleotide substitution model GTR + I + G (proportion of invariable sites: 0.436, gamma shape parameter: 0.671); (d) genotype 2 subtree of the NS5B region (positions 8283 to 8591), nucleotide Date of HCV diagnosis 2001 2002 2001 -F Age (years) 60 51 69 -HCV treatment No No Not eligible for Yes o treatment r Clinical outcome Persistently normal Cirrhosis, portal Pluripathology Sustained
	P e P e P e P e P e P e P e substitution model GTR + I + G (proportion of invariable sites: 0.279, gamma shape parameter: 0.833). Positions are given according to the H77 reference sequence. transaminase hypertension (advanced renal virological P Discharged (2005) Exitus (2005) e levels (esophagic varices) insufficiency) response
	e r e r e r e r e r e r e r Sequences are identified by genotype, subtype and GenBank accession number. City of residence Santa Coloma Badalona Badalona Barcelona e r (province) (Barcelona) (Barcelona) (Barcelona) (Barcelona)
	R R R R R R R Sequences that have not been assigned to any subtype are denoted as "2_". Sequences newly described in this study are underlined. Nodes supported by a bootstrap support R
	e v i e e v i e e v i e e v i e e v i e e v i e e v i e e value >70% are indicated. The scale bar represents number of substitutions per nucleotide position. v i e
	w w w w w w w w

Table II . Identity (%) among genotype 2 subtypes that have been characterized by complete genome sequencing.

 II GenBank accession numbers of reference sequences:Nucleotide identity was computed between nucleotide positions 31 to 9336 according to the H77 reference sequence. Patient ID is indicated in parenthesis.

		F o r
			P
			e
			e r
				R
				e
	D00944 (2a), D10988 (2b), D50409 (2c), AB031663 (2k) and DQ155561 (2i). v i e
					w
		2a	2b	2c	2i	2k	2q (852)
	2a	-
	2b	76.75	-
	2c	79.44	76.09	-
	2i	79.93	77.33	80.29	-
	2k	80.16	76.64	80.09	81.10	-
	2q (852)	79.62	77.42	82.52	80.40	79.47	-
	2q (963)	80.04	77.58	82.23	80.54	80.10	90.34

Table III . Size composition of HCV protein regions among the genotype 2 subtypes that have been characterized by complete genome sequencing

 III . a Based on curated //hcv.lanl.gov/content/sequence/HCV/ToolsOutline.html). b Partial sequence at its 3' end. c Based only on the 963 isolate. GenBank accession numbers of reference

								Page
	alignments	available	at	the	HCV	LANL	Database
	(http:sequences: D00944 (2a), D10988 (2b), D50409 (2c), AB031663 (2k) and DQ155561
	(2i).
					Region length (nt) a
	Core E1 E2	p7 NS2 NS3 NS4A NS4B NS5A NS5B Polyprotein
	2a 573 576 1101 189 651 1893 162	783	1398 1776	9102
	2b 573 576 1101 189 651 1893 162	783	1398 1776	9102
	2c 573 576 1101 189 651 1893 162	783	1410 1776	9114
	2i 573 576 1101 189 651 1893 162	783	1395 1734 b	9057 b
	2k 573 576 1101 189 651 1893 162	783	1398 1776	9102
	2q 573 576 1101 189 651 1893 162	783	1398 1776 c	9102 c

22 of 22 John Wiley & Sons Journal of Medical Virology

Journal of Medical Virology

ACKNOWLEDGEMENTS

This work was supported partially by CIBER Epidemiología y Salud Pública (CIBERESP), projects PI05/1131 (EM, RP, VA), CP09/0004 (EM), CP07/00078 (MAB), and BFU2008-03000 (FG-C, MAB) from "Instituto de Salud Carlos III -Fondo


K Lidbrink

K Krawiec

Sandelin

Catharina Eriksen
email: catharina.eriksen@karolinska.se

Immediate reconstruction with implants in women with invasive breast cancer does not affect oncological safety in a matched cohort study

Keywords: breast cancer, immediate breast reconstruction, recurrence rate, oncological safety, "delay adjuvant treatment", radiotherapy 1

Immediate reconstruction with implants in women with invasive breast cancer does not affect oncological safety in a matched cohort study

