MD Patric Raiss

MD Stefan Kinkel

Ulrich Sauter

PhD Thomas Bruckner

MD Burkhard Lehner
email: burkhard.lehner@ok.uni-heidelberg.de

Replacement of the Proximal Humerus with MUTARS Tumor Endoprostheses

Keywords: arthroplasty, humerus, defect reconstruction, limb salvage, megaprosthesis, MUTARS

Background and Objectives:

The aim of this study was to analyze our results using a modular endoprosthetic replacement system (MUTARS) for bone tumors of the proximal humerus.

Methods

: Thirty-nine patients were treated with a MUTARS endoprosthesis of the proximal humerus. Mean follow-up duration was 38 months (3--138 months). Most operations were necessitated by metastasis (n=30); surgery for a primary tumor (n=9) was less frequent. The Enneking score was recorded and the active ranges of motion for shoulder flexion, abduction, and external rotation. Complete refixation of the rotator cuff was possible in 23 cases. Radiographs of the affected shoulders were obtained in two planes. Results: Patient survival was 77% at 2 years and 45% at 11.5 years after surgery.

The survival rate of the limb was 90% at 11.5 years. The mean Enneking score was 19 points (range 7--27 points). The mean shoulder flexion was 34° (range 0-90°), abduction 33° (range 0-90°), and external rotation 12° (range --10-50°). Patients with a complete rotator cuff repair showed a significant better range of motion compared to patients with a partial or no repair (p<0.015). No signs of implant loosening were observed on postoperative radiographs. Seven complications occurred, five of them leading to implant revision. At 11.5 years after surgery, the survival rate for the whole cohort without complications was 72%, without revision of the implant 70%.

Conclusions: Replacement of the proximal humerus with MUTARS endoprosthesis is a viable treatment option for bone tumors with satisfying results.

Introduction

Malignant bone tumors of the proximal humerus are common, and multiple treatment options have been reported in recent years [START_REF] Hage | Incidence, location, and diagnostic evaluation of metastatic bone disease[END_REF][START_REF] Kumar | Endoprosthetic replacement of the proximal humerus. Long-term results[END_REF][START_REF] Gebhardt | Osteoarticular allografts for reconstruction in the proximal part of the humerus after excision of a musculoskeletal tumor[END_REF][START_REF] Jensen | Proximal humeral reconstruction after excision of a primary sarcoma[END_REF][START_REF] Kocialkowski | Fate of frozen intercalary allograft at one year after implantation with adjuvant chemotherapy treatment. A case report[END_REF][START_REF] Mankin | Longterm results of allograft replacement in the management of bone tumors[END_REF][START_REF] Rodl | Reconstruction of the proximal humerus after wide resection of tumours[END_REF][START_REF] Ross | Endoprosthetic replacement of the proximal humerus[END_REF]. In most cases, surgery comprises an essential element of therapy. The clinical outcome, local recurrence, and survival rates after limb-preserving and other procedures seem to be comparable [START_REF] Malawer | Prosthetic survival and clinical results with use of large-segment replacements in the treatment of high-grade bone sarcomas[END_REF][START_REF] Simon | Limb-salvage treatment versus amputation for osteosarcoma of the distal end of the femur[END_REF][START_REF] Springfield | Surgical treatment for osteosarcoma[END_REF]. To provide a platform for elbow and hand function, reconstructive limb-preserving procedures have been proposed for the proximal humerus [START_REF] Cannon | Functional outcome following endoprosthetic reconstruction of the proximal humerus[END_REF]. Moreover, patient acceptance has been described as higher for limb-preserving treatments. Therefore, amputation of the shoulder girdle is avoided if possible.

In limb salvage procedures, large bone defects may result after resection of the proximal humerus. These defects can be reconstructed with osteoarticular allografts, allograft prosthesis composites, or endoprostheses [START_REF] Cannon | Functional outcome following endoprosthetic reconstruction of the proximal humerus[END_REF][START_REF] Black | Treatment of malignant tumors of the proximal humerus with allograft-prosthesis composite reconstruction[END_REF][START_REF] Wada | Reconstruction and limb salvage after resection for malignant bone tumour of the proximal humerus. A sling procedure using a free vascularised fibular graft[END_REF][15].

Many different endoprosthetic systems for defect reconstruction of the proximal humerus are currently in clinical use. Different implants from different manufactures with a cemented or uncemented fixation concept have been described for replacement [2;8;8;12;16-18]. Little information is available about the modular endoprosthetic system "MUTARS" for replacement of the proximal humerus [19;20]. This implant is available in combination with a Trevira tube for refixation of detached tendons. There is no information available on whether refixation of the detached tendons to a tube leads to a better functional outcome.

Therefore, the aim of the present study was to examine the clinical and oncological outcome using this endoprosthetic system while evaluating the implant survival rate.

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Materials and Methods

Patients

Between 1995 and 2008, 61 patients at our institution were treated by endoprosthetic replacement of the proximal humerus after tumor resection and included in a prospective database. In 43 cases a modular tumor endoprosthesis (MUTARS) was used. Four of these 43 patients were lost to follow-up, so 39 patients were included in this study. Inclusion criteria were: (1) tumor or metastasis of the proximal humerus and (2) treatment with a modular tumor endoprosthesis (MUTARS).

There were 20 women and 19 men, with an average age of 60 years (24--84 years) at the time of implantation. The mean duration of follow-up was 38 months (range 3--138 months). Eighteen patients died during the follow-up period, 7 of them within 3 months of surgery.

Most operations were performed because of metastasis (n=30); surgery for a primary tumor (n=9) was less frequent. The tumor entities were: renal cell cancer (n=9), breast cancer (n=7), lung cancer (n=5), plasmacytoma (n=4), chondrosarcoma (n=4), Ewing sarcoma (n=2), hepatocellular carcinoma (n=2), angiosarcoma (n=2), colon cancer (n=1), Non-Hodgkin lymphoma (n=1), prostate cancer (n=1) and leiomyosarcoma (n=1). The indication for surgery was a pathological fracture in 25 patients and an initial manifestation of a tumor at the proximal humerus in 14 patients.

In one case a previous osteosynthesis had failed.

Clinical and radiographical examination

Radiographs in two planes (antero-posterior and lateral) and MRI´s of the affected shoulder were obtained preoperatively. Postoperatively, all patients were evaluated using the Enneking score. This score was used to assess the function of the upper planes were made for all follow-up examinations (Fig. 1).

Operative technique and implants

An anterior deltopectoral approach was used in all cases. In all but one case, intraarticular resection of the tumor was possible. For the one extraarticular resection, the neck of the glenoid was transected. In all cases the tendons of the rotator cuffs were detached from the proximal humerus. Complete refixation of the cuff was possible in 23 cases. A partial reattachment of the cuff (supra-and infraspinatus tendons) was possible in 6 patients. In 10 patients reattachment of any tendon failed. Incomplete or failed reattachment of the rotator cuff was based due to the desired resection margins and therefore determined by the oncological approach.

In three patients part of the triceps brachii muscle and in three patients part of the deltoid muscle including the axillary nerve had to be resected. After preparation or resection of the soft tissues, the humerus was explored. The osteotomy was performed according to the intramedullary extension of the tumor planned on MRI´s and radiographs in two projections. The mean length of the bone segment resected

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was 13 cm (range 7-19 cm) measured from the apex of the humerus. Depending on the site and length of resection, the required MUTARS modules were selected. The fixation was achieved with an intramedullary stem, which was cemented in 33 cases and inserted cementless in 6 cases. In the cementless version the stem is inserted using a press-fit technique in order to achieve sufficient primary stability. The stem has a rough surface to allow ongrowth of bone. The stem is made of titanium alloy (TiAl6V4) in the cementless and of CoCrMo alloy in the cemented version. After implantation, a Trevira tube for refixation of the tendons was used in all patients. The rotator cuff was repaired if possible.

Rehabilitation protocol

Drains were removed on the second day after operation. To protect the reconstructed tendons the arms were placed in internal rotation in Gilchrist bandages for 6 weeks.

Postoperatively, the shoulders were mobilized passively by a physiotherapist for 6 weeks with 60° shoulder flexion and abduction and 0° external rotation. After 6 weeks patients were asked to support these movements actively with an unlimited range of motion.

Statistics and ethics

Statistical analysis was performed using SAS 9.1. Estimates of survival were calculated by means of the Kaplan--Meier method for the whole cohort of patients, for amputations, for implants, for complications, and for revisions. The Wilcoxon U-test was used to assess differences in the postoperative shoulder motion between patients with or without refixation of the rotator cuff. P<0.05 was considered significant.

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The study was reviewed and approved by the ethics committee of the local university (IRB number 278/99).

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9

Results

Clinical results

The functional score according to Enneking was 19 points (range 7 --27 points) at the last follow-up examination. Figure 2 shows the overall Enneking score and its subgroups for the whole study cohort.

The mean postoperative shoulder flexion of the whole cohort was 34° (range 0-90°), abduction 33° (range 0-90°), and external rotation 10° (range --10-50°).

Patients with a complete refixation of the rotator cuff had better shoulder motion than patients with partial repair of the cuff (Figure 3). These differences were significant (p<0.015).

