BMJ Open. For peer review only - Journal: BMJ Open. Manuscript ID: bmjopen

BMJ Open

Low prevalence of methicillin-resistant Staphylococcus aureus among men who have sex with men attending an STI clinic in Amsterdam, a prospective cross-sectional study

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Manuscript ID: Article Type:

Date Submitted by the Author:

Complete List of Authors:

BMJ Open bmjopen-2012-002505 Research 16-Dec-2012

Primary Subject Heading:

Dermatology, Infectious diseases, Sexual health Infectious diseases & infestations < DERMATOLOGY, Infection control < INFECTIOUS DISEASES, Public health < INFECTIOUS DISEASES, Dermatology < INTERNAL MEDICINE, MICROBIOLOGY, PUBLIC HEALTH

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Keywords:

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Secondary Subject Heading:

Public health

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Joore, Ivo; Public Health Service Amsterdam, cluster infectious diseases, STI Outpatient Clinic van Rooijen, Martijn; Public Health Service Amsterdam, cluster infectious diseases, STI Outpatient Clinic Schim van der Loeff, Maarten; Public Health Service Amsterdam, cluster infectious diseases, research department de Neeling, Han; Center for Infection and Immunology Amsterdam (CINIMA), Academic Medical Center (AMC), University of Amsterdam, National Institute for Public Health and the Environment (CIb/RIVM) van Dam, Alje; Public Health Service Amsterdam, cluster infectious diseases, Public Health Laboratory de Vries, H; Municipal Health Service Amsterdam, STI outpatient clinic; Academic Medical Centre, University of Amsterdam, Dermatology

For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml

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BMJ Open

1 Low prevalence of methicillin-resistant Staphylococcus aureus among men who have sex with men attending an STI clinic in Amsterdam, a prospective cross-sectional study

1

Joore IKCW , van Rooijen MS

1,2,3

2,3

4

, Schim van der Loeff MF , De Neeling AJ , van Dam A

5, 6

, de

Vries HJC1,4,7

1. STI outpatient clinic; Cluster of Infectious Diseases, Public Health Service Amsterdam (GGD),

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Amsterdam, The Netherlands

2. Department of Research; Cluster of Infectious Diseases, Public Health Service Amsterdam (GGD), Amsterdam, The Netherlands 3. Department of Internal Medicine; Center for Infection and Immunology Amsterdam (CINIMA), Academic Medical Center (AMC), University of Amsterdam, Amsterdam, the Netherlands

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4. Centre for Infectious Diseases Control; National Institute for Public Health and the Environment (CIb/RIVM), Bilthoven, the Netherlands

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5. Public Health Laboratory; Cluster of Infectious Diseases, Public Health Service Amsterdam (GGD), Amsterdam, The Netherlands

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6. Department of Medical Microbiology, Onze Lieve Vrouwe Gasthuis general hospital, Amsterdam, The Netherlands

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7. Department of Dermatology; Academic Medical Center (AMC), University of Amsterdam, Amsterdam, the Netherlands.

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Prof. Henry J.C. de Vries, MD, PhD,

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Corresponding author:

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STI outpatient clinic, Cluster of Infectious Diseases, Public Health Service Amsterdam (GGD), 1000 CE Amsterdam, the Netherlands Telephone number: +31(20)5555429 Fax number: +31(20)6960076 Email: [email protected]


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2 Community-acquired

Keywords:

methicillin-resistant

Staphylococcus

aureus,

HIV,

male

homosexuality, sexually transmitted infections Word count body of text: 2075, Word count abstract: 247

ABSTRACT

Objective: Community associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is

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common among men who have sex with men (MSM) in the USA. It is unknown whether this is also the case in Amsterdam, the Netherlands. Design: Prospective cross-sectional study Setting: STI outpatient low-threshold clinic, Amsterdam, the Netherlands Participants: Between October 2008 and April 2010 211 two groups of men were included: (1)

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74 MSM with clinical signs of a skin or soft tissue infection (symptomatic group) and (2) 137 MSM without clinical signs of such infections (asymptomatic group).

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Primary outcome measures: Staphylococcus aureus and MRSA infection and/or colonisation.

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Swabs were collected from the anterior nasal cavity, throat, perineum, penile glans and, if present, from infected skin lesions. Culture for S. aureus was done on blood agar plates and for

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MRSA on selective chromagar plates after enrichment in broth. If MRSA was found, the spa-gene was sequenced.

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Secondary outcome measures: Associated demographic characteristics, medical history, risk factors for colonisation with S. aureus and high-risk sexual behaviour were collected through a self-completed questionnaire.

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Results: The prevalence of S. aureus colonisation in the nares was 37%, the pharynx 11%, the

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perianal region 12%, the glans penis 10%, and in skin lesions 40%. In multivariable analysis adjusting for age, anogenital S. aureus colonisation was significantly associated with the symptomatic group (P=0.01) and marginally with HIV (P=0.06). MRSA was diagnosed in 2 cases: prevalence 0.9% [95%CI 0.1-3.4%]). Neither had CA-MRSA strains. Conclusions: CA-MRSA among MSM in Amsterdam is rare. Genital colonisation of S. aureus is not associated with sexual high-risk behaviour.


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3 ARTICLE SUMMARY Article focus: •

Community associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is common among men who have sex with men (MSM) in the USA.

•

It is unknown whether this is also the case in Amsterdam, the Netherlands.

•

In a prospective cross-sectional study at the STI outpatient low-threshold clinic, Amsterdam, the Netherlands we studied the prevalence of S. aureus and MRSA

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colonisation and infecions among symptomatic and asymptomatic men who have sex with men (MSM)

Key messages:

• Among MSM visiting the STI clinic in Amsterdam CA-MRSA is rare.

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• Genital colonisation of S. aureus is not associated with sexual high risk behaviour.

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Strengths and limitations of this study

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• The study was limited to the Amsterdam MSM population visiting the STI outpatient clinic. Funding: This work was supported by the Research and Development fund of the Public Health

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Service of Amsterdam [Project 2373 Community associated MRSA prevalence and risk factors in male std outpatient clinic visitors who have sex with men]. The funder had no role in study design, data

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collection and analysis, decision to publish, or preparation of the manuscript.

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Competing Interests: The authors certify that they have no affiliation with or financial involvement in any organization or entity with a direct financial interest in the subject matter or materials discussed in the manuscript (e.g., employment, consultancies, stock ownership, or honoraria).

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This work was presented in an oral presentation titled “Community acquired MRSA and MSSA among MSM” during the 17th meeting International Society for STD Research (ISSTDR) in Quebec, Canada, July 10th, 2011

Data Sharing: Technical appendix, statistical code, and dataset available from the corresponding author at Dryad repository, who will provide a permanent, citable and open access home for the dataset.


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4 Author’s contribution IJ analysed the data and wrote the first manuscript draft. MvR collected the data and analysed the data, MSvdL supervised the epidemiological analysis and wrote the article, HdN performed the molecular strain typing and wrote the article, AvD performed the cultivation experiments, designed the study and wrote the aricle, HdV conceptualized and supervised the study and approved the final manuscript. Technical appendix, statistical code, and dataset available from the corresponding author at Dryad repository, who will provide a permanent, citable and open access home for the dataset.

INTRODUCTION

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Staphylococcus aureus is a pathogen that can cause skin and soft-tissue infections. Nasal carriage plays an important role in the epidemiology and pathogenesis of this infection.

1;2

Around

20% (12-30%) of individuals are persistent S. aureus nasal carriers, approximately 30% are

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intermittent carriers (range 16-70%), and about 50% (range 16-69%) non-carriers.

1;3

A causal

relation between S. aureus nasal carriage and infection is supported by the fact that often the

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nasal S. aureus strain and the infecting strain are the same phage type or genotype. Local antibiotic treatment of S. aureus from the nares results in the subsequent disappearance of S. aureus from other parts of the body.

1;2;4-6

include the skin, perineum, and pharynx.7;8

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Extra-nasal sites that typically harbour the organism

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ie Most strains of S. aureus are methicillin-susceptible (MSSA), but some strains are resistant to methicillin: methicillin-resistant Staphylococcus aureus (MRSA). MRSA colonisation used to be a

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nosocomial pathogen (Hospital Acquired MRSA or HA-MRSA).9 The resistance to beta-lactam antibiotics is based on the presence of the mecA gene. This gene encodes for the production of a

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modified penicillin-binding protein, PBP-2a, which has a low affinity for beta-lactam antibiotics.8

Lately, MRSA cases have been reported that appeared to be unrelated to exposure in hospitals.

10;11

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is

distinguished from HA-MRSA by clinical, laboratory, and epidemiologic characteristics.12 A new CA-MRSA clone (USA300), resistant against multiple antibiotics, has been described and was identified in several communities in the USA and Canada.9-11 New clones of CA-MRSA have also


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5 been reported in Europe.13;14 CA-MRSA often produces Panton-Valentine Leukocidin (PVL), a 13;15

toxin that causes polymorphonuclear leukocyte lysis and tissue necrosis

In men who have sex with men (MSM), outbreaks of CA-MRSA skin infections have been reported and the majority of the patients were HIV infected.8;16-18 It has been suggested that CAMRSA might be transmitted in this population from skin to skin during sexual contact.

18;19

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A cross-sectional study among 104 men conducted in Italy did not detect a single case of colonisation with CA-MRSA in HIV-infected MSM.20 No other study in Europe has reported the prevalence of CA-MRSA colonisation in MSM. The purpose of this investigation was to evaluate the prevalence of, and sexual risk factors for S. aureus colonisation (MSSA and MRSA) and CAMRSA infection among MSM visiting the sexual transmitted infection (STI) outpatient clinic in Amsterdam.

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Study population

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METHODS

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Consecutive MSM attending the STI outpatient clinic in Amsterdam, The Netherlands, were invited to participate in this cross-sectional study. We aimed to include both symptomatic men

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(those who had clinical signs of a skin or soft tissue infection [pustules, abscesses, ulcerations, erythematous painful papules or plaques]) and asymptomatic men (those without above-

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mentioned clinical signs). All participants were asked to complete a questionnaire concerning hospital admissions during the past year, factors known to be associated with S. aureus

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colonisation (e.g. sharing razor blades, tending animals), STI risk factors and sexual behaviour. STI risk factors were also obtained from the electronic files of the routine patient history.

Swabs were taken from the anterior nasal cavity, throat, perianal area, and penile glans. From symptomatic men also swabs from suspected infected skin lesions were obtained. If MRSA was detected, the participant was referred to his general practitioner for eradication therapy, and the sexual partners of the patient were invited for MRSA screening. The study was approved by the


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6 ethics committee of the Academic Medical Center of the University of Amsterdam. All participants provided written informed consent.

Laboratory tests Culture for S. aureus was done on blood agar plates and for MRSA on selective chromagar plates after enrichment in broth. S. aureus strains were confirmed by the SA442 (Martineau) nucleic acid amplification test (NAAT) and MRSA strains by the mecA NAAT.21;22 If MRSA was

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23

found, the spa-gene was sequenced as described before.

Statistical analysis

A sample size calculation was done prior to the study. The prevalence of MRSA carriage in the 24

general population was estimated to be 0.03%.

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We aimed to assess whether the MRSA

prevalence among MSM patients of the STI clinic was at least 3%. In order to reject the null hypothesis (prevalence of 0.03% or less among the studied group) with a study power of 95%

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and a significance level of 0.05, a sample size of at least 113 asymptomatic men was needed;

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this was rounded up to 125. As men with signs suggestive of S. aureus skin or soft tissue infection were thought to be more likely to be infected with MRSA, we also aimed to include a

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group of 75 symptomatic men. Because the expected number of asymptomatic men was much larger than that of symptomatic patients, the inclusion period for the symptomatic participants was scheduled to be longer.

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The electronic patient file with routine STI screening data, the questionnaire and the laboratory results were merged into one database. Chi-square test or Fisher’s exact test were used to

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compare categorical variables between groups; the ranksum test was used to compare continuous variables between groups. Data analyses were performed with STATA software (STATA Intercooled, College Station, TX, USA), version 11.0. P values of less than 0.05 were considered statistically significant. Associations between possible risk factors and anogenital S. aureus colonisation were examined with multivariable logistic regression, reporting adjusted odds ratios (OR) with 95% confidence intervals (CI). An initial model included all variables that were


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7 significant in bivariable analyses; variables were dropped one by one from the model based on the likelihood ratio test (if P>0.05); HIV was forced into the model.

