The prognostic role of HER2 expression in ductal breast carcinoma in situ (DCIS); a population-based cohort study

Signe Borgquist
Wenjing Zhou
Karin Jirström
Rose-Marie Amini
Thomas Sollie
Therese Sørlie
Carl Blomqvist
Salma Butt
Fredrik Wärnberg
The prognostic role of HER2 expression in ductal breast carcinoma in situ (DCIS); a population-based cohort study
10.1186/s12885-015-1479-3R E S E A R C H A R T I C L E Open Access
Background: HER2 is a well-established prognostic and predictive factor in invasive breast cancer. The role of HER2 in ductal breast carcinoma in situ (DCIS) is debated and recent data have suggested that HER2 is mainly related to in situ recurrences. Our aim was to study HER2 as a prognostic factor in a large population based cohort of DCIS with long-term follow-up. Methods: All 458 patients diagnosed with a primary DCIS 1986-2004 in two Swedish counties were included. Silver-enhanced in situ hybridisation (SISH) was used for detection of HER2 gene amplification and protein expression was assessed by immunohistochemistry (IHC) in tissue microarrays. HER2 positivity was defined as amplified HER2 gene and/or HER2 3+ by IHC. HER2 status in relation to new ipsilateral events (IBE) and Invasive Breast Cancer Recurrences, local or distant (IBCR) was assessed by Kaplan-Meier survival analyses and Cox proportional hazards regression models. Results: Primary DCIS was screening-detected in 75.5 % of cases. Breast conserving surgery (BCS) was performed in 78.6 % of whom 44.0 % received postoperative radiotherapy. No patients received adjuvant endocrine-or chemotherapy. The majority of DCIS could be HER2 classified (N = 420 (91.7 %)); 132 HER2 positive (31 %) and 288 HER2 negative (69 %)). HER2 positivity was related to large tumor size (P = 0.002), high grade (P < 0.001) and ER-and PR negativity (P < 0.001 for both). During follow-up (mean 184 months), 106 IBCRs and 105 IBEs were identified among all 458 cases corresponding to 54 in situ and 51 invasive recurrences. Eighteen women died from breast cancer and another 114 had died from other causes. The risk of IBCR was statistically significantly lower subsequent to a HER2 positive DCIS compared to a HER2 negative DCIS, (Log-Rank P = 0.03, (HR) 0.60 (95 % CI 0.38-0.94)). Remarkably, the curves did not separate until after 10 years. In ER-stratified analyses, HER2 positive DCIS was associated with lower risk of IBCR among women with ER negative DCIS (Log-Rank P = 0.003), but not for women with ER positive DCIS. Conclusions: Improved prognostic tools for DCIS patients are warranted to tailor adjuvant therapy. Here, we demonstrate that HER2 positive disease in the primary DCIS is associated with lower risk of recurrent invasive breast cancer.

Background

In general, patients with ductal breast carcinoma in situ (DCIS) have an excellent prognosis in terms of survival [1,2]. However, the rate of new ipsilateral events (IBE) is high, even higher than following invasive breast cancer [2]. Almost half of all IBEs subsequent to a DCIS are invasive cancer and survival after an invasive IBE has, not surprisingly, been reported to be worse than after a non-invasive recurrence [3]. The knowledge about clinical and pathological predictors of recurrence following DCIS is currently limited [4,5]. Certain subtypes of DCIS might be more prone to recur as invasive cancer and new biological markers may provide additional prognostic and/or treatment predictive value [6][7][8][9]. Recently, genomic-based data from a study using the Oncotype DX® indicated that it was possible to predict the local relapse risk for a primary DCIS independently from classical factors [10].

HER2 is an established negative prognostic factor in invasive breast cancer [11]. The prognostic significance of HER2 status in DCIS is, however, less clear [7,12]. Both the relation of HER2 to risk of recurrence and its role in the progression from in situ to invasive cancer have been debated [12]. HER2 over-expression is reported to be more frequent in DCIS than in invasive cancer [13]. This may seem counterintuitive as HER2 is proposed to play a role in tumour progression. Some studies report an even higher proportion of HER2 positivity in micro-invasive cancer [14,15] and, in preoperative tumour biopsies displaying DCIS, HER2 overexpression has been related to a co-existing invasive component in the surgical specimen [16,17]. Furthermore, HER2 is associated with high histopathological grade both in invasive cancer and in DCIS [18].

HER2 amplification is an important factor in the classification of molecular subtypes of invasive breast cancer [19]. An approximation of the gene expression based subtypes using immunohistochemical (IHC) detection of HER2, estrogen receptor-(ER) and progesterone receptor (PR) expression has been suggested for invasive breast cancer [20]. DCIS has been divided into the same surrogate molecular subtypes as invasive breast cancer [6,[21][22][23] but the prognostic value of these subgroups has not been convincing in DCIS [8,24].

We have previously studied the surrogate molecular subgroups in this population-based DCIS cohort [8,24]. Those studies indicated a lower risk of invasive recurrence in HER2-positive DCIS, although not statistical significant. We therefore conducted this updated analysis on the same patient population with further extended follow-up time and pooled HER2 data focusing on the role of HER2 status in DCIS and its relation to prognosis.

Methods

Patients

All patients diagnosed with a primary DCIS between 1986 and 2004 in two Swedish counties (Uppland and Västmanland) were included in this DCIS cohort (n = 458). The majority underwent breast-conserving surgery (79 %), and clear surgical margins were obtained in 88 % among all cases irrespective of surgical intervention. All cases were histopathologically reexamined. Tumour tissue biopsies of 1.0 mm in duplicate from paraffin blocks of samples taken as part of standard care were used to construct tissue microarrays (TMA). Follow up was complete up to December 15 th , 2013. This study was approved by the Ethics Committee at Uppsala University, Sweden (Dnr 2005:118) and no informed consent was needed.

