Frederic Pozzetto

Lucht

Philippe Pr

PhD, MPH Berthelot

PhD, MPH Philippe Berthelot
email: philippe.berthelot@chu-st-etienne.fr

MD Florence Grattard

MD, PhD Celine Cazorla

MD Jean-Paul Paul Passot

MD; Jean Philippe Fayard

MD Roland Meley

MD Jacques Bejuy

MD Frederic Farizon

MD Bruno Pozzetto

MD, PhD Frederic Lucht

Roland Fayard

Jacques Meley

Frederic Bejuy

Bruno Farizon

F Berthelot

C Grattard

B Cazorla

pathway for surgical-site infection in orthopaedic surgery? 2

Keywords: Staphylococcus aureus, nasal carriage, carrier, surgical site infection, hospital infection, orthopaedic surgery Keywords. Staphylococcus aureus, nasal carriage, carrier, surgical site infection, hospital 22 infection, orthopaedic surgery 23

Is nasal carriage of the main acquisition pathway for

surgical-site infection in orthopaedic surgery?

It is unfortunate that they did not screen other sites such as the throat and perineum.

We agree with this comment. Initially, the protocol included a rectal swabbing but many patients were reluctant to this proposal. Due to a high rate of missing data during the initial step of the study, we decided to stop the sampling of this site. The following sentence was added in the discussion section of the new manuscript (page 11; lines 12 to 14): "Our protocol included initially a rectal swabbing before surgery; however, since many patients denied this sample, the data regarding rectal specimens could not be taken into consideration". Concerning the throat sampling, when the study was designed in 2002, sampling this site was not recommended since this specimen was not considered at this time to increase significantly the sensitivity of the recovery of S. aureus from the upper respiratory tract as shown later by Metz et al. in 2007 [ref 23] (this point is discussed page 11, lines 14 to 17).

They could point out that sole carriage at these sites is rare (perhaps 10%) and add this to their estimates of the possible size of the problem (20% larger!). Nasal carriage rather than carriage should be in the text.

We also agree with this comment, which is emphasised in the new discussion section (page 11; lines 14 to 17): "With a single nasal sample and in the absence of additional throat and rectal specimens, the frequency of carriage of S. aureus in our cohort was certainly underestimated, as illustrated by the figure of 20.2% of nasal carriers in this study compared to 37.1% in the one of Mertz et al [23] that combined nasal and throat samplings."

Comment 1 about CNS data: 1) Table 3 has overall data. They could compare the antibiograms of the individual isolates and see if there are very different strains. I doubt the organisms are available for molecular analysis (if so this could be "future work" for the discussion) but at least the antibiograms could be compared by ward and centre and see if there are any similarities and differences and informed guesses made as to the likelihood of cross infection e.g. quinolone resistance is usually mutational 2) All the other infected organism data. Again what about antibiograms and possible cross infection?

The goal of the study was to address the potential link between S. aureus nasal carriage and occurrence of S. aureus SSI, and not to study CNS or other bacterial cross infections. Strains other than S. aureus were not kept for further analysis. By contrast, the potentiality of cross infections with S. aureus was investigated and presented with some details in the revised manuscript (see below).

3) S aureus data. We need to see their rule set for deciding why the extra 5 strains were different or similar. This is a difficult area and must be explained.

The way used to compare antibiotypes is reported in the method section (page 6, lines 12 to 16). As required by the referee, additional information regarding the comparison of antibiotic profiles has been added in the footnotes of Table III.

They should compare the 13 SA from non carriers (and indeed the carriers) to see if there are possible issues of cross infection in terms of time person and place of the affected patients. Are these strains similar by PFGE? Are they similar to any of the carriers?

From the 13 S. aureus SSI strains isolated from non nasal carriers, 8 were available for comparison by PFGE: all these strains exhibited independent profiles, also different from those of strains isolated from the nose of colonised patients. The 5 remaining strains were isolated from non nasal carriers hospitalized in 5 different centres, excluding cross transmission. These data were added in the result section page 9 lines 10 to 13.

Table IV only has death related risk analysis not as stated on page 10 lines 23-24 for SSI and SA nasal carriage.

Because of the low number of SSI infections, the multivariate analysis was run with a maximum of 3 covariates selected among the most significant in the univariate analyses. This sentence was added in the method section (page 8 lines 8 to 10). We also modified the sentence presenting Table IV (page 10 lines 14 to 15). 4) There are also the therapeutic issues raised by the AST results for all categories of infections above. These are perhaps a little peripheral but I think it would add to the paper or perhaps be another one if there are clinical outcome data? Data regarding therapeutic issues are not presented in this paper. Another clinical paper focusing on these results is in preparation Other points How were nasal swabs taken: were the swabs moistened?

The precision regarding moistened transport medium was added in the method section (page 5, line 1).

Was the PFGE protocol validated to be able to distinguish strains and how?

The criteria for PFGE interpretation are described page 6 lines 10 to 12 "For a same patient, according to Tenover's criteria [18], two S. aureus strains were classified as epidemiologically distinct if a difference of more than three bands was ascertained between the PFGE profiles performed in a same run".

Reviewer 2

First this was a multicenter study and in these kind of studies cluster effects may occur. In the multivariate analysis, adjustment for cluster effects was not included. This would strengthen the conclusions.

As pointed by the reviewer it is important to look for cluster effect. This was investigated by adding a "centre" variable in the different statistical analyses. This variable did not remain in the final model used for multivariate analysis. But as required by the referee, we performed another multivariate analysis using windows SAS® 9.1 software to adjust for cluster effect. This was added in the method section (page 7, line 25). We replaced the results of the previous multivariate analysis performed using SPSS software by the results of this multivariate analysis adjusted for cluster effect (Table IV).

Second, the authors conclude that the majority of the S. aureus strains are of exogenous origin. They put it quite strong in the conclusions that 'at least 16 were not endogenous'. In my opinion this is not correct. The nasal cultures were negative but this may have been a false-negative result, e.g. due to the sampling technique. As the carriage rate was only 20% it is clear that the culture technique was not very sensitive (normally 30% of the population is nasal carrier). Therefore, I suggest to modify this conclusion. In the majority of cases an endogenous origin could not be demonstrated would be more correct.

We agree with this comment. The discussion section was amended accordingly (see answer to reviewer 1 and additional sentence in page 11; lines 14 to 17)). As suggested by the referee, we also modified the conclusion; the sentence "In most cases of S. aureus SSI, pre-operative nasal colonization was not documented or retrieved a different strain from the infecting pathogen" was replaced by the following one in the new manuscript (page 12, lines 20 to 22 and in the abstract): "In most cases of S. aureus SSI, either an endogenous origin could not be demonstrated, or pre-operative nasal colonisation retrieved a strain different from the one recovered from the surgical site" Also, it would be important to provide information on the typing of the strains from non-carriers as well. I wonder whether there were many similar strains within centers suggesting a common source? We conducted a multicentre prospective cohort study to analyse the respective part of 6 exogenous contamination and endogenous self-inoculation by S. aureus during elective 7 orthopaedic surgery. The nose of each consecutive patient was sampled before surgery. 8 Strains of S. aureus isolated from the nose and the wound, in case of SSI, were compared by 9 antibiotypes or pulsed field gel electrophoresis (PFGE). 10 Results 11 3908 consecutive patients undergoing orthopaedic surgery were included. Seventy seven 12 patients developed an SSI (2%), including 22 related to S. aureus (0.6%). S. aureus was 13 isolated from the nose of 790 patients (20.2%) at the time of surgery. In multivariate analysis, 14 S. aureus nasal carriage was found as a risk factor for S. aureus SSI in orthopaedic surgery. 15 However, only 9 subjects exhibiting S. aureus SSI had been found carriers before surgery: 16 when compared, 3 pairs of strains were considered different and 6 similar. 17

Conclusion 18

In most cases of S. aureus SSI, either an endogenous origin could not be demonstrated, or 19 pre-operative nasal colonisation retrieved a strain different from the one recovered from the 20 surgical site 21

Introduction 1 2
The risk of prosthetic joint infection is less than 2 percent after joint replacement [1-3] or after 3 implantation of internal fracture-fixation devices [4]. However, the large number of such 4 procedures performed annually makes these infections highly significant in terms of 5 mortality, morbidity and costs, as far as the elderly population is increasing in industrialized 6 countries [5][6][7]. It is a well-known fact that S. aureus carriage predisposes to post-operative 7 staphylococcal infection in general surgery [8], in heart surgery [9-10], as in orthopaedic 8 surgery [11]. However, Kalmeijer et al. [11], in orthopaedic surgery, realized their study in 9 only one centre, with a limited number of patients. Moreover, the studies showing a 10 substantial reduction of SSI among patients receiving mupirocin were all compared to 11 historical control [8, 12-14], whilst Kalmeijer et al.[15], in a double-blind, randomised, 12 placebo-controlled study, in orthopaedic surgery, failed to show any beneficial effect. So, the 13 exogenous pathway of contamination in orthopaedic surgery remains an important hypothesis 14 and we undertook a large prospective multicenter cohort study, to determine whether the nasal 15 carriage of S. aureus is the main pathway responsible for infection in orthopaedic SSI. 16 17

Patients and methods

19

We conducted a prospective epidemiological multicentre observational study to estimate the 20 relative part of subjects with S. aureus nasal carriage in patients with SSI following prosthetic 21 and internal fracture-fixation device surgery, and in non-infected patients. We compared, in 22 SSI patients, the strains of S. aureus colonizing the anterior nares and that isolated from the 23 site of infection. 24 Patients. All consecutive patients undergoing scheduled orthopaedic surgery with 1 implantation of prosthesis or internal fracture-fixation device, in seventeen centres (see 2 appendix), were included from June 2003 to January 2007. A control visit was performed by 3 the surgeon at least one month and one year after surgery. Exclusion criteria were non-4 programmed surgery and suspicion of infection at the site of surgery. 5 A written informed consent was obtained for all patients. The study protocol was approved by 6 the regional ethical research committee (Comité de Protection des Personnes Rhône-Alpes I). 7

The preparation of patients' skin included at least one preoperative shower with a soap 8 containing either chlorhexidine or polyvidone iodine, cleansing of the skin with an antiseptic 9 soap followed by a disinfection with an alcoholic antiseptic in the operative theatre. Antibiotic 10 prophylaxis was done with cefazolin, 2 grams at the anaesthetic induction. Mupirocin nasal 11 decontamination was not used whatever the results of the sampling. 12 One to three months before surgery, the study was explained to the patient during surgical 13 consultation. Written information was delivered. The nose of the patient was sampled at the 14 admission. To ensure performance and reproducibility of the nasal sampling, nurses were 15 trained and a written protocol was available in each participating unit. The patient's 16 practitioner was informed of the study and asked to refer the patient to the surgeon if a SSI 17 was suspected. In the latter case, at least 3 bacteriological samples were taken at the site of the 18 suspected infection either per-operatively (without antimicrobial prophylaxis) or by aseptic 19 aspiration using ultrasound guidance. Direct examination and bacteriological culture were 20 systematically performed. 21