RESULTS

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In total 214 men were included, starting November 2008. The inclusion of asymptomatic men was completed in December 2008 and of symptomatic men in April 2010. From three participants laboratory results were not available and these were excluded, leaving 211 MSM for analysis

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(137 asymptomatic and 74 symptomatic participants). The median age of men in the two groups was similar (38 and 41 years, respectively, p=0.32, see Table 1). Men in the two groups were

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comparable regarding most non-sexual risk factors, sexual risk factors and STI diagnoses, but

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symptomatic men were more often HIV infected (53% vs. 31%; p=0.002) and were more often diagnoses with syphilis (12% vs. 1%; p<0.001). Also, use of some drugs (cannabis, cocaine, and

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methamphetamine) was significantly more common in the symptomatic group.

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The prevalence of S. aureus colonisation (including MSSA and MRSA) was 37% (78/211) in the nose, 11% (23/211) in the throat, 12% (26/210) in the perineum, and 10% (22/211) on the glans

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penis. The prevalences were similar between symptomatic and asymptomatic men for nose and throat, but the symptomatic group had significantly higher penile and perineal colonisation

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prevalences (Table 1). In the symptomatic group 40% (27/67) of men had an S. aureus infected skin lesion. In one symptomatic and one asymptomatic case MRSA was detected, giving an overall prevalence of MRSA carriage of 0.9% (95% binomial exact confidence interval [95%CI] 0.1-3.4%), and a prevalence of 0.7% (95%CI 0.02-4.0%) among asymptomatic MSM. Details of these two cases are described below. No known CA-MRSA nor PVL toxin-producing clones were detected.


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8 S. aureus colonisation of the ano-genital area (based on penile and perianal swabs) was found in 18% (38/211) of the participants. Participants with and without ano-genital S. aureus colonisation were similar in respect to sexual risk behaviour, drug use, history or diagnosis of sexual transmitted diseases, antibiotic exposure, circumcision status and hygiene behaviour, but men with ano-genital S. aureus colonisation were older (42 vs. 38 years; P=0.05), and they were more often HIV infected (58% vs. 34%, p=0.007; see Table 2). In a multivariable logistic regression analysis, adjusting for age, HIV status (aOR 2.2, 95%CI 0.96-4.9), belonging to the symptomatic

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group (aOR=2.7, 95%CI 1.2-5.9) and having had few sexual partners (aOR 3.1, 95%CI 1.2-8.3 for those with <5 sexual partners in previous year compared to those with over 20 sexual partners) were independently and significantly associated with anogenital S. aureus colonisation.

We examined associations between nasal carriage of S. aureus and a long list of possible non-

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sexual risk factors, like admissions, operations, employment in health care sector, shaving habits, participation in team sports, sauna visits, and other factors; none of these was associated with S. aureus colonisation of the nose.

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Asymptomatic carrier of MRSA

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An HIV infected 40-year-old, asymptomatic, Dutch MSM was positive for MRSA at the perineum. The MRSA had spa type t064 and was resistant to ciprofloxacin, gentamicin, oxacillin, penicillin

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and trimethoprim/sulfamethoxazole. Further genetic analysis revealed mecA+ and Martineau+; pUSA03 and PVL were both negative. Recently, the patient was diagnosed with a hepatitis C

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infection. In the preceding year he had been hospitalized for an elective operation. He had travelled abroad frequently, including to the United States of America. He regularly used

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recreational drugs and visited a public gym and sauna. Furthermore, he engaged in high risk sex, including unprotected anal intercourse, active and passive fisting, group sex, sex at sex parties and sex with partners met through the internet. We attempted to trace six of his sexual partners of whom four were screened. None had MRSA and none had clinical signs of a skin infection.

Symptomatic carrier of MRSA


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9 In an HIV infected, 46-year old Dutch MSM MRSA was detected on swabs taken from a penile ulcer, the nasal cavity, the perineum and the glans penis. The MRSA had spa type t002 and was resistant to ciprofloxacin, erythromycin, oxacillin and penicillin. Further genetic analysis revealed mecA+ and Martineau+; pUSA03 and PVL were both negative. In the preceding year the patient had not been hospitalized, nor operated upon, nor had he travelled abroad. He did not use recreational drugs, and did not visit public gyms or saunas. He had not engaged in high risk sex; he had had sex with one partner in the preceding year. This partner could not be traced for

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further screening. DISCUSSION

The prevalence of MRSA among MSM clients of the STI outpatient clinic in Amsterdam was 0.9% (95%CI 0.1-3.4%); among clients without skin lesions this was 0.7% (95%CI 0.02-4.0%). This is

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similar to the overall prevalence in Dutch hospitals (1.8%).25 No CA-MRSA associated clones were found in this population. These findings are in contrast to reports from the USA where

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considerable CA-MRSA prevalences were observed among MSM attending STI screening sites.

18;26

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Although some MSM residing in Amsterdam frequently travel to the North American

continent, this has not resulted in an extensive introduction of CA-MRSA within the Amsterdam

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population yet. The low MRSA prevalence among the Dutch population in general and among the MSM STI clinic population may be attributed to the strict Dutch search–and-destroy policy and 24

restrictive antibiotic prescription policy.

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At present, routine screening of MSM STI clinic visitors

for CA-MRSA seems not indicated in the Netherlands.

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We observed similar prevalences of S. aureus nasal colonisation (37%) in MSM as observed in

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the general population. Genital S. aureus colonisation was associated with HIV infection; although HIV infection may be considered a proxy for high risk sexual behaviour, other markers for risk behaviour were not significantly associated with S. aureus colonisation. We attribute the more common colonisation among HIV positive participants to their immunocompromised status. Unfortunately, we did not have data on virological, immunological or clinical parameters of HIV infection, nor about use of antiretroviral drugs. We did not observe an increased risk of S. aureus nasal carriage among HIV infected, as was reported earlier.

27


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10

In contrast to Southern European countries, the Netherlands has a low rate of health careassociated MRSA (HA-MRSA), probably due to the above-mentioned search-and-destroy policy. The restricted use of betalactam antibiotics outside of hospitals, in the community, might explain the low prevalence of CA-MRSA. Europe has not yet been confronted with CA-MRSA on a wide 20

scale , contrary to Canada and the USA where high prevalences are found.

9-11;18-20

Yet, in

contrast to low prevalences of HA-MRSA and CA-MRSA in the Netherlands, screening of Dutch

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workers with livestock has revealed that 39% of slaughterhouse pigs and >20% of pig farmers are positive for the Lifestock Associated strain of methicillin-resistant Staphylococcus aureus (LAMRSA) belonging to sequence type (ST) 398.

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The 2 MRSA strains isolated in our study were

both unrelated to this LA-MRSA epidemic.

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In conclusion, CA-MRSA among MSM visiting the STI outpatient clinic in Amsterdam is rare. Although genital S. aureus colonisation was more common among HIV infected MSM, we found

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no association between high risk sexual behaviour and genital colonisation with S. aureus. In the

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Netherlands there is no indication for MRSA screening of MSM attending STI clinics.

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11 Table 1. Demographic, behavioural and clinical characteristics, and S. aureus colonisation of five anatomical locations, of 211 MSM attending the STI clinic, Amsterdam, 2008-1010, according to symptomatology of skin or soft tissue infection. Asymptomatic N = 137 n %

Symptomatic N = 74 n %

P-value

A. Demographics Median age in years (IQR) Dutch ethnicity

38 (31-45) 111 81.0

41 (31-48) 54 73.0

0.32 0.18

B. Recreational drugs use None Alcohol Cannabis XTC GHB Poppers (alkyl nitrites) Cocaine Ketamine Amphetamine Methamphetamine

21 92 34 40 36 76 29 14 8 3

15.3 67.2 24.8 29.2 26.3 55.5 21.2 10.2 5.8 2.2

7 47 33 27 25 44 27 13 7 6

9.5 63.5 44.6 36.5 33.8 59.5 36.5 17.6 9.5 8.1

0.23 0.60 0.003 0.28 0.25 0.58 0.02 0.13 0.33 0.04

13.8 21.9 14.6 20.4 19.0 10.2 71.5 57.7 49.6 40.9 15.3 10.2 43.8 62.0 22.6 54.7 57.7

9 14 11 15 12 13 48 58 32 37 15 11 38 47 17 45 45

12.2 18.9 14.9 20.3 16.2 17.6 64.9 78.4 43.2 50.0 20.3 14.9 51.4 63.5 23.0 60.8 60.8

0.32 0.003 0.38 0.20 0.36 0.32 0.29 0.83 0.95 0.40 0.66

Characteristic

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19 30 20 28 26 14 98 79 68 56 21 14 60 85 31 75 79

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C. Sexual history Number of sexual partners § 0 1-4 5-9 10-19 20-39 40 or more Active anal sex with condom # Passive anal sex with condom # Active anal sex without condom # Passive anal sex without condom # Active fisting # Passive fisting # Had group sex § Visited a sex club § Visited a sex party § Met sex partner through internet § Had sex abroad §

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D. STI: history & diagnoses History of STI HIV infected* Syphilis diagnosis Gonorrhoea diagnosis Chlamydia diagnosis

34 42 1 9 23

24.8 30.9 0.7 6.6 16.8

24 38 9 5 14

32.4 52.8 12.2 5.4 18.9

0.24 0.002 <0.001 0.74 0.70

E. S. aureus colonisation Nares Pharynx

50 13

36.5 9.5

28 10

37.8 13.5

0.85 0.37


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12 Perineum Glans penis Other locations †

9 10 --

6.6 7.3 --

17 12 27

23.3 6.2 40.3

<0.001 0.043 --

* HIV status missing for 3 patients who did not want to be tested; § During the previous year; # In the past 6 months. † Data available from 67/74 only. Abbreviations: HIV: Human immunodeficiency virus; STI: sexually transmitted infection; XTC: MDMA/methamphetamine; GHB: GammaHydroxyButyricAcid; MSM: men who have sex with men; IQR: interquartile range

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13 Table 2. Demographic, behavioural and clinical characteristics of 211 MSM attending the STI clinic in Amsterdam, 2008-10, by S. aureus ano-genital colonisation status S. aureus neg

S. aureus pos.

N = 173

N =38

n

%

n

P-value %

A. Demographics Median age in years (IQR)

38 (30-45)

Dutch Ethnicity

138

42 (35-49) 79.8

27

0.05 71.1

0.24

B. Recreational drug use None Alcohol Cannabis XTC GHB Poppers Cocaine Ketamine Amphetamine Methamphetamine

Number of sexual partners § 0

7

18.4

0.30

21

55.3

0.13

57

33.0

10

26.3

0.43

53

30.6

14

36.8

0.46

49

28.3

12

31.6

0.69

96

55.5

24

63.2

0.39

44

25.4

12

31.6

0.44

23

13.3

4

10.5

0.64

12

6.9

3

7.9

0.84

4.1

2

5.3

0.74

7

0.08

18

10.4

10

26.3

35

20.2

9

23.7

16.8

2

5.3

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1-4

12.1 68.2

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C. Sexual history

21 118

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5-9

29

10-19

35

20.2

8

21.1

20-39

33

19.1

5

13.2

40 or more

23

13.3

4

10.5

Active anal sex with condom #

121

69.9

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110

63.6

85

49.1

Passive anal sex without condom #

74

42.8

25

65.8

0.62

27

71.1

0.38

15

39.5

0.28

19

50.0

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Passive anal sex with condom # Active anal sex without condom #

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0.42

18.4

0.81

7.9

0.41

18

47.4

0.90

63.0

23

60.5

0.78

20.8

12

31.6

0.15

102

59.0

18

47.4

0.19

105

60.7

19

50.0

0.23

51

29.5

7

18.4

0.17

Active fisting #

29

16.8

7

Passive fisting #

22

12.7

3

Had group sex §

80

46.2

Visited a sex club §

109

Visited a sex party §

36

Met sex partner through internet§ Had sex abroad § D. STI: history & diagnoses History of STI