All patients were able to reach their mouth with the ipsilateral hand.

Survival and complications

For the whole cohort the overall survival was 77% at 2 years and 45% at 11.5 years (Fig. 4). The survival rate of the limb without amputation was 90% at 11.5 years.

Twenty-seven of the monitored patients had metastases at the last follow-up examination. Local recurrences had been detected in 2 patients, one of these leading to an interscapulothoracic amputation. The overall 5-year survival of patients with and without metastases was 48% and 64%, respectively. There was no significant difference between the survival curves (log-rank-test: p=0.192).

The local recurrences developed after resection of metastases of the proximal humerus without pathological fractures. The overall 5-year survival of patients with and without pathological fractures of the proximal humerus was 42% and 66%, respectively. There was no significant difference between the survival curves (log- In total, 7 complications in 39 proximal humeral replacements were recorded. Out of these seven patients, five underwent revision surgery. The most common complication was dislocation of the humeral head (n=4). In three of these cases the rotator cuff was completely resected, and in one case the supra-and infraspinatus muscles were resected. Implant revision was necessary for two of the patients with dislocation. Retroversion of the humeral head was changed from 40° to 60° in both cases, without any re-dislocation. In two patients deep wound infection occurred, 4 and 5 years after surgery. In both cases the prosthesis was explanted. In one of theses cases re-implantation was possible 7 months after explantation following two further revisions. Implant loosening was observed in one patient with a cementless implant. After explantation of the prosthesis the defect was treated with an autologous fibula graft. In one case an intraoperative fracture of the humeral shaft was observed and treated with osteosynthesis during the same session. All complications with radiographic results and treatments are shown in table 1.

The survival rate for the whole cohort without any complication was 82% at 2 years and 72% at 11.5 years.

The survival rate without revision of the implant was 90% at 2 years and 70% at 11.5 years (Fig. 5).

Imaging results

Implant loosening was seen in only one patient, who underwent revision surgery (see above). No further obvious radiological signs of implant loosening were observed.

Cranial migration of the humeral head was observed in 14 cases. In 6 of these patients a partial (n=3) or complete (n=3) resection of the rotator cuff was performed. small bone defects, the clavicula pro humero procedure has been employed with an acceptable outcome but also with a high complication rate [START_REF] Rodl | Reconstruction of the proximal humerus after wide resection of tumours[END_REF]. Biologic reconstruction procedures such as osteoarticular allografts, prosthesis allograft composites, and fibula autografts, which can be implanted vascularized or non-vascularized, have been described as viable treatment options [14;22-24]. As a non-biologic reconstruction, endoprosthetic replacement of the proximal humerus with megaimplants has been described in several studies [2;8;9;12;16-20].

Endoprosthetic replacement of the proximal humerus

Cannon et al. recently published a series of 83 patients treated with endoprosthetic replacement for defects of the proximal humerus [START_REF] Cannon | Functional outcome following endoprosthetic reconstruction of the proximal humerus[END_REF]. In all of these cases an intraarticular resection of the tumor was performed, with sparing of the deltoid muscle and axillary nerve. Mean shoulder flexion was 42° and abduction 41°, with a mean Musculoskeletal Tumor Society Score (MSTS) of 63%. Loosening of the implant was not observed, and the overall complication rate was low. One hundred patients with endoprosthetic replacement of the proximal humerus were followed up by Kumar et al. [START_REF] Kumar | Endoprosthetic replacement of the proximal humerus. Long-term results[END_REF]. The authors reported mean shoulder flexion of 55° and abduction of 45° in combination with a mean MSTS of 79%. The overall survival at 10 years was 42%, and the survival of the implant without further surgery for

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13 mechanical failure was 86.5% at 20 years. In 9 patients an implant-related revision was necessary, and in 8 patients a forequarter amputation was performed.

Complications of endoprosthetic replacement of the proximal humerus

Divergent rates of complications and revision have been reported in further studies [2;8;12;16;17;19]. Revision rates of up to 67% [17] and complication rates of up to 72% [START_REF] Ross | Endoprosthetic replacement of the proximal humerus[END_REF] have been described. The complication rate in our study seems to be acceptable compared with the above-mentioned trials. In only one case aseptic loosening of an uncemented implant was observed. Therefore, we do not further use uncemented humeral stems. We did not observe any signs of lossening in cases with cemented stems. Hence, we routinely use this fixation technique for MUTARS shoulders in our institution.

Results with the MUTARS system

The use of MUTARS tumor endoprostheses in the lower limb is well documented

The role of the rotator cuff and discussion of results

The clinical results of the current investigation seem to be comparable with those of the above-mentioned studies. The mean Enneking score of 19 points represents an overall good result for this procedure. The mean degree of pain after surgery was between no pain and non-disabling pain (Figure 2). Emotional acceptance after surgery was satisfying. The function of the affected limb was acceptable, although mean shoulder elevation was restricted to 33°. The shoulder motion results reported by Cannon et al. [START_REF] Cannon | Functional outcome following endoprosthetic reconstruction of the proximal humerus[END_REF] and Kumar et al. [START_REF] Kumar | Endoprosthetic replacement of the proximal humerus. Long-term results[END_REF] are superior to our findings. A possible reason for these divergent results is the degree of refixation of the rotator cuff and the degree of resection of the deltoid muscle and axillary nerve. We demonstrated that the degree of refixation of the rotator cuff to a Trevira tube has an influence on postoperative shoulder function (Figure 3). In other endoprosthetic systems, the sutures of the armed rotator cuffs can be fixated by special eyelets. We believe that a Trevira or Dacron tube [16] offers more advantages for refixation of tendons compared to eyelets because fibrous ingrowths of the tissue into the tube is possible.

Moreover, the area for refixation of the tissue is much greater by using a tube. Using an eyelet, only a punctual refixation without ingrowths is possible.

The shoulder motion in patients with preserved rotator cuffs in the current study was comparable with the shoulder motion reported in the above-mentioned trials.

Possibly, the use of a reversed tumor-implant for the proximal humerus could lead to better functional results in cases when the deltoid muscle and axillary nerve can be preserved.

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15 Failed refixation of the rotator cuff and/or the deltoid muscle often leads to cranial migration of the humeral head. Cannon et al. found cranial migration of the prosthesis in 22 out of 76 patients with adequate postoperative radiographs. In our study this was encountered in 14 cases. However, refixation of the rotator cuff had been performed in only 8 of the 14 patients, and it seems that there is no strict coherence between cranial migration and refixation of the rotator cuff. Moreover, caudal migration of the prosthesis was found in three cases after resection of parts of the deltoid muscle and axillary nerve.

Hand function after endoprosthetic replacement of the proximal humerus

It has been reported that replacement of the proximal humerus provides an excellent platform for function of the elbow and the hand [START_REF] Cannon | Functional outcome following endoprosthetic reconstruction of the proximal humerus[END_REF]. In this study all the patients had good elbow and hand function with very satisfying manual dexterity, and they were able to reach their mouth with the ipsilateral hand. In the current study, most operations were performed due to metastases. In this light the patient survival rate of 45% at 11.5 years appears acceptable, as does the local recurrence rate of 5%.

Implant survival

Survival of the implant was lower in our study than in other trials. Mayilvahanan et al.

[18] and Kumar et al. [START_REF] Kumar | Endoprosthetic replacement of the proximal humerus. Long-term results[END_REF] described survival rates of 83% and 87% after 10 years, respectively. Our finding of implant survival of 70% after 11.5 years may be explained by the different indications for surgery: a relatively high proportion of patients in the present study were treated because of a metastasis or pathological fracture of the proximal humerus.

  Enneking score is divided in 6 subgroups: pain, function, emotional acceptance, hand position, manual dexterity and lifting ability [21]. For each subgroup a minimum of 0 points and a maximum of 5 points can be reached. An overall result of 30 points represents a normal function without pain of the upper extremity, whereas 0 points represents no function and disabling pain. Additionally, the active range of motion in degrees was recorded for shoulder flexion, abduction and external rotation with the hanging arm in neutral position and elbow flexion of 90°. All patients were routinely evaluated according to the treatment plan for each tumor entity at defined intervals. Neoadjuvant or adjuvant therapy was performed according to the diagnosis and the corresponding protocol. Radiographs in two

  M A N U S C R I P T A C C E P T E D ARTICLE IN PRESS10 rank-test p=0.379). There was no further evidence of neoplastic disease in nine patients.