22

Bacteriological methods 23

Nasal cultures: One swab was used to sample both nares and plated onto sheep blood agar 24 plate or onto specific S. aureus Chromastaph medium (Biomérieux, Marcy l'Etoile, France) 25 within 24 hours after sampling if a moistened transport medium was used (Transtube®, 1 Medical Wire and Equipment Co., Corsham, England or Amies agar gel, Copan, Brescia, 2 Italy) or within 2 hours in the opposite case. Plates were incubated up to 48h at 36°C. The 3 identification of S. aureus isolates and their sensitivity to antibiotics were determined by 4 standard microbiological procedures. 5

As for nasal samples, swabs were cultured within 2 hours after sampling if no transport 6 medium was used, and within 24 hours in the opposite case. Fluid samples were examined by 7 light microscopy after Gram staining. They were cultured both aerobically and anaerobically 8 into blood culture bottles and monitored in an automated system. Solid samples were 9 dissected into tiny pieces with a lancet in a safety cabinet and then ground down. The 10 homogenate was cultured onto aerobic and anaerobic media. The media were incubated at 11 36°C for at least 48 hours for plate cultures and 15 days for liquid broths. 12 In addition to phenotypic characteristics, all the available couples of strains of S. aureus 13 isolated from the nose and the infected surgical site were compared by PFGE using SmaI. The 14 available strains were centralised in a single laboratory where PFGE was performed. Plugs 15 containing DNA were prepared following a rapid lysis procedure [16]. Bacterial cells from an 16 overnight broth culture were mixed with 1.6% of SeaPlaque agarose (Cambrex, Rockland, 17 USA). The plugs were incubated in lysis buffer containing 250 IU of lysostaphin (AMBI Inc, 18 Lawrence, NY, USA) for 2 hours at 37°C, followed by a one-hour incubation at 50°C with 19 ESP buffer containing 100 mg/L of proteinase K. Plugs were rinsed with TE buffer, incubated 20 in TE with PMFS for 30 min at 37°C before final washing in TE buffer. The genomic DNA 21 inserts were digested at 25°C for 2 h with 20 UI of SmaI enzyme. The fragments were 22 separated by PFGE with a CHEF-DrII apparatus (Biorad, Ivry sur Seine, France) in a 1.1% 23 agarose gel (Gigaphor, Promega, Les Ullis, France) in TBE buffer, at 14°C under a field 24 strength of 6 V/cm and a linear ramp of 1s-28s for 21 hours. 25 For a same patient, according to Tenover's criteria [18], two S. aureus strains were classified 10 as epidemiologically distinct if a difference of more than three bands was ascertained between 11 the PFGE profiles performed in a same run. When the S. aureus strain isolated from the 12 surgical site was not available for typing, the antimicrobial patterns were used to compare the 13 strains isolated in a same patient: a difference in antimicrobial susceptibility of two or more 14 molecules signed different strains; in case of difference concerning one or no antibiotic, 15 strains were considered phenotypically similar. 16 17

Data recorded 18

The following variables were recorded for each patient: age, sex, Body Mass Index (BMI), 19 tobacco use, underlying diseases (malignancy, diabetes, malnutrition, immunodeficiency, 20 renal deficiency), medications (particularly antibiotics one month before surgery and 21 immunosuppressive drugs as corticosteroids), rheumatoid arthritis, kind of procedure 22 performed and type of prosthesis, cement use, antimicrobial prophylaxis, date of the 23 admission to the hospital, date of surgery, date of discharge, dates of follow-up, class of 24 wound contamination, American Society of Anaesthesiologist (ASA) score, duration of 25 surgery, prosthesis first implantation or revision, blood transfusion, hematologic diseases and 1 notable blood collection after surgery. The data of nasal samplings and their results were also 2 recorded. In case of SSI, the following data were collected: date of infection, localization of 3 infection (superficial or deep), clinical symptoms, microbiological identification and 4 treatment. 5 6 Outcomes 7

The primary outcome was to identify risk factors of SSI due to S. aureus by using 8 multivariate analysis with special emphasis to the variable "nasal carriage of S. aureus". The 9 secondary outcomes were (i) to evaluate the proportion of S. aureus strains isolated in a same 10 patient from the nose and SSI found similar by microbiological methods, (ii) to estimate the 11 frequency of S. aureus nasal carriage in our cohort of patients, and (iii) to estimate the risk of 12 S. aureus SSI. 13 14

Statistical analyses 15

We estimated that SSI would occur in 1% of hip prosthesis surgery and in 2% of knee 16 prosthesis surgery and that the proportion of S. aureus responsible for infection would be 40 17 %. Overall, we calculated that 4200 patients were needed for this study to detect a 20% 18 difference of S. aureus nasal carriage between patients without and with SSI due to S. aureus, 19 given a two-tailed alpha level of 5% and a statistical power of 80%. It was hypothesized that 20 an attending number of 40 SSI due to S. aureus would give sufficient discriminative power to 21 compare pairs of strains (nose and SSI) by molecular techniques. 22

The software used for the collection of the recorded data was Epi-info, version 6.04d Fr 23 (CDC-WHO). The SPSS software, version 16.0 (Chicago, Illinois, USA) was used for 24 univariate and multivariate analyses. Windows SAS® 9.1 software was used to adjust for 25 cluster effect in the multivariate analysis. The rates of SSI and SSI due to S. aureus were 1 calculated globally and according to patients' nasal carriage of S. aureus and to the type of 2 device implemented. These rates were also stratified according to the NNIS index. 3 Fisher's exact test was used for analysis of categorical variables. The Wilcoxon non 4 parametric test was used for mean comparisons. Two multivariate analyses were carried out to 5 evaluate (i) risk factors for SSI and (ii) risk factors for SSI due to S. aureus. To adjust for 6 confounding factors, variables with a P value below the 0.05 significance level in univariate 7 analysis were entered into a multiple logistic regression model. Because of the low number of 8 SSI infections, the multivariate analysis was run with a maximum of 3 covariates selected 9 among the most significant in the univariate analyses. P values below the 5% level were 10 considered statistically significant. 11 12

Results

14

Demographic and clinical data 15

The total duration of the study was 4 years (3 years of inclusion and one year of 16 supplementary follow-up; end at January 2008). Four thousand and forty-six patients were 17 included and 3908 (96.6%) patients were evaluated. Demographic characteristics of the 18 patients are shown in Table I. The causes for exclusion (138 patients) are described in Figure 19 1. Seventy-seven patients (2%) were considered as victims of SSI. The overall infection rate 20 was 2.2% for total hip replacement, 1.7% for knee replacement and 1.6% for partial prosthesis 21 and fracture-fixation devices. 22 23 24 25

Microbiological data 1

The most common infecting micro-organisms were coagulase-negative Staphylococcus spp. 2 [0.9% of patients], followed by Staphylococcus aureus [0.6%], and Gram negative bacteria 3 [0.4%]. Of these infections, 61% arose in the first three months following surgery. There was 4 no significant change in the infection rate or type of infecting micro-organism over the course 5 of the study. As shown in Table II, 94 micro-organisms were found responsible for SSI. 6

Fourteen SSI were polymicrobial. 7

Twenty-two SSI due to S. aureus were documented; 13 of these patients were classified as 8 nasal non carriers and 9 as nasal carrier. 9

Within the strains isolated from the 13 patients classified as nasal non carriers, 8 were 10 available for typing by PFGE. All of these strains exhibited independent profiles (data not 11 shown). The 5 remaining strains were isolated from patients hospitalized in 5 different 12 centres, excluding cross transmission. 13

In patients classified as nasal carriers, the 9 couples of strains isolated from SSI and nasal 14 specimen were compared according to their genotype (4 cases) and/or their antimicrobial 15 susceptibility (5 cases). From the 4 pairs of strains compared by PFGE, 2 pairs exhibited 16 different profiles whereas 2 other pairs were shown to share a similar profile according to 17 Tenover's criteria (Figure 2). All of these strains were different from those isolated from 18 infected patients classified as non carriers (data not shown). For the 5 remaining cases, nasal 19 samples were not available for typing in 2 cases and, in 3 cases, strains recovered from the 20 surgical site were not stored because isolated in laboratories located outside an university 21 hospital; when compared according to antibiotypes, one of these 5 pairs was classified 22 different whereas the four others were classified similar because of identical or minor 23 differences in antimicrobial susceptibility (see footnotes of Table III

for details). 24

Overall, by combining the results of genotypic and phenotypic typing methods, from a total of 1 22 S. aureus SSI, no S. aureus nasal carriage was noted in 13 patients; in the 9 remaining 2 patients, 3 pairs of strains were different and 6 pairs were similar (Table III). So, with the 3 hypothesis of maximum bias, at least 16 of the 22 cases of S. aureus SSI were independent of 4 nasal carriage of S. aureus at the time of surgery. 5 6 SSI and nasal carriage 7 S. aureus was isolated in the nose of 790 patients at the time of surgery (20.2%). Methicillin 8 resistant S. aureus (MRSA) strains were isolated in only 0.6% of patients. The overall risk of 9 SSI was measured at 2% (CI 95% [1.6 -2.4], range 0 -7.6 according to the centre). The 10 prevalence of SSI due to S. aureus was 0.6 % (CI 95% [0.36 -0.84]). 11 12

Risk factors for SSI 13

Univariate and multivariate analyses were performed to assess the risk factors for SSI (Table 14 IV). In multivariate analysis, significant risk factors were tobacco use, wound haematoma and 15 NNIS score for the occurrence of SSI., nasal carriage together with tobacco use and NNIS 16 score for SSI due to S. aureus (Table IV). 17 The present study is, to our knowledge, the largest evaluation of nasal carriage of S. aureus in 22 patients undergoing orthopaedic surgery. It confirms that nasal carriage of S. aureus at the 23 time of surgery is a risk factor for S. aureus SSI in orthopaedic surgery. However 13 out of 22 24 S. aureus SSI occurred in non carriers, and the majority (16/22) of S. aureus SSI were 25 independent of nasal carriage, suggesting an exogenous pathway of contamination. Recently, 1 two studies performed in cardiothoracic surgery [19] and in general surgery [20] showed that 2 endogenous nasal contamination was the major acquisition pathway for methicillin-3 susceptible S. aureus (MSSA), whereas exogenous acquisition pathway was essential for 4 methicillin-resistant S. aureus (MRSA), underscoring the crucial role of hospital infection 5 control measures. 6