Silver-enhanced In Situ Hybridization (SISH) and IHC

Scoring and IHC protocols are previously described in detail [21,25]. SISH was performed on the automated instrument, Ventana Benchmark (Ventana Medical Systems, Tucson, AZ), as per the manufacturer's protocols for the INFORM HER2 DNA probe and chromosome 17 probes. Testing for the HER2 gene and chromosome 17 was performed on sequential sections. Both probes are labelled with dinitrophenol and denaturation occurred on the instrument with enzyme digestion in protease 3 for eight minutes. The detection system used a multimer labelled with goat anti-rabbit antibody horseradish peroxidase as the linking step. Visualization occurred with the sequential addition of silver acetate as the source of ionic silver, hydroquinone, and hydrogen peroxide to give black metallic silver precipitate at the probe site. Counterstaining was performed with hematoxylin II on the instrument. The time taken for the complete run was 6.5 h. Both HER2 and chromosome 17-detection were performed on the same slide run. Gene amplification was assessed using the American Society of Clinical Oncology/College of American Pathologists guideline and Australian HER2 Advisory Board criteria for single HER2 probe testing (diploid, 1 to 2.5 copies/nucleus; polysemy >2.5 to 4 copies/nucleus; equivocal, >4 to 6 copies/ nucleus; low-level amplification, >6 to 10 copies/ nucleus; and high-level amplification >10 copies/nucleus) and for dual HER2/CHR17 probe testing (non-amplified ratio <1.8; equivocal ratio, 1.8 to 2.2; gene amplification, >2.2) [26]. The HER2 status was predominantly relying on the SISH data. For those cases in which SISH failed, HER2 status was based on the IHC data and cases were considered HER2 positive if the IHC score was 3+, using the HercepTest©. As for hormone receptors (estrogen receptor alpha (ER) and progesterone receptor (PR)), tumours with 10 % or more nuclei stained were considered ER and PR positive, respectively. Staining intensity was not taken into consideration.

Statistic analyses

Baseline characteristics among women with different HER2 status were compared by Chi-square test for categorical variables or analysis of variance for continuous variables. Survival analyses were performed using Kaplan-Meier curves, including the Log-Rank test. Cox proportional hazards regression models were used to generate hazard ratios (HRs) with 95 % confidence intervals (CIs), with adjustment for radiotherapy, age at diagnosis (continuous), tumour size (two categories: ≤ 25 mm; >25 mm or multifocal), and ER status. Stratification analyses were performed by mode of detection, type of surgery and ER in different multivariate models. All statistical tests were two-sided, and p-values less than 0.05 were considered significant. Data were analysed using the SPSS Statistics, version 19 (IBM, Chicago, IL, USA) and SAS 9.3 (Cary, NC, USA).

Primary endpoints were Ipsilateral Breast cancer Events (IBE) and Invasive Breast Cancer Recurrences (IBCR). IBEs were divided in new in situ events and invasive IBEs. As for IBCR, an event was defined as an invasive IBE, a regional recurrence, a contralateral invasive breast cancer or distant metastasis. IBCR free survival was calculated based on the time from primary surgery to the date of any invasive breast cancer event, date of death or date of last follow-up as by December 15 th 2013. For an event to be classified as IBE or IBCR, a minimum of 3 months had to pass after the primary surgery.

Results

Patient and tumour characteristics

Baseline characteristics by HER2 status are presented in Table 1. The mean age at diagnosis was 58.6 years, ranging from 30 to 90 years of age. A total of 202 women were diagnosed in Västerås, whereas the remaining 256  Table 1). None of the patients received hormonal therapy or chemotherapy according to Swedish clinical guidelines.

Follow-up data

The mean follow up time was 183.5 months (range 3-329 months). A total of 105 IBEs were identified of which 54 were a new in situ and 51 an invasive IBE in the entire cohort of 458 patients. Among women treated with BCS (N = 324), recurrences were detected among 95 women comprising 49 in situ and 46 invasive cases. Eleven of the in situ IBEs were followed by a subsequent invasive IBE. One hundred and six IBCR were identified. The first invasive event was an invasive IBE in 50 cases, regional axillary metastasis in four, locoregional together with distant metastases or distant metastasis only in twelve cases and a contralateral invasive cancer in 40 cases. In total, 32 patients developed generalized disease; fifteen subsequently to an invasive IBE, eight after an invasive contralateral cancer and nine had no reported prior local or regional recurrence. Eighteen women died from breast cancer and another 114 had died from other causes.  (Table 2).

HER2 and survival analyses

Among the 420 women with available HER2 status, a total of 102 women experienced recurrences during follow-up. HER2 positivity in the primary DCIS was not a risk factor for IBE in women undergoing BCS (Log-Rank P = 0.40, HR 1.20 (95 % CI, 0.78-1.85)), (Fig. 1a and Table 3). Inter  Table 3). Results from the multivariate analyses did not differ substantially from the univariate analyses (Table 3). The risk of IBCRs was statistically significantly lower subsequent to a HER2 positive DCIS compared to a HER2 negative primary DCIS (Log-Rank P = 0.03, HR 0.60 (95 % CI, 0.38-0.94)) ( Fig. 2 and Table 3). Remarkably, the curves in the Kaplan-Meier plot did not separate until after almost 10 years (Fig. 2). In the multivariate analyses, HRs after adjustments were very similar to the crude analyses ( Table 3).

The survival analyses were stratified for ER-status, and for patients with an ER negative DCIS, HER2 positivity predicted a significantly lower risk of IBCR (Log-Rank, P = 0.003), (Fig. 3a), which was not the case for ER positive DCIS patients (Log-Rank, P = 0.76), (Fig. 3b). In analyses restricted to women who undergoing BCS, ER status remained an effect modifier for the association between HER2 status and IBCR.

The prognostic value of age was studied in survival analysis using age as at dichotomous variable of ≤ 50 years (N = 141, 31 %) or > 50 years (N = 317, 69 %). Age was not proven to be a prognostic indicator of neither invasive nor in situ recurrence (data not shown). However, age added substantial information to the prognostic value of HER2 status. For presumably premenopausal women aged 50 or below, HER2 expression in the DCIS revealed no prognostic information (Fig. 3c). In contrast, for women who were older than 50 years of age at diagnosis, HER2 positivity was associated with a markedly improved recurrence-free survival for invasive disease (Log-Rank, P = 0.002), (Fig. 3d). The age-stratified analyses for in situ IBEs did not reveal any significant results.

Discussion

In this long-term follow-up DCIS cohort, positive HER2 status in the primary lesion predicted lower risk of late invasive breast cancer recurrence compared to negative HER2 status in the primary DCIS. HER2 positivity did on the other hand indicate a non-significant higher risk of local in situ IBEs.

DCIS is by definition a non-invasive disease exerting a rather high risk of local recurrence but an especially low risk of lethal outcome from the disease [27]. However, an increasing rate of early-stage disease, including DCIS, is observed due to mammography screening [28]. The ideal screening method aims at fractioning indolent disease from cancers that will ultimately cause harm if undetected at an early stage. Current mammography methods are incapable of characterising the aggressiveness of a given breast tumor, and tissue based assessment of biological entities is warranted for improved clinical decision-making [1,29,30]. DCIS includes a plethora of diseases ranging from conditions where active surveillance might be adequate, to conditions where tumors should be surgically removed followed by adjuvant treatment. Prediction of recurrences is thus a high priority; both prognostic and treatment predictive factors should be identified [29].