12 Discussion

 12 the humeral head was detected in 3 cases after resection of the deltoid muscle and axillary nerve.In no case was secondary wear of the glenoid cartilage and bone found on the most recent follow-up radiographs in two planes.Treatment options for defect reconstructionBone tumors of the proximal part of the humerus often necessitate combined procedures such as chemotherapy, radiotherapy, and surgery. If surgery is required, large bone defects of the proximal humerus can result. Different interventions have been proposed for bridging the resulting defects. In children and in patients with

[

  19;25-27]. To our knowledge, however, only a few studies have been published on the use of this modular system for the treatment of large bone defects of the proximal humerus [19;20]. Gosheger et al. reported endoprosthetic replacement in 250 patients with sarcoma [19]. Out of this cohort, 39 patients were treated with replacement of the proximal humerus. The 5-year survival of the endoprosthesis was 93.6%. The mean Enneking score was 21 points, and the authors stated that the MUTARS systems have some advantages over other endoprosthetic systems. They concluded that the use of a Trevira tube makes muscle and tendon refixation easier, resulting in a reduced dislocation rate and better functional results in the lower extremity. They did not describe if refixation of the rotator cuff using the Trevira tube leads to a better functional outcome in cases of replacement of the proximal M A N U S C R I P T A C C E P T E D ARTICLE IN PRESS 14 humerus. Overall, a high success rate and good functional results were stated. This system was also introduced by Gosheger et al. for a combined procedure with replacement of the proximal humerus and a trapezius--latissimus dorsi transfer [20].

ConclusionFig. 1 :

 1 Fig. 1: Radiograph of a 35-year-old man 2 years after resection of a chondrosarcoma

Fig. 2 :

 2 Fig. 2: The graph is showing the results (points) of the Enneking score and its

Fig. 3 :

 3 Fig. 3: The graph is showing the normal Range of Motion (ROM) of the shoulder for

Fig. 4 :

 4 Fig. 4: Kaplan--Meier curve showing overall patient survival.

Fig. 5 :

 5 Fig. 5: Kaplan--Meier curve showing implant survival without further surgery.

Table 1 :

 1 Early and late complications with imaging results and treatments

	Time	Complications	Imaging results	Treatment
	Early (< 3 months after surgery)	Dislocation (n=4) Intraoperative shaft fracture (n=1)	Conservative (n=2) Re-operation with changing the retroversion Fracture of the humeral shaft Osteosynthesis Anterior dislocation (n=3) Posterior dislocation (n=1)
	Late (>3 months	Infection (n=2)	Correct positioning of the implants, no signs for loosening	Removal of the implant (n=2), re-implantation (n=1)
	after surgery	Aseptic loosening (n=1)	Loosened cementless stem	Removal of the implant, autologous fibula graft


Mohammed R S Keshtgar
email: mo.keshtgar@royalfree.nhs.uk

N Aresti

F Macneil

Establishing axillary Sentinel Lymph Node Biopsy (SLNB) for early breast cancer in the United Kingdom: A survey of the national training program

operative diagnosis, of which the majority (6%) use touch imprint cytology. 31% included their results in their most recent surgical appraisal.

Conclusions

The majority of breast surgeons in the UK are convinced by the evidence for SLNB, and most use SLNB in their practice for staging. Reasons for not conducting SLNB are logistical rather than lack of belief in the procedure.

The majority of respondents completed their training within the anticipated time line. The majority of centres do not perform intra-operative assessment.

Introduction

The UK National training programme (NEWSTART) for SLNB in breast cancer was established in 2004, aimed at providing structured, standardised training with a focus on multidisciplinary team (MDT) delivery.

Methodology

A questionnaire was devised and after approval by the Association of Breast Surgeons (ABS) executive committee they were sent to all full members of the ABS.

Results

Most (97%) of breast surgeons are convinced by the evidence for SLNB as standard of care for early breast cancer. 64% use SLNB to stage clinically node negative patients, of whom 23% use it as a standalone procedure.

38% of surgeons were dissatisfied with the time it takes to complete the in house training, and 87% with the time it takes to complete the validation phase. Logistical and funding issues were the main problems cited.

The majority of surgeons (86%) use the recommended combined technique, with 47% continuing to use the dual localisation method. 14% use either blue dye or isotope alone, without scintiscan. Only 10% offer intra Introduction Sentinel node biopsy (SLNB) is now the recognised standard of care for axillary staging in patients with early breast cancer. There is level 1 evidence to support its staging accuracy as well as lower morbidity when compared to axillary lymph node dissection (ALND) [START_REF] Veronesi | A randomized comparison of sentinel-node biopsy with routine axillary dissection in breast cancer[END_REF][START_REF] Purushotham | Morbidity after sentinel lymph node biopsy in primary breast cancer: results from a randomized controlled trial[END_REF][START_REF] Mansel | Randomized multicenter trial of sentinel node biopsy versus standard axillary treatment in operable breast cancer: the ALMANAC Trial[END_REF][START_REF] Bianco | Morbidity comparison of sentinel lymph node biopsy versus conventional axillary lymph node dissection for breast cancer patients: results of the sentinella-GIVOM Italian randomised clinical trial[END_REF][START_REF] Gill | The SNAC Trial Group of the Royal Australasian College of Surgeons (RACS) and NHMRC Clinical Trials Centre. Sentinel-Lymph-Node-Based Management or Routine Axillary Clearance? One-Year Outcomes of Sentinel Node Biopsy Versus Axillary Clearance (SNAC): A Randomized Controlled Surgical Trial[END_REF] .

The UK SLNB training program (NEW START), a collaborative project between the Department of Health, the University of Cardiff and the Royal College of Surgeons of England, was launched in October 2004. This unique national educational initiative aimed at providing structured, standardised training with a focus on multidisciplinary team (MDT) delivery. Patient safety was the primary aim and central audit and validation was integral to the program.

The training program was offered regionally and at a modest cost to breast teams. It was promoted through professional associations, the Royal College of Surgeons and government bodies. The target of the campaign was consultant and associate specialist surgeons, breast teams, hospital senior managers and cancer networks.

The plan was to ensure a rapid, national coverage and uptake. The aim of the project was to train 80% of UK breast teams over a 4 year period. The targeted duration of completion of the program was 24 months per team.

The steering group anticipated the main barriers to delivering a uniform national SLNB service would be logistical and financial. Prior to the launch of the SLNB program, a proportion of UK surgeons offered axillary sampling (four node sampling) as an alternative to full dissection for patients with early disease. Sampling represented a pragmatic solution to axillary management for the rising numbers of early breast cancers as it offered staging with lower morbidity, comparable early recurrence and survival rates.

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of the SLNB procedure, most surgeons performing axillary sampling saw little need to change their practice, whereas surgeons performing axillary clearance were more enthusiastic advocates of the targeted approach to axillary staging. This survey was initiated with the aim of obtaining a snap shot of the training program and identifying if there are any common barriers that might limit participation and progression in SLNB training.

UK SLNB training

The NEW START program consists of three phases:

(1) Theory day: Covers the practical aspects of performing the SLNB procedure, incorporating skills workshops. This is delivered by 6 regional centres.

(2) Proctored training: Supervision of the first 5 SLNB procedures in the participant's own hospital and operating room.

(3) Validation: The team performs 25 further cases (total of 30) with prospective centralised data collection.

There are two training routes:

(A) Model A -for SLNB naïve breast units and surgeons. A minimum of 30 SLNB cases immediately followed by the units/surgeons standard axillary staging procedure (sampling or clearance): the first 5 cases are supervised by an NEW START accredited trainer in the trainee's own hospital. Out of thirty cases a minimum of 10 must be node positive to allow false negative ascertainment. • Localisation rate 90% (27/30) -data collected for models A and B.

• False negative rate for node positive cases 10% (1/10)-data collected for model A only.

METHODS

A questionnaire was created and received approval from the executive committee of the Association of breast Surgery (ABS) at the British Association of Surgical Oncology (BASO). Electronic and paper copies were sent to the ABS's fully registered members and their responses kept anonymous.

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The questionnaire was divided into three sections: Section 1 -"The Concept" -In this section, surgeons conveyed their preferences for axillary staging clinically node-negative breast cancer patients, and addressed their views on the validity and use of SLNB.

RESULTS

A total of 206 questionnaires were returned, which equates to a total response rate of 52%. The results were blinded from the authors and analysed.

Section 1 -The Concept

The first question addressed surgeons' views on the validity of SLNB. 88% agreed that SLNB is the best surgical option for axillary staging of clinically node negative breast cancer patients. Of the remaining 12%, 3% do not foresee it becoming standard procedure at their centre (table 1). Several surgeons expressed concerns regarding detection of micro-metastatic disease within the SLN and its management, and also variation between surgical approaches leading to the excision of the wrong node; hence false negative SLNB.

The second question explored preferences for the staging of node negative breast cancer patients. A total of 64% of responding surgeons indicated that SLNB is performed as standard practise at their place of work. SLNB is used as a standalone procedure by 23% of surgeons, whilst 41% use it in combination with other treatments, e.g. axillary dissection/sampling. Axillary sampling is performed by 36% of surgeons, axillary radiotherapy performed by 10%, and axillary lymph node dissection performed by 76% (figure 1).

Section 2 -Training for SLNB

A total of 86% of surgeons have completed a NEW START theory day, the majority having done so in 2006.

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38% of respondents were dissatisfied with the time it took to complete the 'in house' training. Logistical issues were the main reasons cited, for example a lack of on-site nuclear medical facilities and equipment, including gamma detection probes. Difficulties with arranging a regulatory ARSAC licence (Administration of Radioactive Substances Advisory Committee), difficulties within teams and funding issues were other points highlighted by a significant number of respondents (table 2).