Several criticisms can be addressed to our study. First, we have limited the sampling to the 7 anterior nares since this site is considered to be the primary colonization site of S. aureus [21, 8 22]. Recently [23], a large study has highlighted the importance of the throat as a significant 9 site of S. aureus carriage, with an additional sensitivity of 25.7% when combined to nasal 10 sampling. It confirmed, however, that the anterior nares are the most colonized site with S. 11 aureus. Our protocol included initially a rectal swabbing before surgery; however, since many 12 patients denied this sample, the data regarding rectal specimens could not be taken into 13 consideration. With a single nasal sample and in the absence of additional throat and rectal 14 specimens, the frequency of carriage of S. aureus in our cohort was certainly underestimated, 15 as illustrated by the figure of 20.2% of nasal carriers in this study compared to 37.1% in the 16 one of Mertz et al [23] that combined nasal and throat samplings. Second, we obtained only 17 one qualitative (and not quantitative) nasal sample from each patient. However, the most 18 predictive factor for S. aureus infections is the persistent carriage pattern [24]. Getting up to 19 seven nasal swab cultures to accurately segregate non-carriers from intermittent carriers, as 20 suggested by Nouwen et al. [25], was not practically feasible for such a large effective. 21 Indeed, most S. aureus screening programs require only one swabbing of the anterior nares [8, 22 13, 15]. Third, with an end-point at 1 year after the device implantation, we missed late 23 infections (> 2 years) [26]. However, the latter infections are not systematically attributed to a 24 per-operative contamination and are mostly caused by organisms other than S. aureus [4]. 25 Fourth, and this is the major weakness of our study, many strains of S. aureus were not 1 available for molecular typing because infection was often detected outside the surgical 2 setting and the corresponding laboratory omitted to keep the strain. 3 Despite these criticisms, in our study, with the hypothesis of maximum bias, at least 16 of the 4 22 cases of S. aureus SSI were not related to S. aureus nasal carriage before surgery. The 5 origin of S. aureus may be another site of carriage, such as throat [23], perineum or digestive 6 tract [27,28]. Indeed, throat or intestinal carriage without nasal carriage occurs relatively 7 frequently [23,27], which can provide a rationale to investigate this reservoir for 8 endogenous origin of S. aureus infection. Orthopaedic SSI can also result from exogenous 9 transmission, as it has been already shown, notably from the hospital environment and from 10 health-care workers [29,30]. It is noteworthy that, in our study, no cross transmission of S. 11 aureus within centres was documented. Our results could explain why in general surgery [9] 12 and in orthopaedic surgery [15], randomised controlled trials with mupirocin, eliminating 13 solely the nasal carriage, were unable to reduce S. aureus SSI. Concerning the strategies to 14 prevent SSI, in the more recent SHEA/IDSA practice recommendations, the routine screening 15 for S. aureus carriage or attempts to decolonize surgical patients with an antistaphylococcal 16 agent in the preoperative setting remains unsolved issues [31]. 17

In conclusion, nasal carriage of S. aureus at the time of surgery, as investigated in this study, 18 is a risk factor for S. aureus SSI in orthopaedic surgery. However, screening for S. aureus of 19 the nares only, did not reliably predict S. aureus SSI. In most cases of S. aureus SSI, either an 20 endogenous origin could not be demonstrated, or pre-operative nasal colonisation retrieved a 21 strain different from the infecting pathogen.

Reply to reviewers

Thank you for these interesting comments. Please find below our answers to the queries of the two reviewers.

Reviewer 1

It is unfortunate that they did not screen other sites such as the throat and perineum.

We agree with this comment. Initially, the protocol included a rectal swabbing but many patients were reluctant to this proposal. Due to a high rate of missing data during the initial step of the study, we decided to stop the sampling of this site. The following sentence was added in the discussion section of the new manuscript (page 11; lines 12 to 14): "Our protocol included initially a rectal swabbing before surgery; however, since many patients denied this sample, the data regarding rectal specimens could not be taken into consideration". Concerning the throat sampling, when the study was designed in 2002, sampling this site was not recommended since this specimen was not considered at this time to increase significantly the sensitivity of the recovery of S. aureus from the upper respiratory tract as shown later by Metz et al. in 2007 [ref 23] (this point is discussed page 11, lines 14 to 17).

They could point out that sole carriage at these sites is rare (perhaps 10%) and add this to their estimates of the possible size of the problem (20% larger!). Nasal carriage rather than carriage should be in the text.

We also agree with this comment, which is emphasised in the new discussion section (page 11; lines 14 to 17): "With a single nasal sample and in the absence of additional throat and rectal specimens, the frequency of carriage of S. aureus in our cohort was certainly underestimated, as illustrated by the figure of 20 The goal of the study was to address the potential link between S. aureus nasal carriage and occurrence of S. aureus SSI, and not to study CNS or other bacterial cross infections. Strains other than S. aureus were not kept for further analysis. By contrast, the potentiality of cross infections with S. aureus was investigated and presented with some details in the revised manuscript (see below).

3) S aureus data. We need to see their rule set for deciding why the extra 5 strains were different or similar. This is a difficult area and must be explained.

The way used to compare antibiotypes is reported in the method section (page 6, lines 12 to 16). As required by the referee, additional information regarding the comparison of antibiotic profiles has been added in the footnotes of Table III.

They should compare the 13 SA from non carriers (and indeed the carriers) to see if there are possible issues of cross infection in terms of time person and place of the affected patients. Are these strains similar by PFGE? Are they similar to any of the carriers?

From the 13 S. aureus SSI strains isolated from non nasal carriers, 8 were available for comparison by PFGE: all these strains exhibited independent profiles, also different from those of strains isolated from the nose of colonised patients. The 5 remaining strains were isolated from non nasal carriers hospitalized in 5 different centres, excluding cross transmission. These data were added in the result section page 9 lines 10 to 13. Because of the low number of SSI infections, the multivariate analysis was run with a maximum of 3 covariates selected among the most significant in the univariate analyses. This sentence was added in the method section (page 8 lines 8 to 10). We also modified the sentence presenting Table IV (page 10 lines 14 to 15).

4) There are also the therapeutic issues raised by the AST results for all categories of infections above. These are perhaps a little peripheral but I think it would add to the paper or perhaps be another one if there are clinical outcome data?

Data regarding therapeutic issues are not presented in this paper. Another clinical paper focusing on these results is in preparation Other points How were nasal swabs taken: were the swabs moistened?

The precision regarding moistened transport medium was added in the method section (page 5, line 1).

Was the PFGE protocol validated to be able to distinguish strains and how?

The criteria for PFGE interpretation are described page 6 lines 10 to 12 "For a same patient, according to Tenover's criteria [18], two S. aureus strains were classified as epidemiologically distinct if a difference of more than three bands was ascertained between the PFGE profiles performed in a same run".

Reviewer 2

First this was a multicenter study and in these kind of studies cluster effects may occur. In the multivariate analysis, adjustment for cluster effects was not included. This would strengthen the conclusions.

As pointed by the reviewer it is important to look for cluster effect. This was investigated by adding a "centre" variable in the different statistical analyses. This variable did not remain in the final model used for multivariate analysis. But as required by the referee, we performed another multivariate analysis using windows SAS® 9.1 software to adjust for cluster effect. This was added in the method section (page 7, line 25). We replaced the results of the previous multivariate analysis performed using SPSS software by the results of this multivariate analysis adjusted for cluster effect (Table IV).

Second, the authors conclude that the majority of the S. aureus strains are of exogenous origin. They put it quite strong in the conclusions that 'at least 16 were not endogenous'. In my opinion this is not correct. The nasal cultures were negative but this may have been a false-negative result, e.g. due to the sampling technique. As the carriage rate was only 20% it is clear that the culture technique was not very sensitive (normally 30% of the population is nasal carrier). Therefore, I suggest to modify this conclusion.

In the majority of cases an endogenous origin could not be demonstrated would be more correct.

We agree with this comment. The discussion section was amended accordingly (see answer to reviewer 1 and additional sentence in page 11; lines 14 to 17)).

As suggested by the referee, we also modified the conclusion; the sentence "In most cases of S. aureus SSI, pre-operative nasal colonization was not documented or retrieved a different strain from the infecting pathogen" was replaced by the following one in the new manuscript (page 12, lines 20 to 22 and in the abstract): "In most cases of S. aureus SSI, either an endogenous origin could not be demonstrated, or pre-operative nasal colonisation retrieved a strain different from the one recovered from the surgical site" Also, it would be important to provide information on the typing of the strains from non-carriers as well. I wonder whether there were many similar strains within centers suggesting a common source?

This point was also addressed by reviewer 1. No cross transmission of S. aureus was documented in this cohort. This important point was added in the result and discussion section, page 9 lines 10 to 13. The following sentence was added page 12 line 11 to 12 "It is noteworthy that, in our study, no cross transmission of S. aureus within centres was documented".

Figure

  Figure

Definitions 1 7

 1 Nasal carriage of S. aureus: 2Carriage was considered if a strain of S. aureus was recovered at one sampling. Patients 3 without documented S. aureus carriage were classified non carriers. 4SSI: 5Definitions for SSI were those edited by the Centers for Disease Control (CDC)[17]. 6Classification of pairs of S. aureus strains isolated from the nose and the SSI in a same 8 patient 9

  Further studies are necessary to elucidate the role 22 of extra-nasal S. aureus carriage and the potential role of exogenous sources of contamination 23 in elective orthopaedic surgery. As pointed out by our results, non S. aureus nasal carriers 24 must also be taken into account arguing for the importance of implementing robust standard have contributed to this clinical trial. The authors thank : the University Hospital of 19 Saint-Etienne (MH.Fessy 1 , F.Farizon 1 , AC.Vautrin 2 ); the "Clinique Mutualiste" of Saint-Etienne (JP.Passot 1 , 20 JP.Fayard 1 , F.Chalencon 1 , JM.Semay 1 , L.Dupre La Tour 1 , L.Beguin 1 , R.Meley 2 ); the "Centre Hospitalier Privé de 21 la Loire of Saint-Etienne" (A.Mesguich 1 , DG.Avet 1 , PH.Charlier 1 , M.Rousson 2 ); the "Clinique du Parc" of Saint-22 Etienne (L.Charmion 1 , E.Puech 2 ); the Military Hospital Desgenettes of Lyon (JP.Romanet 1 , F.Rongiéras 1 , 23 P.Lainé 1 , F.Chauvin 1 , M.Branfaux 1 , B.Chevalier 2 ); the University Hospital of Lyon (J.Bejuin-Hugues 1 , JP.Carret 1 , 24 P.Neyret 1 , F.Lecuire 1 , J.Rubini 1 , M.Basso 1 , I.Benareau 1 , P.Girardo 2 , S.Tigaud 2 , J.Carrere 2 , C.Galabert 2 , D. 25 Peyramond 3 ); the "Clinique Sainte-Anne" of Lyon (M.Bonnin 1 , JP.Hager 1 , P.Chambat 1 , A.François 2 ); the 26 University Hospital of Grenoble (JP.Stahl 1 , P.Merloz 1 , J.Croize 2 , JP Stahl 3 ); the "Clinique des Cévennes" of 27 Annonay (A.Mironneau 1 , E.Forestier 2 ); the Hospital of Montbrison (P.Girardin 1 , Dr F.Crepet 2 ); the Hospital of 28 Roanne (A.Durand 1 , W.Tohoubi 1 , Dr D.Raou 1 , Dr M.Boyer 2 ); the "Clinique Orthopédique Médico-Chirurgicale" 29 of Dracy-le-Fort (Dr P.Deroche 1 , G.Deschamps 1 , S.Rizk 1 , C.Chol 1 , P.Ragois 1 , C.Pomel 2 ); the University Hospital 30 of Nimes (G.Asencio 1 , R.Bertin 1 , N.Bouziges 2 , A.Sotto 3 ); the "Clinique Clairval" of Marseille (D.Prost 1 , 31 JF.Thiery 1 , M.Chickly 1 , A.Merabet-Sigwalt 2 ) and the University Hospital of Nice (P.Boileau 1 , G.Armando 1 , 32 H.Carsenti 2 , P.Dellamonica 3 ).