Previous studies have shown increased risk of DCIS recurrences among patients with HER2 positive disease in the primary DCIS [31,32]. These findings were vaguely indicated but not statistically significantly confirmed in this study. More importantly, HER2 status was predictive of invasive recurrences in this study showing that HER2 positive tumors were less reluctant to recur as an invasive tumor. The positive prognostic value of HER2 in DCIS demonstrated here might seem counterintuitive given the data showing HER2 to be positively related to tumor size, multifocality and nuclear grade and may reflect an independent role of HER2 in DCIS as a long-term prognostic biomarker. This study includes an extensive long-term follow-up, which enabled the detection of the lower incidence of late-recurrences among patients with HER2 positive DCIS. The Kaplan-Meier curves for invasive recurrences were concordant for several years, and not until 10 years after the primary DCIS, did the curves separate. The discordances between our results and the results generated by Kerlikowske et al.

[32] may well be explained by the differences in follow-up time. More importantly, the endpoints applied in this study are not identical to previous studies among which any ipsilateral recurrence regardless of type has served as endpoint. Our results stating that HER2 positive DCIS is a beneficial prognostic marker for invasive recurrent disease highlights the need for studying several different endpoints to understand the prognostic value of a given biological marker. Presumably the key endpoint should be invasive recurrence representing the actual threat to the patient's life as discussed in the review by Benson et al. stating the main goals of DCIS treatment to be prevention of recurrent invasive disease, minimised treatment-related morbidity, and optimised cosmetics [29]. In a previous publication, we evaluated the prognostic impact of molecular subtypes defined by the immunohistochemical surrogate classification [24]. The subtypes were based on the proposed classification for invasive breast cancer according to the St Gallen consensus meeting in 2011 [20]. HER2 cases were thus presented in two subtypes, i.e. Luminal B(HER2+) and HER2 + (ER-/PR-) leaving few cases in each subtype, which might explain the non-significant data generated in that study. Herein, we report significant results for HER2 as a prognostic marker for recurrent invasive disease, presumable prone to the facts that, in this study, all HER2 positive DCIS were analysed together and moreover, the follow-up period has been extended.

The significantly reduced risk estimates for patients with HER2 positive DCIS might help identifying a lowrisk group for whom adjuvant treatment after surgical excision could safely be omitted. To keep overtreatment to an innocuous minimum is essential for DCIS-patients among whom a substantial amount of the lesions may be of limited clinical importance [28].

This study has several strengths, such as its relatively large sample size and confirmation of all events by a physician. Another important strength was the longterm follow-up of more than 15 years (mean). During follow-up invasive recurrences were identified in 23 % (106/458) of the women with a previous DCIS. From a biological point of view, the identified regional or distant metastases have most likely evolved from an invasive foci in the breast, which could have occurred at any time between the DCIS diagnosis and the diagnosis of metastatic disease, although not detected and therefore not possible to report in this study. The number of invasive recurrences does, however, correspond with numbers from previous publications on DCIS patients showing a recurrence rate of 25 % within 10 years of follow-up, 50 % of these being invasive recurrences [33], and 18 % during 5 years of follow-up [34]. Unequal recurrenceestimates may be explained be differences in follow-up length and the fact that no systemic endocrine treatment is recommended Swedish DCIS patients. Some considerations are worth discussing for the interpretation of our results. All evaluated tumor biomarkers were investigated on TMAs, which may have limited the assessment of heterogeneous expression compared to whole sections. Previous validation studies of TMA-based biomarker assessment, however, show accurate concordance between TMAs and whole sections motivating use of TMAs for routine breast biomarker (ER, PR, HER2) analyses in the clinical setting [35]. A previous publication including a subset of this study population, showed an overall concordance of 80 % between TMAs and whole sections for IHC assessment of ER, PR, and HER2, and notably the prognostic value of these markers was similar irrespective of assessment based on TMA or whole sections [36]. This study is predominantly based on SISH regarding HER2 status with an anticipated high accuracy compared to IHC [37]. Breast conserving surgery represents the surgical intervention predominantly used in our cohort. Compared to mastectomy, the outcome following this surgical approach requires higher accuracy in pre-surgical imaging of the tumor extend. No data on presurgical imaging was however available for this study and differences could not be addressed further, The challenge of surgical margins differ depending of surgical method as evidenced in this cohort with clear margins obtained in 94 % of cases undergoing mastectomy compared to 86 % among the women who underwent breast conserving surgery. However, no association between surgical method and HER2 status was found, and the risk of confounding by surgical method is considered small.

HER2 positive disease is a strong predictor for impaired recurrence-free survival in invasive breast cancer, opposing the results on DCIS in this study. This controversy may reflect the diverse expression of HER2 in invasive lesions and adjacent DCIS components as recently discussed in a review by Cowell et al. [30]. The tentative explanations given are that HER2 amplification may either have been lost in the progression from ductal carcinoma in situ to invasive breast cancer, or that the invasive component has arisen from a DCIS clone without HER2 Fig. 3 Invasive recurrence-free-survival according to HER2 of the primary DCIS, stratified for ER status and age, respectively. Kaplan-Meier analyses stratified for ER status of the primary DCIS, demonstrating invasive recurrence-free survival (IBCR) with respect to HER2 status of the primary DCIS among ER negative (a) and ER positive DCIS patients (b). Additional Kaplan-Meier curves show analyses stratified for age at diagnosis (≤50 years/> 50 years) displaying invasive recurrence-free survival (IBCR) according to HER2 status of the primary DCIS among young patients (≤50 years at diagnosis), (c) and older patients with age 50 years or above at diagnosis (d) amplification [30]. Biologically, the striking issue is why progression to invasive disease would imply loss of HER2 amplification, which warrants further attention.

Although this study implies, that patients with HER2 positive DCIS are less likely to experience recurrent invasive disease, the HER2 positive DCIS disease has substantial clinical interest as a targetable early-stage disease. This has been acknowledged by recently conducted clinical trials testing the efficacy of HER2 targeting therapy with lapatinib in DCIS patients (ClinicalTrials.gov NCT00555152 and NCT00857714, respectively). The results of these trials are currently not published, however the trial results by Estevés et al. assessing lapatinib in the pre-surgical setting reported significant inhibition of HER2 signalling and reduced tumor size following 4 weeks of treatment in HER2 positive DCIS patients [38]. Further, other biomarker trials have assessed the effects of trastuzumab in DCIS patients, among which, one trial has reported the results, showing no significant tumor effects in terms of proliferation and apoptosis after a single-dose monotherapy trastuzumab pre-surgically [39]. None of these studies have addressed the long-term benefits of HER2 targeted therapy in DCIS. However, a currently recruiting phase III randomized trial prescribing trastuzumab versus placebo concomitantly with radiotherapy, may shed light over the long-term effects (ClinicalTrials NCT00769379).