Responding surgeons', who were within the validation phase, had performed a median of 12 procedures. Only 13% were satisfied with the time it took to complete this phase. The main reasons for dissatisfaction included the long delay in setting up the program locally, lack of suitable patients, consent issues, and difficulties obtaining equipment (table 2).

The validation phase has been completed by 41% of responding surgeons, with the highest completion rate being in 2007 (35%). Since validation, 17% have performed more than 100 cases, 1% have performed 51-100 and 13% have performed less than 50 SLNB procedures.

Section 3 -SLNB post training

The third section of the questionnaire concentrated on the technical aspects of SLNB following the completion of training. The majority (47%) continue to use dual localisation technique using blue dye and isotope with scintiscan imaging. 39% use isotope and blue dye without imaging, 11% use blue dye alone and 3% use isotope alone (figure 3).

Only 10% offer intra-operative SLN assessment to their patients. Of these, 6% use touch imprint cytology, 3% use frozen sections and 1% use molecular diagnosis with PCR (polymerase chain reaction) technology. The most commonly stated reasons for not performing intra-operative SLNB assessment were logistical constraints (23%), for example lack of an onsite pathologist, a lack of funding and a lack of time. Concerns were also raised about the accuracy of intra-operative diagnosis (table 2). However, several centres reported to be in the process of setting up the necessary infrastructure.

Following completion of the validation phase, surgeons are encouraged by the ABS and NEW START to continue to collect the audit data on their stand alone SLNB procedures. When asked whether they do, 33% of surgeons declined to answer and a further 12% stated that they did not perform ongoing audit. Of the 55% who do continue to collect audit data, 31% included their results in their most recent annual appraisal.

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DISCUSSION

In the UK alone, widespread use of SLNB could potentially save over 20,000 women unnecessary axillary dissection and its associated morbidity per year. However by 2003, for a variety of reasons outlined earlier, UK adoption of SLNB was limited to a few specialist centres and training opportunities were restricted.

The NEW START program introduced and set a standard of care in a systematic method. It was unique in the sense that training was validated across a comprehensive national health service, translating specialist standards to every unit in the country, over a relatively short time frame. The NEWSTART progress reports demonstrated excellent early engagement by the surgical community, but progression from theory day training to the validation phase and then to completion was slower than anticipated by the participants. [START_REF] Macneill | NEW START: The UK SLNB Training Program -A Progress Report[END_REF] Anecdotal feedback suggested that despite central government support and communication with individual hospital chief executives, local managerial commitment and lack of resources was an issue even for enthusiastic early adopters. This survey was conducted 4 years after the introduction of the UK national training program with the aim of assessing how the program was being perceived and adopted, as well as identifying any problems that might hinder uptake and national roll out. It was anonymous and conducted through postal and electronic questionnaires. The 52% response rate was lower than expected, but reflected similar return rates for previous national ABS surveys. It has been noted that those surgeons who have taken part in the NEW START program and are practising SLNB are more likely to have returned the questionnaire.

The data suggests that the majority (97%) of breast surgeons are convinced by the evidence for SLNB, and so most now use SLNB in their practice for staging clinically node negative breast cancer. Other international studies have suggested similar rates of conviction. [START_REF] Quan | National Adoption of Sentinel Node Biopsy for Breast Cancer: Lessons Learned from the Canadian Experience[END_REF] The 3% who do not envisage SLNB becoming standard practise are concerned regarding micro-metastatic deposits and how to manage them, and high false negative rates.

There are no clear guidelines in the literature with regards to micro metastatic disease and its management.

The indications are that the presence of micro metastases represents an incremental detriment to prognosis with increased risk of non-SLN involvement. There is lack of level 1 evidence concerning its management and current recommendations are that each case requires individual discussion regarding tumour and patient related factors, in the context of the multidisciplinary team. [START_REF] Patani | The clinical significance of sentinel lymph node micrometastasis in breast cancer[END_REF] There is evidence to support that with adequate training, the false negative rate can be kept at approximately 5%, which is no higher than the false negative rate in routine level 1 and 2 axillary lymph node dissection. [START_REF] Miltenburg | Meta-analysis of sentinel lymph node biopsy in breast cancer[END_REF] False negative rates can be influenced by tumour and patient demographics: Increasing body mass index (BMI), tumour location other than the upper outer quadrant and non-visualisation of hot nodes on the pre-

M A N U S C R I P T A C C E P T E D ARTICLE IN PRESS

operative lymphoscintiscan all decrease SLNB success. Complete involvement of the SLN with metastatic deposits is another recognised pitfall for false negative SLNB. [START_REF] Keshtgar | Clinical Role of Sentinel Lymph Node Biopsy in Breast Cancer[END_REF] The accuracy of SLNB can be improved using the combined blue dye and isoptope technique. [START_REF] Goyal | Factors affecting failed localisation and false-negative rates of sentinel node biopsy in breast cancer --results of the ALMANAC validation phase[END_REF] This was used by 86% of the respondents, which indicates that the majority of centres followed the recommended guidelines. This is much higher then the NHS breast screening programme (NHSBSP) audit of 2007-08 figures, which report 58% use the combined technique. However, 32% of those audited in the NHSBSP did not indicate the type of SLNB technique used, meaning the figure could be as high as 90%. [START_REF]West Midlands Cancer Intelligence Unit -NHS breast screening programme and Association of Breast Surgery at BASO -An audit of screen detected breast cancers for the year of screening April 2007 to[END_REF] Due to lack of on-site nuclear medical facilities several centres could not offer the SLNB imaging in their practice. In a national survey conducted by Quan et al on the national adaptation of SLNB in Canada, lack of access to nuclear medicine facilities and difficulty in obtaining gamma detection probe were similarly amongst the commonest reasons for delay in adaptation of the technique. [START_REF] Quan | National Adoption of Sentinel Node Biopsy for Breast Cancer: Lessons Learned from the Canadian Experience[END_REF] According to this survey, 64% of surgeons offer SLNB to their patients. There is clear evidence in literature of the increasing use of this procedure. A survey in 2001/02 demonstrated only 17% of surgeons used SLNB routinely in the UK, in combination with other procedures. [START_REF] Benson | Management of the axilla in women with breast cancer[END_REF] A 2006 study showed 52% of surgeons used the technique, suggesting a 12% increase in the use of SLNB over two years, which demonstrates the impact of the NEWSTART national training program. These figures also correlate with international figures, for example a Canadian study on the uptake of SLNB suggested a rate of use of 62% in Canada. [START_REF] Quan | National Adoption of Sentinel Node Biopsy for Breast Cancer: Lessons Learned from the Canadian Experience[END_REF]15 Intra-operative diagnosis (IOD) of the SLN is currently only performed by a small number of respondents (10%), despite increasing evidence suggesting its efficacy. 16 This probably reflects the lack of UK experience in SLNB as intra-operative assessment represents a natural progression in the maturation of SLN biopsy world-wide.

IOD requires considerable pathological expertise and time -for centres struggling to introduce basic SLNB, this may be one step too far. Reasons described by respondents for not performing IOD included an absence of onsite pathologists and lack of evidence in its cost effectiveness.

Those who have completed the in house proctoring phase are generally satisfied with the time it takes to complete the phase; however the majority of validated surgeons are dissatisfied with the time it takes to become fully validated. The reported delay in completion of the program is mainly due to logistical issues, with problems such as lack of facilities, equipment and funding being encountered. Lack of on-site nuclear medical facilities and equipment, including gamma detection probes were amongst common reasons expressed by the respondents.

These issues cannot be resolved or altered by NEW START. Instead, the Department of Health and the National Health Service (NHS) Trusts need to look closely at the logistics and funding of breast units to enable surgeons to train and carry out the SLNB procedure as recommended. This will ensure that this powerful technique is

M A N U S C R I P T A C C E P T E D ARTICLE IN PRESS

universally available to all breast cancer patients. Furthermore, the expansion and implementation of the NEW START program may help guide future postgraduate surgical education programs.

CONCLUSION

The NEW START program introduced and set a standard of care in a systematic method. It was unique in the sense that training was validated across a comprehensive national health service, translating specialist standards to every unit in the country, over a relatively short time frame. The majority of breast surgeons in the UK are convinced by the evidence for SLNB, and so most now use SLNB in their practice for staging clinically node negative breast cancer. Reasons for not conducting SLNB or IOD are logistical rather than lack of belief in the procedure. Barriers to uptake and progress were as expected -logistical and financial rather than attitudinal. The majority of respondents who completed their training did so within the anticipated time line.

The majority of centres do not perform intra-operative assessment of the SLN due to lack of resources which means node positive patients have to undergo a second operation.

(

  B) Model B -for SLNB naïve surgeons in breast units where SLNB is the standard of care. 30 cases are performed under direct supervision of a NEW START accredited trainer who provides written assessment. This model was developed to allow experienced SLNB centres to offer one standard of care during surgeons SLNB training. It represents the more traditional surgical training model but with the addition of built in quality assurance. Surgeons do not pass or fail the NEW START program. They continue in validation until the set standards are achieved. The validation standards set by the steering committee, based on published data, were:

Section 2 -

 2 "Training for SLNB" -This section assessed surgeons' progress through the NEW START program, their current training stage, and any problems encountered with the training system. Section 3 -"SLNB post training" -Concentrated on the various technical aspects of SLNB post training and addressed ongoing audits.