Introduction

European guidelines are in agreement that breast reconstruction should be an integral part of breast cancer treatment [START_REF]National guidelines for breast cancer treatment in Sweden www.swebcg.roc.se 2. European society of breast cancer specialists www[END_REF][2][START_REF][END_REF][START_REF] Baildam | Oncoplastic breast surgery-A guide to good practice[END_REF]. Previous studies have suggested that after breast reconstruction, women with breast cancer have a better quality of life [START_REF] Elder | Quality of life and patient satisfaction in breast cancer patients after immediate reconstruction: a prospective study[END_REF][START_REF] Rowland | Psychological response to breast reconstruction. Expectations for and impact on postmastectomy functioning[END_REF][START_REF] Rosenqvist | Patients psychological and cosmetic experience after immediate breast reconstruction[END_REF][START_REF] Brandberg | A prospective and randomized study, "SVEA", comparing effects of three methods for delayed breast reconstruction on quality of life, patient-defined problem areas of life, and cosmetic result[END_REF]. The timing and type of reconstructions can vary with local traditions but the demand for reconstruction is growing. Traditionally, women with ductal carcinoma in situ and T1-2 tumors are offered immediate breast reconstruction (IBR), while those presumed to receive radiotherapy or those with more advanced breast cancer usually are recommended to have delayed procedures [START_REF] Slavin | Skin-sparing mastectomy and immediate reconstruction: oncologic risks and aesthetic results in patients with early-stage breast cancer[END_REF][START_REF] Rainsbury | Skin-sparing mastectomy[END_REF][START_REF] Kroll | Local recurrence risk after skin-sparing and conventional mastectomy: a 6-year follow up[END_REF]. The latter patients are often advised to wait for 1-2 years after adjuvant treatment before starting the reconstruction. This recommendation is often based on an increased risk of local recurrence during the first two years and concern that a reconstruction could hamper detection of a local recurrence. Another issue has been that IBR, especially autologous reconstructions, may influence the oncological treatment in that more extensive surgery with more potential complications such as an increased number of infections could delay adjuvant treatment [START_REF] Lewis | Autologous tissue immediate breast reconstruction: desired but oncologically safe?[END_REF][START_REF] Taylor | Oncological aspects of breast reconstruction[END_REF]. Other reasons for postponing immediate breast reconstruction are unsatisfactory cosmetic results in connection with radiotherapy and its administration [START_REF] Lee | Postmastectomy radiation therapy and breast reconstruction: an analysis of complications and patient satisfaction[END_REF][START_REF] Motwani | The impact of immediate breast reconstruction on the technical delivery of postmastectomy radiotherapy[END_REF]. IBR with implants has several advantages over late breast reconstructions, such as fewer operations, no breast loss, less morbidity (no donor site), simpler surgical techniques and less expense [START_REF] Khoo | a comparison of resource costs of immediate and delayed breast reconstruction[END_REF].

Notwithstanding the lack of randomized controlled studies, breast reconstructions are increasing [START_REF]National guidelines for breast cancer treatment in Sweden www.swebcg.roc.se 2. European society of breast cancer specialists www[END_REF][2][START_REF][END_REF][START_REF] Baildam | Oncoplastic breast surgery-A guide to good practice[END_REF][START_REF] Rainsbury | Skin-sparing mastectomy[END_REF]. Studies addressing the safety of IBR have shown varying rates of local recurrence [START_REF] Vandeweyer | Immediate breast reconstruction with saline-filled implants: no interference with the oncologic outcome?[END_REF][START_REF] Johnson | Oncological aspects of immediate breast reconstruction following mastectomy for malignancy[END_REF][START_REF] Slavin | Recurrent breast cancer following immediate reconstruction with myocutaneous flaps[END_REF][START_REF] Noon | Recurrence of breast carcinoma following immediate reconstruction: A 13-year review[END_REF][START_REF] Newman | Feasibility of immediate breast reconstruction for locally advanced breast cancer[END_REF][START_REF] Allweis | Immediate reconstruction after mastectomy for breast cancer does not prolong the time to starting adjuvant chemotherapy[END_REF][START_REF] Langstein | Breast cancer recurrence after immediate reconstruction: Patterns and significance[END_REF][START_REF] Murphy Rx | Impact of immediate reconstruction on the local recurrence of breast cancer after mastectomy[END_REF][START_REF] O"brian | Comparison of postoperative wound complications and early cancer recurrence between patients undergoing mastectomy with or without immediate breast reconstruction[END_REF][START_REF] Knottenbelt | The oncological implications of immediate breast reconstruction[END_REF]. As there may be a considerable interval before recurrences occur, confirmation of these results requires a long-term follow-up. There is also a lack of studies in this area, especially in patients receiving neoadjuvant chemotherapy and postoperative radiotherapy [START_REF] Patani | Oncological and aesthetic considerations of skin-sparing mastectomy[END_REF]. Karolinska University Hospital has 20 years of experience of implant-based IBR and two previous studies showed low rates of local recurrences [START_REF] Sandelin | Management, morbidity and oncologic aspects in 100 consecutive patients with immediate breast reconstruction[END_REF][START_REF] Sandelin | Oncological outcome after immediate breast reconstruction for invasive breast cancer: a long term study[END_REF]. The patients have also been investigated regarding postoperative complications, aesthetic results and quality of life [START_REF] Elder | Quality of life and patient satisfaction in breast cancer patients after immediate reconstruction: a prospective study[END_REF][START_REF] Lagergren | Technical aspects of immediate breast reconstruction with implants: Five year follow up[END_REF].