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14

HIV infected *

59

34.3

21

58.3

0.007

Syphilis diagnosis

10

5.8

0

0

0.13

Gonorrhoea diagnosis

13

7.5

0

0

0.08

Chlamydia diagnosis

30

17.3

7

18.4

0.87

Regularly attends the gym §

109

63.0

20

52.6

0.24

Regularly plays in a team sport §

13

7.5

0

0

0.08

Regularly visits the sauna §

110

63.6

23

60.5

0.72

Regularly takes a public shower §

103

59.5

25

65.8

0.48

149

86.1

32

84.2

0.76

E. Other possible risk factors

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Regularly travels abroad § Regularly shaves: Facial hair

165

95.4

38

100

0.18

Pubic hair

149

86.1

31

81.6

0.47

Body hair

110

63.6

20

52.6

0.21

45

26.0

11

29.0

0.71

14

8.1

3

7.9

0.97

9

5.2

4

10.5

0.22

23

13.5

8

21.6

0.21

Has pets

Works in health care

Was admitted to hospital § Had surgery § Had a blood transfusion § Had enemas §

Frequent bites his fingernails

6

3.5

1

2.6

0.79

49

28.3

14

36.8

0.30

55

31.8

11

29.0

0.73

11

29.0

0.28

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Has been circumcised

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36

20.8

* HIV status missing for 3 patients who did not want to be tested; § During the previous year; # In the past 6 months. Abbreviations: HIV: Human immunodeficiency virus; STI: sexually transmitted infection; XTC: MDMA/methamphetamine; GHB: Gamma hydroxybutyric acid; MSM: men who have sex with men; IQR: interquartile range

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15 REFERENCE LIST 1. Wertheim HF, Melles DC, Vos MC et al. The role of nasal carriage in Staphylococcus aureus infections. Lancet Infect Dis 2005; 5: 751-62. 2. von Eiff C, Becker K, Machka K et al. Nasal carriage as a source of Staphylococcus aureus bacteremia. Study Group. N Engl J Med 2001; 344: 11-6. 3. Nouwen JL, Ott A, Kluytmans-Vandenbergh MF et al. Predicting the Staphylococcus aureus nasal carrier state: derivation and validation of a "culture rule". Clin Infect Dis 2004; 39: 806-11. 4. Parras F, Guerrero MC, Bouza E et al. Comparative study of mupirocin and oral co-trimoxazole plus topical fusidic acid in eradication of nasal carriage of methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother 1995; 39: 175-9.

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5. Casewell MW, Hill RL. Elimination of nasal carriage of Staphylococcus aureus with mupirocin ('pseudomonic acid')--a controlled trial. J Antimicrob Chemother 1986; 17: 365-72. 6. Reagan DR, Doebbeling BN, Pfaller MA et al. Elimination of coincident Staphylococcus aureus nasal and hand carriage with intranasal application of mupirocin calcium ointment. Ann Intern Med 1991; 114: 101-6.

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7. Eriksen NH, Espersen F, Rosdahl VT et al. Carriage of Staphylococcus aureus among 104 healthy persons during a 19-month period. Epidemiol Infect 1995; 115: 51-60.

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8. Lowy FD. Staphylococcus aureus infections. N Engl J Med 1998; 339: 520-32. 9. Moran GJ, Krishnadasan A, Gorwitz RJ et al. Methicillin-resistant S. aureus infections among patients in the emergency department. N Engl J Med 2006; 355: 666-74.

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10. Gilbert M, MacDonald J, Gregson D et al. Outbreak in Alberta of community-acquired (USA300) methicillin-resistant Staphylococcus aureus in people with a history of drug use, homelessness or incarceration. CMAJ 2006; 175: 149-54.

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11. Hota B, Ellenbogen C, Hayden MK et al. Community-associated methicillin-resistant Staphylococcus aureus skin and soft tissue infections at a public hospital: do public housing and incarceration amplify transmission? Arch Intern Med 2007; 167: 1026-33.

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12. Mediavilla JR, Chen L, Mathema B et al. Global epidemiology of community-associated methicillin resistant Staphylococcus aureus (CA-MRSA). Curr Opin Microbiol 2012. 13. Tristan A, Bes M, Meugnier H et al. Global distribution of Panton-Valentine leukocidin-positive methicillin-resistant Staphylococcus aureus, 2006. Emerg Infect Dis 2007; 13: 594-600.

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14. Petersen A, Stegger M, Heltberg O et al. Epidemiology of methicillin-resistant Staphylococcus aureus carrying the novel mecC gene in Denmark corroborates a zoonotic reservoir with transmission to humans. Clin Microbiol Infect 2012. 15. Boyle-Vavra S, Daum RS. Community-acquired methicillin-resistant Staphylococcus aureus: the role of Panton-Valentine leukocidin. Lab Invest 2007; 87: 3-9. 16. Shastry L, Rahimian J, Lascher S. Community-associated methicillin-resistant Staphylococcus aureus skin and soft tissue infections in men who have sex with men in New York City. Arch Intern Med 2007; 167: 854-7.


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16 17. Senthilkumar A, Kumar S, Sheagren JN. Increased incidence of Staphylococcus aureus bacteremia in hospitalized patients with acquired immunodeficiency syndrome. Clin Infect Dis 2001; 33: 1412-6. 18. Diep BA, Chambers HF, Graber CJ et al. Emergence of multidrug-resistant, communityassociated, methicillin-resistant Staphylococcus aureus clone USA300 in men who have sex with men. Ann Intern Med 2008; 148: 249-57. 19. Lee NE, Taylor MM, Bancroft E et al. Risk factors for community-associated methicillinresistant Staphylococcus aureus skin infections among HIV-positive men who have sex with men. Clin Infect Dis 2005; 40: 1529-34. 20. Giuliani M, Longo B, Latini A et al. No evidence of colonization with community-acquired methicillin-resistant Staphylococcus aureus in HIV-1-infected men who have sex with men. Epidemiol Infect 2010; 138: 738-42.

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21. Klaassen CH, de Valk HA, Horrevorts AM. Clinical Staphylococcus aureus isolate negative for the Sa442 fragment. J Clin Microbiol 2003; 41: 4493. 22. Shrestha NK, Tuohy MJ, Hall GS et al. Rapid identification of Staphylococcus aureus and the mecA gene from BacT/ALERT blood culture bottles by using the LightCycler system. J Clin Microbiol 2002; 40: 2659-61.

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23. Harmsen D, Claus H, Witte W et al. Typing of methicillin-resistant Staphylococcus aureus in a university hospital setting by using novel software for spa repeat determination and database management. J Clin Microbiol 2003; 41: 5442-8.

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24. Wertheim HF, Vos MC, Boelens HA et al. Low prevalence of methicillin-resistant Staphylococcus aureus (MRSA) at hospital admission in the Netherlands: the value of search and destroy and restrictive antibiotic use. J Hosp Infect 2004; 56: 321-5.

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25. Bode LG, Wertheim HF, Kluytmans JA et al. Sustained low prevalence of meticillin-resistant Staphylococcus aureus upon admission to hospital in The Netherlands. J Hosp Infect 2011; 79: 198-201.

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26. Anonymous. Skin infection spreads among gay men. AIDS Read 2003; 13: 108. 27. Melles DC, Pauw E, van den BL et al. Host-microbe interplay in persistent Staphylococcus aureus nasal carriage in HIV patients. Microbes Infect 2008; 10: 151-8.

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28. van L, I, Huijsdens X, Tiemersma E et al. Emergence of methicillin-resistant Staphylococcus aureus of animal origin in humans. Emerg Infect Dis 2007; 13: 1834-9.

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STROBE 2007 (v4) checklist of items to be included in reports of observational studies in epidemiology*Checklist for cohort, case-control, and cross-sectional studies (combined) Section/Topic Title and abstract

Introduction Background/rationale Objectives

Item # 1

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Methods Study design

4

Setting

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Participants

Recommendation (a) Indicate the study’s design with a commonly used term in the title or the abstract (b) Provide in the abstract an informative and balanced summary of what was done and what was found Explain the scientific background and rationale for the investigation being reported State specific objectives, including any prespecified hypotheses Present key elements of study design early in the paper Describe the setting, locations, and relevant dates, including periods of recruitment, exposure, followup, and data collection (a) Cohort study—Give the eligibility criteria, and the sources and methods of selection of participants. Describe methods of follow-up Case-control study—Give the eligibility criteria, and the sources and methods of case ascertainment and control selection. Give the rationale for the choice of cases and controls Cross-sectional study—Give the eligibility criteria, and the sources and methods of selection of participants (b) Cohort study—For matched studies, give matching criteria and number of exposed and unexposed Case-control study—For matched studies, give matching criteria and the number of controls per case Clearly define all outcomes, exposures, predictors, potential confounders, and effect modifiers. Give diagnostic criteria, if applicable For each variable of interest, give sources of data and details of methods of assessment (measurement). Describe comparability of assessment methods if there is more than one group Describe any efforts to address potential sources of bias Explain how the study size was arrived at Explain how quantitative variables were handled in the analyses. If applicable, describe which groupings were chosen and why (a) Describe all statistical methods, including those used to control for confounding (b) Describe any methods used to examine

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Reported on page # 2 2

4 5

5 5

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Data sources/ measurement

8*

Bias

9

Study size Quantitative variables

10 11

Statistical methods

12

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Variables

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2

subgroups and interactions (c) Explain how missing data were addressed (d) Cohort study—If applicable, explain how loss to follow-up was addressed Case-control study—If applicable, explain how matching of cases and controls was addressed Cross-sectional study—If applicable, describe analytical methods taking account of sampling strategy (e) Describe any sensitivity analyses Results Participants

13*

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Descriptive data

7

n.a n.a. 11

15*

n.a.

16

n.a.

n.a.

8/13/14 13/14

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Main results

n.a.

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Outcome data

14*

(a) Report numbers of individuals at each stage of study—eg numbers potentially eligible, examined for eligibility, confirmed eligible, included in the study, completing follow-up, and analysed (b) Give reasons for non-participation at each stage (c) Consider use of a flow diagram (a) Give characteristics of study participants (eg demographic, clinical, social) and information on exposures and potential confounders (b) Indicate number of participants with missing data for each variable of interest (c) Cohort study—Summarise follow-up time (eg, average and total amount) Cohort study—Report numbers of outcome events or summary measures over time Case-control study—Report numbers in each exposure category, or summary measures of exposure Cross-sectional study—Report numbers of outcome events or summary measures (a) Give unadjusted estimates and, if applicable, confounder-adjusted estimates and their precision (eg, 95% confidence interval). Make clear which confounders were adjusted for and why they were included (b) Report category boundaries when continuous variables were categorized (c) If relevant, consider translating estimates of relative risk into absolute risk for a meaningful time period Report other analyses done—eg analyses of subgroups and interactions, and sensitivity analyses

n.a. 6

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17

Discussion Key results

18

Limitations

19

Interpretation

20

13/14 n.a.

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Summarise key results with reference to study objectives Discuss limitations of the study, taking into account sources of potential bias or imprecision. Discuss both direction and magnitude of any potential bias Give a cautious overall interpretation of results considering objectives, limitations, multiplicity of analyses, results from similar studies, and other

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Generalisability

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Other information Funding

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relevant evidence Discuss the generalisability (external validity) of the study results

10

Give the source of funding and the role of the 3 funders for the present study and, if applicable, for the original study on which the present article is based *Give information separately for cases and controls in case-control studies and, if applicable, for exposed and unexposed groups in cohort and cross-sectional studies. Note: An Explanation and Elaboration article discusses each checklist item and gives methodological background and published examples of transparent reporting. The STROBE checklist is best used in conjunction with this article (freely available on the Web sites of PLoS Medicine at http://www.plosmedicine.org/, Annals of Internal Medicine at http://www.annals.org/, and Epidemiology at http://www.epidem.com/). Information on the STROBE Initiative is available at www.strobe-statement.org.