Figure 2

 2 Figure 2 illustrates the year and distribution of centres the surgeons attended. Following the theory day, 29% entered the apprentice model of training (model B). Of the remaining surgeons, 49% have completed the 'in house training' of 5 cases and 41% have completed the validation phase of 30 patients. The remaining 8% of surgeons were either still undergoing in house training, or did not clearly indicate their stage of training. Of the validated surgeons, 96% have achieved the minimum standard, 93% are offering SLNB as a standalone procedure and 48% have applied to the Royal College of Surgeons of England (RCS) to become trainers.

  L, Sosa I, Dionello R, Subramanian A, Devalia H, Mokbel K. Current management of the axilla in patients with clinically node-negative breast cancer: a nationwide survey of United Kingdom breast surgeons. Int Semin Surg Oncol. 2007; 4: 4 16. Gawlick U, Mone MC, Hansen HJ, Connors RC, Nelson EW. Selective use of intraoperative sentinel lymph node pathological evaluation in breast cancer. American Journal of Surgery (2008) 196: 851-856 17. MacNeill F. NEW START: The UK SLNB training programme -a progress report. Ann R Coll Surg Engl (Suppl) 2007; 89:60-61 18. Clarke D, Newcombe RG, Mansel RE. The learning curve in sentinel node biopsy: the ALMANAC experience. Ann Surg Oncol 2004;11(Suppl 3):211S-215S. 19. Tafra L. The learning curve and sentinel node biopsy. Am J Surg 2001;182(4):347-50 20. Keshtgar M, Chicken D, Waddington W, Raven W, Ell P. A training simulator for sentinel node biopsy in breast cancer: a new standard. EJSO 2005; 31:134-140 M A N U S C R I P T A C C E P T E D ARTICLE IN PRESS

Figure 1 :

 1 Figures and Tables

Figure 2 :

 2 Figure 2: Distribution of centres and dates where theory day was completed

  Of Surgeons Using Various SLNB Methods Problems with the in house training and validation phases Proportion of surgeons using intra-operative diagnosis

	In house training	Lack of nuclear medical facilities	34%
		Lack of equipment		31%
		Difficulties with ARSAC licence	19%
		Difficulties within team		17%
		Funding issues		15%
	Validation phase	Time to set up program		40%
		Lack of suitable patients		38%
		Difficulties obtaining equipment	30%
		Not able to achieve minimum standard	10%
		Funding			5%
	TnQTable1Table 1: Proportion of surgeons using	Yes 10%	Touch imprint cytology	6%
	intra operative diagnosis		Frozen section	3%
				Molecular diagnosis	1
			No 90%	Logistical issues	23%
				Funding	19%
				Too much time	18%
				Lack of accuracy	15%
	TnQTable2Table 2:

Figure3:


K Miragaia

M Hyde

H Aires-De-Sousa

Lencastre

T Conceição

A Tavares

M Miragaia

K Hyde

M Aires-De-Sousa

H De Lencastre

: A Tavares

: H De Lencastre

Prevalence and clonality of methicillin-resistant Staphylococcus aureus (MRSA) in the Atlantic Azores islands: predominance of SCCmec types IV, V and VI

Prevalence and clonality of methicillin-resistant (MRSA)

in the Atlantic Azores islands: predominance of SCC types IV, V and VI

Introduction

Methicillin-resistant Staphylococcus aureus (MRSA) is a major pathogen associated with both nosocomial-and community-acquired infections (hospital-acquired [HA] MRSA and CA-MRSA, respectively) [START_REF]European Antimicrobial Resistance Surveillance System[END_REF]. MRSA are believed to have emerged by the acquisition of the staphylococcal cassette chromosome mec (SCCmec), the mobile genetic element carrying the determinant of methicillin resistance (mecA) into methicillin-susceptible S. aureus (MSSA). Eight different SCCmec types (I to VIII) have been described so far in S. aureus, differing in their structure and size. Epidemiological studies using molecular typing methods showed that the massive geographical spread of MRSA in hospitals results from the dissemination of a few highly epidemic clones. These major epidemic clones resulted from a limited number of independent acquisitions of SCCmec into well-defined genetic backgrounds: (i) New York/Japan clone, sequence type (ST) 5, SCCmec II; (ii) Pediatric clone, ST5-IV/VI; (iii) Brazilian clone, ST239-III/IIIA; (iv) Iberian clone, ST247-IA; (v) EMRSA-15, ST22-IV; (vi) EMRSA-16, ST36-II; and (vii) Berlin clone, ST45-IV [START_REF] Oliveira | Secrets of success of a human pathogen: molecular evolution of pandemic clones of meticillin-resistant Staphylococcus aureus[END_REF][START_REF] Enright | The evolution of a resistant pathogen-the case of MRSA[END_REF][START_REF] Nübel | Frequent emergence and limited geographic dispersal of methicillin-resistant Staphylococcus aureus[END_REF].

The MRSA prevalence in European hospitals varies from less than 3.0% in the Nordic Countries and The Netherlands to over 50% in southern European countries and the United Kingdom. Despite the significant decrease in MRSA incidence in a few countries (France, Slovenia, Belgium, Cyprus and Turkey) due to improved infection control, the incidence has increased in other countries, including Portugal, where the MRSA proportion is, nowadays, over 50%, being one of the highest in Europe [START_REF]European Antimicrobial Resistance Surveillance System[END_REF][START_REF] Aires-De-Sousa | Changing patterns in frequency of recovery of five methicillin-resistant Staphylococcus aureus clones in Portuguese hospitals: surveillance over a 16-year period[END_REF].

Surveillance studies have been conducted in Portugal since the early 1990s, showing temporal waves of MRSA clonal prevalence. In the most recent survey conducted in 2006, EMRSA-15 (ST22-IVh) was found to be the most prevalent clone in Portuguese hospitals, followed by the New York/Japan clone (ST5-II) [START_REF] Aires-De-Sousa | Changing patterns in frequency of recovery of five methicillin-resistant Staphylococcus aureus clones in Portuguese hospitals: surveillance over a 16-year period[END_REF][START_REF] Amorim | Changes in the clonal nature and antibiotic resistance profiles of methicillin-resistant Staphylococcus aureus isolates associated with spread of the EMRSA-15 clone in a tertiary care Portuguese hospital[END_REF].

Information about the epidemiology of CA-MRSA is still scarce and non-existent in Portugal. Unlike HA-MRSA, CA-MRSA are usually only resistant to beta-lactams and one or two additional antimicrobial classes, and carry mainly SCCmec types IV and V, the smallest in size, which are believed to be more mobile. CA-MRSA frequently harbour Panton-Valentine leukocidin (PVL), but the toxin has also been detected in the hospital environment [START_REF] Tristan | Virulence determinants in community and hospital meticillin-resistant Staphylococcus aureus[END_REF][START_REF] Grundmann | Geographic distribution of Staphylococcus aureus causing invasive infections in Europe: a molecular-epidemiological analysis[END_REF]. Similarly to HA-MRSA, CA-MRSA epidemic clones have been described [START_REF] Chambers | Waves of resistance: Staphylococcus aureus in the antibiotic era[END_REF]. However, a much larger genetic diversity in the genetic backgrounds of CA-MRSA has been observed, suggesting a high number of SCCmec acquisitions in the community. Recent data indicates that MRSA epidemiology is suffering a new change and that the boundaries between the hospital and the community are blurring. As a result, clones traditionally linked to the hospital, like the EMRSA-15 clone, were found to be aetiologic agents of infection in the community [START_REF] O'brien | Diversity among community isolates of methicillinresistant Staphylococcus aureus in Australia[END_REF] and clones specific to the community were observed as causing infections in hospitals, as is the case of the USA300 strain [START_REF] Saiman | Hospital transmission of community-acquired methicillin-resistant Staphylococcus aureus among postpartum women[END_REF][START_REF] Seybold | Emergence of community-associated methicillin-resistant Staphylococcus aureus USA300 genotype as a major cause of health care-associated blood stream infections[END_REF]. A common trait to these clones that can survive in both environments appears to be the presence of SCCmec type IV that is, nowadays, prevalent among the current overall MRSA population [START_REF] Aires-De-Sousa | Changing patterns in frequency of recovery of five methicillin-resistant Staphylococcus aureus clones in Portuguese hospitals: surveillance over a 16-year period[END_REF][START_REF] Amorim | Changes in the clonal nature and antibiotic resistance profiles of methicillin-resistant Staphylococcus aureus isolates associated with spread of the EMRSA-15 clone in a tertiary care Portuguese hospital[END_REF][START_REF]International Working Group on the Classification of Staphylococcal Cassette Chromosome Elements (IWG-SCC) (2009) Classification of staphylococcal cassette chromosome mec (SCCmec): guidelines for reporting novel SCCmec elements[END_REF][START_REF] Alcoceba | Molecular epidemiology of methicillin-resistant Staphylococcus aureus in Majorcan hospitals: high prevalence of the epidemic clone EMRSA-15[END_REF][START_REF] Vindel | Methicillin-resistant Staphylococcus aureus in Spain: molecular epidemiology and utility of different typing methods[END_REF].