The principal aim of the study was to analyze the differences in local, regional and distant recurrences between women undergoing IBR and women operated with mastectomy alone. Oncological safety in terms of time to oncological treatment, disease-free survival and breast cancer specific survival in both groups was also evaluated.

Study design

The study is a matched retrospective cohort study.

Ethical approval

The study was approved by the Ethics Committee 2008, number 2008/767-31/4.

Patients and methods

Data collection: During the period 1990-2004, 475 consecutive patients operated with IBR at Karolinska University Hospital were prospectively registered in a hospital database. Only patients with invasive breast cancer and implant-based reconstruction were included, n=300. Patients operated with primary flap reconstruction were excluded and patients with earlier ipsilateral breast cancer were classified as local recurrence and were also excluded from the study. The patient match was performed 1:1 and concerned four categories: age, tumor size (0-20 mm, 21-50 mm, and > 50 mm), nodal status (0, 1-3, ≥4) and year of operation (+/-3 years), Table I. The matched control group were identified via the population-based Regional Breast Cancer Register of the Stockholm-Gotland health-care region and consisted of 300 women undergoing mastectomy alone.

The patients were followed until the end of June 2008. The median follow-up time for the IBR group was 12 years (range 4-18) and for the control group it was 11, 5 years (range 2-20 years). The median follow-up for both groups was 11.5 years (range 2-20). Type of breast cancer (ductal, lobular and other), hormonal status (ER,PR +/-), time to oncological treatment, type of chemotherapy, radiotherapy and recurrence, time to recurrence, detection mode, disease free survival and breast cancer specific survival were registered and evaluated. Elston grade (I-III) was registered but not evaluated since the analysis was not performed on the majority of the tumors during the study period [START_REF] Elston | Pathological prognostic factors in breast cancer. I. The value of histological grade in breast cancer: experience from a large study with long-term follow-up[END_REF].

Early complications (< 30 days) like infections treated with intravenous antibiotics, reoperations due to bleeding or wound heeling problems were registered. Decision process for IBR: All newly diagnosed breast cancer patients were discussed preoperatively and postoperatively at the multidisciplinary team conference. Patients with inflammatory breast cancer and tumors adhering to the pectoral muscle were not offered IBR. Similarly, women with a high body mass index and thus considered unsuitable for implant reconstruction and those who were heavy smokers were not recommended immediate reconstruction. Remaining cases where the woman wanted IBR, proceeded to operation. Clinical information, including recurrences and death, was retrieved from medical records (surgery, plastic surgery and oncological journals) and from the National Causes of Death Register. A local recurrence was defined as a relapse of cancer in the skin and the myocutaneous tissue of the chest wall. Regional recurrences were defined as a relapse in the ipsilateral axilla or in the supraclavicular nodes. All other relapses were defined as distant metastases. Surgical procedure: Initially the mastectomy incisions aimed to remove as much skin over the tumor as possible when reconstruction was concurrently done. After 1994, more tailor-made skin incisions were performed. Earlier scars and the nipple-areola complex were, however, always excised and the inner layer of the pectoral fascia was left intact in both groups unless there were macroscopic signs of tumor engagement of the muscle. Axillary surgery was performed as level I and II dissections and in 1998 the sentinel lymph node biopsy technique was introduced. Surgical-reconstructive procedure: All patients in the IBR group were operated with permanent or expandable implants. The implants were placed submuscularly and a full muscle cover was always aimed for. All operations were performed by a limited number of breast as well as reconstructive plastic surgeons with extensive experience. One preoperative dose of penicillin V (Cloxacillin) and penicillin G (Benzyl penicillin) or Clindamycin was given exclusively to all patients in the IBR group and they also received low-molecularweight heparin postoperatively. Oncological treatment: Oncological treatment was based on recommendations from the Regional Stockholm-Gotland Breast Cancer Study Group.