Conclusions

Clinical decision making for DCIS patients is still challenged by the inability to predict recurrence or, most importantly, progression to invasive disease. In line with the models developed for invasive breast cancer, such as Adjuvant! [40], a comprehensive set of solid biomarkers for DCIS prognosis and treatment prediction has repeatedly been requested. In this large DCIS cohort with extensive follow-up, we demonstrate significantly improved long-term invasive disease-free survival for patients with HER2 positive disease in the primary DCIS. These results indicate that HER2 status could add significant information in future development of prognostic and predictive DCIS models.

Competing interests

S. Borgquist declares that she has previously received lecturer salary from Roche. The other authors declare that they have no competing interests.

Authors' contributions

SBo and WZ were responsible for data analyses, and manuscript preparation and editing. KJ was involved in the pathology review and construction of tissue microarrays. RMA was responsible for HER2 scoring of stains and SISH together with WZ. TS and SBu up-dated the follow up in the cohort. CB and TS helped with the interpretation of the results and with drafting the manuscript. FW designed the overall study, coordinated the study, and helped draft and finalize the manuscript. All authors contributed in the preparation of the manuscript and all authors read and approved the final manuscript.

estingly, divided by type of IBE, HER2 positivity showed a borderline statistically significant increased risk of in situ IBEs (Log-Rank P = 0.09, HR 1.63 (95 % CI, 0.92-2.89), and no association with risk of invasive IBEs (Log-Rank p = 0.48, HR 0.78 (95 % CI, 0.40-1.55)), (Fig. 1b-c and

Fig. 1
1Ipsilateral Breast cancer Events (IBE) according to HER2 status of the primary DCIS. Kaplan-Meier plots showing ipsilateral recurrence-free survival analyses (IBE) among women with DCIS treated with breast conserving surgery (BCS) with respect to HER2 status of the primary DCIS regarding all ipsilateral events (a), ipsilateral in situ events (b), and ipsilateral invasive events (c)

Fig. 2
2Invasive recurrence-free-survival according to HER2 status of the primary DCIS. Kaplan-Meier plot showing invasive recurrence-free survival analyses (IBCR) among women with a DCIS with respect to HER2 status of the primary DCIS

Table 1
1Baseline characteristics by HER2 status in 458 women with a primary DCISCharacteristics
All
HER2 positive
HER2 negative
HER2 missing

(n = 458)
(n = 132)
(n = 288)
(n = 38)

n (%)
n (%)
n (%)
P-value a
n (%)

Age (years) mean (n = 458)
58.6
57.4
59.2
0.13
58.8

Detection mode (n = 457)

Screening
345 (75.5 %)
108 (81.8 %)
210 (73.2 %)
27 (71.1 %)

Clinically
112 (24.5 %)
24 (18.2 %)
77 (26.8 %)
0.05
11 (28.9 %)

Type of Surgery (n = 457)

BCS
359 (78.6 %)
98 (74.2 %)
233 (81.2 %)
28 (73.7 %)

Mastectomy
98 (21.4 %)
34 (25.8 %)
54 (18.8 %)
0.11
10 (26.3 %)

Radiotherapy (n = 359)

Yes
158 (44.0 %)
49 (50.0 %)
100 (42.9 %)
9 (32.1 %)

No
201 (56.0 %)
49 (50.0 %)
133 (57.1 %)
0.32
19 (67.9 %)

Tumor size (mm) (n = 409)

≤ 25
294 (71.9 %)
68 (61.3 %)
203 (77.2 %)
23 (65.7 %)

> 25 or multifocal
115 (28.1 %)
43 (38.7 %)
60 (22.8 %)
0.002
12 (34.3 %)

Nuclear grade (n = 458)

Grade 1
42 (9.2 %)
1 (0.8 %)
34 (11.8 %)
7 (18.4 %)

Grade 2
176 (38.4 %)
20 (15.1 %)
145 (50.3 %)
11 (28.9 %)

Grade 3
240 (52.4 %)
111 (84.1 %)
109 (37.8 %)
<0.001
20 (52.6 %)

ER status (n = 419)

Positive
307 (73.3 %)
59 (45.7 %)
243 (86.5 %)
5 (55.6 %)

Negative
112 (26.7 %)
70 (54.3 %)
38 (13.5 %)
<0.001
4 (44.4 %)

PR status (n = 409)

Positive
213 (52.1 %)
35 (28.2 %)
175 (62.9 %)
4 (57.1 %)

Negative
196 (47.93 %)
89 (71.8 %)
103 (37.1 %)
<0.001
3 (42.9 %)

BCS breast conserving surgery, RT postoperative radiotherapy

a

Comparisons between HER2-negative and HER2-positive DCIS were made by χ 2 -test, except for age and size that was compared by T-test

Table 2
2Patient-and tumor characteristics of the primary DCIS in relation to risk of a breast cancer event. Risk of an ipsilateral breast cancer event (IBE) including risk of in situ IBEs and invasive ipsilateral recurrence, respectively, and any invasive recurrence (IBCR) (univariate Cox regression analyses)RT radiotherapy, BCS breast conserving surgery, IBE ipsilateral breast events, IBCR invasive breast cancer recurrence HER2 status and patientand tumor characteristicsCharacteristics
Ipsilateral Breast Events (IBE)
in situ IBEs
invasive IBEs
Invasive Breast Cancer Recurrence (IBCR)

Among BCS (n = 359)
BCS
BCS
Among all (n = 458)

HR (95 % CI)
HR (95 % CI)
HR (95 % CI)
HR (95 % CI)

Age (years)

< 50
Reference
Reference
Reference
Reference

50-65
0.77 (0.44-1.31)
0.92 (0.41-2.05)
0.63 (0.30-1.32)
0.68 (0.41-1.12)

> 65
1.19 (0.67-2.12)
1.31 (0.55-3.11)
1.09 (0.50-2.37)
0.98 (0.57-1.66)