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Community associated MRSA prevalentie en risicofactoren onder mannen die seks hebben met mannen Hoofdonderzoeker: dr. H. J. C. de Vries, dermatoloog, SOA polikliniek, cluster infectieziekten, GGD Amsterdam tevens afd. Dermatologie, AMC Amsterdam en Centrum Infectieziekten bestrijding, RIVM, Bilthoven. Overige onderzoekers: dr. M Schim van der Loeff, arts-epidemioloog, afdeling onderzoek, infectieziekten, GGD Amsterdam drs. P Gruteke, arts-microbioloog, streeklaboratorium, cluster infectieziekten, GGD Amsterdam, tevens afd. microbiologie, OLVG, Amsterdam dr. A van Dam, arts-microbioloog, streeklaboratorium, cluster infectieziekten, GGD Amsterdam, tevens afd. microbiologie, OLVG, Amsterdam mw. G.B. Linde, hoofdanalist, streeklaboratorium, cluster infectieziekten, GGD Amsterdam dr. A de Neeling, microbioloog, projectleider antimicrobiële resistentie, Centrum Infectieziekten bestrijding, RIVM, Bilthoven. dr. J.S.A. Fennema,. hoofd, cluster infectieziekten, GGD Amsterdam. research verpleegkundige, vacature

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Versie 1For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Pagina 1 16-12-2012

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Inhoud

1. Contact informatie ......................................................................................................... 4 2. Protocol ondertekeningpagina .................................................................................. 5 3. Samenvatting .................................................................................................................. 6 4. Achtergrond ..................................................................................................................... 7 5. Relevantie ......................................................................................................................... 8 7. Studie opzet...................................................................................................................... 9 8. Behandeling bij MRSA dragerschap/symptomatische MRSA infectie ....... 10 9. Sample-size berekening en statistiek................................................................... 11 9. Referenties ..................................................................................................................... 12

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Appendix I: Patientinformatie en toestemmingsformulier

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1. Contact informatie Hoofdonderzoeker dr. H.J.C. de Vries, SOA polikliniek, Weesperplein 1 1018 WZ Amsterdam Cluster Infectieziekten, GGD Amsterdam, Tel: +31-20-555 5429 Email: [email protected] en

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afdeling Dermatologie, Academisch Medisch Centum, Universiteit van Amsterdam Meibergdreef 9 1105 AZ Amsterdam Tel: +31-20-566 9111

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afdeling Dermatologie, Academisch Medisch Centum, Universiteit van Amsterdam

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Studielokaties SOA polikliniek, Cluster Infectieziekten, GGD Amsterdam,

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Laboratoria streeklaboratorium, cluster infectieziekten, GGD Amsterdam Centrum Infectieziekten bestrijding (CIb), RIVM, Bilthoven

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2. Protocol ondertekeningpagina Community associated MRSA prevalentie en risicofactoren onder mannelijke soa poli bezoekers die seks hebben met mannen Handtekening hoofd onderzoeker: H.J.C. de Vries, MD, PhD, SOA polikliniek, Cluster Infectieziekten, GGD Amsterdam

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________________________ Handtekening

________________________ Datum

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3. Samenvatting Methicilline Resistente Staphylococcus Aureus (MRSA) is een Ziekenhuis bacterie die tot grote behandelproblemen leidt. Sinds 1996 wordt een Community Associated of Acquired MRSA (CA-MRSA) beschreven die buiten ziekenhuizen in de gezonde populatie voorkomt. In Nederland wordt CA-MRSA regelmatig aangetroffen onder varkenshouders en in de Verenigde Staten ook onder mannen die seks hebben met mannen (MSM, homo- en biseksuele mannen). CA-MRSA veroorzaakt huid en weke delen infecties zoals abcessen, impetigo, en folliculitis. Begin 2008 is een nog resistenter en virulenter CA-MRSA kloon beschreven onder MSM, de USA 300 kloon, eerst in San Francisco en Boston, en recent in Duitsland. Ook op de SOA polikliniek van de GGD Amsterdam is het eerste geval van USA 300 CAMRSA onder een MSM bezoeker gevonden. In deze studie willen we de volgende vragen beantwoorden: 1) Wat is de prevalentie van CA-MRSA asymptomatisch dragerschap onder MSM SOA poli bezoekers? 2) Wat zijn risicofactoren voor CA-MRSA dragerschap en/of infecties onder MSM? 3) Wordt CA-MRSA onder MSM verspreid via seksuele netwerken? 4) Wat is de prevalentie van MRSA in MSM bezoekers van de GGD SOA poli en de AMC polikliniek dermatologie met afwijkingen passend bij bacteriële huidinfecties? Bij de GGD Soa poli zullen 125 MSM (voldoende power om 3% prevalentie mee aan te tonen) nagekeken worden op dragerschap (asymptomatische infecties) dmv afname van kweken van neusholte, perianaal en van de glans penis. Daarnaast zullen bij de GGD Soa poli en AMC dermatologie poli 75 MSM met aanwijzing van huidinfectie gerichte kweken plus kweken van de neusholte, perianaal en van de glans penis worden afgenomen. Bij alle deelnemers wordt een risico anamnese voor CA_MRSA afgenomen. De verwachting is dat in totaal 40 asymptomatische S. aureus infecties (20-30% algehele bevolking), en 40 symptomatische S. aureus infecties zullen worden opgespoord. Indien MRSA word gevonden volgt uitgebreid moleculair onderzoek: MLVA, PCR en DNA sequencing ism het CIb/RIVM, dmv Bayesiaanse Monte Carlo Markov chain (MCMC) fylogenetische analyse. Met de sequence gegevens wordt getracht een seksueel netwerk in kaart te brengen naast risicofactor analyse.

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4. Achtergrond Methicilline Resistente Staphylococcus Aureus (MRSA) is een bacterie die clinici voor grote behandelproblemen plaatst doordat er voor de eerste keuze antibiotische opties veelal resistentie bestaat. In ziekenhuizen kan MRSA moeilijk behandelbare infecties veroorzaken onder ernstig zieke patiënten. Omdat transmissie verloopt via huid-huid contact is strenge isolatie vervolgens geboden om transmissie te voorkomen. Om diezelfde reden worden met enige regelmaat afdelingen gesloten om uitbraken van MRSA te kunnen elimineren (zo is december j.l. in het Boven IJ-ziekenhuis nog voor zes van de elf afdelingen een opnamestop afgekondigd). Sinds 1996 wordt melding gemaakt van MRSA infecties onder ogenschijnlijk gezonde individuen die buiten het ziekenhuis, in de gemeenschap zijn opgelopen. Deze infecties werden het eerst gesignaleerd onder veehouders (met name varkenshouders), “Native Americans” in de VS en worden Community Associated of Acquired MRSA (CA-MRSA) genoemd. CA-MRSA veroorzaakt meestal huid en weke delen infecties zoals abcessen, impetigo (krentenbaard), folliculitis (haarzakontstekingen) maar daarnaast ook longontstekingen. Daar veel eerste keuze antibiotica onwerkzaam zijn kan vertraging in de behandeling optreden waardoor de infectie kans ziet zich verder te verspreiden en aanleiding kan geven tot systemische infecties zoals sepsis, hartklepontstekingen en andere orgaanproblemen. Van later datum zijn meldingen van uitbraken van CA-MRSA onder gevangenen, sporters die intensief huid-huid contact hebben (worstelaars) en sinds 2003 ook onder mannen die seks hebben met mannen (MSM).[1] Binnen de groep van CA-MRSA zijn nog gevaarlijker varianten aanwijsbaar zoals de multi-resistente USA300 kloon die resistent is tegen een nog groter deel van de eerste keuze preparaten voor huid en wekedelen infecties zoals clindamycine, tetracycline, co-trimoxazol, en beta-lactam preparaten. Voortschrijdende resistentie van de USA300 kloon is gemeld doordat extra resistentie genen via incorporatie van het plasmide pUSA03 heeft plaats gevonden. Hierdoor bestaat er tevens resistentie tegen macroliden, clindamycine en mupirocine.[2] pUSA03 positieve USA300 CA_MRSA wordt gerapporteerd onder MSM in San Francisco en Boston. Naast eerdere MRSA infectie, clindamycine gebruik, vormt man-man seks de sterkste risicofactor voor een CA-MRSA infectie met deze USA300 kloon. Overigens is hiv status in deze studie niet geassocieerd met CA-MRSA infectie. Ook in Europa is de USA300 positieve CA-MRSA gemeld in een HIV positieve Duitse MSM.[3] In maart j.l. is ook op de SOA polikliniek een HIV positieve MSM gediagnosticeerd met een huidinfectie op basis van MRSA die viel binnen de US300 groep (SPA 8). Verder is gerapporteerd dat sommige USA300 klonen virulentiegenen meedragen die coderen voor het cytotoxine Panton-Valentine Leukocidin (PVL) en fenol-oplosbare peptiden die er voor zorgen dat deze stammen aanleiding kunnen geven tot meer invasieve en ernstiger infecties, zoals de potentieel lethale necrotiserende fasciitis.[4] PVL positieve CA-MRSA wordt gerapporteerd onder 2 HIV positieve MSM in Amsterdam.

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De meeste meldingen van CA-MRSA in MSM betreffen symptomatische infecties, veelal van de huid en wekedelen in de genitaal streek, de billen en het perineum.[5] Daarnaast zijn CA-MRSA infecties onder HIV positieve mannen geassocieerd met seksueel risicogedrag.[6] Op basis van deze bevinding is seksuele overdracht van CA-MRSA voorstelbaar. Er heeft echter nog geen goed gestructureerd onderzoek plaats gevonden om deze aanname verder te onderbouwen. Daarnaast is er nog geen zicht op asymptomatisch dragerschap van MRSA stammen onder MSM en de relevantie van dragerschap voor de verdere verspreiding van de infectie binnen de gemeenschap. In een recente Rotterdamse studie naar de prevalentie van S. aureus positieve neus swabs onder 443 HIV patiënten kwam en prevalentie van 0,45% MRSA positieve patiënten aan het licht waarbij geen PVL positieve stammen werden gevonden.[7] Dit betreft de Rotterdamse situatie en daarnaast is het onderzoek beperkt tot neus uitstrijkjes waardoor geen uitspraak gedaan kan worden over dragerschap in de anogenitale regio.

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5. Relevantie CA-MRSA vormt in de VS een toenemende bedreiging voor risico populaties zoals mensen die leven in instituties bijvoorbeeld bewoners van zorginstellingen en gevangenen, immuun gecompromitteerden, en verder ook minderheden en MSM.[8] Naast de resistentie en samenhangende behandelproblematiek zijn er aanwijzingen dat CA-MRSA ook meer virulente varianten omvat die aanleiding kunnen geven tot aanzienlijke morbiditeit zoals longontstekingen en ernstige wekendelen infecties. Er bestaat op dit moment geen inzicht in de verspreiding van CA-MRSA binnen de Amsterdamse populatie. Het ligt voor de hand eerst te onderzoeken of CAMRSA in Amsterdam onder een van de reeds geïdentificeerde risicogroepen (MSM) voorkomt. Daarnaast is het vanuit de preventie belangrijk te weten hoe CA-MRSA zich binnen een populatie verspreidt en dienen netwerken en risicofactoren te worden blootgelegd. Als blijkt dat CA-MRSA transmissie onder MSM binnen seksuele netwerken plaatsvindt, dan zal de SOA polikliniek een belangrijke taak hebben in verder onderzoek, behandeling, voorlichting en preventie. Daarnaast is het uit oogpunt van de medewerkers van Soa poliklinieken van belang te weten in hoeverre zij kans lopen op besmetting met MRSA. Dit heeft directe implicaties voor de uitoefening van hun werk aangezien MRSA dragers in de gezondheidzorg niet mogen werken totdat dragerschap is geëlimineerd.

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6. Doelstellingen 1. Wat is de prevalentie van community associated MRSA asymptomatisch dragerschap onder mannelijke SOA poli bezoekers die seks hebben met mannen? 2. Wat zijn risicofactoren voor community associated MRSA dragerschap en/of infecties onder mannen die seks hebben met mannen? 3. Wordt community associated MRSA onder mannen die seks hebben met mannen verspreid via seksuele netwerken? 4. Wat is de prevalentie van community associated MRSA veroorzaakte huidinfecties onder MSM bezoekers van de GGD SOA poli en de AMC polikliniek dermatologie?