  particularly Nicolas Eydoux, Estelle Badel and Fabrice Di Palma for the collection and 37 recording of data. 38 39 1 = Orthopedic Surgeon; 2 = Microbiologist; 3 = Infectious Diseases Consultant 40 41 Contributors 1 PB, CC and FL participated in the design of the study, in the collection, analysis, and interpretation of the clinical 2 data, in the writing of the report and the decision to submit the paper for publication. FG and BP participated in the 3 collection, analysis and interpretation of the microbiological data. JPP, JPF, JB, MHF, FF participated in various 4 aspects of trial conduct and patient referral. All authors had full access to all of the data in this study and take 5 complete responsibility for the integrity of the data and the accuracy of the data analysis. MG, Bonten MJ, Hayden MK (1996) Rapid preparation of bacterial DNA for pulsed-field gel 1 electrophoresis. J Clin Microbiol. 34:2598-600. 2 3 17. Horan TC, Gaynes RP, Martone WJ, Jarvis WR, Emori TG (1992) CDC definitions of nosocomial 4 surgical site infections, 1992: a modification of CDC definitions of surgical wound infections. FC, Arbeit RD, Goering PA , Mickelsen PA, Murray BE, Persing DH, Swaminathan B (1995) 8 Interpretating chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis : 9 criteria for bacterial strain typing. J Clin Microbiol 33:2233-9. 10 11 19. San Juan R, Chaves F, Lopez Gude J, Díaz-Pedroche C, Otero J, Cortina Romero JM, Rufilanchas JJ, 12 Aguado JM (2007) Staphylococcus aureus poststernotomy mediastinitis: description of two distinct 13 acquisition pathways with different potential preventive approaches. J Thorac Cardiovasc Surg 104:670-S, Huttner B, Gervaz P, Fankhauser C, Chraiti MN, Schrenzel J, Licker M, Pittet D (2008) 17 Risk factors for methicillin-resistant Staphylococcus aureus surgical site infection. Infect Control Hosp 18 Epidemiol 29: 890-3. 19 20 21. Kluytmans J, van Belkum A, Verbrugh H. Nasal carriage of Staphylococcus aureus: epidemiology, 21 underlying mechanisms, and associated risks (1997) Clin Microbiol Rev 10:505-20. 22 23 22. Lowy FD (1998) Staphylococcus aureus infections. N Engl J Med 339:520-32. 24 25 23. Mertz D, Frei R, Jaussi B, Tietz A, Stebler C, Flückiger U, Widmer AF (2007) Throat swabs are 26 necessary to reliably detect carriers of Staphylococcus aureus. Clin Infect Dis 45:475-7 27 28 24. Van Belkum A, Verkaik NJ, de Vogel CP, Boelens HA, Verveer J, Nouwen JL, Verbrugh HA, 29 Wertheim HF (2009) Reclassification of Staphylococcus aureus nasal carriage types. J Infect Dis.

Figure 1 : 4 Figure 2 :

 142 Figure 1: Frequency of surgical site infection according to the type of prosthesis implemented 3

Table I :

 I Main characteristics of the 3908 patients included in the study according to the type of osteosynthetic surgery

	Type of osteosynthetic surgery Hip prosthesis	Knee	Uni	Others	Total
				prosthesis	compartmental
					knee prosthesis
	Number of patients (%)	2321 (59.4%)	1162 (29.7%)	292 (7.5%)	133 (3.4%)	3908 (100%)
	Mean age (+/-SD)	67.7 (+/-10.8)	72.9 (+/-7.9)	72.7 (+/-7.2)	76.6 (+/-10.3)	69.9(+/-10.0)
	Male (%)		1121 (48.3%) 408 (35.1%)	91 (31.2%)	58 (43.0%)	1678 (43.0%)
	Diabetes (%)	211 (9.0%)	159 (13.7%)	37 (12.7%)	13 (9.6%)	420 (10.7%)
	Immunocompromised patients a 5 (0.2%)	5 (0.4%)	1 (0.3%)	0 (0%)	11 (0.3%)
	Cigarette use	445 (19.2%)	152 (12.9%)	28 (9.6%)	17 (12.6%)	642 (16.4%)
	Rheumatoid polyarthritis	50 (2.1%)	31 (2.7%)	2 (0.7%)	7 (5.2%)	90 (2.3%)
	Antibiotic use one month prior	97 (4.2%)	49 (4.2%)	10 (3.4%)	8 (5.9%)	164 (4.2%)
	Corticosteroids use	75 (3.2%)	35 (3.0%)	6 (2.1%)	6 (4.4%)	122 (3.1%)
	Cancer		227 (9.8%)	130 (11.2%)	35 (12.0%)	16 (11.9%)	408 (10.4%)
	Renal disease	8 (0.3%)	2 (0.2%)	0 (0%)	0 (0 %)	10 (0.3%)
	Bacteriuria	50 (2.2%)	23 (2.0%)	4 (1.4%)	3 (2.2 %)	80 (2.1%)
	Blood transfusion	182 (7.8%)	156 (13.4%)	1 (0.3%)	24 (17.8%)	363 (9.3%)
	Notable hematoma	28 (1.2%)	16 (1.4%)	4 (1.4%)	0 (0%)	48 (1.2%)
	Antimicrobial prophylaxis	2310 (99.5%) 1159 (99.7%) 292 (100%)	133 (100 %)	3895 (99.7%)
	Cement use for prosthesis
	1-cement		58 (2.5%)	418 (36.0%)	25 (8.6%)	47 (33.8%)	548 (14.0%)
	2 -partially cemented	248 (10.7%)	359 (30.9%)	7 (2.4%)	9 (6.8%)	623 (16.0%)
	3 -no cement	2017 (86.8%)	384 (33.0%)	260 (89.0 %)	76 (57.1%)	2737 (70.0%)
	Primo implementation	2254 (97.1%) 1134 (97.6%) 288 (98.6%)	13 (9.6%)	3689 (94.4%)
	SSI frequency	2.2%	1.7%	0.7%	3.8%	2.0%
	SSI frequency according to
	NNIS index
		0	1.5%	0.9%	1.0%	2.2%	1.3%
	1	3.8%	3.3%	0%	5.3%	3.3%
		2	4.9%	5.6%	0%	20.0%	6.6%
	Frequency of SSI due to S.	0.8%	0.2%	0%	2.3%	0.6%
	aureus
	Frequency of SSI due to S.
	aureus according to NNIS
	index	0	0.3%	0.1%	0%	2.3%	0.3%
		1	2.0%	0.3%	0%	0%	1.3%
		2	1.6%	0%	0%	20.0%	1.9%

a Immunocompromised patients: malignancy, transplantation, immunosuppressive therapy, HIV infection Table

Table II : Microbiological data Bacterium Number

 II

	Coagulase negative Staphylococcus spp.	36
	Methicillin susceptible S. aureus	18
	Methicillin resistant S. aureus	4
	Enterobacteriacae	11
	Enterococcus spp.	7
	Streptococcus spp.	6
	Propionibacterium acnes	3
	Pseudomonas aeruginosa	2
	Corynebacterium spp.	2
	Pseudomonas paucimobilis	1
	Clostridium perfringens	1
	Fusobacterium spp.	1
	Peptostreptococcus spp.	1
	Acinetobacter spp.	1
	Including polymicrobial infections	14

Table III :

 III Detailed results of the 22 cases of SSI due to S. aureus and their relationship with nasal carriage. Figures in brackets correspond to the numbering of S. aureus isolates in Fig.2b patient 3 harbored nasal and SSI MRSA strains that were both sensitive to all aminoglycosides but resistant to erythromycin and lincomycin. c patient 5 harbored distinct S. aureus strains: the nasal strain was only resistant to penicillin G and intermediate to norfloxacin whereas the SSI strain was resistant to penicillin G and erythromycin and sensitive to norfloxacin. d patient 11 harbored nasal and SSI MSSA strains that were both sensitive to all tested antibiotics except penicillin G. e patient 18 harbored nasal and SSI MSSA strains both only resistant to penicillin G. f patient 19 Nasal strain was only resistant to penicillin G although the SSI strain was resistant to penicillin G and intermediate to pristinamycin

	Patient	Type of surgery	No. of days between surgery and SSI	Bacterial strain responsible for SSI a	Nasal sampling before surgery a	Other nasal samplings a	Classification of pairs of S. aureus isolated from the nose and the SSI
	1	Hip prosthesis	15	MSSA (I4)	Positive (N3)		different (PFGE and AP)
	2	Knee prosthesis	15	MSSA	Negative
	3	Hip prosthesis	26	MRSA	Positive	positive one month after SSI diagnosis	similar (AP) b
	4	Hip prosthesis	22	MSSA	Negative
	5	Hip prosthesis	25	MSSA	Positive	positive 2 months after SSI diagnosis	different (AP) c
	6	Hip prosthesis	25	MSSA	Negative	positive at the time of SSI diagnosis
	7	Hip prosthesis	30	MSSA	Negative
	8	Hip prosthesis	20	MRSA	Negative
		Partial hip
	9	prosthesis	24	MSSA	Negative
		replacement
	10	Hip prosthesis	19	MSSA	Negative
	11	Hip prosthesis	22	MSSA	Positive	positive 2 months after SSI diagnosis	similar (AP) d
	12	Hip prosthesis	43	MSSA (I9)	Positive (N8)	nose sampling number 2 positive	similar (PFGE and AP)
	13	Hip prosthesis	35	MSSA	Negative
	14	Hip prosthesis	20	MSSA	Negative
	15	Hip prosthesis	23	MSSA (I2)	Positive (N1)		similar (PFGE and AP)
	16	Hip prosthesis	62	MSSA	Negative	2 negative samplings
		Knee
	17	prosthesis	82	MRSA	Negative
		replacement
	18	Knee prosthesis	41	MSSA	Positive	positive 13 days after SSI diagnosis	similar (AP) e
	19	Hip prosthesis	42	MSSA	Positive	negative 2 months after SSI diagnosis	similar (AP) f
	20	Hip prosthesis	73	MSSA	Negative
	21	Hip prosthesis	41	MSSA pneumoniae + Klebsiella	Negative
	22	Hip prosthesis	63	MRSA (I7) + Pseudomonas aeruginosa	Positive (MSSA) (N5)	positive MRSA (S6) in stools five days before SSI	Different (PFGE and AP)
	SSI = surgical site infection; AP = antimicrobial profile; MSSA = Methicillin susceptible S. aureus; MRSA =
	Methicillin resistant S. aureus ; PFGE = Pulsed Field Gel Electrophoresis.

a

Table IV :

 IV Univariate and multivariate analysis of risk factors for SSI overall and SSI due to S. aureus Please find enclosed a revised manuscript of our study entitled "Is nasal carriage of Staphylococcus aureus the main acquisition pathway for surgical-site infection in orthopaedic surgery?" Ms. No. EJCMID-D-09-00479 and the answers, point by point, to the queries of the two reviewers that are detailed below. The main changes done are highlighted in yellow in one of the copy of the new manuscript.