Detection mode

Screening
Reference
Reference
Reference
Reference

Clinically
1.78 (1.04-3.07)
1.78 (0.81-3.91)
2.09 (0.98-4.42)
1.47 (0.93-2.35)

Type of Surgery

BCS
-
-
-
Reference

Mastectomy
-
-
-
0.55 (0.29-1.03)

RT after BCS

No
Reference
Reference
Reference
Reference

Yes
0.53 (0.33-0.86)
0.50 (0.25-1.01)
0.51 (0.27-0.98)
0.91 (0.59-1.41)

Tumor size (mm)

≤ 25
Reference
Reference
Reference
Reference

> 25 / multifocal
1.14 (0.71-1.83)
1.88 (0.97-3.62)
0.64 (0.30-1.34)
0.89 (0.55-1.45)

Nuclear grade

Grade 1
Reference
Reference
Reference
Reference

Grade 2
0.90 (0.41-1.96)
1.05 (0.30-3.63)
0.73 (0.27-1.99)
0.82 (0.38-1.77)

Grade 3
0.83 (0.39-1.79)
1.03 (0.30-3.49)
0.63 (0.23-1.69)
0.64 (0.30-1.37)

ER status

Negative
Reference
Reference
Reference
Reference

Positive
0.90 (0.52-1.55)
0.80 (0.38-1.71)
1.02 (0.47-2.22)
1.16 (0.70-1.93)

PR status

Negative
Reference
Reference
Reference
Reference

Positive
0.83 (0.52-1.32)
1.04 (0.53-2.02)
0.66 (0.35-1.27)
1.06 (0.69-1.63)

Table 3
3Cox regression analyses by HER2 status in 458 women with a primary DCISDCIS

HER2 positive
HER2
negative

HR (95 % CI)
HR (95 % CI)

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Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution. I A Olivotto, C D Bajdik, P M Ravdin, C H Speers, A J Coldman, B D Norris, J Clin Oncol. 23Population-based validation of the prognostic model ADJUVANT! for early breast cancer. Submit your manuscript at www.biomedcentral.com/submitOlivotto IA, Bajdik CD, Ravdin PM, Speers CH, Coldman AJ, Norris BD, et al. Population-based validation of the prognostic model ADJUVANT! for early breast cancer. J Clin Oncol. 2005;23:2716-25. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit


The clinical and molecular epidemiology of Staphylococcus aureus infections in Fiji

Adam Jenney
Deborah Holt
Roselyn Ritika
Paul Southwell
Shalini Pravin
Eka Buadromo
Jonathan Carapetis
Steven Tong
Andrew Steer
The clinical and molecular epidemiology of Staphylococcus aureus infections in Fiji
R E S E A R C H A R T I C L E Open Access
Background: There are few data describing the microbiology and genetic typing of Staphylococcus aureus that cause infections in developing countries. Methods: In this study we observed S. aureus infections in Pacific Island nation of Fiji in both the community and hospital setting with an emphasis on clonal complex (CC) genotyping and antimicrobial susceptibility. Results: S. aureus was commonly found in impetigo lesions of school children and was recovered from 57% of impetigo lesions frequently in conjunction with group A streptococcal infection. Methicillin-resistant S. aureus (MRSA) comprised 7% (20/299) of isolates and were all non-multi-resistant and all genotyped as CC1. In contrast, there was a diverse selection of 17 CCs among the 105 genotyped methicillin-susceptible S.aureus (MSSA) strains. Isolates of the rare, phylogenetically divergent and non-pigmented CC75 lineage (also called S.argenteus) were found in Fiji. From hospitalized patients the available 36 MRSA isolates from a 9-month period were represented by five CCs. The most common CCs were CC1 and CC239. CC1 is likely to be a community-acquired strain, reflecting what was found in the school children, whereas the CC239 is the very successful multi-drug resistant MRSA nosocomial lineage. Of 17 MSSA isolates, 59% carried genes for Panton-Valentine leukocidin. The S. aureus bacteraemia incidence rate of 50 per 100,000 population is among the highest reported in the literature and likely reflects the high overall burden of staphylococcal infections in this population. Conclusions: S. aureus is an important cause of disease in Fiji and there is considerable genotypic diversity in community skin infections in Fijian schoolchildren. Community acquired-(CA)-MRSA is present at a relatively low prevalence (6.7%) and was solely to CC1 (CA-MRSA). The globally successful CC239 is also a significant pathogen in Fiji.

Background

Staphylococcus aureus is one of the most successful human pathogens. The dispersion of certain successful lineages can be tracked across the globe [1]. Since first being recognised in the early 1960s, methicillin-resistant S. aureus (MRSA) emerged as an important worldwide pathogen associated with nosocomial infections in the developed world, so-called healthcare-associated MRSA (HA-MRSA) [2]. In the last two decades however, community-associated MRSA (CA-MRSA) strains have been observed to cause disease in people without healthcare contact. At-risk groups have been identified, including sports teams, prisoners, military personnel, and Indigenous population groups. In addition, S. aureus lineages have demonstrated a degree of geographical restriction [3].

While extensive data on the burden of disease due to S. aureus are available from many industrialised countries, there are few data from developing countries. In an analysis of 220 papers concerning hospital-acquired infection in developing countries, S. aureus was the second most recognised cause after the Enterobacteriaceae; antibiotic resistance was recorded in just eight of the studies reviewed and, of these, 54% of the S.aureus isolates were found to be methicillin resistant [4]. In a study of S. aureus from rural Thailand, S.aureus caused similar clinical manifestations of disease as those seen in more developed countries but an increased mortality was noted [5]. Within developed countries, S. aureus has been found to cause significant disease amongst certain disadvantaged populations, including Indigenous communities [6]. For example, in a study of impetigo in an urbanised population of Indigenous Australians in Far North Queensland in Australia, S. aureus was isolated from 92 of 118 cases (78%) of which 16% were CA-MRSA [7]. In a study of impetigo in remote Aboriginal communities in northern Australia, S. aureus was isolated in 59% of impetigo lesions and 23% were CA-MRSA [8]. Furthermore, in northern Australia, the incidence of S. aureus bacteraemia in the Indigenous population was 5.8 times that of non-Indigenous Australians [9].

Infection due to S. aureus is thought to be the cause of significant disease in Pacific Island Countries (PICs) [10]. There is a large burden of impetigo due to S. aureus and Streptococcus pyogenes in many PICs, often due to secondary infection of scabies [11]. Although there are few population-based studies of S. aureus in the Pacific region, CA-MRSA is known to occur in the region. One particular strain of S. aureus, the so-called South-West Pacific (SWP) CA-MRSA clone, has been prevalent in in Samoan patients living in New Zealand, and also Pacific Island people populations in Australia [12]. Outbreaks of CA-MRSA have also been described in Pacific Island people populations in Hawaii [13], and amongst Samoans living in Alaska [14] and in Samoa itself [15].