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7. Studie opzet Tijdens het hoogrisico MSM spreekuur op de soa polikliniek (waarbij op afspraak MSM worden gezien met meerdere bewezen SOA in de voorgeschiedenis), en tijdens de reguliere spreekuren zullen 125 MSM gevraagd worden deel te nemen aan de studie. De huid zal worden nagekeken (mn genitaal, perianaal en de nates) op tekenen van huidinfecties (folliculitis, cellulitis, carbunkels, impetiginisatie, abcessen en fistels) en indien aanwezig zullen hiervan gericht kweken worden afgenomen. Standaard worden banale en MRSA specifieke kweken afgenomen uit de neusholte, de keelholte, perianaal en van de glans penis. Daarnaast zullen bij 1) de GGD SOA poli, 2) de AMC polikliek dermatologie bij 75 MSM bezoekers met afwijkingen passend bij een bacteriële huidinfectie een lesionale banale kweek worden afgenomen, en standaard kweken perianaal, van de glans penis en van de neusholte. Bij alle deelnemers zal een vragenlijst afgenomen worden gericht op het achterhalen van risicofactoren samenhangend met dragerschap of symptomatische infecties met S. aureus Naar verwachting zal bij 40 deelnemers asymptomatisch dragerschap worden gevonden (dit komt voor bij 20-30% van de algemene bevolking). In de groep met huidafwijkingen komen naar verwachting 40 positieve S. aureus kweken aan het licht. De volgende risicofactoren zullen worden onderzocht: reisgedrag, (seksuele)partner selectie, seksuele technieken, hygiëne technieken (o.m. klysma gebruik, nagelbijten, scheergedrag, gebruik en delen van huidproducten), huidinfecties bij personen in de directe omgeving, contact met dieren, druggebruik, bezoek aan en behandeling in zorginstellingen, bezoek/verblijf in openbare gelegenheden en penitentiaire inrichtingen, beroep, hobbies, sportuitoefening, saunabezoek, massage, antibioticagebruik, eerdere (MRSA) infecties en hiv status (indien positief eveneens laatste CD4 getal en viral load). Voorafgaande aan de studie zal getracht worden dmv enkele kwalitatieve interviews overige relevante risicofactoren voor transmissie van huidinfecties te achterhalen. Indien een kweek positief is voor S. aureus volgt uitgebreid resistentieonderzoek. Indien MRSA wordt aangetoond volgt ism het CIb/RIVM (dr. A de Neeling) moleculair onderzoek bestaande uit: MultipleLocus Variable-Number Tandem Repeat Analysis (MLVA), polymerase chain

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reaction (PCR) en DNA sequencing. Hiermee zullen genen betrokken bij antibiotica resistentie (o.m. USA300, pUSA03, ermC, mupA, msrA,en mphB) en virulentie (o.m. PVL en fenol oplosbare peptiden) in kaart worden gebracht. Indien bij een deelnemer MRSA wordt aangetoond dan wordt hij hiervan op de hoogte gesteld. Tevens moeten alle hulpverleners die tijdens het consult in aanraking zijn gekomen (dwz. huid/huid contact hebben gehad) met deze deelnemer worden nagekeken op MRSA dragerschap door middel van het afnemen van een neus, keel en perineumkweek conform de richtlijnen van commissie ziekenhuishygiëne en infectiepreventie van het AMC of de microbiologen van het streeklaboratorium van de GGD Amsterdam, naar gelang waar de deelnemer is onderzocht.

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8. Behandeling bij MRSA dragerschap/symptomatische MRSA infectie Indien bij een deelnemer of een hulpverlener die betrokken is bij het onderzoek MRSA wordt aangetoond dan worden de aanbevelingen ten aanzien van MRSA dragerschapbehandeling, zoals beschreven in de richtlijn “Optimaliseren van het antibioticabeleid in Nederland XI SWAB richtlijn Behandeling MRSA dragers”van de Stichting Werkgroep Antibioticabeleid (SWAB), Maart 2007 in acht genomen.[9] De aanbevelingen zijn verschillend voor ongecompliceerd en gecompliceerd MRSA dragerschap. Men spreekt van ongecompliceerd MRSA-dragerschap , wanneer dit voldoet aan onderstaande punten: individu zonder actieve infectie met MRSA en MRSA is in vitro gevoelig voor de toe te passen antibiotica en er zijn geen actieve huidlaesies en er is geen lichaamsvreemd materiaal dat een verbinding vormt tussen milieu interieur en milieu exterieur (bijvoorbeeld urine catheter, fixateur externa en dragerschap is in de neus gelokaliseerd. Men spreekt van compliceerde MRSA-dragerschap , wanneer dit voldoet aan minstens één van onderstaande punten: er zijn actieve huidlaesies en/of er is lichaamsvreemd materiaal dat een verbinding vormt tussen milieu interieur en milieu exterieur en/of MRSA is in vitro ongevoelig voor mupirocine, en/of eerdere behandelingen volgens de adviezen bij ongecompliceerd dragerschap hebben gefaald en/of dragerschap bevindt zich uitsluitend op andere plaatsen dan de neus, zoals keel, perineum, of huidlesies. Ongecompliceerd dragerschap: Mupirocine neuszalf 3 dd gedurende 5 dagen. Gedurende de behandeling worden huid en haren dagelijks met een desinfecterende zeep (Chloorhexidine zeep oplossing 40 mg/ml of betadine shampoo 75 mg/ml) gewassen, bij voorkeur onder de douche (niet in bad). Dagelijks schoon ondergoed, schone kleding, schone washandjes en handdoeken gebruiken. Op dag 1, 2 en 5 van de kuur beddengoed volledig verschonen. Bij het naar bed gaan dient tevens gedurende de behandeling schoon ondergoed dan wel pyjama te worden aangetrokken. Bij therapie falen: Nagaan of er sprake is van een reservoir in de thuissituatie (mens of dier). Als er een reservoir in de thuis situatie aanwezig is, dan dient deze gelijktijdig te worden meebehandeld.

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Bij tweede keer falen is er sprake van een gecompliceerd MRSA dragerschap: Indien er sprak is van actieve huidlaesies, eerst de huidlaesies behandelen, zonodig in overleg met dermatoloog. Is er na afloop van de behandeling sprake van ongecompliceerd dragerschap dan kan de behandeling die daar is aangegeven worden ingesteld. Controle kweken worden afgenomen en verder bewerkt volgens de richtlijnen van de Nederlandse Vereniging voor Medische Microbiologie (www.nvmm.nl). De eerste kweken ter beoordeling van de effectiviteit van de behandeling worden tenminste 48 uur na het beëindigen van de behandeling afgenomen. De frequentie van verdere kweekafname is onder andere afhankelijk van de gevolgen voor het betrokken individu. In de richtlijnen van de WIP zijn deze gevolgen vastgelegd (www.wip.nl).

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9. Sample-size berekening en statistiek De prevalentie MRSA dragerschap in de algemene bevolking wordt geschat op 0,03%.7 De hypothese luidt dat de MRSA prevalentie onder MSM bezoekers van de SOA polikliniek ten minste 3% bedraagt. Om de null hypothese (0,03% prevalentie onder de onderzochte groep) te verwerpen moeten ten minste 113 mannen worden geïncludeerd om een valide uitspraak met een power van 95% en een significantie level van 0.05 te kunnen doen. Met behulp van Bayesiaanse Monte Carlo Markov chain (MCMC) fylogenetische analyse van MRSA genen zoals SPA zullen clusters en associaties worden geanalyseerd. In combinatie met de uitgevraagde risicofactoren zal getracht worden inzicht te krijgen in de bijdrage van seksuele netwerken in de transmissie van MRSA onder MSM.

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9. Referenties (1) Outbreaks of community-associated methicillin-resistant Staphylococcus aureus skin infections--Los Angeles County, California, 2002-2003. MMWR Morb Mortal Wkly Rep 2003 Feb 7; 52(5):88. (2) Diep BA, Chambers HF, Graber CJ, et al. Emergence of multidrugresistant, community-associated, methicillin-resistant Staphylococcus aureus clone USA300 in men who have sex with men. Ann Intern Med 2008 Feb 19; 148(4):249-57.

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(3) Witte W, Braulke C, Strommenger B. Community-associated methicillinresistant Staphylococcus aureus ST8 ("USA300") in an HIV-positive patient in Cologne, Germany, February 2008. Euro Surveill 2008; 13(13). (4) Tiemersma E, Wannet W, op de Coul E, de Neeling AJ, van de Laar M. Introduction of surveillance of infections with Staphylococcus aureus containing the Panton-Valentine Leukocidin gene in the Netherlands. Euro Surveill 2003; 4(16).

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(5) Diep BA, Chambers HF, Graber CJ, et al. Emergence of multidrugresistant, community-associated, methicillin-resistant Staphylococcus aureus clone USA300 in men who have sex with men. Ann Intern Med 2008 Feb 19; 148(4):249-57.

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(6) Lee NE, Taylor MM, Bancroft E, et al. Risk factors for communityassociated methicillin-resistant Staphylococcus aureus skin infections among HIV-positive men who have sex with men. Clin Infect Dis 2005 May 15; 40(10):1529-34.

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(7) Melles DC, Pauw E, van den BL, et al. Host-microbe interplay in persistent Staphylococcus aureus nasal carriage in HIV patients. Microbes Infect 2008 Feb; 10(2):151-8.

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(8) Outbreaks of community-associated methicillin-resistant Staphylococcus aureus skin infections--Los Angeles County, California, 2002-2003. MMWR Morb Mortal Wkly Rep 2003 Feb 7; 52(5):88.

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(9) Stichting Werkgroep Antibioticabeleid (SWAB). Optimaliseren van het antibioticabeleid in Nederland XI SWAB richtlijn Behandeling MRSA dragers. 2007.


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Low prevalence of methicillin-resistant Staphylococcus aureus among men who have sex with men attending an STI clinic in Amsterdam, a cross-sectional study

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Manuscript ID: Article Type:

Date Submitted by the Author:

Complete List of Authors:

BMJ Open bmjopen-2012-002505.R1 Research 31-Jan-2013

Primary Subject Heading:

Dermatology, Infectious diseases, Sexual health Infectious diseases & infestations < DERMATOLOGY, Infection control < INFECTIOUS DISEASES, Public health < INFECTIOUS DISEASES, Dermatology < INTERNAL MEDICINE, MICROBIOLOGY, PUBLIC HEALTH

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Keywords:

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Secondary Subject Heading:

Public health

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Joore, Ivo; Public Health Service Amsterdam, cluster infectious diseases, STI Outpatient Clinic van Rooijen, Martijn; Public Health Service Amsterdam, cluster infectious diseases, STI Outpatient Clinic Schim van der Loeff, Maarten; Public Health Service Amsterdam, cluster infectious diseases, research department de Neeling, Han; Center for Infection and Immunology Amsterdam (CINIMA), Academic Medical Center (AMC), University of Amsterdam, National Institute for Public Health and the Environment (CIb/RIVM) van Dam, Alje; Public Health Service Amsterdam, cluster infectious diseases, Public Health Laboratory de Vries, H; Municipal Health Service Amsterdam, STI outpatient clinic; Academic Medical Centre, University of Amsterdam, Dermatology


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1 Low prevalence of methicillin-resistant Staphylococcus aureus among men who have sex with men attending an STI clinic in Amsterdam, a cross-sectional study

1


1,2,3

2,3

4


5, 6

, de

Vries HJC1,4,7


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Keywords:


Staphylococcus

aureus,

HIV,

male


ABSTRACT


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common among men who have sex with men (MSM) in the USA. It is unknown whether this is also the case in Amsterdam, the Netherlands. Design: Cross-sectional study

Setting: STI outpatient low-threshold clinic, Amsterdam, the Netherlands Participants: Between October 2008 and April 2010, a total of 211 men were included, in two

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groups: (1) 74 MSM with clinical signs of a skin or soft tissue infection (symptomatic group) and (2) 137 MSM without clinical signs of such infections (asymptomatic group).

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•


•

In a cross-sectional study at the STI outpatient low-threshold clinic, Amsterdam, the Netherlands we studied the prevalence of S. aureus and MRSA colonisation and

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infections among symptomatic and asymptomatic men who have sex with men (MSM)

Key messages:

• Among MSM visiting the STI clinic in Amsterdam CA-MRSA is rare. • Genital colonisation of S. aureus is not associated with sexual high risk behaviour.

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• The study was limited to the Amsterdam MSM population visiting the STI outpatient clinic.