	Dear Editor,
	Risks factors	Univariate analysis	Multivariate analysis
	For SSI overall	P	P	Exp (B) CI 95%
	Centre	< 0.01	0.0379	Adjustment factor
	Age We hope that this new version will be found suitable for publication in your journal. 0.03
	BMI I look forward to hearing from you soon. < 0.001
	Tobacco use Sincerely yours,	0.003	0.0018 2.244 [1.352 -3.726]
	Diabetes	0.04
	Cancer Pr Berthelot	0.06
	Corticosteroids MD, PhD.	0.1
	First implantation	0.07
	Duration of surgery	0.004
	Hematoma	0.002	0.0026 4.665 [1.714 -12.695]
	Nasal carriage of S. aureus	0.3
	NNIS	< 0.001	< 0.0001 3.073 [1.874 -5.038]
	ASA score > 2	< 0.01
	For SSI due to S.aureus
	Centre	NS	0.9978	Adjustment factor
	ASA score > 2	< 0.01
	BMI	0.2
	Tobacco use	0.005	0.0024 3.907 [1.621 -9.420]
	Diabetes	0.025
	Cancer	0.02
	Duration of surgery	0.02
	Nasal carriage of S. aureus	0.02	0.0208 2.786 [1.169 -6.640]
	NNIS	< 0.001	0.0007 5.205 [2.013 -13.455]

about CNS data: 1) Table 3 has overall data. They could compare the antibiograms of the individual isolates and see if there are very different strains. I doubt the organisms are available for molecular analysis (if so this could be "future work" for the discussion) but at least the antibiograms could be compared by ward and centre and see if there are any similarities and differences and informed guesses made as to the likelihood of cross infection e.g. quinolone resistance is usually mutational 2) All the other infected organism data. Again what about antibiograms and possible cross infection?

.2% of nasal carriers in this study compared to 37.1% in the one of Mertz et al [23] that combined nasal and throat samplings." Comment 1

Table IV only has death related risk analysis not as stated on page 10 lines 23-24 for SSI and SA nasal carriage.

 IV

The authors have received funding support from the Ministère de la Santé-France-Programme Hospitalier de 21 Recherche Clinique for this study (PHRC Dépistaph). No conflict of interest. No financial interest in a


Dr Michael A Borg
email: michael.a.borg@gov.mt

Are healthcare economics a factor behind European MRSA rates

Keywords: MRSA, infant mortality rate, gross domestic product, Europe, EARSS MRSA, infant mortality rate, gross domestic product, Europe, EARSS behind national European MRSA rates? MRSA, infant mortality rate, gross domestic product, Europe, EARSS

This study assessed the relationship between MRSA prevalence in 26 European countries with two key socioeconomic parameters, namely gross domestic product per capita (GDP) as well as infant mortality rate (IMR). Whilst no significant relationship was identified between MRSA and GDP (Spearman correlation: -0.387; 95%CI: -0.674 to 0.000; p = 0.0528), a significant correlation was evident between MRSA and IMR (Spearman correlation: 0.545; 95% CI: 0.208 to 0.766; p = 0.005). Elimination of a widely outlying data point retained significance. Various studies been shown IMR to be a good marker of healthcare expenditure and it is therefore reasonable to postulate that one possible factor in the multifactorial epidemiology of MRSA in Europe is the level and effectiveness of healthcare expenditure within the individual countries.

Thank you for your letter and request for revision of my manuscript. I have performed a thorough revision based on the referee's comments as well as the specific requests as per below:

Two version are being forwarded; one with track changes to allow you to identify the changes made in line with the referee's requests and one in which the track changes have been removed. I hope that the new amended version will be found suitable for publication.

Yours sincerely

Michael A. Borg

Reviewer #1:

1. EARSS data do not represent MRSA prevalence but only MRSA proportion of Staphylococcus aureus bloodstream isolates, as author correctly has said in the lines 34-36. This should be consistently used throughout the article.

I would respectfully disagree with the referee on this point. The EARSS group has published evidence of a clear correlation between proportion and incidence (de Kraker et al 2006). By extrapolation, I believe that the inferences to MRSA prevalence & incidence

within the manuscript are therefore justified. The reference has been added to the manuscript to clarify similar doubts in the readers.

2. The addition to the abstract of Spearman correlation data after eliminating widely outlying data point would give the reader better insight into the problem even after reading the abstract.

This has been added to the abstract.

Reviewer #3:

One major concern I have with his Methods is the use of multiple linear regression with MRSA proportions as the dependent variable. Observing the X axis of Figure 1, it is clear that MRSA prevalence is not normally distributed and that it is skewed to the right.

Using a non-normally distributed dependent variable in linear regression may lead to false conclusions. The author will need to either transform MRSA proportion into a normally distributed variable, or rely on Spearman's rho alone as his main analyses.

All references to the linear regression calculation have been removed.

The author also notes some valid limitations to his study. One major limitation he does not address is the generalizability of his analyses outside of Europe. For example, the United States has high per capita healthcare expenditures, but a high infant mortality rate relative to per capita GDP and high MRSA prevalence. The author should note this is an additional limitation to his analyses.

I thank the referee for pointing out this valid comment; as a result, the title has been amended to specify the European focus of the paper. A comment has also been added in the discussion.

(MRSA) has increased significantly. One reason for this has been the greater availability of surveillance data at pan-European level through good quality comparable data from the successful European Antimicrobial Resistance Surveillance System (EARSS) [www.rivm.nl/earss] [1]. This preliminary study attempted to determine relationships between MRSA prevalence at country level and two key socioeconomic parameters: gross domestic product per capita (GDP) as well as infant mortality rate (IMR). The GDP of a country is defined as the market value of all final goods and services produced within that nation in a given period of time. It is used by economists as a way of measuring the size of a country's economic strength and purchasing power. IMR refers to the number of newborns dying within their first twelve months of age, divided by the number of live births during that year. IMR has been used as a useful indicator of a country's level of health development; in fact, public spending on health and better access to healthcare facilities are critical determinants of IMR [2,3].

Data was obtained from publically available documents. MRSA figures for the year 2005 were sourced from the EARSS annual report 2005 (National Institute for Public Health and the Environment, Bilthoven, The Netherlands). This reported, amongst others, the proportion of Staphylococcus aureus, isolated from routine blood cultures, that were identified as meticillin resistant by the participating laboratories within the EARSS project during the year 2005. In turn, MRSA proportions have been shown to be strongly correlated to incidence [4]. GDP and IMR statistics for the same year were obtained from the CIA world handbook 2005 (Central Intelligence Agency Washington, USA). Respective data per country were then inputted in Excel (Microsoft Excel 2003, Microsoft Corp) for preliminary evaluation. Spearman correlation was used to compare the results of countries, arranged hierarchically according to their MRSA proportions, versus their respective GDP and IMR statistics. Analyses were performed in Medcalc, version 9.2.1.0 (Medcalc Software, Mariakerke, Belgium). A p value of 0.05 was taken to indicate statistical significance.

MRSA proportions from the 27 countries varied from 0.01 to 71.7% (mean: 23.41%) whilst GDP for the same countries ranged from $8200 to $55600 (mean: $26351). Although the more affluent nations in Europe appeared to be the ones with better MRSA control, a substantial degree of heterogeneity was evident. Several countries with relatively high GDP still had MRSA proportions that were much higher than those for less affluent nations. Rank correlation between MRSA proportions for the different European countries and the GDP per capita for that same country yielded a Spearman correlation index (rho) of -0.387 (95%CI: -0.674 to 0.000) and a p-value of 0.0528.

On the other hand, when the same MRSA proportions were plotted against IMR, goodness of fit was much more in evidence with a substantial proportion of the points falling close to the line of best fit (Figure 1). This was confirmed by a Spearman correlation index (rho) of 0.545 (95% Confidence Interval for rho = 0.208 to 0.766) and p=0.005. In order to ensure that the widely outlying data point was not unduly influencing the correlation, the analysis was repeated by eliminating this data set from the analysis. Nevertheless, a statistically significant result was still obtained, albeit slightly inferior to that of the complete range; the Spearman's coefficient of rank correlation (rho) = 0.491 (95% Confidence Interval for rho = 0.127 to 0.738) and a p-value of 0.014.

The lack of significant relationship between the levels of GDP and MRSA proportions in the individual countries suggests that economic strength and purchasing power does not automatically result in improved prevention of healthcare associated infections such as MRSA. This is not surprising since the mere economic affluence of a nation would not necessarily translate into better healthcare outcomes unless that same economic effluence is channelled appropriately into effective expenditure and use. It is in this context that the strong relationship between IMR rates and MRSA proportions in European countries is very interesting. Indeed, Elola et al found that infant mortality was a better predictor of health care expenditure than was GDP [START_REF] Elola | Health indicators and the organization of health care systems in Western Europe[END_REF]. Various elements of health care expenditure and quality have been linked to IMR by other authors. Babazono and Hillman suggested that perinatal and infant mortality was related to the number of hospital beds [2], whilst the number of physicians per capita was found to be a significant variable for infant mortality in 12 countries within the Organisation for Economic Co-operation and Development (OECD) [6].

Furthermore, Wolf and Gabay showed that increased medical expenditure led to improvements in several health outcomes, including infant mortality rate [7]. This would be more relevant in a region or continent, such as Europe, where public health has made substantial progress over the past century. To a reasonable degree, these countries exhibit are no cataclysmic shifts in general living conditions between each other, as evident from the GDP per capita of all the studied countries, which was within 2 standard deviations of the median. In such a background, a major input to reducing infant mortality would therefore be the ability to increase survival in low birthweight infants where improved neonatal and obstetric services are important contributors [8].

Just as it seems to be a determinant for IMR, the degree of investment in healthcare systems would almost certainly play a critical role in determining the success or otherwise of MRSA control in the different European countries. Whilst direct references to the role of health care expenditure for improved MRSA control at a pan-national level have not been highlighted in medical literature, both capital and manpower investment within hospitals are critical to obtain effective MRSA control [9].

Despite the rationality of this argumentation, it would be unacceptably simplistic to assume that the only determinant for successful MRSA control is just the level and effectiveness of funds pumped into the country's healthcare system. This is evident from the correlation coefficient of 0.545. In addition, both sets of statistics are recognised to possess inherent sources of error. There is considerable debate on the different practices and cut-offs to define infant mortality in different countries, which of course have implications on the rate itself [10]. The MRSA proportions reported by EARSS are based on diverse population coverage in each of the participating European country.

Potential bias may be present if the proportion of the population under study is either low or partial. Furthermore the model may not be applicable in other continents outside of Europe, especially if the GDP varies substantially from the European median.

Nevertheless, despite these limitations, the identified correlation between MRSA and IMR cannot be easily ignored and would justify a more extensive study into health care expenditure and utilisation and its impact on MRSA and other healthcare associated infections. MRSA proportions from the 27 countries varied from 0.01 to 71.7% (mean: 23.41%) whilst GDP for the same countries ranged from $8200 to $55600 (mean: $26351). Although the more affluent nations in Europe appeared to be the ones with better MRSA control, a substantial degree of heterogeneity was evident. Several countries with relatively high GDP still had MRSA proportions that were much higher than those for less affluent nations. Rank correlation between MRSA proportions for the different European countries and the GDP per capita for that same country yielded a Spearman correlation index (rho) of -0.387 (95%CI: -0.674 to 0.000) and a p-value of 0.0528.