Given the paucity of data regarding the clinical microbiological features as well as the molecular epidemiology of S. aureus from the Pacific Islands, the aim of this study was to describe the clinical and microbiologic epidemiology of infections amongst school children and hospital inpatients with S. aureus in the Republic of Fiji. Other aims included an examination of the incidence of S.aureus bacteraemia amongst different populations of Fijian residents. The study also sought to describe the frequency of MRSA infections among those with impetigo in schools to determine if a change was required from the current practice of using empiric beta lactam antibiotics for suspected S.aureus infections.

Methods

Setting

Fiji has a population of approximately 837,000 people living in some of its 330 islands in the tropical Western Pacific. The majority of the population is i-Taukei (Indigenous Fijian) with a large minority (37%) being Fijians of Indian descent [16]. We conducted two separate studies: the first was a prospective cohort survey of impetigo in 450 children over a period of 10 months in 3 primary schools in Fiji [11], and the second was a retrospective study of S. aureus isolated from clinical specimens sent to the microbiology laboratory at the Colonial War Memorial Hospital (CWMH) in the capital city of Suva.

Community study

This was a prospective cohort study to investigate the part played by S. aureus in the causation of impetigo in school children aged 5 -15 years in three schools conducted in 2006 [11]. Two of these were schools located in a rural area where the population was predominantly iTaukei, whilst the third (and largest) school was located in Suva with a population predominantly of Fijians of Indian descent. Each school was visited six times over a ten-month period at two monthly intervals and at each visit the children were examined for impetigo. Infected skin lesions were swabbed and swabs were placed in airtight bags with desiccant for transportation back to the laboratory in Suva. The swabs were plated onto sheep blood agar and incubated at 37°C for 24-48 hours [5]. S. aureus was identified by colony morphology and a latex slide agglutination test [Staphaurex Remel, Lenexa, KS, USA]. Isolates were stored in glycerol and transferred frozen to Darwin, Australia for further testing (described below).

Hospital study

In this study isolates were collected from the diagnostic microbiology laboratory of the CWMH, Suva, the country's largest hospital serving the Central Division of Fiji, which is inhabited by approximately one third of the nation's population. Over a nine-month period September 2006 to May 2007, consecutive isolates of MRSA were collected from sterile and non-sterile sites and where possible, clinical and demographic data were collected from the patients' medical record. During this time a small proportion (approximately 5% of the total identified in the laboratory) of MSSA isolates were collected (each MSSA isolate selected was identified temporally as close as possible to each MRSA isolate collected). This was done to ascertain some molecular epidemiological data of the MSSA isolates causing symptoms at the same time as the MRSA isolates that were being investigated. This yielded a similar number of MSSA as MRSA isolates, over the same time period. [From the four annual surveys, prior to this study, conducted by the scientist at the CWMH laboratory, approximately 85% of the S.aureus isolates came from swabs and 3-5% were MRSA (S. Pravin, personal communication)]. Also during this time the blood culture isolates of S.aureus were enumerated to calculate the incidence of S.aureus bacteraemia. We used the population of the Central Division of Fiji from the 2007 national census as the basis for denominator calculations. Because surveillance occurred during 2006 (4 months) and 2007 (5 months), we extrapolated a total population figure for 2006 using the pro rata difference between the last official census in 1996 and the latest census in 2007. In calculating average annualized rates, the denominator of person-months was calculated by multiplying each year's population by the number of months of surveillance for each year (i.e. 2006 population by 4 months and 2007 population by 5 months) and adding these totals. The total number of cases over the 9 months was then divided by the total personmonths and multiplied by 12 to give average annualized incidence rates with binomial exact 95% confidence intervals (CI). Incidence rate ratios were used to compare rates between ethnic groups.

Antimicrobial susceptibility testing

Antimicrobial susceptibility testing was undertaken using the Vitek2 automated system [BioMerieux, Mary l'etoile, France] as per the manufacturer's instructions. We considered non-multidrug-resistant MRSA (nmMRSA) isolates to represent CA-MRSA strains and considered multidrugresistant MRSA (mMRSA) isolates to represent HA-MRSA strains [17]. nmMRSA isolates were defined phenotypically as those isolates resistant to beta-lactam antibiotics but resistant to no more than one other class of antibiotic, and mMRSA isolates were defined as those resistant to two or more classes of antibiotic in addition beta-lactam antibiotics. Antibiotic phenotype has previously been shown to accurately predict the genotype of CA-MRSA strains [18], and this result has been validated in various studies from Australia [6,9,17,19,20].

Molecular typing

Using real-time polymerase chain reaction (PCR), S. aureus was verified by confirming the presence of nucA gene, MRSA was confirmed by the presence of mecA gene. The presence Panton Valentine leukocidin (PVL) has been associated with particularly virulent disease in some studies of CA-MRSA particularly. The ability to produce PVL was detected by the presence of lukS-PV-lukF-PV genes [21]. A proportion of the collected isolates were genotyped by means of a single-nucleotide polymorphism (SNP) genotyping system based on the multilocus sequence type (MLST) database, as previously elsewhere [8,21]. Of the 303 isolates 105 were selected for typing. These isolates came from throughout the whole collection period, and though not randomly chosen, there was no regard to individual isolate selection thereby avoiding bias as much as possible. In short, a kinetic PCR method was used to interrogate 8 highly discriminatory SNPs, allowing isolates to be assigned into eBURST [22] derived clonal complexes (CCs). Sequencing of selected MLST loci of some isolates was carried out using standard MLST primers and methodology for S. aureus [23]. Investigation of spa type diversity was performed using a high-resolution melting (HRM) assay of the spa locus [24].

Statistical analysis

Data were analysed using non-parametric formulae in Stata version 9.1 (Stata-Corp, College Station, Texas, USA). The population-based incidence of S. aureus bacteraemia was calculated using the census data for the Central Division of Fiji as the denominator.

Ethical approval

Ethical approval for this study was obtained from the Fiji National Research Ethics Review Committee, the Fiji National Health Research Committee and the Human Research Ethics Committee at the University of Melbourne. Children were only enrolled if written consent from a parent or guardian was obtained, and in the case of children aged ten years or older, if written assent was also obtained.