Chemotherapy: The standard adjuvant chemotherapy was CMF (cyclophosphamid, 5floururacil, and methotrexate) initially; after 1995 the standard regime was antracyclinebased therapy FEC (5-floururacil, epirubicin and cyclophosphamid). A small group received Taxane-based treatment, Table II. The treatment was given every third week in 6 cycles. Neoadjuvant chemotherapy was given to patients with large tumors (> 4 cm) and/or with clinically positive axillary nodes to decrease tumor burden and facilitate loco regional therapy. Based on clinical, pathological and radiological data, no response was defined as progressive or stabile disease, partial response as a decrease of the tumor and/or axillary nodes and complete response was defined as undetectable disease in the breast and the axilla. Radiotherapy: Local (chest wall) and locoregional (chest wall, axilla and supraclavicular lymph nodes) radiotherapy of 46 Gy was given in daily 2-Gy fractions with mostly tangential fields and in some cases with electron beams. Hormonal treatment: Patients with hormone receptor-positive tumors were offered Tamoxifen or Aromatase inhibitors in the latter part of the study. In the early stage of the study, patients were offered megestolacetate (Megace) as part of a study. Megace and Goserelin (Zoladex) are referred to as "other hormonal treatment" in Table II. Follow-up: The patients were reexamined at the Oncological Department every 3 months for the first 2 years and then every 6-12 months for a total of five years. Thereafter, patients with no sign of recurrences were discharged and referred to the general practitioners for further follow-up. Reconstructive revisions and assessments were performed by the reconstructive plastic surgeons.

Statistics

The McNemar test and the Stuart-Maxwell test were used for pairwise comparison of categorical variables. When pairwise comparison was not appropriate, Person"s chisquare test for categorical variables and the Mann-Whitney-Wilcoxon test for continuous variables were performed. The Kaplan-Meier method was used to calculate disease-free survival as well as breast cancer specific survival. Hazard ratios and 95 % confidence intervals were estimated with the Cox proportional hazard regression model with stratification on the matched pairs. All statistical analyses were performed in STATA/IC statistical software, version 10.0 [START_REF] Elston | Pathological prognostic factors in breast cancer. I. The value of histological grade in breast cancer: experience from a large study with long-term follow-up[END_REF].

Results

The median age was 48 years in both groups and median tumor size was 19 mm in the IBR group and 20 mm in the control group. Node positive disease in the IBR and the control group was 36% and 36.7%, respectively. Lobular cancers were significantly more prevalent in the IBR group and there were also more hormone receptor-positive tumors in the IBR group, Table I. There were no significant differences between the two groups in the proportion of patients treated with neoadjuvant chemotherapy (13% versus 15,7%), or in those receiving adjuvant chemotherapy and postoperative radiotherapy. Patients in the IBR group received more hormonal treatment. Furthermore, there were no differences between the groups in postoperative early complication rates or in time to oncological treatment, Table II. Among the radiated patients, there were no significant differences in recurrences between the groups (Fig. 1).

The analysis of first events showed that the incidence of total recurrences was 28.4% in the IBR cohort and 32.8 % in the control group. Regarding total number of event there were no significant differences in either local recurrences in the breast (8.2% in the IBR group, 9.0% in the control group) or in the regional recurrence rate (8.2% and 9.7%, respectively) although distant metastases were significantly more frequent in the control group, p=0.049. Median time to detection of local recurrence was 1.3 years in the IBR group versus 2.2 years in the control group, Table III. The majority of local recurrences were detected by clinical examination (94%) and there were no significant differences between the two groups in the way regional recurrences and distant metastases were detected, Table III. The IBR group showed better survival than the controls, (Fig. 2 and3). The differences between breast cancer mortality and all-causes mortality, were statistically significant, p=0.038 and 0.026, Table IV.