On the other hand, when the same MRSA proportions were plotted against IMR, goodness of fit was much more in evidence with a substantial proportion of the points falling close to the line of best fit (Figure 1). This was confirmed by a Spearman correlation index (rho) of 0.545 (95% Confidence

Interval for rho = 0.208 to 0.766) and p=0.005. In order to ensure that the widely outlying data point was not unduly influencing the correlation, the analysis was repeated by eliminating this data set from the analysis. Nevertheless, a statistically significant result was still obtained, albeit slightly inferior to that of the complete range; the Spearman's coefficient of rank correlation (rho) = 0.491 [9]. These include the availability of sufficient beds to avoid overcrowding [8], as well as the provision of the sufficient resources, such as alcohol rub, to maximize hand hygiene and therefore improve MRSA control [9].

(
Despite the rationality of this argumentation, it would be unacceptably simplistic to assume that the only determinant for successful MRSA control is just the level and effectiveness of funds pumped into the country's healthcare system. This is evident from the correlation coefficient of 0.545. In addition, both sets of statistics are recognised to possess inherent sources of error. There is considerable debate on the different practices and cut-offs to define infant mortality in different countries, which of course have implications on the rate itself [10]. The MRSA proportions reported by EARSS are based on diverse population coverage in each of the participating European country.

Potential bias may be present if the proportion of the population under study is either low or partial.

Furthermore the model may not be applicable in other continents outside of Europe, especially if the GDP varies substantially from the European median.

Nevertheless, despite these limitations, the identified correlation between MRSA and IMR cannot be easily ignored and would justify a more extensive study into health care expenditure and utilisation so as to develop possible causal models which estimate more effectivelyand its the impact of distribution of income and services on MRSA and other healthcare associated infections.

Formatted: Indent: Left: 0"

Figure 1 :

 1 Figure 1: Scatterplot of Infant Mortality rate (IMR) per thousand live births vs %MRSA proportion (MRSA) for countries participating in the EARSS network in 2005 with line of best fit (solid line) and 95% confidence intervals (dotted lines)

  Data was obtained from publically available documents. MRSA figures for the year 2005 were sourced from the EARSS annual report 2005 (National Institute for Public Health and the Environment, Bilthoven, The Netherlands). This reported, amongst others, the proportion of Staphylococcus aureus, isolated from routine blood cultures, that were identified as meticillin resistant by the participating laboratories within the EARSS project during the year 2005. In turn, MRSA proportions have been shown to be strongly correlated to incidence [4]. GDP and IMR statistics for the same year were obtained from the CIA world handbook 2005 (Central Intelligence Agency Washington, USA). Respective data per country were then inputted in Excel (Microsoft Excel 2003, Microsoft Corp) for preliminary evaluation. Spearman correlation was used to compare the results of countries, arranged hierarchically according to their MRSA proportions, versus their respective GDP and IMR statistics. Multiple linear regression was then carried out using the backward stepwise technique, utilizing MRSA proportions as the dependant variables. Analyses were performed in Medcalc, version 9.2.1.0 (Medcalc Software, Mariakerke, Belgium). A p value of 0.05 was taken to indicate statistical significance.

Figure 1 :

 1 Figure 1: Scatterplot of Infant Mortality rate (IMR) per thousand live births vs %MRSA proportion (MRSA) for countries participating in the EARSS network in 2005 with line of best fit (solid line) and 95% confidence intervals (dotted lines)

*Manuscript Revised with Track changes Click here to download Manuscript: MRSA vs IMR paper -EJCMID revised TC.doc Click here to view linked References

  IMR). The GDP of a country is defined as the market value of all final goods and services produced within that nation in a given period of time. It is used by economists as a way of measuring the size of a country's economic strength and purchasing power. IMR refers to the number of newborns dying within their first twelve months of age, divided by the number of live births during that year. IMR has been used as a useful indicator of a country's level of health

	Summary: Over recent years, public awareness and interest in meticillin resistant Staphylococcus aureus
	(MRSA) has increased significantly. One reason for this has been the greater availability of
	This study assessed the relationship between MRSA prevalence in 26 European countries with two
	surveillance data at pan-European level through good quality comparable data from the successful
	key socioeconomic parameters, namely gross domestic product per capita (GDP) as well as infant
	European Antimicrobial Resistance Surveillance System (EARSS) [www.rivm.nl/earss] [1]. This
	mortality rate (IMR). Whilst no significant relationship was identified between MRSA and GDP
	preliminary study attempted to determine relationships between MRSA prevalence at country level
	(Spearman correlation: -0.387; 95%CI: -0.674 to 0.000; p = 0.0528), a significant correlation was
	and two key socioeconomic parameters: gross domestic product per capita (GDP) as well as infant
	evident between MRSA and IMR (Spearman correlation: 0.545; 95% CI: 0.208 to 0.766; p = 0.005).
	mortality rate (
	Elimination of a widely outlying data point retained significance. Various studies been shown IMR to
	be a good marker of healthcare expenditure and it is therefore reasonable to postulate that one
	possible factor in the multifactorial epidemiology of MRSA in Europe is the level and effectiveness of
	healthcare expenditure within the individual countries.

development; in fact, public spending on health and better access to healthcare facilities are critical determinants of IMR

[2,3]

.

  It is in this context that the strong relationship between IMR rates and MRSA proportions in European countries is very interesting. Indeed, Elola et al found that infant mortality was a better predictor of health care expenditure than was GDP[45]. Various elements of health care expenditure and quality have been linked to IMR by other authors. Babazono and Hillman suggested that perinatal and infant mortality was related to the number of hospital beds[2], whilst the number of physicians per capita was found to be a significant variable for infant mortality in 12 countries within the Organisation for Economic Co-operation and Development (OECD)[56].Furthermore, Wolf and Gabay showed that increased medical expenditure led to improvements in several health outcomes, including infant mortality rate [67]. This would be more relevant in a region or continent, such as Europe, where public health has made substantial progress over the past century. To a reasonable degree, these countries exhibit are no cataclysmic shifts in general living conditions between each other, as evident from the GDP per capita of all the studied countries, which was within 2 standard deviations of the median. In such a background, a major input to reducing infant mortality would therefore be the ability to increase survival in low birthweight infants where improved neonatal and obstetric services are important contributors[78].Just as it seems to be a determinant for IMR, the degree of investment in healthcare systems would almost certainly play a critical role in determining the success or otherwise of MRSA control in the

	95% Confidence Interval for rho = 0.127 to 0.738) and a p-value of 0.014. Multiple linear regression
	generated a final model in which only IMR was independently and positively associated with MRSA
	proportions (regression coefficient = 2.38; p = 0.005).
	The lack of significant relationship between the levels of GDP and MRSA proportions in the
	individual countries suggests that economic strength and purchasing power does not automatically
	result in improved prevention of healthcare associated infections such as MRSA. This is not
	surprising since the mere economic affluence of a nation would not necessarily translate into better
	healthcare outcomes unless that same economic effluence is channelled appropriately into effective
	expenditure and use.

different European countries. Whilst direct references to the role of health care expenditure for improved MRSA control at a pan-national level have not been highlighted in medical literature, various studies have shown the importance of both capital and manpower investment within individual hospitals are critical to obtain effective MRSA control


Mattia Brambilla
email: brambilla.mattia@gmail.com

Severino Vitulano

Andrea Ferri

Fernando Spina

Ettore Elena Fabbri

Randi M Brambilla

E Randi

What are we dealing with? An explicit test reveals different levels of taxonomical diagnosability in the Sylvia cantillans species complex

Keywords:

Diagnosability is our ability in discriminating between similar taxa, including sibling or cryptic ones. We made an explicit test of diagnosability using the Sylvia cantillans species complex as a model, comparing phenotypic with genetic identifications. Individual warblers sampled during migration in Central Italy were (sub)specifically identified using putatively diagnostic plumage traits. Nucleotide sequences of a fragment (598 bp) of the mtDNA cytochrome b were then used to assign each individual to distinct phylogenetic clades, as determined by reference haplotypes sequenced in breeding individuals, defining well-distinct clades corresponding to known taxa of the complex. The new haplotypes were assigned to one of the previously identified groups (corresponding to three different taxa); no sample was assigned outside them. In contrast, when using plumage traits 11 out of 58 birds were classified as "uncertain/intermediate" among two taxa, while 5 were differently classified under the two methods. Only for one taxon (Sylvia subalpina, syn. S. moltonii) there was a perfect concordance between the two methods. For the other two taxa of the complex, diagnosability is not guaranteed and their field and hand identification should be carefully addressed. Our work provides an example for an explicit test of taxa diagnosability, where the use of two or more "markers" for diagnosis may point out discordant identification and thus miss unambiguous diagnosability. Our results outline the importance of considering different features for taxa diagnosis and point out the weakness of visual appearance-based identification (currently widely used for taxa determination) in our study complex.

Introduction

Evolution promotes speciation, which means increasing diversity through divergence among taxa that become progressively distinguishable to us in genotype and phenotype [START_REF] De Queiroz | The general lineage concept of species and the defining properties of the species category In Wilson RA (ed) Species, New Interdisciplinary Essays[END_REF]. Diagnosability (see [START_REF] Helbig | Guidelines for assigning species rank[END_REF][START_REF] Watson | Diagnosable versus Distinct: Evaluating Species Limits in Birds[END_REF]) is our ability to discriminate between similar taxa based on characters or character state. It is an obvious requirement for taxon distinction, and a necessary condition for assigning species rank (Helbig et al. 2002, and references therein). A taxon is "diagnosable" when individuals of at least one age/sex class can be distinguished from individuals of the same class(es) of all other taxa by at least one qualitative difference, or at least one class is separated by a complete discontinuity in at least one continuously varying character from the same class(es) of other taxa [START_REF] Helbig | Guidelines for assigning species rank[END_REF]. These diagnostic traits may be of various origins, as e.g. external and internal morphology or morphometrics, molecular data and karyotypes, distribution range and habitat preferences, behavioural and reproduction traits [START_REF] Watson | Diagnosable versus Distinct: Evaluating Species Limits in Birds[END_REF]. Suitable diagnostic traits in taxonomy should have strong genetic and low environmental components of variation [START_REF] Helbig | Guidelines for assigning species rank[END_REF].

Diagnosability has often been claimed for assigning taxonomic rank (particularly in birds), and is considered essential to all species concepts at the operational level [START_REF] Li | Species delimitation in the Hwamei Garrulax canorus[END_REF].

However, tests of taxa diagnosability are scarce. In this study we aim to show the potential importance of such a test with a simple worked example. We carried out an explicit test of diagnosability in a complex of closely related taxa and evaluated the reliability of current field diagnosis among the taxa of the complex.