Results

Community study

A total of 563 swabs were taken from 455 children (108 children had two swabs taken at the same visit from two infected sites) and there was bacterial growth from 522 swabs (92.7%). There was growth of beta-haemolytic streptococci (predominantly Streptococcus pyogenes) from 491 swabs (87.2%), and S. aureus was isolated from 323 swabs (57.4%), co-existing with beta-haemolytic streptococci in 292 swabs (51.9%).

Antimicrobial resistance testing was performed on 299 of the 323 isolates. 20 isolates (6.7%, 95% CI 4.1 -10.1) from 14 children were found to be nmMRSA, 17 underwent SNP-typing, and all were CC1 and PVL negative.

Of the remaining 303 MSSA isolates, 105 underwent SNP-typing. There was a wide distribution of CCs among the MSSA isolates ( Figure 1) with the most common being CC5, 7, 14, 75 and 121. As the CC75 lineage has been noted to be non-pigmented in collections from northern Australia, we also demonstrated that all CC75 strains from this study were also non-pigmented when grown in LB broth. This CC75 lineage has provisionally been named S. argenteus given its divergent phylogenetic position [25] and non-pigmented phenotype and will herein be referred to as S. argenteus. Two isolates produced a previously undescribed SNP profile and full MLST revealed that they were a new MLST sequence type, which is a single locus variant of ST96. PVL positive strains comprised 26 of the 105 isolates and were all of the CC30 (7), CC119 (1) and CC121 (12) isolates and 6 of the 20 CC7 isolates.

Hospital setting

Of the 36 MRSA isolates that underwent antimicrobial susceptibility testing and genotyping, 14 (39%) were mMRSA and all of these were of the globally recognised HA-MRSA lineage (ST239) and were PVL negative. The other 22 MRSA isolates were of the nmMRSA susceptibility phenotype, of which 5 (23%) were PVL positive (all were CC30). Of the remainder, fifteen were CC1, one was CC59 and one was CC101 (Figure 2).

Of the 27 MSSA isolates analysed, there were nine different CCs, and 59% (16/27) of isolates were PVL positive. To determine if a particular sub-lineage of the community-based CC1 strains was causing hospital infections we investigated diversity at the spa locus with HRM. We found that 3 HRM curves were present in similar proportions in both community and hospital isolates (community strains: HRM spa 1 (2 isolates), HRM spa 2 (14), HRM spa 3 (3); hospital strains: HRM spa 1 (1), HRM spa 2 (14), HRM spa 3 (3), nontypeable (2)).

Clinical features of patients with MRSA infection

Clinical information was available for 22 patients that had MRSA isolates. Eight of these 22 isolates were from infected surgical wounds, seven from non-surgical skin infections, two from CSF and one from blood culture. The median age was 43.5 years (range, 2 months-77 years); 55% were female; 16 were iTaukei, three were Fijians of Indian descent and 3 were 'other' ethnicity. Eight had had a history of hospitalisation or surgical procedures in the 12 months prior to the isolate being identified and 15 (68%) had received antibiotics (from CWMH) in the previous year. Eleven had diabetes mellitus and of these, five had MRSA isolated from diabetic foot infections. The median length of hospitalisation was 22 days (interquartile range, 15 to 36 days).

Incidence of S aureus bacteraemia

There were 128 separate episodes of S. aureus bacteraemia over the 9-month study period. This equated to an all-ages incidence figure for the Central Division of Fiji of S. aureus bacteraemia of 50.1 cases per 100,000 population (95% confidence interval, CI, 41.8 -59.5) with peaks in children aged less than 5 years and adults aged 55 to 64 years ( Figure 3). The incidence was highest in iTaukei (all-ages incidence 66.2 per 100,000, 95% CI 54.2 -80.1), and iTaukei were three times as likely to have S. aureus bacteraemia compared with people of other ethnic backgrounds (incidence rate ratio 3.0, 95% CI 1.9 -5.1). Only 3 of the 128 isolates were MRSA (2.3%). No further clinical information was available.

Discussion

Although this study found a relatively low incidence of MRSA in Fiji in the community setting, the overall incidence of S. aureus bacteraemia was very high. There was considerable diversity of CC types seen amongst the CA-MRSA and the MSSA isolates, and this diversity was seen in both the community and hospital settings. While CC1 was the most common lineage observed and found both in the community and hospital studies, the CC239 clone was found to be well-established amongst hospital patients similar to more developed nations. CC1 has also been found to represent 26% of MRSA strains in the Pacific Island nation of Samoa [15]. Further sub-typing of CC1 strains demonstrated that there was no  over-representation of any particular community-based sub-lineage as a cause of hospital infections. CC239 is the typical HA-MRSA lineage found in Australia and Asia [26], and is evidently present in Fiji as a cause of nosocomial infections. With regards to CC30 MRSA, although the name SWP lineage would suggest an origin from this region of the world, it only comprised five of the 22 nmMRSA of hospital isolates and is most likely not a dominant lineage in either Fiji or Samoa [15].

Perhaps most surprising was the presence of S. argenteus strains. This divergent and early-branching lineage of S. aureus is prevalent in Indigenous populations in northern Australia, [8,27] and in an Amazonian tribe in French Guinea [28]. S. argenteus typically lacks the genes for production of the carotenoid pigment staphyloxanthin [25] and is hypothesised to be well adapted to causing skin infections and to be less virulent than other S. aureus lineages [29]. Findings from our current study extend the geographical distribution of this unusual lineage and confirm its non-pigmented phenotype. In addition, we provide supportive evidence regarding its virulence and epidemiological niche in that it represented 8.2% of community based impetigo isolates but only 1.6% of hospital based clinical isolates.

Our study found a prevalence of CA-MRSA in impetigo lesions among Fiji children of 6.7%, which is similar to a reported prevalence of 9% in Samoa [15]. This is in striking contrast to reports of high prevalence of CA-MRSA among Pacific Island peoples and Australian Aboriginal populations living in developed country settings [9,30,31]. The reason for the lower prevalence in Fiji is unclear, particularly as many of the conditions thought to contribute to the emergence of CA-MRSA in Indigenous populations in the developed world setting [6] are also present in Fiji. These factors are likely to include overcrowding, inadequate health-hardware for washing of clothes and people, poor skin hygiene and high rates of antibiotic use and further research specific to Fiji to investigate these is required. The prevalence of MRSA among hospital isolates of S. aureus in Fiji is also low; in the year prior to this study the proportion of all S.aureus isolates from the CWMH laboratory that were methicillin-resistant was 5% (personal communication, S. Pravin).