Whereas the epidemiology of nosocomial MRSA has been very well characterised in the Portuguese continental territory, there is no information concerning the prevalence and clonality of MRSA in the Portuguese islands, namely in the Azores archipelago.

The aim of the present study was to identify the MRSA clonal types currently circulating in the Azores archipelago and compare them with the major MRSA clones described in continental Portugal, Europe and the United States.

Materials and methods

Hospital setting

Hospital do Divino Espírito Santo (HDES) is a 390-bed central hospital located in Ponta Delgada, São Miguel island, that services an outpatient population of 138,000 inhabitants, from the two most populous Azorean islands, S. Miguel and Santa Maria. The hospital, the largest in the archipelago, includes medicine and surgery as principal wards, and a large outpatient ambulatory unit.

Bacterial isolates

Between January 2007 and February 2008, a total of 106 MRSA isolates were collected from both inpatients (n=93, 87.7% of the isolates) and outpatients (n = 13, 12.3% of the isolates) attending HDES. The collection included singlepatient isolates recovered from several sources, including sputum (n=35, 33%), urine (n = 22, 21%), swabs from various origins (n = 19, 18%), exudates from wounds, one abscess and one ear infection (n = 16, 15%), blood (n = 10, 9.5%), catheters (n = 2, 1.9%) and auricular and peritoneal fluids (n = 1, 0.9% each). Most of the isolates (95%, n = 101) were from infection. The majority of the patients (73.5%) were inpatients hospitalised in three wards: medicine (n=42, 39.6%), surgery (n = 24, 22.6%) and pneumology (n = 12, 11.3%). Among the 13 outpatients, three attended the haemodialysis unit.

Antimicrobial susceptibility testing and mecA detection Susceptibility testing was performed with the semiautomatic Vitek system (bioMérieux, SA, France) according to the manufacturer's instructions for a panel of 12 antibiotics: ciprofloxacin, clindamycin, erythromycin, gentamicin, linezolid, oxacillin, penicillin, rifampicin, trimethoprim-sulfamethoxazole, teicoplanin, tetracycline and vancomycin. Bacterial isolates were considered to belong to different antibiotypes if at least one difference was observed in the antibiotic resistance profile.

The presence of the mecA gene was confirmed in all oxacillin-resistant isolates by polymerase chain reaction (PCR) [START_REF] Okuma | Dissemination of new methicillin-resistant Staphylococcus aureus clones in the community[END_REF].

PFGE

Pulsed-field gel electrophoresis (PFGE) was performed as described by Chung et al. [START_REF] Chung | Molecular typing of methicillin-resistant Staphylococcus aureus by pulsedfield gel electrophoresis: comparison of results obtained in a multilaboratory effort using identical protocols and MRSA strains[END_REF] on all 106 isolates. The resulting SmaI restriction patterns were analysed by both visual inspection using the criteria of McDougal et al. [START_REF] Mcdougal | Pulsed-field gel electrophoresis typing of oxacillin-resistant Staphylococcus aureus isolates from the United States: establishing a national database[END_REF] and automatically with the BioNumerics software version 4.61 (Applied Maths, Sint-Martens-Latem, Belgium). Dendrograms were generated as previously described [START_REF] Faria | Analysis of typing methods for epidemiological surveillance of both methicillin-resistant and methicillin-susceptible Staphylococcus aureus strains[END_REF] using an optimisation of 0.50% and a tolerance of 1.25%. A similarity coefficient of 80% was used to define the PFGE type clusters [START_REF] Faria | Analysis of typing methods for epidemiological surveillance of both methicillin-resistant and methicillin-susceptible Staphylococcus aureus strains[END_REF].

spa typing and MLST spa typing was carried out on at least one representative of each PFGE subtype (n = 26) and spa types were assigned through the Ridom web server (http://spaserver.ridom.de) [START_REF] Aires-De-Sousa | High interlaboratory reproducibility of DNA sequence-based typing of bacteria in a multicenter study[END_REF]. Multilocus sequence typing (MLST) was conducted as described [START_REF] Crisóstomo | The evolution of methicillin resistance in Staphylococcus aureus: similarity of genetic backgrounds in historically early methicillin-susceptible and -resistant isolates and contemporary epidemic clones[END_REF] for selected strains previously typed by PFGE and spa typing. The allelic profiles and sequence types (ST) were defined using the MLST database (http://www.mlst.net).

SCCmec and ccr typing

SCCmec was typed by the multiplex PCR strategy described by Milheiriço et al. [START_REF] Milheiriço | Update to the multiplex PCR strategy for assignment of mec element types in Staphylococcus aureus[END_REF]. Additionally, all type IV isolates were further subtyped as previously described [START_REF] Milheiriço | Multiplex PCR strategy for subtyping the staphylococcal cassette chromosome mec type IV in methicillin-resistant Staphylococcus aureus: 'SCCmec IV multiplex[END_REF]. Type VI isolates were confirmed by amplification of the cassette chromosome recombinase genes ccrAB4 [START_REF] Oliveira | Redefining a structural variant of staphylococcal cassette chromosome mec, SCCmec type VI[END_REF]. Type V was confirmed by PCR amplification of ccrC and mec complex C, as previously described [START_REF] Okuma | Dissemination of new methicillin-resistant Staphylococcus aureus clones in the community[END_REF][START_REF] Ito | Novel type V staphylococcal cassette chromosome mec driven by a novel cassette chromosome recombinase, ccrC[END_REF]. Nontypeable isolates by the methods mentioned above were characterised by ccrB sequencing [START_REF] Oliveira | Assessment of allelic variation in the ccrAB locus in methicillinresistant Staphylococcus aureus clones[END_REF].

PVL detection

All 106 MRSA isolates were screened for the presence of PVL lukS/lukF genes by PCR, as described previously [START_REF] Lina | Involvement of Panton-Valentine leukocidin-producing Staphylococcus aureus in primary skin infections and pneumonia[END_REF]. Positive results were confirmed by sequencing of the PCR amplicon.

Results

Antimicrobial susceptibility

The 106 isolates were assigned to eight different antibiotypes (Table 1) with a prevalent non-multiresistant pattern.

The majority of the isolates (n = 91, 85.9%) showed antibiotype 1 (resistance to oxacillin, penicillin, ciprofloxacin and erythromycin) or 2 (resistance to oxacillin, penicillin and ciprofloxacin). The isolate with antibiotype 7 was unique in showing resistance to gentamicin. Antibiotype 8, which showed resistance to six different antimicrobials, included a single isolate.

All 106 isolates harboured the mecA gene and, therefore, were classified as MRSA.

Predominance of EMRSA-15 clone

The characterisation of the 106 MRSA strains by PFGE, spa typing, MLST and SCCmec typing clustered the isolates into four clonal types (Fig. 1). The majority of the isolates (n=92, 87%) belonged to PFGE type A. PFGE type B included 11% (n = 12) of the isolates and types C and D were represented by single isolates only (1% each).

PFGE type A was subdivided into nine subtypes; subtypes A1 (n=40) and A2 (n = 22) represented 67% of all type A isolates. The representative isolate of PFGE type A (subtype A1) showed spa type t032, ST22 and SCCmec type IVh, which are characteristics of the internationally disseminated EMRSA-15 clone [START_REF] Moore | Molecular characterisation of the dominant UK methicillin-resistant Staphylococcus aureus strains, EMRSA-15 and EMRSA-16[END_REF]. Interestingly, subtype A4 showed the same restriction profile as the EMRSA-15 clone reference strain, HAR22, but a different spa type (t032), including one additional repeat sequence (Fig. 1). All representative isolates of type A shared the same spa type t032, except one isolate (t910), which differs from t032 by the deletion of five repeats. HDES85 was the unique isolate of PFGE type A1 defined as non-typeable by spa typing, since no PCR product was obtained. Another 1).

Evidence of horizontal gene transfer among strains of the Pediatric clone PFGE type B was the second most common PFGE type and included three subtypes, of which B1 represented 75% (n=9) of type B isolates (Fig. 1). Subtypes B1 and B2 showed ST5, SCCmec VI and spa types t062, t2049, t2724 (B1)/t311 (B2), which are typical of the Pediatric clone. SCCmec type VI was confirmed in five isolates by amplification of the characteristic ccrAB4 genes. The remaining type VI isolates did not amplify the ccrAB4 genes by uniplex PCR but showed a ccrB sequence with 100% homology with the Pediatric reference strain HDE288 [START_REF] Sá-Leão | Detection of an archaic clone of Staphylococcus aureus with low-level resistance to methicillin in a pediatric hospital in Portugal and in international samples: relics of a formerly widely disseminated strain?[END_REF]. Interestingly, subtype B3 carried SCCmec type IVc instead of the SCCmec type VI, which may indicate the occurrence of independent acquisition of SCCmec in the Pediatric clone background. Moreover, we found that three isolates belonging to PFGE subtype B1 (ST5-VI) carried the PVL genes.