Our model species complex, the subalpine warbler [Sylvia cantillans], is a Mediterranean passerine bird. It has formerly been considered a polytypic species (Cramp 1992), but actually it is better regarded as a complex of different taxa (traditionally ranked as subspecies, but including at least two different species; Brambilla et al. 2008a), closely related to each other but with distinctive characteristics. The main differences among the taxa of the complex rely on contact/alarm calls [START_REF] Brambilla | Quando la voce è tutto: l"identificazione delle sottospecie di sterpazzolina Sylvia cantillans[END_REF] and on plumage, especially underpart colours in males [START_REF] Shirihai | Sylvia Warblers[END_REF]. Song perception has been shown to be the most likely underlying mechanism of reproductive isolation between two taxa of the complex which occur in sympatry and parapatry but do not hybridise (Brambilla et al. 2008a;Brambilla et al. 2008b). Call differences are rather diagnostic and are handily being used to identify three differentiated taxa [START_REF] Shirihai | Sylvia Warblers[END_REF][START_REF] Brambilla | Quando la voce è tutto: l"identificazione delle sottospecie di sterpazzolina Sylvia cantillans[END_REF], of which one (the nominate subspecies S. c. cantillans) has been shown to be polyphyletic, including two genetically well-diverged clades (Brambilla et al. 2008a). In contrast, morphometrics are of rather limited use, with only S. c. albistriata being on average larger than other taxa [START_REF] Shirihai | Sylvia Warblers[END_REF]. Most ringed birds belonging to the S. cantillans group are trapped during migration, when they are rarely vocal, and are currently identified mainly on the basis of their visual appearance. According to literature, males (especially individuals at least two years old) are often easily identified at the (sub)specific level based on their plumage colours, while juveniles and females are often indistinguishable, with rather few exceptions (see [START_REF] Shirihai | Sylvia Warblers[END_REF].

We aimed to assess the reliability of plumage diagnosability in males of the subalpine warbler species complex. Plumage-based recognition has a major role in bird identification, including (sub)specific attribution of taxa belonging to the S. cantillans complex, and thus the implications of our experiments could be useful for field observers and ringers. Moreover, plumage-based identification is particularly interesting for explicitly testing diagnosability, because of its wide use in bird diagnosis. In our study, plumage identification is compared with genetic classification, obtained using independent data (mtDNA haplotypes).

We decided to work on the cytochrome b, one of the most used genes for phylogeny and phylogeography of passerine birds [START_REF] Wink | Use of DNA markers to study bird migration[END_REF], as it was known to show diagnostic variations within our study species complex (Brambilla et al. 2008a). We considered a taxon "diagnosable" if all the individuals classified on the basis of plumage features belong to the relevant mtDNA clade, and vice-versa (all individuals of a mtDNA clade showed the plumage characters of such clade). Otherwise, taxa were considered to be not diagnosable (i.e. when some individuals resulted in discordant identifications).

Methods

Study area and samples collection

All samples used in this study were collected during spring migration at Ventotene island (40.48°N, 13.25°E), a ringing station in the central Tyrrhenian Sea. Samples were collected from 58 trapped individuals, caught through mist netting during the "Progetto Piccole Isole", between 31 st March and 28 th May 2006.

Preliminary data indicate that trapping birds at this stopover site should likely lead to capture the following recognized taxa of the complex: the nominate race Sylvia cantillans cantillans, which breeds in the western Mediterranean to south Italy [START_REF] Shirihai | Sylvia Warblers[END_REF][START_REF] Brambilla | Geographical distribution of Subalpine Warbler Sylvia cantillans subspecies in mainland Italy[END_REF]; now regarded as including two distinct groups: see below and Brambilla et al. 2008a); S. c. albistriata, an eastern subspecies, which breeds from Trieste (NE Italy) to Greece and western Turkey; Moltoni"s warbler S. subalpina (synonym S. moltonii) endemic to a restricted area in the central Mediterranean (Sardinia, Corsica, Balearics, and few regions in CN Italy, [START_REF] Brambilla | Geographical distribution of Subalpine Warbler Sylvia cantillans subspecies in mainland Italy[END_REF][START_REF] Brambilla | Habitat use by Moltoni"s Warbler Sylvia cantillans moltonii in Italy[END_REF]; now considered as a full species, Brambilla et al. 2008a, b). Virtually, we could miss only S. c. inornata, the subspecies inhabiting northern Africa [START_REF] Shirihai | Sylvia Warblers[END_REF], and the western (Spain, France) populations formerly belonging to S. c. cantillans, which are genetically rather diverged from southern (Italian) cantillans, despite morphological and vocal similarity (Brambilla et al. 2008a), and probably perform a more western route towards the breeding grounds, crossing Gibraltar (see Cramp 1992, and references therein; [START_REF] Shirihai | Sylvia Warblers[END_REF].

Studying migrants rather than breeding birds, we cannot a priori presume what (sub)species are we dealing with (with the exception of the contact zone between cantillans and subalpina, all taxa of the complex are mainly allopatric, and thus breeding birds in many cases could be assigned to a given taxon simply based on their geographical origin), hence the (sub)specific identification should not be biased by known distribution data.

Plumage-based (sub)specific attribution of trapped birds A minimum of four pictures were taken for each trapped bird. All pictures were carefully examined and subspecies assigned by judging and comparing the photographs. Birds were (sub)specifically determined based on the features of breast, belly, submoustachial stripe and head (table 1). It should be noted that Italian cantillans breeders are generally similar to western cantillans, but they often have wide white moustache, a rather large white belly patch, sometimes quite dark lores and also slightly washed-out tinge on flanks [START_REF] Brambilla | Distribuzione, ecologia e differenziazione in Sylvia cantillans[END_REF], thus being in these traits a bit more reminiscent of albistriata (although not reaching its typical appearance) with respect to western cantillans; notably, descriptions of cantillans in classical handbook usually refer to the latter populations. Anyway, southern cantillans birds typically have a definitely orange-red tinge on underparts, rather different from the brick-red or vinaceous colour of albistriata, and the red colour on breast is not sharply demarcated against the white belly (Brambilla 2007). However, some birds were classified as uncertain/intermediate, as they appeared somewhat between cantillans and albistriata. In most cases, they had a pronounced submoustachial stripe, a separation between red breast and white belly but just approaching the characteristic neat demarcation of typical albistriata, a deep red (but not vinaceous or brick-red) colouration on throat and breast, and flank colour slightly paler than breast and belly, not almost uniform as in typical cantillans neither clearly different as in typical albistriata.

Genetic identification

DNA was extracted from feathers stored in ethanol, following laboratory procedures described by [START_REF] Randi | Phylogeography of the rock partridge (Alectoris graeca)[END_REF] and Brambilla et al. (2008a). PCR primers (named 4L and 662H) used to amplify the first half of the mtDNA cytochrome b gene were the same of Brambilla et al. (2008a). A potential problem with phylogenetic analyses based on mtDNA data is the occurrence and amplification of mitochondrial DNA homologues from the nuclear genome (numts) (e.g. [START_REF] Sorenson | Multiple independent transpositions of mitochondrial DNA control region sequences to the nucleus[END_REF]Fleischer 1996, Kidd and[START_REF] Kidd | Sequence variation in the Guillemot (Alcidae: Cepphus) mitochondrial control region and its nuclear homolog[END_REF]. All the cyt b sequences we obtained could be functionally translated without premature terminations and there was no evidence suggesting that the presence of numts may bias our analyses on mtDNA.

A contiguous fragment of 598 bp was obtained from each sample. Sequences of migrant haplotypes have been deposited in GenBank under Accession Nos. XXXXXXXX-XXXXXXXX. Individuals were assigned to a subspecies/group using a phylogenetic approach. A mtDNA cytochrome b phylogenetic tree (Brambilla et al. 2008a) revealed the existence of large phylogenetic distinctions among albistriata, subalpina and two (western and southern) cantillans mtDNA clades. These clades showed genetic distances D comprised between 1.7% and 5.0%. We used a subset of reference haplotypes determined in breeding individuals caught in the range of the four clades, and thus representing cantillans (southern and western clades; see above), subalpina (both mainland and insular populations) and albistriata (both from the western and eastern portions of its range). For each group, we selected the four commonest haplotypes (as found in Brambilla et al. 2008a); for subalpina, we selected the two commonest mainland haplotypes and two insular haplotypes, the latter representing Corsican and Balearic populations. The tree resulting from the reference haplotypes showed a clear distinction among the three subspecies and strong statistical support for each group (bootstrap values for each group 99%), consistent with the wider analysis of breeding birds (Brambilla et al. 2008a). Together with the reference haplotypes we then included all migrants, classified on the basis of their plumage features (see above) and re-built the phylogenetic tree, recording the placement in the tree of the newly included (migrant) samples (see Fig. 1). On the basis of the assignment (no sample was placed outside the previously identified groups; see Results and Fig. 1) we determined the (sub)specific attribution based on mtDNA. One sequence of Sylvia atricapilla (GenBank accession no. Z73494.2) was used as outgroup. Phylogenetic relationships were inferred using: 1) neighbour-joining (NJ) method, based on number of differences among sequences (using MEGA 3.1; [START_REF] Kumar | MEGA3: Integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment[END_REF]); bootstrap tests with 1000 replicates were performed to assess the robustness of the clades (i.e., bootstrap support); 2) heuristic search strategy, carried out in PAUP* 4.08b [START_REF] Swofford | PAUP*: Phylogenetic Analysis Using Parsimony (and other methods) Version 408b[END_REF], with exclusion of all uninformative nucleotide positions, unordered and equally weighted characters, starting trees obtained via random stepwise addition, random haplotype additions with 10 replicates, multiple minimal trees swapped by TBR (tree-bisection-reconnection) branch swapping, collapsed zero length branches; MulTrees option in effect; Steepest descent option not in effect, gaps treated as missing data, 300 replicates for assessing bootstrap values (10 6 rearrangements tried); 3) Maximum Likelihood (ML) method, carried out in PAUP* 4.08b [START_REF] Swofford | PAUP*: Phylogenetic Analysis Using Parsimony (and other methods) Version 408b[END_REF]) and bootstrap values assessed by means of 100 replicates on 100 equiprobable trees; 4) Bayesian inference using MrBayes 3.1.2 [START_REF] Huelsenbeck | MrBayes: Bayesian inference of phylogeny[END_REF]www.mrbayes.net). The choice of model (HKY+G) mirrored the results of the previous phylogenetic study (Brambilla et al. 2008a).

Posterior probabilities were calculated in MrBayes for cytochrome b under a Hasegawa-Kishino-Yano with gamma-shaped variation across sites (HKY+G) model (see above and Brambilla et al. 2008a). Four Metropolis-coupled MCMC chains were run for 10 6 generations and sampled every 100 generations. The temperature was set to 0.1 to improve the mixing of the chains, given that it was found to be poor at the default temperature 0.2 (cf. [START_REF] Olsson | Non-monophyletic taxa and cryptic species -evidence from a molecular phylogeny of leaf-warblers (Phylloscopus, Aves)[END_REF]. Other settings were kept as default values. The first 200,000 generations, before the chain reached apparent stationarity (burn-in), were discarded and the posterior probability estimated for the remaining 800,000 generations. Stationarity was confirmed by potential scale reduction factors (all the parameters comprised between 1.000 and 1.013) and by the plot looking like "white noise", with no tendency of increase or decrease over time.

In all cases, trees were collapsed to obtain strict and 50% majority-rule consensus trees and the results obtained through the three different procedures were compared.

Results

The reference haplotypes lead to a well-resolved phylogenetic tree, according to all methods: the breeding birds of the four clades were grouped into four different and clearly separated groups.