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Funding: This work was supported by the Research and Development fund of the Public Health Service of Amsterdam [Project 2373 Community associated MRSA prevalence and risk factors in male

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std outpatient clinic visitors who have sex with men]. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Competing Interests: The authors certify that they have no affiliation with or financial involvement in any organization or entity with a direct financial interest in the subject matter or materials discussed in

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the manuscript (e.g., employment, consultancies, stock ownership, or honoraria).

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4 INTRODUCTION


1;2

Around

20% (12-30%) of individuals are persistent S. aureus nasal carriers, approximately 30% are intermittent carriers (range 16-70%), and about 50% (range 16-69%) non-carriers.

1;3

A causal


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1;2;4-6



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Most strains of S. aureus are methicillin-susceptible (MSSA), but some strains, called methicillinresistant Staphylococcus aureus (MRSA) are resistant to methicillin and all beta-lactam

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antibiotics with the exception of the novel cephalosporin, ceftarolin, that can bind to penicillin-

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binding-protein PBP2a. MRSA has become a major nosocomial infection control problem (known as health care-associated MRSA, or HA-MRSA) in many parts of the world with the exception of

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the Netherlands and of Scandinavian countries.9,24,25 Later on, independent from health care institutions, MRSA emerged in the community (CA-MRSA) and livestock (LA-MRSA).

10;11,28

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Resistance to methicillin and other ß-lactams in S.aureus is based on an additional penicillin8

binding-protein (PBP) which is coded by a mec gene such as mecA. Homologues of mec A such as mecC were described recently.

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CA-MRSA is distinguished from HA-MRSA by clinical, laboratory, and epidemiologic 12

characteristics.

A new CA-MRSA clone (USA300), has been described and was identified in

several communities in the USA and Canada.9-11 New clones of CA-MRSA have also been reported in Europe.

13;14

CA-MRSA often produces Panton-Valentine Leukocidin (PVL), a toxin

that causes polymorphonuclear leukocyte lysis and tissue necrosis13;15


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5 In men who have sex with men (MSM), outbreaks of CA-MRSA skin infections have been reported and the majority of the patients were HIV infected.

8;16-18

It has been suggested that CA-

MRSA might be transmitted in this population from skin to skin during sexual contact.18;19

A cross-sectional study among 104 men conducted in Italy did not detect a single case of colonisation with CA-MRSA in HIV-infected MSM.

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No other study in Europe has reported the

prevalence of CA-MRSA colonisation in MSM. The purpose of this investigation was to evaluate

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the prevalence of, and sexual risk factors for S. aureus colonisation (MSSA and MRSA) and CAMRSA infection among MSM visiting the sexual transmitted infection (STI) outpatient clinic in Amsterdam.

METHODS

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Study population

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Consecutive MSM attending the STI outpatient clinic in Amsterdam, The Netherlands, were

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invited to participate in this cross-sectional study. We aimed to include both symptomatic men (those who had clinical signs of a skin or soft tissue infection [pustules, abscesses, ulcerations,

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erythematous painful papules or plaques]) and asymptomatic men (those without abovementioned clinical signs). All participants were asked to complete a questionnaire concerning

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hospital admissions during the past year, factors known to be associated with S. aureus colonisation (e.g. sharing razor blades, tending animals), STI risk factors and sexual behaviour.

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STI risk factors were also obtained from the electronic files of the routine patient history.

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Swabs were taken from the anterior nasal cavity, throat, perianal area, and penile glans. From symptomatic men also swabs from suspected infected skin lesions were obtained. If MRSA was detected, the participant was referred to his general practitioner for eradication therapy, and the sexual partners of the patient were invited for MRSA screening. The study was approved by the ethics committee of the Academic Medical Center of the University of Amsterdam. All participants provided written informed consent.


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6 Laboratory tests Culture for S. aureus was done on blood agar plates and for MRSA on selective chromagar plates after enrichment in broth. S. aureus strains were confirmed by the SA442 (Martineau) nucleic acid amplification test (NAAT) and MRSA strains by the mecA NAAT.

21;22

If MRSA was

found, the spa-gene was sequenced as described before.23


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A sample size calculation was done prior to the study. The prevalence of MRSA carriage in the general population was estimated to be 0.03%.24 We aimed to assess whether the MRSA prevalence among MSM patients of the STI clinic was at least 3%. In order to reject the null hypothesis (prevalence of 0.03% or less among the studied group) with a study power of 95% and a significance level of 0.05, a sample size of at least 113 asymptomatic men was needed;

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group of 75 symptomatic men. Because the expected number of asymptomatic men was much

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larger than that of symptomatic patients, the inclusion period for the symptomatic participants was scheduled to be longer.

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The electronic patient file with routine STI screening data, the questionnaire and the laboratory

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results were merged into one database. Chi-square test or Fisher’s exact test were used to compare categorical variables between groups; the ranksum test was used to compare

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continuous variables between groups. Data analyses were performed with STATA software (STATA Intercooled, College Station, TX, USA), version 11.0. P values of less than 0.05 were

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considered statistically significant. Associations between possible risk factors and anogenital S. aureus colonisation were examined with multivariable logistic regression, reporting adjusted odds ratios (OR) with 95% confidence intervals (CI). An initial model included all variables that were significant in bivariable analyses; variables were dropped one by one from the model based on the likelihood ratio test (if P>0.05); HIV was forced into the model.


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7

RESULTS

In total 214 men were included, starting November 2008. The inclusion of asymptomatic men was completed in December 2008 and of symptomatic men in April 2010. From three participants

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laboratory results were not available and these were excluded, leaving 211 MSM for analysis (137 asymptomatic and 74 symptomatic participants). The median age of men in the two groups was similar (38 and 41 years, respectively, p=0.32, see Table 1). Men in the two groups were comparable regarding most non-sexual risk factors, sexual risk factors and STI diagnoses, but symptomatic men were more often HIV infected (53% vs. 31%; p=0.002) and were more often

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diagnosed with syphilis (12% vs. 1%; p<0.001). Also, use of some drugs (cannabis, cocaine, and methamphetamine) was significantly more common in the symptomatic group.

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prevalences (Table 1). In the symptomatic group 40% (27/67) of men had an S. aureus infected skin lesion. In one symptomatic and one asymptomatic case MRSA was detected, giving an

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overall prevalence of MRSA carriage of 0.9% (95% binomial exact confidence interval [95%CI] 0.1-3.4%), and a prevalence of 0.7% (95%CI 0.02-4.0%) among asymptomatic MSM. Details of

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these two cases are described below. No known CA-MRSA nor PVL toxin-producing clones were detected.

S. aureus colonisation of the ano-genital area (based on penile and perianal swabs) was found in 18% (38/211) of the participants. Participants with and without ano-genital S. aureus colonisation were similar in respect to sexual risk behaviour, drug use, history or diagnosis of sexual transmitted diseases, antibiotic exposure, circumcision status and hygiene behaviour, but men


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8 with ano-genital S. aureus colonisation were older (42 vs. 38 years; P=0.05), and they were more often HIV infected (58% vs. 34%, p=0.007; see Table 2). In a multivariable logistic regression analysis, adjusting for age, HIV status (aOR 2.2, 95%CI 0.96-4.9), belonging to the symptomatic group (aOR=2.7, 95%CI 1.2-5.9) and having had few sexual partners (aOR 3.1, 95%CI 1.2-8.3 for those with <5 sexual partners in previous year compared to those with over 20 sexual partners) were independently and significantly associated with anogenital S. aureus colonisation.

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We examined associations between nasal carriage of S. aureus and a long list of possible nonsexual risk factors, like admissions, operations, employment in health care sector, shaving habits, participation in team sports, sauna visits, and other factors; none of these was associated with S. aureus colonisation of the nose.

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An HIV infected 40-year-old, asymptomatic, Dutch MSM was positive for MRSA at the perineum.

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The MRSA had spa type t064 and was resistant to ciprofloxacin, gentamicin, oxacillin, penicillin

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recreational drugs and visited a public gym and sauna. Furthermore, he engaged in high risk sex, including unprotected anal intercourse, active and passive fisting, group sex, sex at sex parties

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and sex with partners met through the internet. We attempted to trace six of his sexual partners of whom four were screened. None had MRSA and none had clinical signs of a skin infection.

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Symptomatic carrier of MRSA In an HIV infected, 46-year old Dutch MSM MRSA was detected on swabs taken from a penile ulcer, the nasal cavity, the perineum and the glans penis. The MRSA had spa type t002 and was resistant to ciprofloxacin, erythromycin, oxacillin and penicillin. Further genetic analysis revealed mecA+ and Martineau+; pUSA03 and PVL were both negative. In the preceding year the patient had not been hospitalized, nor operated upon, nor had he travelled abroad. He did not use


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9 recreational drugs, and did not visit public gyms or saunas. He had not engaged in high risk sex; he had had sex with one partner in the preceding year. This partner could not be traced for further screening.

DISCUSSION

The prevalence of MRSA among MSM clients of the STI outpatient clinic in Amsterdam was 0.9%

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(95%CI 0.1-3.4%); among clients without skin lesions this was 0.7% (95%CI 0.02-4.0%). This is similar to the overall prevalence in Dutch hospitals (1.8%).25 No CA-MRSA associated clones were found in this population. These findings are in contrast to reports from the USA where considerable CA-MRSA prevalences were observed among MSM attending STI screening sites.

18;26


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continent, this has not resulted in an extensive introduction of CA-MRSA within the Amsterdam population yet. The low MRSA prevalence among the Dutch population in general and among the

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MSM STI clinic population may be attributed to the strict Dutch search–and-destroy policy and 24


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We observed similar prevalences of S. aureus nasal colonisation (37%) in MSM as observed in 1

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the general population. Genital S. aureus colonisation was associated with HIV infection; although HIV infection may be considered a proxy for high risk sexual behaviour, other markers

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for risk behaviour were not significantly associated with S. aureus colonisation. We attribute the more common colonisation among HIV positive participants to their immunocompromised status.

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Unfortunately, we did not have data on virological, immunological or clinical parameters of HIV infection, nor about use of antiretroviral drugs. We did not observe an increased risk of S. aureus nasal carriage among HIV infected, as was reported earlier.

27

In contrast to Southern European countries, the Netherlands has a low rate of health careassociated MRSA (HA-MRSA), probably due to the above-mentioned search-and-destroy policy. The restricted use of betalactam antibiotics outside of hospitals, in the community, might explain


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10 the low prevalence of CA-MRSA. Europe has not yet been confronted with CA-MRSA on a wide 20


9-11;18-20

Yet, in

contrast to low prevalences of HA-MRSA and CA-MRSA in the Netherlands, screening of Dutch workers with livestock has revealed that 39% of slaughterhouse pigs and >20% of pig farmers are asymptomatic carriers of LA-MRSA belonging to sequence type (ST) 398.28 The 2 MRSA strains isolated in our study were both unrelated to this LA-MRSA epidemic.

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In conclusion, CA-MRSA among MSM visiting the STI outpatient clinic in Amsterdam is rare. Although genital S. aureus colonisation was more common among HIV infected MSM, we found no association between high risk sexual behaviour and genital colonisation with S. aureus. In the Netherlands there is no indication for MRSA screening of MSM attending STI clinics.

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P-value


38 (31-45) 111 81.0

41 (31-48) 54 73.0

0.32 0.18


21 92 34 40 36 76 29 14 8 3

15.3 67.2 24.8 29.2 26.3 55.5 21.2 10.2 5.8 2.2

7 47 33 27 25 44 27 13 7 6

9.5 63.5 44.6 36.5 33.8 59.5 36.5 17.6 9.5 8.1

0.23 0.60 0.003 0.28 0.25 0.58 0.02 0.13 0.33 0.04

13.8 21.9 14.6 20.4 19.0 10.2 71.5 57.7 49.6 40.9 15.3 10.2 43.8 62.0 22.6 54.7 57.7

9 14 11 15 12 13 48 58 32 37 15 11 38 47 17 45 45

12.2 18.9 14.9 20.3 16.2 17.6 64.9 78.4 43.2 50.0 20.3 14.9 51.4 63.5 23.0 60.8 60.8

0.32 0.003 0.38 0.20 0.36 0.32 0.29 0.83 0.95 0.40 0.66

Characteristic

0.77

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


34 42 1 9 23

24.8 30.9 0.7 6.6 16.8

24 38 9 5 14

32.4 52.8 12.2 5.4 18.9

0.24 0.002 <0.001 0.74 0.70


50 13

36.5 9.5

28 10

37.8 13.5

0.85 0.37


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9 10 --

6.6 7.3 --

17 12 27

23.3 6.2 40.3

<0.001 0.043 --


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S. aureus pos.