The remaining two isolates belonged to PFGE types C and D and were, respectively, recovered from an outpatient and an inpatient. PFGE C isolate, spa type t004 and ST45-IVa showed a single band difference relative to HAR38, the prototype isolate of the Berlin clone (spa type t004, ST45-IVa) [START_REF] Murchan | Harmonization of pulsed-field gel electrophoresis protocols for epidemiological typing of strains of methicillin-resistant Staphylococcus aureus: a single approach developed by consensus in 10 European laboratories and its application for tracing the spread of related strains[END_REF][START_REF] Cookson | Evaluation of molecular typing methods in characterizing a European collection of epidemic methicillinresistant Staphylococcus aureus strains: the HARMONY collection[END_REF]. PFGE type D isolate showed spa type t1839, a novel sequence type (ST1339) not related to any major clonal complex (CC) and a variant of SCCmec type V.

High genetic diversity and high frequency of SCCmec types IV, V and VI SCCmec type IV was found to be the prevalent type (n = 13) among the 24 isolates tested. Three different SCCmec IV subtypes were found in this collection, associated to three unrelated genetic backgrounds (ST5: SCCmec IVc, ST22: SCCmec IVh/IVnon-subtypeable, and ST45: SCCmec IVa). SCCmec type VI was the second most prevalent cassette type, which appeared to be associated to isolates with PFGE types B1 and B2. A variant of SCCmec type V was shown on a single isolate that harboured the mec complex C and ccrC genes, but missed the specific type V J1 region. No isolates carrying SCCmec types I, II or III were identified in this collection.

Discussion

In order to describe the MRSA population structure in the Atlantic Azores islands, a collection of 106 isolates recovered during a 13-month period was characterised by a combination of up-to-date molecular typing methods. The analysis showed the predominance of two major international MRSA clones: the EMRSA-15 clone and the Pediatric clone.

Massive dominance of the EMRSA-15 clone

In the present study, 87% of the isolates showed molecular characteristics of the highly internationally disseminated EMRSA-15 clone (ST22-IVh) [START_REF] Moore | Molecular characterisation of the dominant UK methicillin-resistant Staphylococcus aureus strains, EMRSA-15 and EMRSA-16[END_REF], including a common spa type t032. In addition, a single PFGE type A2 isolate showed t910, a spa type already reported in a CC22 isolate from Germany [START_REF] Strommenger | spa typing of Staphylococcus aureus as a frontline tool in epidemiological typing[END_REF]. This fact is not surprising, since the spa gene is a variable repeat region prone to the introduction or deletion of repeats. Regional spa clusters frequently include highly related spa types characterising clonal related isolates, which translates a local evolution of the spa locus [START_REF] Grundmann | Geographic distribution of Staphylococcus aureus causing invasive infections in Europe: a molecular-epidemiological analysis[END_REF][START_REF] Harmsen | Typing of methicillin-resistant Staphylococcus aureus in a university hospital setting by using novel software for spa repeat determination and database management[END_REF]. Unusually, a non-typeable isolate was found in the collection, which should be due to a mutation or insertion/deletion in spa rather than non-existence of the gene [START_REF] Baum | Non-spa-typeable clinical Staphylococcus aureus strains are naturally occurring protein A mutants[END_REF]. Interestingly, the majority of the EMRSA-15 isolates described in this study were recovered from sources other than blood (sputum, swabs, exudates and urine), corresponding to the major pathologies presented by the patients attending HDES (respiratory tract and skin related). The non-multiresistant EMRSA-15 clone emerged in the United Kingdom in 1991 and rapidly became a dominant clone worldwide, particularly in Europe [START_REF] Grundmann | Geographic distribution of Staphylococcus aureus causing invasive infections in Europe: a molecular-epidemiological analysis[END_REF][START_REF] Johnson | Surveillance and epidemiology of MRSA bacteraemia in the UK[END_REF]. Recently, an EMRSA-15 variant was reported in central United States [START_REF] Wolter | Isolation and characterization of an epidemic methicillin-resistant Staphylococcus aureus 15 variant in the central United States[END_REF]. In the Azores islands, the clear dominance of the EMRSA-15 clone is, in part, in agreement with the situation in continental Portugal [START_REF] Aires-De-Sousa | Changing patterns in frequency of recovery of five methicillin-resistant Staphylococcus aureus clones in Portuguese hospitals: surveillance over a 16-year period[END_REF][START_REF] Amorim | Changes in the clonal nature and antibiotic resistance profiles of methicillin-resistant Staphylococcus aureus isolates associated with spread of the EMRSA-15 clone in a tertiary care Portuguese hospital[END_REF]. In fact, in a study involving isolates recovered in the same period from 11 hospitals scattered over continental Portugal, EMRSA-15 was found to be the most prevalent clone. However, and in contrast with the present study, the New York/Japan clone (ST5-II) was shown to represent a new wave of MRSA in the country, whereas the Pediatric clone (ST5-VI/IV) was exceedingly rare [START_REF] Aires-De-Sousa | Changing patterns in frequency of recovery of five methicillin-resistant Staphylococcus aureus clones in Portuguese hospitals: surveillance over a 16-year period[END_REF]. In the Portuguese neighbouring country Spain, EMRSA-15 was found in continental territory in co-existence with EMRSA-16 and two CC5 clones (ST125-IV and ST146-IV), but not as a major clone as in the Spanish islands (Majorca and Canarias) and as in our study [START_REF] Alcoceba | Molecular epidemiology of methicillin-resistant Staphylococcus aureus in Majorcan hospitals: high prevalence of the epidemic clone EMRSA-15[END_REF][START_REF] Vindel | Prevalence and evolution of methicillin-resistant Staphylococcus aureus in Spanish hospitals between 1996 and 2002[END_REF][START_REF] Pérez-Roth | Tracking methicillin-resistant Staphylococcus aureus clones during a 5-year period (1998 to 2002) in a Spanish hospital[END_REF]. The frequent touristic flux from the United Kingdom, hypothesised as the cause of the importation of EMRSA-15 to the Mediterranean island of Malta [START_REF] Scicluna | Characterisation of MRSA from Malta and the description of a Maltese epidemic MRSA strain[END_REF], could also support, in part, the emergence of this clone in the most touristic Azores islands. In parallel, the regular exchange of inhabitants between the islands and the continent seem to be an important vehicle of clonal importation.

Emergence of PVL and SCCmec variability: evidence for local evolution

Recent studies revealed that the Pediatric MRSA clone, isolated for the first time in 1992 in a Portuguese hospital [START_REF] Sá-Leão | Detection of an archaic clone of Staphylococcus aureus with low-level resistance to methicillin in a pediatric hospital in Portugal and in international samples: relics of a formerly widely disseminated strain?[END_REF], was no longer present in that hospital in 2006 [START_REF] Aires-De-Sousa | Changing patterns in frequency of recovery of five methicillin-resistant Staphylococcus aureus clones in Portuguese hospitals: surveillance over a 16-year period[END_REF]. However, it appeared in the present collection as the second major clone.

Interestingly, in this study, we observed that strains of the Pediatric clone differing in three to five bands in PFGE pattern, isolated in the same hospital, carried either the typical SCCmec types VI or IVc. ST5-VI MRSA isolates, which had been restricted to isolates from continental Portugal, were recently found in a collection of nosocomial isolates recovered from 23 hospitals located throughout France [START_REF] Oliveira | Redefining a structural variant of staphylococcal cassette chromosome mec, SCCmec type VI[END_REF][START_REF] Dauwalder | Epidemiology of invasive methicillin-resistant Staphylococcus aureus clones collected in France in 2006 and 2007[END_REF]. The hypothesis of a recent introduction of this clone from continental Portugal seems to be quite remote, since, in a recent national study, this clone was isolated from a single patient only in a hospital in the South of Portugal [START_REF] Aires-De-Sousa | Changing patterns in frequency of recovery of five methicillin-resistant Staphylococcus aureus clones in Portuguese hospitals: surveillance over a 16-year period[END_REF]. On the other hand, the Azores islands have a high emigrant community in the United States, who frequently travel to the national territory, constituting also possible vehicles of dissemination of the Pediatric clone strains already reported in this country.

ST5 lineage is known to frequently acquire mobile genetic elements, such as different SCCmec, antibiotic resistance and toxin genes [START_REF] Nübel | Frequent emergence and limited geographic dispersal of methicillin-resistant Staphylococcus aureus[END_REF][START_REF] Enright | The evolutionary history of methicillin-resistant Staphylococcus aureus (MRSA)[END_REF]. In a recent study, Nübel et al. showed that at least 23 SCCmec acquisitions events have occurred within ST5 lineage, on multiple and independent occasions, showing evidence that the MRSA emerged in numerous circumstances and in distinct locations rather than by the geographic dispersal of isolates [START_REF] Nübel | Frequent emergence and limited geographic dispersal of methicillin-resistant Staphylococcus aureus[END_REF]. Moreover, besides being originally identified in a community-associated USA400 strain, the first introduction of SCCmecIV in S. aureus might have occurred in the ST5 Pediatric clone background that was circulating in hospitals in the 1990s [START_REF] Chambers | Waves of resistance: Staphylococcus aureus in the antibiotic era[END_REF]. Similarly, our results suggest that SCCmec cassettes VI and IVc might have been introduced de novo and recently in these ST5 strains and that the Pediatric clone may be evolving locally. Another observation that supports the hypothesis of recent local evolution of the Pediatric clone is the presence of PVL-positive and PVLnegative strains with closely related PFGE profiles. Noteworthy, this is the first report of PVL among isolates belonging to the SCCmec type VI Pediatric clone. The introduction of the PVL genes in a genetic background with such a high epidemic potential is of particular concern. However, we cannot exclude the reverse hypothesis of a local evolution through the acquisition of SCCmec type VI by a PVL-positive MSSA. Only the analysis of the genetic background of susceptible S. aureus in the Azores islands would clarify the chronological acquisition of PVL and SCCmec in these Pediatric clone strains.