The incidence of SAB of 50.1 per 100,000 overall and 66.2 per 100,000 for the i-Taukei (Indigenous Fijian) population is considerably higher than that reported from developed countries [32] and is similar to that seen in Indigenous populations in both northern Australia (65 per 100,000) [9] and for the whole of Australia (62.5 per 100,000) [33]. The age distribution is also similar to that in Indigenous Australian populations with a peak incidence in the 45-65 age group. This is in stark contrast to that seen in developed countries, including the non-Indigenous population of Australia, where incidence increases with age [32,33]. Although we cannot infer a causative link, there is likely to be an association between high rates of skin and soft tissue infections and the high incidence of SAB in both Fiji and Indigenous Australian populations. Community-based efforts to improve skin health among these populations [34,35] will hopefully result in reduced rates of skin and soft tissue infections, and it will be important to determine if there is a concurrent reduction in incidence rates of communityonset SAB.

The hospital study identified consecutive patients that had MRSA isolated from clinical specimens sent to the hospital laboratory for culture, and in this small sample MRSA was primarily isolated from wounds (post-surgical or community acquired injuries). From the molecular typing data it appears that CC239 is commonly observed in hospital in Fiji and given the experience worldwide it would be expected that this is a nosocomial isolate. The nmMRSA CC1 isolates that were identified in the hospital setting however were most likely acquired in the community prior to admission, as the only MRSA detected in community study (in children that had likely not been to hospital) was CC1. However, these data come from a very brief snapshot of S. aureus infections at CWMH. It has been noted elsewhere that CA-MRSA isolates most frequently associated with community-associated infections can become established as the aetiology of nosocomial infections, [36] and therefore we cannot rule out this phenomenon occurring at CWMH.

Notably, of the hospital-based MSSA isolates, 59% carried pvl genes. While this represented only 16 of 27 isolates it is both surprising and concerning but is similar to findings in Africa [37] and Indigenous populations in northern Australia [29] where PVL was present in 57% and 40% of MSSA isolates respectively. Although we did not collect clinical information on the infections caused by these isolates, it nonetheless suggests that there is a large burden of PVL + MSSA disease given that MSSA represents >95% of S. aureus isolates at CWMH.

There are a number of limitations to our study. The community portion of our study investigated one type of clinical infection (impetigo) in one demographic group (school children) in two closely related regions of Fiji. A broader investigation of staphylococcal infections that includes older members of the community and a wider geographic region would be valuable. Within this school group there may also be may be an under-estimation of staphylococcal disease in this population as the surveys took place at two-monthly intervals it is likely that some episodes of impetigo occurred between visits and therefore were missed. The hospital portion of our study had a narrow time frame and a small numbers of cases (especially those where clinical information was available) and so the conclusions from this part of the study are limited.

Conclusions

Despite these limitations it is clear that S. aureus is an important cause of disease in Fiji and there is considerable genotypic diversity in community skin infections in Fijian schoolchildren. CA-MRSA is present at a relatively low prevalence (6.7%) and in this survey was found to be due solely to CC1 (CA-MRSA). Isolates typically associated with both CA-MRSA and HA-MRSA isolates were found in the hospital setting with CC239 responsible for all of the HA-MRSA in this series. Beta-lactam agents, such as cloxacillin, remain the first choice for community-acquired S. aureus infections in Fiji. However, if the initial clinical response is poor then appropriate therapy for MRSA should be considered, such as clindamycin or vancomycin.

Competing interests

Authors declare that they have no competing interests.

Authors' contributions AJ helped to devise the study and made substantial contributions to the acquisition of data, bacterial isolation and identification and writing the manuscript, DH carried out the molecular genetic studies, RR processed the specimens and isolated the bacteria in Fiji and assisted with molecular studies in Darwin, PS performed the antimicrobial susceptibility testing, SS supervised and assisted bacterial isolation, identification and susceptibility testing in Fiji, EB provided local Fiji data invaluable to the study design, JC significantly contributed to the study design and the drafting of manuscript, ST made major contribution to study design, analysis of data, and prepared most of the manuscript, AS devised the study, arranged the collection of samples, and gave significant assistance towards the drafting of manuscript. All authors read and approved the final manuscript.

Figure 1
1Clonal complex types of S.aureus isolated from Fijian schoolchildren's impetigo lesions. Clonal complexes identified from SNP-typing of Staphylococcus aureus isolates collected from impetigo lesions in school children in Fiji (MSSA: methicillin sensitive Staphylococcus aureus; nmMRSA: non-multidrug-resistant methicillin resistant Staphylococcus aureus; PVL: Panton Valentine Leukocidin).

Figure 2
2Clonal complex types of S.aureus isolated from patients at CWMH, Fiji. Clonal complexes identified from SNP typing of Staphylococcus aureus isolates collected from the Colonial War Memorial Hospital, Fiji (MSSA: methicillin sensitive Staphylococcus aureus; nmMRSA: non-multidrug-resistant methicillin resistant Staphylococcus aureus; mMRSA: multidrug-resistant MRSA; PVL: Panton Valentine Leukocidin).

Figure 3
3Annualised incidence of S.aureus bacteraemia at CWMH, Fiji. Number of cases and annualised incidence of cases where Staphylococcus aureus was cultured from blood at the Colonial War Memorial Hospital, Central Division, Fiji, May 2006 -September 2007 showing a predominance of cases amongst the iTaukei (Indigenous Fijian) population.

Abbreviations CC: Clonal complex; MRSA: Methicillin-resistant S. aureus; HA-MRSA: Healthcareassociated MRSA; CA-MRSA: Community-associated MRSA; PICs: Pacific island countries; SWP: South-West Pacific; CWMH: Colonial war memorial hospital; nmMRSA: Non-multidrug-resistant MRSA; mMRSA: Multidrug-resistant MRSA; PCR: Polymerase chain reaction; PVL: Panton-Valentine leukocidin; SNP: Single-nucleotide polymorphism; MLST: Multilocus sequence type; HRM: High-resolution melting.
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AcknowledgementsThe authors acknowledge the assistance of the Fiji GrASP team, the Ministry of Health, Fiji, Professor Trevor Duke and the CICH team in Melbourne but especially the students and staff of the Schools involved in the study.Author details 1Submit your next manuscript to BioMed Central and take full advantage of:
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The clinical and molecular epidemiology of Staphylococcus aureus infections in Fiji. Jenney, BMC Infectious Diseases. 14160Cite this article asCite this article as: Jenney et al.: The clinical and molecular epidemiology of Staphylococcus aureus infections in Fiji. BMC Infectious Diseases 2014 14:160.