Discussion

Since the introduction of IBR, oncological safety has been a debatable issue among physicians and patients and the information given to patients has varied with local traditions regarding knowledge, competence and financial resources. In order to evaluate the possible influence of a reconstructive procedure together with breast cancer surgery, a matched group of women with breast cancer without primary reconstruction was chosen as control. This long-term follow-up study provides evidence that IBR with implants can be considered a safe procedure regarding all oncological aspects. In this matched cohort study, no significant inter-group differences were found in the rates of local recurrence and regional recurrence. Neither were there any differences in the time to oncological treatment or in detection modes. The rate of distant metastases was significantly higher in the control group and the survival figures were better for the reconstructed group. These results are in line with other studies, possibly as a consequence of confounding factors such as socioeconomic status and selection bias in inclusion criteria as IBR was usually not recommended for overweight patients and smokers [START_REF] Rothman | Modern Epidemiology. 3 rd[END_REF][START_REF] Mccarthy | Breast cancer recurrence following prosthetic postmastectomy reconstruction: Incidence, detection, and treatment[END_REF]. Our results also show that the reconstruction did not influence the start of oncological treatment or given dosages. In both groups the local recurrences were superficial and easy to detect, which is in accordance with other studies [START_REF] Langstein | Breast cancer recurrence after immediate reconstruction: Patterns and significance[END_REF][START_REF] Rothman | Modern Epidemiology. 3 rd[END_REF][START_REF] Bezuhly | Immediate postmastectomy reconstruction is associated with improved breast cancerspecific survival: Evidence and new challenges from the surveillance, epidemiology, and end results database[END_REF]. The study is retrospective and while the two groups were well-matched, there were more lobular cancers and a higher rate of hormone receptor-positive tumors among the IBR group. This could indicate an overrepresentation of tumors with a favorable prognosis in the IBR group. On the other hand, lobular carcinoma does not have a better prognostic clinical outcome or survival rate compared to ductal carcinoma [START_REF] Petit | Oncological results of immediate breast reconstruction: long term follow-up of a large series at a single institution[END_REF]. We also performed a statistical correction for hormonal receptorstatus which did not alter the results regarding recurrences, but the difference in breast cancer mortality risk was no longer significant. Another limitation is that complications are difficult to evaluate retrospectively and in the control group patients were discharged early and minor complications may have been taking care of by general practitioners. On the other hand the IBR group has been reviewed several times and clinical information was readily accessible from the charts [START_REF] Lagergren | Technical aspects of immediate breast reconstruction with implants: Five year follow up[END_REF]. We choose to report on early complications in order to show any possible effect on time to onset of oncological treatment in both groups.

A review from 2005 concluded that there was a lack of good-quality evidence of how to advise women considering IBR concerning the possible impact on adjuvant treatment and prognosis [START_REF] Taylor | Oncological aspects of breast reconstruction[END_REF]. Since then, two long-term follow-up studies have been published with a median follow-up time of 5.7 years. In both these studies recurrence rates did not differ significantly between the IBR group and patients undergoing mastectomy alone [START_REF] Rothman | Modern Epidemiology. 3 rd[END_REF][START_REF] Bezuhly | Immediate postmastectomy reconstruction is associated with improved breast cancerspecific survival: Evidence and new challenges from the surveillance, epidemiology, and end results database[END_REF]. These results have since then been corroborated by two review articles, including a metaanalysis of nine non-randomized trials [START_REF] Patani | Oncological and aesthetic considerations of skin-sparing mastectomy[END_REF][START_REF] Arpino | Infiltrating lobular carcinoma of the breast: tumor characteristics and clinical outcome[END_REF]. The strength of our study is the long follow-up time with a median of 11.5 years. The study was a matched cohort group of patients where both groups had similar rates of adjuvant treatment in terms of chemoand/or radiotherapy. We also made a separate comparison of recurrences among the patients who had received radiotherapy and found no significant differences regarding local and regional recurrences between the IBR and the control group as this has been a concern [START_REF] Lee | Postmastectomy radiation therapy and breast reconstruction: an analysis of complications and patient satisfaction[END_REF][START_REF] Motwani | The impact of immediate breast reconstruction on the technical delivery of postmastectomy radiotherapy[END_REF][START_REF] Lanitis | Comparison of skin-sparing mastectomy versus non-skin-sparing mastectomy for breast cancer: A meta-analysis of observational studies[END_REF]. Many authors favor autologous tissue reconstructions for primary or delayed procedures. However, these techniques are less well investigated regarding oncological safety and more complications are known to occur [START_REF] Lewis | Autologous tissue immediate breast reconstruction: desired but oncologically safe?[END_REF][START_REF] Taylor | Oncological aspects of breast reconstruction[END_REF][START_REF] Kronowitz | Radiation therapy and breast reconstruction: a critical review of the literature[END_REF]. Sentinel lymph node biopsy can be used to predict the need for postoperative radiotherapy and as a guideline in decision-making regarding reconstructive method and timing [START_REF] Pomahac | New trends in breast cancer management: is the era of immediate breast reconstruction changing?[END_REF].