N = 173

N =38

n

%

n

P-value %


38 (30-45)

Dutch Ethnicity

138

42 (35-49) 79.8

27

0.05 71.1

0.24



7

18.4

0.30

21

55.3

0.13

57

33.0

10

26.3

0.43

53

30.6

14

36.8

0.46

49

28.3

12

31.6

0.69

96

55.5

24

63.2

0.39

44

25.4

12

31.6

0.44

23

13.3

4

10.5

0.64

12

6.9

3

7.9

0.84

4.1

2

5.3

0.74

7

0.08

18

10.4

10

26.3

35

20.2

9

23.7

16.8

2

5.3

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12.1 68.2

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C. Sexual history

21 118

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5-9

29

10-19

35

20.2

8

21.1

20-39

33

19.1

5

13.2

40 or more

23

13.3

4

10.5


121

69.9

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110

63.6

85

49.1


74

42.8

25

65.8

0.62

27

71.1

0.38

15

39.5

0.28

19

50.0

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0.42

18.4

0.81

7.9

0.41

18

47.4

0.90

63.0

23

60.5

0.78

20.8

12

31.6

0.15

102

59.0

18

47.4

0.19

105

60.7

19

50.0

0.23

51

29.5

7

18.4

0.17

Active fisting #

29

16.8

7

Passive fisting #

22

12.7

3

Had group sex §

80

46.2


109


36



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14

HIV infected *

59

34.3

21

58.3

0.007

Syphilis diagnosis

10

5.8

0

0

0.13


13

7.5

0

0

0.08

Chlamydia diagnosis

30

17.3

7

18.4

0.87


109

63.0

20

52.6

0.24


13

7.5

0

0

0.08


110

63.6

23

60.5

0.72


103

59.5

25

65.8

0.48

149

86.1

32

84.2

0.76


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165

95.4

38

100

0.18

Pubic hair

149

86.1

31

81.6

0.47

Body hair

110

63.6

20

52.6

0.21

45

26.0

11

29.0

0.71

14

8.1

3

7.9

0.97

9

5.2

4

10.5

0.22

23

13.5

8

21.6

0.21

Has pets




6

3.5

1

2.6

0.79

49

28.3

14

36.8

0.30

55

31.8

11

29.0

0.73

11

29.0

0.28

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20.8


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Author’s contribution IJ analysed the data and wrote the first manuscript draft. MvR collected the data and analysed the data, MSvdL supervised the epidemiological analysis and wrote the article, HdN performed the molecular strain typing and wrote the article, AvD performed the cultivation experiments, designed the study and wrote the aricle, HdV conceptualized and supervised the study and approved the final manuscript. Technical appendix, statistical code, and dataset available from the corresponding author at Dryad repository, who will provide a permanent, citable and open access home for the dataset.


Page 17 of 51

Reply to REVIEWERS COMMENTS MRSA BMJ OPEN From the managing editor: A cross-sectional study is not usually considered a prospective study design. This seems to have been more of a case series. Can you double-check that cross-sectional study is what you mean?" We fully agree that this is a cross-sectional study and have removed the phrase “prospective” from the title, abstract, and article focus sections.

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Reviewer: Marco Cusini Head STI Clinic UO Dermatologia Fondazione IRCCS Ca' Granda Ospedale Maggiore Milano Italy No conflict of interest

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The paper is very interesting and deal with a very actual topic. Few points: Abstract: Line six: the number of total patients 211 is not well integrated in the sentence. Point taken, we altered the sentence into: “Between October 2008 and April 2010, a total of 211 men were included, in two groups: (1) 74 MSM with clinical signs of a skin or soft tissue infection (symptomatic group) and (2) 137 MSM without clinical signs of such infections (asymptomatic group).”

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tables. they are quite long and difficult to read: recreational drugs may be grouped and also some parametres of sekual history We agree that the tables are quite long. In the abstract and the results section of the manuscript we grouped and condensed the key findings. Yet we believe that the current table provides important extensive information for those readers who are interested to go in depth.

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Reviewer: Prof.Dr. Wolfgang Witte RKI Fellow Robert Koch Institute Institute, Wernigerode Branch Wernigerode Germany There are no competing interests When emergence and dissemination of community associated MRSA in the USA were described at the beginning of this millenium


BMJ Open

MSM were identified as a particular human population at particular risk. Although “import” , especially of “USA300” into European countries has been observed for several times, they remained infrequent here so far. Because of the well known promiscuity of MSM it is of interest to know about the CA-MRSA situation among this group in Europe. The manuscript reports a study which addresses this issue in MSM in Amsterdam. The study is based on diagnostics of swabs from various sites from persons with affections of skin and from persons without skin-symptoms together with a very thorough record of background data. Although the “sample” studied is comparably small, it has power enough for showing low prevalence of MRSA among MSM. The manuscript is well written, only one section in the introduction will need attention (line 37 at page 4. Here my suggestion “ Methicillin resistance has become a major nocomial infection control problem in many parts of the world with the exception of the Netherlannds and of Scandinavian countries (HA-MRSA). Later on independent upon hospitals MRSA emerged in the community (CA-MRSA) and livestock (LA-MRSA). Resistance to methicillin and other ß-lactams in S.aureus is based on an additional penicillin-binding-protein (PBP) which is coded by a mec gene such as mecA and PBP2a. Furtermore, homologues of mecA such as mecC were described recently”. We like to thank the reviewer for his kind words and his suggestion. We have changed the second paragraph of the introduction accordingly.

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Reviewer: Michael Z. David, M.D., Ph.D. University of Chicago I have no competing interests.

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Thank you for the opportunity to review the manuscript “Low prevalence of methicillin-resistant Staphylococcus aureus among men who have sex with men attending an STI clinic in Amsterdam, a prospective cross-sectional study” by Joore et al. The authors studied S. aureus colonization at several body sites in men who have sex with men (MSM) who presented with (n=74) and without (n=137) a skin infection. These 2 groups were compared. The authors found that colonization of the perineum or genitals was associated with having an active skin infection, older age, and having had fewer sexual partners. HIV trended toward an association with colonization at these sites, although there was no association between HIV and increased risk of nasal S. aureus colonization. MRSA was identified in only 0.9% (n=2) of the studied MSM, which was a much lower prevalence than found in similar studies performed in the U.S. Furthermore, both of the subjects who carried

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Page 18 of 51


Page 19 of 51

MRSA carried PVL-negative strains with spa types not associated with CA-MRSA. Again, this finding differed from similar studies in the U.S. in which CA-MRSA strains types were isolated from MSM. Also the authors found that S. aureus was found most commonly in the nares and less than 1/3 as frequently in cultures of the pharynx, in the perianal region or on the glans penis. The findings of the manuscript are of interest because it is often thought that MSM are at especially high risk of MRSA colonization and infection. This study does not support this contention for MSM attending a clinic in Amsterdam. It is not so surprising that, compared with the U.S., there were fewer MRSA carriers among MSM in the Netherlands, where the percentage of MRSA isolates among unselected S. aureus isolates obtained from infections is very low. However, the results of the manuscript concerning risk factors for S. aureus colonization are surprising. It has been hypothesized that S. aureus may be transmitted by sexual activity in the manner of STIs. It is believed that skin-to-skin contact is the primary modality of transmission of S. aureus. If this were true, one might expect anogenital colonization to be increasingly prevalent in MSM with a greater number of reported sexual partners. The authors found the opposite to be true.

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I have several comments, questions, and suggested revisions for the authors:

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1. Article summary, page 3, line 17. “infections”. Point taken

“infecions” should be

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2. Introduction, page 4, line 39. “methicillin” This statement may be misleading to the reader. MRSA strains are resistant to all beta-lactam antibiotics with the exception of the novel cephalosporins that can bind to PBP2a. We have altered the text accordingly: “resistant to methicillin and all beta-lactam antibiotics with the exception of the novel cephalosporin ceftarolin”

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3. Introduction, page 4, line 41. HA-MRSA is usually an abbreviation for “health care-associated MRSA” because HA-MRSA strain types are not limited to acute care hospitals, but more generally to health care facilities. Point taken 4. Introduction, page 4, lines 50-56. The CA-MRSA clone with multidrug resistance identified in 2 studies in the U.S. from Boston and San Francisco but rarely in other studies was a


BMJ Open

strain type that was categorized as USA300 by PFGE. This strain is not at all typical of most USA300 isolates. In fact, USA300 isolates are generally susceptible to clindamycin, trimethoprim-sulfamethoxazole, the tetracyclines, rifampin, gentamicin, mupirocin, vancomycin, daptomycin, and linezolid. USA300 is variably susceptible to fluoroquinolones. It is very commonly resistant to erythromycin and usually has a negative D-test. The statement here implies that all USA300 isolates are typically resistant against multiple antibiotics, but this is incorrect. We have now removed the phrase: “reistant against multiple antibiotics”.

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5. Results, page 7, line 33. “diagnosed”. Point taken

“diagnoses”

should be

6. Discussion, page 10, line 18. “Lifestock” should be “livestock”, and “Staphylococcus aureus” should be italicized. Also, does this sentence mean by “are positive for” that these pigs and pig farmers have asymptomatic colonization with MRSA? The phrase “livestock” is now introduced for the first time in the introduction and correctly spelled. We have rephrased “positive for” into “asymptomatic carriers of”

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Thank you for the opportunity to review this interesting manuscript. Thank you for reviewing our manuscript and your kind words.

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1 Low prevalence of methicillin-resistant Staphylococcus aureus among men who have sex with men attending an STI clinic in Amsterdam, a cross-sectional study

1


1,2,3

2,3

4


5, 6

, de

Vries HJC1,4,7


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Keywords:


Staphylococcus

aureus,

HIV,

male


ABSTRACT


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common among men who have sex with men (MSM) in the USA. It is unknown whether this is also the case in Amsterdam, the Netherlands. Design: Cross-sectional study

Setting: STI outpatient low-threshold clinic, Amsterdam, the Netherlands Participants: Between October 2008 and April 2010, a total of 211 men were included, in two

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groups: (1) 74 MSM with clinical signs of a skin or soft tissue infection (symptomatic group) and (2) 137 MSM without clinical signs of such infections (asymptomatic group).

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•


•

In a cross-sectional study at the STI outpatient low-threshold clinic, Amsterdam, the Netherlands we studied the prevalence of S. aureus and MRSA colonisation and

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infections among symptomatic and asymptomatic men who have sex with men (MSM)

Key messages:

• Among MSM visiting the STI clinic in Amsterdam CA-MRSA is rare. • Genital colonisation of S. aureus is not associated with sexual high risk behaviour.

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• The study was limited to the Amsterdam MSM population visiting the STI outpatient clinic.

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Funding: This work was supported by the Research and Development fund of the Public Health Service of Amsterdam [Project 2373 Community associated MRSA prevalence and risk factors in male

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std outpatient clinic visitors who have sex with men]. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Competing Interests: The authors certify that they have no affiliation with or financial involvement in any organization or entity with a direct financial interest in the subject matter or materials discussed in

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the manuscript (e.g., employment, consultancies, stock ownership, or honoraria).

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4 INTRODUCTION


1;2

Around

20% (12-30%) of individuals are persistent S. aureus nasal carriers, approximately 30% are intermittent carriers (range 16-70%), and about 50% (range 16-69%) non-carriers.

1;3

A causal


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1;2;4-6



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Most strains of S. aureus are methicillin-susceptible (MSSA), but some strains, called methicillinresistant Staphylococcus aureus (MRSA) are resistant to methicillin and all beta-lactam

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antibiotics with the exception of the novel cephalosporin, ceftarolin, that can bind to penicillin-

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binding-protein PBP2a. MRSA has become a major nosocomial infection control problem (known as health care-associated MRSA, or HA-MRSA) in many parts of the world with the exception of

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the Netherlands and of Scandinavian countries.9,24,25 Later on, independent from health care institutions, MRSA emerged in the community (CA-MRSA) and livestock (LA-MRSA).