Of the 58 photographed and examined birds, 29 were classified as cantillans, 10 as albistriata, 8 as subalpina based on plumage traits, while 11 were classified as "uncertain/intermediate" cantillans/albistriata, the latter showing intermediate features between the typical appearance of the two taxa, not allowing us to unequivocally assign them to one of the two subspecies. Moreover, some cantillans birds showed a paler colour on flanks and a rather wide submustachial stripe, yet possessing all the other typical cantillans traits and were assigned to this group. The latter all resulted as belonging to this group according to the cyt b.

Genetic analysis revealed that all birds belonged to three distinct clades perfectly associated with the "reference" haplotypes. As expected, no western cantillans was sampled (see above).

Bootstrap values of the three methods confirmed the validity of the tree, as all the groups had a support equal to or greater than 87% for bootstrap procedure in the neighbour joining tree, equal to or greater than 78% according to the maximum parsimony tree, equal to or greater than 74% according to the maximum likelihood tree, and had a posterior probability equal to or larger than 0.88 in the Bayesian analysis (see Fig. 1). Therefore, each group received a good statistical support from bootstrap and posterior probability values.

However, the position in the phylogenetic tree imperfectly matched identification based on photographs, and thus genotypic identification in some cases differ from phenotypic attribution. In fact, out of ten birds determined as albistriata, only five showed an albistriatalike cyt b sequence, while the remaining five were grouped together with cantillans. Three of these misclassified birds showed a nearly "unmistakable" albistriata plumage, with all typical features of this subspecies (large white moustache, vinaceous-red tinge restricted to the upper breast, faint red colour on flanks, dark lores).

Of the 11 intermediate cantillans/albistriata birds, one proved to have an albistriata-like cyt b, while the other ten were genetically cantillans.

The 29 males classified as cantillans were actually all grouped in the phylogenetic tree with the reference cantillans breeders.

All the eight males classified as subalpina were identically classified by the genetic data, being grouped with the respective reference haplotypes in a unique group (but evidently mutually with "mainland" or "insular" haplotypes, respectively), and no subalpina mtDNA was found among differently classified birds.

Discussion

Diagnosability is a discussed topic in current ornithology [START_REF] Watson | Diagnosable versus Distinct: Evaluating Species Limits in Birds[END_REF]. Correct identification of taxa is required both in taxonomic research and field ornithology [START_REF] Helbig | Guidelines for assigning species rank[END_REF]. However, although a variety of different suites of traits are potentially diagnostic (i.e., morphometrics, external or internal morphology, behavioural or ecological traits, molecular data, etc.), most of them have unknown taxonomical value and weight [START_REF] Helbig | Guidelines for assigning species rank[END_REF]. Thus, explicit tests of diagnosability are needed, particularly in assemblages including sibling or cryptic taxa. In this study we test the strength of diagnoses based on plumage in a group of strictly related taxa, currently mainly identified based on male plumage, as contrasted with mtDNA-based diagnosablity, by analysing trapped males.

The results of this study show that different levels of diagnosability apply to the different taxa comprised within the Sylvia cantillans complex. The haplotypes of migrant warblers were assigned to the distinct haplogroups as defined based on the reference breeding birds: there were three well-distinct groups; no haplotype showed problematic position and all groups had a good statistical support. However, plumage-based identification was not always consistent with the classification provided by mtDNA. Intermediate birds (not safely classifiable based on their plumage) were placed in both the albistriata and (especially) cantillans groups and, more importantly, albistriata-like birds, as identified on the basis of plumage traits, were not all classified as belonging to this subspecies, as some of them were determined as cantillans according to their cyt b. Therefore, weakness of plumage-based identification currently complicates field and hand identification of these taxa, whose subspecific attribution requires caution. MtDNA did not allow us to check for the occurrence of birds originating from a secondary contact area with mixed mating between cantillans and albistriata; such birds would probably show intermediate or even "wrong" plumages when compared with mtDNA.

Intermediate and misclassified birds could be hybrids of cantillans and albistriata, or could result from (possibly unilateral) introgression, or they could be southern cantillans with an outer appearance similar to albistriata. However, these ambiguities could be resolved only with the use of a nuclear marker and a larger sample.

On the other side, subalpina clearly stands out as the most differentiated taxon; all birds assigned to this subspecies by plumage were placed together with breeders of this race, and no subalpina haplotype was found among birds otherwise identified. Therefore, according to our sample this species appears as 100% diagnosable on the basis of males" plumage features (and of mtDNA too). Although the low sample prevents us from drawing definitive conclusions, the perfect diagnosability of subalpina in the Ventotene sample confirms the strong differentiation between this and the other taxa of the complex (Brambilla et al. 2008a), as demonstrated also by placement in the mtDNA tree (Figs. 12).

In conclusion, plumage-and mtDNA-based diagnosability of taxa belonging to the Sylvia cantillans complex may be considered as straightforward for subalpina, but unreliable for many individuals, which despite a rather typical "albistriata" appearance showed a cantillans mtDNA; other traits should be studied in order to develop a safe method for hand diagnosability of the latter taxa. Under a taxonomic point of view, the splitting of subalpina (cf. [START_REF] Shirihai | Sylvia Warblers[END_REF]) into another different species (Sylvia subalpina, or S. moltonii), as recently proposed on the basis of song perception (Brambilla et al. 2008b) and genetic divergence among breeding populations (Brambilla et al. 2008a), is acknowledged also by males" diagnosability, other than by strong differences in vocalizations [START_REF] Brambilla | Quando la voce è tutto: l"identificazione delle sottospecie di sterpazzolina Sylvia cantillans[END_REF] and particular distribution pattern [START_REF] Brambilla | Geographical distribution of Subalpine Warbler Sylvia cantillans subspecies in mainland Italy[END_REF].

At the same time, our study provides a worked example of an explicit test of taxa diagnosability. In particular, our results outline the importance of considering different features for taxa diagnosis: if we only consider mtDNA, also cantillans and albistriata may appear fully diagnosable. The use of two or more "markers" for diagnosis may point out discordant identification and thus lack of unambiguous diagnosability. Procedures based upon two or more "independent" traits should be suggested for judging diagnosability among closely related taxa in taxonomic studies. With this respect, [START_REF] Alström | Species delimitation based on multiple criteria: the Spotted Bush Warbler Bradypterus thoracicus complex (Aves: Megaluridae)[END_REF] provided with a fine example of how the use of multiple criteria can greatly improve assessment of taxonomic status in a bird species complex, the Bradypterus thoracicus complex, and similar multidimensional analyses greatly enhanced taxonomic diagnosibilty in Asian Phylloscopus Warblers [START_REF] Martens | Intraspecific differentiation of Sino-Himalayan bushdwelling Phylloscopus leaf warblers, with description of two new taxa (P. fuscatus, P. fuligiventer, P. affinis, P. armandii, P. subaffinis)[END_REF] and Canary Island Goldcrests [START_REF] Päckert | Radiation of Atlantic goldcrests Regulus regulus spp.: evidence of a new taxon from the Canary Islands[END_REF]. In this species complex, mtDNA and song were the most useful traits for taxonomic assessment [START_REF] Alström | Species delimitation based on multiple criteria: the Spotted Bush Warbler Bradypterus thoracicus complex (Aves: Megaluridae)[END_REF], similarly to the Sylvia cantillans complex, where mtDNA identified main clades (Brambilla et al. 2008a) and song seemed to represent the most important isolation mechanism (Brambilla et al. 2008b).

Genetic attribution may be a powerful tool if a clear pattern of genetic divergence among populations is known; the use of finer molecular markers, such as microsatellites or nuclear genes, may help in determining the occurrence of hybridisation and introgression among related taxa, which was not possible in our analysis based on mtDNA. Table 1 Characters and character states adopted for (sub)specific identification of studied birds of the S cantillans complex (Cramp 1992;[START_REF] Shirihai | Sylvia Warblers[END_REF][START_REF] Brambilla | Distribuzione, ecologia e differenziazione in Sylvia cantillans[END_REF].

Zusammenfassung

  Worum handelt es sich? Ein expliziter Test offenbart verschiedene Niveaus der Artenbestimmung im Artenkomplex Sylvia cantillans "Diagnosability" ist unsere Fähigkeit zwischen ähnlichen Taxa zu unterscheiden, Schwester-oder kryptische Taxa eingeschlossen. Wir haben die "Diagnosability" am Modell-Artenkomplex Sylvia cantillans explizit getestet und die Bestimmung mittels phänotypischer mit der mittels genetischer Merkmale verglichen. Individuelle Weißbartgrasmücken, gefangen während der Zugperiode in Zentralitalien, wurden bis zur Unterart identifiziert mittels vermeintlich artbestimmender Merkmale des Federkleides. Mittels Referenzhaplotypen, die in brütenden Individuen sequenziert wurden und eindeutige Stammbäume entsprechend der bekannten Taxone dieses Komplexes lieferten, wurden Nukleotidsequenzen des mtDNS Cytochrom b Fragmentes (598 bp) verwandt um jedem Individuum ein eindeutiges phylogenetische Taxon zuzuordnen. Diese neuen Haplotypen wurden einer der vorher identifizierten Gruppen (entsprechend drei verschiedenen Taxa) zugeordnet. Bei dieser molekularen Methode fiel keine Probe außerhalb besagter Taxa. Trotzdem wurden bei der Bestimmung mittels Gefiedermerkmalen 11 von 58 Vögel als "unsicher/intermediär" zwischen zwei Taxa eingeordnet, und 5 Vögel wurden von beiden Methoden unterschiedlich klassifiziert. Nur bei einem Taxon (Sylvia subalpina, syn. S. moltonii) stimmten die Resultate beider Methoden perfekt überein. Für die anderen zwei Taxa dieses Artenkomplexes kann die "Diagnosability" nicht garantiert werden, und die Bestimmung im Feld sollte mit Vorsicht betrachtet werden. Unsere Arbeit liefert ein Beispiel für einen expliziten Test von "Diagnosability". Wir konnten zeigen, dass die Nutzung von zwei oder mehr Merkmalen zur Artbestimmung kontroverse Ergebnisse liefert, und damit der Anspruch auf Eindeutigkeit verfehlt wird. Unsere Ergebnisse betonen die Bedeutung der Nutzung mehrerer, unterschiedlicher Merkmale zur Artbestimmung und zeigen, in dem von uns studierten Artenkomplex, die Schwächen der visuellen, auf äußere Erscheinung basierten Identifikation auf.

Fig. 1

 1 Fig. 1 Phylogenetic tree for migrant attribution with node estimates based on (in this order)

Acknowledgments We are very grateful to all the people who helped us with field and laboratory work, and especially to F. Akriotis, P. Arrojo, N. Baccetti, J. Blondel, R. Carini, J.

Cecere, F. Davoli, A. De Faveri, A. Flitti, P. Giusti, F. Guidali, O. Hameau, K. Kravos, D. Iavicoli, N. Mucci, P. Perret, E. Savo, D. Sere, A. Sorace, E. Strinella, P. Sunyer. A special thank goes to O. Janni, M. Wink and an anonymous reviewer for very helpful comments and to all volunteer ringers at Ventotene, which were partly supported by a grant from the Riserva Naturale Statale Isole di Ventotene e Santo Stefano. We also thank the Municipality of Ventotene and the Centro di Educazione Ambientale Luca Bertolaccini for support and logistics. Risultati del "Progetto Piccole Isole" ISPRA: paper n.