According to other studies presenting positive data regarding oncological safety and IBR, our experience is that IBR with implants can be used in combination with irradiation from an oncological point of view, although the aesthetic results tend to be inferior compared to non irradiated reconstructions [START_REF] Christante | Using Complications Associated With Postmastectomy Radiation and Immediate Breast Reconstruction to Improve Surgical Decision Making[END_REF]41,42]. Prospective studies are therefore still desirable, especially regarding advantages and disadvantages of breast reconstructions, radiation and which method can be considered oncological safe and aesthetically favorable in the long run.

Conclusion

This long-term follow-up study (median 11.5 years) with a well-matched control group demonstrates that IBR with implants can be offered to and performed on patients with invasive breast cancer without any negative effect on oncological safety.

Fig. 1

 1 Fig.1 Recurrences in patients treated with radiotherapy. Immediate breast reconstruction (IBR) (n=119) and controls (n=115).

  Kaplan-Meier disease free survival curves. IBR=Immediate breast reconstructions. (IBR followed for 18 years and controls followed for 20 years) Kaplan-Meier breast cancer specific survival curves. IBR=Immediate breast reconstructions. (IBR followed for 18 years and controls followed for 20 years) McNemar chi-square test unless otherwise stated, 2 Person"s chi-square test for categorical variables; Mann-Whitney-Wilcoxon test for continuous variables Table III. Incidence, detection mode and time to detection of recurrence Total number of events Immediate breast reconstruction (IBR). 1 Memar chi-square unless otherwise stated, 2 Person"s chi-square test for categorical variables; Mann-Whitney-Wilcoxon test for continuous variables Table IV. Hazard ratio and confidence interval in total number of events