The Berlin MRSA clone (ST45-IVa), first observed in Berlin hospitals in 1993, is currently spread in some European countries [START_REF] Grundmann | Geographic distribution of Staphylococcus aureus causing invasive infections in Europe: a molecular-epidemiological analysis[END_REF][START_REF] Wannet | Widespread dissemination in The Netherlands of the epidemic Berlin methicillin-resistant Staphylococcus aureus clone with low-level resistance to oxacillin[END_REF], showing a high epidemic potential not only in the hospital environment but also in the community [START_REF] Witte | Changing pattern of antibiotic resistance in methicillin-resistant Staphylococcus aureus from German hospitals[END_REF][START_REF] Regev-Yochay | Prevalence and genetic relatedness of community-acquired methicillin-resistant Staphylococcus aureus in Israel[END_REF]. This clone was found in a single MRSA isolate in the Azores island collection. The same genetic background (ST45) had been previously described in Portuguese hospitals, but exclusively among MSSA isolates [START_REF] De Sousa | Comparison of genetic backgrounds of methicillin-resistant and -susceptible Staphylococcus aureus isolates from Portuguese hospitals and the community[END_REF]. The collection of a single MRSA isolate carrying SCCmec IVa suggests another de novo SCCmec acquisition, probably in an already established MSSA clone.

Likewise, the introduction of a variant of SCCmec type V seems to have occurred in an isolate with spa type t1839 and a novel sequence type (ST1339) that is a singleton. This strain is not related to the single SCCmec type V variant isolate (ST45) previously reported in Portugal [START_REF] Aires-De-Sousa | Changing patterns in frequency of recovery of five methicillin-resistant Staphylococcus aureus clones in Portuguese hospitals: surveillance over a 16-year period[END_REF]. Interestingly, this isolate was recovered from a patient within the first 48 h of hospitalisation, from a skin and soft tissue infection (erysipelas), the main source of CA-MRSA isolates. However, the isolate could not be considered as having a community origin, since no additional information regarding patient risk factors was available.

Emergence of HA-MRSA isolates with community traits SCCmec type IV is one of the most frequent SCCmec types found in the community and is, nowadays, becoming the predominant type also in the nosocomial setting [START_REF] Aires-De-Sousa | Changing patterns in frequency of recovery of five methicillin-resistant Staphylococcus aureus clones in Portuguese hospitals: surveillance over a 16-year period[END_REF][START_REF] Amorim | Changes in the clonal nature and antibiotic resistance profiles of methicillin-resistant Staphylococcus aureus isolates associated with spread of the EMRSA-15 clone in a tertiary care Portuguese hospital[END_REF][START_REF] Chambers | Waves of resistance: Staphylococcus aureus in the antibiotic era[END_REF][START_REF]International Working Group on the Classification of Staphylococcal Cassette Chromosome Elements (IWG-SCC) (2009) Classification of staphylococcal cassette chromosome mec (SCCmec): guidelines for reporting novel SCCmec elements[END_REF][START_REF] Alcoceba | Molecular epidemiology of methicillin-resistant Staphylococcus aureus in Majorcan hospitals: high prevalence of the epidemic clone EMRSA-15[END_REF][START_REF] Vindel | Methicillin-resistant Staphylococcus aureus in Spain: molecular epidemiology and utility of different typing methods[END_REF][START_REF] Witte | Community-acquired methicillin-resistant Staphylococcus aureus: what do we need to know?[END_REF]. Moreover, this SCCmec type has been usually associated to a pattern of susceptibility to almost all antimicrobial classes. These same characteristics were observed in isolates from our study: SCCmec type IV was found to be the most frequent SCCmec type and all isolates were highly susceptible. At least three different SCCmec subtypes were identified (IVh: EMRSA-15, IVc: Pediatric clone, IVa: Berlin clone), showing evidence of a high genetic diversity among the pool of SCCmec type IV in the Azores islands. Interestingly, the detection of one isolate belonging to the EMRSA-15 clone, SCCmec type IV nonsubtypeable (only positive for the ccrB2 allele in the subtyping multiplex), suggests the presence of a new uncharacterised SCCmec type IV subtype in addition to the ones already described [START_REF] Milheiriço | Multiplex PCR strategy for subtyping the staphylococcal cassette chromosome mec type IV in methicillin-resistant Staphylococcus aureus: 'SCCmec IV multiplex[END_REF].

Besides SCCmec type IV, the only SCCmec types found among the whole collection were the small SCCmec V and VI, reflecting the actual tendency for the dominance of low resistant and fitness cost clones within the hospital setting, which is parallel with what is observed in the community environment [START_REF] Chambers | Waves of resistance: Staphylococcus aureus in the antibiotic era[END_REF]. The introduction of well-established community isolates into the hospital setting is not new, as observed in Taiwan, where the ST59 clonal type, highly prevalent in the community, emerged in the hospital setting as the second most prevalent clone [START_REF] Pérez-Roth | Tracking methicillin-resistant Staphylococcus aureus clones during a 5-year period (1998 to 2002) in a Spanish hospital[END_REF][START_REF] Montesinos | Evolution of the MRSA clones at a university hospital in the Canary islands[END_REF][START_REF] Huang | Changing molecular epidemiology of methicillin-resistant Staphylococcus aureus bloodstream isolates from a teaching hospital in Northern Taiwan[END_REF]. Although ST22-IV EMRSA-15-related isolates as well as ST5-IV were already reported in the community [START_REF] O'brien | Diversity among community isolates of methicillinresistant Staphylococcus aureus in Australia[END_REF][START_REF] Witte | Community-acquired methicillin-resistant Staphylococcus aureus: what do we need to know?[END_REF], no conclusions can be drawn for the flow direction in the present study. Even if a high percentage (30%) of the isolates studied was recovered within the first 48 h of admission or in an outpatient unit, no data of previous hospital contact was available for the patients.

In the present study, we provided the first description of the population structure of MRSA in the Azores islands and showed a prevalence of the epidemic EMRSA-15 clone in co-existence with the Pediatric clone. Our findings were in line with recent reports from continental Portuguese and European hospitals. The horizontal gene transfer and the de novo acquisition of mobile genetic elements seem to have contributed to the local genetic diversity and clonal evolution. Moreover, the population exchange between the islands and continental Portugal or the United States, in addition to the high flux of tourists, particularly from the United Kingdom, may explain the dominance and spread of the EMRSA-15 and Pediatric clones. Further surveillance studies concerning both HA-MRSA and MSSA populations and CA-MRSA in the geographically restricted Azores islands will be fundamental to support the S. aureus localised evolution, as well as to understand the links between the hospital and the community.

Fig. 1

 1 Fig. 1 Molecular characterisation of representative methicillin-resistant Staphylococcus aureus (MRSA) isolates of each pulsed-field gel electrophoresis (PFGE) subtype and comparison with MRSA pandemic clones

Table 1

 1 Resistance profile of the 106 methicillin-resistant Staphylococcus aureus (MRSA) isolates and associated pulsed-field gel electrophoresis (PFGE) types

	Antibiotype Resistance profile a

a S, susceptible; I, intermediate; R, resistant Antibiotic abbreviations: OXA, oxacillin; PEN, penicillin; CIP, ciprofloxacin; ERY, erythromycin; CLI, clindamycin; RIF, rifampicin; GEN, gentamicin; LZD, linezolid; SXT, trimethoprim-sulfamethoxazole; TEC, teicoplanin; TET, tetracycline; VAN, vancomycin single type A1 isolate, HDES73, showed a SCCmec cassette type IV non-subtypeable. PFGE type A included isolates from six out of the eight antibiotypes described, but 95% (n=53) of the isolates belonged to antibiotypes 1 and 2 (Table

Eur J Clin Microbiol Infect Dis (2010) 29:543-550

Eur J Clin Microbiol Infect Dis (2010) 29:543-550

Acknowledgements This work was supported by project TROCAR (contract HEALTH-F3-2008-223031) and CONCORD (contract HEALTHF3-2008-222718) from the European Commission. T. Conceição and A. Tavares were supported by grants SFRH/BD/21424/2005 and SFRH/BD/44220/2008, respectively, from Fundação para a Ciência e a Tecnologia, Lisbon, Portugal.

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