	Lobular Table II. Oncological treatment, time to treatment and complications 27.2 16.2
	6.1% Local recurrence 7.6% IBR Controls 0 2 4 0 2 4 Fig. 3 Table I. Patients" clinical and histopathological characteristics 10.1% 9.8% 0 10 20 30 40 % Regional recurrence IBR Controls 0.00 0.20 0.40 0.60 0.80 1.00 6 8 10 12 Years IBR Controls Fig. 2 0.00 0.20 0.40 0.60 0.80 1.00 6 8 10 12 Years IBR Controls IBR Controls n = 300 n = 300 p-value Distant metastases 30.3% 33.7% IBR Controls 14 16 18 20 14 16 18 20 Age (%) 21-40 21.7 20.3 41-60 71.3 72.3 61-70 7.0 7.3 Median, years 48 48 Range, years 23-70 28-69 Year of operation (%) IBR Controls 1990-1994 1988-1994 33.7 34.0 1995-2000 1995-2000 51.0 46.0 2001-2004 2001-2006 15.3 20.0 Tumor size (%) ≤ 20 63.7 61.0 21-50 33.0 35.0 > 50 3.3 4.0 Median (mm) 19 20 Range (mm) 0-100 0-90 Lymph nodes (%) 0 64.0 63.3 Other 6.5 3.9 Hormone receptor status (%) n = 274 n = 287 ER + 79.9 68.6 0.003 2 n = 251 n = 275 PR + 71.3 61.1 0.008 2 Immediate breast reconstruction (IBR), estrogen receptor (ER), progesterone receptor (PR). 1 Stuart-Maxwell test, IBR Controls n = 300 n = 300 p-value 1 Neoadjuvant chemotheraphy (%) 13.0 15.7 0.346 CMF 1.7 0.0 0.025 FEC 10.7 13.7 0.249 Taxane-based 0.0 1.0 0.083 Other 1.0 1.3 0.706 Response (%) n = 37 n = 44 No 13.5 15.9 1.000 2 Partial 78.4 77.3 Complete 8.1 6.8 Adjuvant chemotherapy (%) n = 292 n = 290 Adjuvant chemotherapy 45.2 47.6 0.431 CMF 18.8 15.5 0.325 FEC 21.9 23.8 0.558 Taxane-based 2.7 3.8 0.285 Other 1.4 2.1 0.480 Completed chemotherapy (%) n = 122 n = 119 Full dose 94.3 92.4 0.569 2 Time to chemotherapy (weeks) n = 112 n = 105 Median 5.0 5.1 0.376 2 Range 2-22 2-33 Hormonal treatment (%) n = 283 n = 273 Hormonal treatment 73.9 66.3 0.103 Tamoxifen 68.2 60.4 0.100 Aromatase inhibitors 4.6 5.1 0.842 Other 12.0 12.1 0.456 Completed hormonal treatment (%) n = 198 n = 171 88.9 85.4 0.313 2 Radiotherapy (%) n = 289 n = 284 Radiotherapy 34.3 32.8 0.825 Local 5.2 1.4 0.046 Loco regional 28.4 29.6 0.808 Localization unknown 0.6 1.8 Time to radiotherapy (weeks) n = 86 n = 70 Median 24.1 24.7 0.902 2 Range 6-48 5-42 Complications < 30 days (%) 7.3 6.3 0.622 Immediate breast reconstruction (IBR), cyclophosphamid, 5-floururacil, and methotrexate (CMF), 5-floururacil, IBR Controls p-value 1 IBR Controls p-value 1 Overall recurrence (%) 28.4 32.8 0.239 Local recurrence (%) 8.2 9.0 0.879 6.9 6.4 0.590 Time to detection (years) Median 1.3 2.2 0.763 2 1.3 1.6 0.673 2 Range 0.2-11.9 0.5-11.6 0.2-11.9 0.5-11.6 Regional recurrence (%) 8.2 9.7 0.555 6.5 6.4 1.000 Time to detection (year) Range 0.1-12.5 0.3-11.0 0.1-11.5 0.3-5.5 Distant metastases (%) 20.3 27.1 0.049 14.8 20.1 0.081 Time to detection (year) Median 2.7 3.3 0.372 2 2.3 3.3 0.288 2 Range Recurrence detected with (%) Image guided radiology Clinical examination 0.0-12.4 53.8 46.2 0.0-13.9 63.2 36.8 0.241 2 0.0-12.4 0.0-13.9 IBR Controls Hazard rate ratio * (95% CI) p-value Total number of events Overall recurrence (%) 28.4 32.8 1.2 (0.9-1.7) 0.301 Local recurrence (%) 8.2 9.0 1.0 (0.5-1.8) 1.000 Regional recurrence (%) 8.2 9.7 1.5 (0.8-2.8) 0.173 Distant metastases (%) 20.3 27.1 1.4 (1.0-2.1) 0.070 Death, all causes (%) 22.0 28.0 1.5 (1.0-2.1) 0.038 epirubicin and cyclophosphamid (FEC). 1 First event Median 4.8 1.6 0.008 2 2.6 1.5 0.014 2 Death, breast cancer (%) 17.0 23.0 1.6 (1.1-2.4) 0.026
	1-3	23.0	22.0
	≥4	13.0	14.7
	Morphology (%)
		n = 291	n = 283
	Ductal	66.3	79.9	0.003 1

* Controls versus immediate breast reconstruction (IBR)

McNemar chi-square test

Acknowledgements

The study was supported by grants from BRO (Swedish Breast Cancer Association) and from Olle Engkvist Foundation. The authors are grateful to Associate Professor Birgit Stark Department of Reconstructive and Plastic Surgery for valuable comments.