10;11,28

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Resistance to methicillin and other ß-lactams in S.aureus is based on an additional penicillin8

binding-protein (PBP) which is coded by a mec gene such as mecA. Homologues of mec A such as mecC were described recently.

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CA-MRSA is distinguished from HA-MRSA by clinical, laboratory, and epidemiologic 12

characteristics.

A new CA-MRSA clone (USA300), has been described and was identified in

several communities in the USA and Canada.9-11 New clones of CA-MRSA have also been reported in Europe.

13;14

CA-MRSA often produces Panton-Valentine Leukocidin (PVL), a toxin

that causes polymorphonuclear leukocyte lysis and tissue necrosis13;15


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5 In men who have sex with men (MSM), outbreaks of CA-MRSA skin infections have been reported and the majority of the patients were HIV infected.

8;16-18

It has been suggested that CA-

MRSA might be transmitted in this population from skin to skin during sexual contact.18;19

A cross-sectional study among 104 men conducted in Italy did not detect a single case of colonisation with CA-MRSA in HIV-infected MSM.

20

No other study in Europe has reported the

prevalence of CA-MRSA colonisation in MSM. The purpose of this investigation was to evaluate

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the prevalence of, and sexual risk factors for S. aureus colonisation (MSSA and MRSA) and CAMRSA infection among MSM visiting the sexual transmitted infection (STI) outpatient clinic in Amsterdam.

METHODS

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Study population

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Consecutive MSM attending the STI outpatient clinic in Amsterdam, The Netherlands, were

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invited to participate in this cross-sectional study. We aimed to include both symptomatic men (those who had clinical signs of a skin or soft tissue infection [pustules, abscesses, ulcerations,

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erythematous painful papules or plaques]) and asymptomatic men (those without abovementioned clinical signs). All participants were asked to complete a questionnaire concerning

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hospital admissions during the past year, factors known to be associated with S. aureus colonisation (e.g. sharing razor blades, tending animals), STI risk factors and sexual behaviour.

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STI risk factors were also obtained from the electronic files of the routine patient history.

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Swabs were taken from the anterior nasal cavity, throat, perianal area, and penile glans. From symptomatic men also swabs from suspected infected skin lesions were obtained. If MRSA was detected, the participant was referred to his general practitioner for eradication therapy, and the sexual partners of the patient were invited for MRSA screening. The study was approved by the ethics committee of the Academic Medical Center of the University of Amsterdam. All participants provided written informed consent.


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6 Laboratory tests Culture for S. aureus was done on blood agar plates and for MRSA on selective chromagar plates after enrichment in broth. S. aureus strains were confirmed by the SA442 (Martineau) nucleic acid amplification test (NAAT) and MRSA strains by the mecA NAAT.

21;22

If MRSA was

found, the spa-gene was sequenced as described before.23


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A sample size calculation was done prior to the study. The prevalence of MRSA carriage in the general population was estimated to be 0.03%.24 We aimed to assess whether the MRSA prevalence among MSM patients of the STI clinic was at least 3%. In order to reject the null hypothesis (prevalence of 0.03% or less among the studied group) with a study power of 95% and a significance level of 0.05, a sample size of at least 113 asymptomatic men was needed;

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group of 75 symptomatic men. Because the expected number of asymptomatic men was much

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larger than that of symptomatic patients, the inclusion period for the symptomatic participants was scheduled to be longer.

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The electronic patient file with routine STI screening data, the questionnaire and the laboratory

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results were merged into one database. Chi-square test or Fisher’s exact test were used to compare categorical variables between groups; the ranksum test was used to compare

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continuous variables between groups. Data analyses were performed with STATA software (STATA Intercooled, College Station, TX, USA), version 11.0. P values of less than 0.05 were

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considered statistically significant. Associations between possible risk factors and anogenital S. aureus colonisation were examined with multivariable logistic regression, reporting adjusted odds ratios (OR) with 95% confidence intervals (CI). An initial model included all variables that were significant in bivariable analyses; variables were dropped one by one from the model based on the likelihood ratio test (if P>0.05); HIV was forced into the model.


Page 27 of 51

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7

RESULTS

In total 214 men were included, starting November 2008. The inclusion of asymptomatic men was completed in December 2008 and of symptomatic men in April 2010. From three participants

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laboratory results were not available and these were excluded, leaving 211 MSM for analysis (137 asymptomatic and 74 symptomatic participants). The median age of men in the two groups was similar (38 and 41 years, respectively, p=0.32, see Table 1). Men in the two groups were comparable regarding most non-sexual risk factors, sexual risk factors and STI diagnoses, but symptomatic men were more often HIV infected (53% vs. 31%; p=0.002) and were more often

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diagnosed with syphilis (12% vs. 1%; p<0.001). Also, use of some drugs (cannabis, cocaine, and methamphetamine) was significantly more common in the symptomatic group.

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prevalences (Table 1). In the symptomatic group 40% (27/67) of men had an S. aureus infected skin lesion. In one symptomatic and one asymptomatic case MRSA was detected, giving an

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overall prevalence of MRSA carriage of 0.9% (95% binomial exact confidence interval [95%CI] 0.1-3.4%), and a prevalence of 0.7% (95%CI 0.02-4.0%) among asymptomatic MSM. Details of

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these two cases are described below. No known CA-MRSA nor PVL toxin-producing clones were detected.

S. aureus colonisation of the ano-genital area (based on penile and perianal swabs) was found in 18% (38/211) of the participants. Participants with and without ano-genital S. aureus colonisation were similar in respect to sexual risk behaviour, drug use, history or diagnosis of sexual transmitted diseases, antibiotic exposure, circumcision status and hygiene behaviour, but men


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Page 28 of 51

8 with ano-genital S. aureus colonisation were older (42 vs. 38 years; P=0.05), and they were more often HIV infected (58% vs. 34%, p=0.007; see Table 2). In a multivariable logistic regression analysis, adjusting for age, HIV status (aOR 2.2, 95%CI 0.96-4.9), belonging to the symptomatic group (aOR=2.7, 95%CI 1.2-5.9) and having had few sexual partners (aOR 3.1, 95%CI 1.2-8.3 for those with <5 sexual partners in previous year compared to those with over 20 sexual partners) were independently and significantly associated with anogenital S. aureus colonisation.

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We examined associations between nasal carriage of S. aureus and a long list of possible nonsexual risk factors, like admissions, operations, employment in health care sector, shaving habits, participation in team sports, sauna visits, and other factors; none of these was associated with S. aureus colonisation of the nose.

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An HIV infected 40-year-old, asymptomatic, Dutch MSM was positive for MRSA at the perineum.

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The MRSA had spa type t064 and was resistant to ciprofloxacin, gentamicin, oxacillin, penicillin

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recreational drugs and visited a public gym and sauna. Furthermore, he engaged in high risk sex, including unprotected anal intercourse, active and passive fisting, group sex, sex at sex parties

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and sex with partners met through the internet. We attempted to trace six of his sexual partners of whom four were screened. None had MRSA and none had clinical signs of a skin infection.

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Symptomatic carrier of MRSA In an HIV infected, 46-year old Dutch MSM MRSA was detected on swabs taken from a penile ulcer, the nasal cavity, the perineum and the glans penis. The MRSA had spa type t002 and was resistant to ciprofloxacin, erythromycin, oxacillin and penicillin. Further genetic analysis revealed mecA+ and Martineau+; pUSA03 and PVL were both negative. In the preceding year the patient had not been hospitalized, nor operated upon, nor had he travelled abroad. He did not use


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9 recreational drugs, and did not visit public gyms or saunas. He had not engaged in high risk sex; he had had sex with one partner in the preceding year. This partner could not be traced for further screening.

DISCUSSION

The prevalence of MRSA among MSM clients of the STI outpatient clinic in Amsterdam was 0.9%

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(95%CI 0.1-3.4%); among clients without skin lesions this was 0.7% (95%CI 0.02-4.0%). This is similar to the overall prevalence in Dutch hospitals (1.8%).25 No CA-MRSA associated clones were found in this population. These findings are in contrast to reports from the USA where considerable CA-MRSA prevalences were observed among MSM attending STI screening sites.

18;26


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continent, this has not resulted in an extensive introduction of CA-MRSA within the Amsterdam population yet. The low MRSA prevalence among the Dutch population in general and among the

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MSM STI clinic population may be attributed to the strict Dutch search–and-destroy policy and 24


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We observed similar prevalences of S. aureus nasal colonisation (37%) in MSM as observed in 1

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the general population. Genital S. aureus colonisation was associated with HIV infection; although HIV infection may be considered a proxy for high risk sexual behaviour, other markers

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for risk behaviour were not significantly associated with S. aureus colonisation. We attribute the more common colonisation among HIV positive participants to their immunocompromised status.

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Unfortunately, we did not have data on virological, immunological or clinical parameters of HIV infection, nor about use of antiretroviral drugs. We did not observe an increased risk of S. aureus nasal carriage among HIV infected, as was reported earlier.

27

In contrast to Southern European countries, the Netherlands has a low rate of health careassociated MRSA (HA-MRSA), probably due to the above-mentioned search-and-destroy policy. The restricted use of betalactam antibiotics outside of hospitals, in the community, might explain


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10 the low prevalence of CA-MRSA. Europe has not yet been confronted with CA-MRSA on a wide 20


9-11;18-20

Yet, in

contrast to low prevalences of HA-MRSA and CA-MRSA in the Netherlands, screening of Dutch workers with livestock has revealed that 39% of slaughterhouse pigs and >20% of pig farmers are asymptomatic carriers of LA-MRSA belonging to sequence type (ST) 398.28 The 2 MRSA strains isolated in our study were both unrelated to this LA-MRSA epidemic.

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In conclusion, CA-MRSA among MSM visiting the STI outpatient clinic in Amsterdam is rare. Although genital S. aureus colonisation was more common among HIV infected MSM, we found no association between high risk sexual behaviour and genital colonisation with S. aureus. In the Netherlands there is no indication for MRSA screening of MSM attending STI clinics.

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P-value


38 (31-45) 111 81.0

41 (31-48) 54 73.0

0.32 0.18


21 92 34 40 36 76 29 14 8 3

15.3 67.2 24.8 29.2 26.3 55.5 21.2 10.2 5.8 2.2

7 47 33 27 25 44 27 13 7 6

9.5 63.5 44.6 36.5 33.8 59.5 36.5 17.6 9.5 8.1

0.23 0.60 0.003 0.28 0.25 0.58 0.02 0.13 0.33 0.04

13.8 21.9 14.6 20.4 19.0 10.2 71.5 57.7 49.6 40.9 15.3 10.2 43.8 62.0 22.6 54.7 57.7

9 14 11 15 12 13 48 58 32 37 15 11 38 47 17 45 45

12.2 18.9 14.9 20.3 16.2 17.6 64.9 78.4 43.2 50.0 20.3 14.9 51.4 63.5 23.0 60.8 60.8

0.32 0.003 0.38 0.20 0.36 0.32 0.29 0.83 0.95 0.40 0.66

Characteristic

0.77

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34 42 1 9 23

24.8 30.9 0.7 6.6 16.8

24 38 9 5 14

32.4 52.8 12.2 5.4 18.9

0.24 0.002 <0.001 0.74 0.70


50 13

36.5 9.5

28 10

37.8 13.5

0.85 0.37


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9 10 --

6.6 7.3 --

17 12 27

23.3 6.2 40.3

<0.001 0.043 --


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S. aureus pos.

N = 173

N =38

n

%

n

P-value %


38 (30-45)

Dutch Ethnicity

138

42 (35-49) 79.8

27

0.05 71.1

0.24



7

18.4

0.30

21

55.3

0.13

57

33.0

10

26.3

0.43

53

30.6

14

36.8

0.46

49

28.3

12

31.6

0.69

96

55.5

24

63.2

0.39

44

25.4

12

31.6

0.44

23

13.3

4

10.5

0.64

12

6.9

3

7.9

0.84

4.1

2

5.3

0.74

7

0.08

18

10.4

10

26.3

35

20.2

9

23.7

16.8

2

5.3

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12.1 68.2

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C. Sexual history

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5-9

29

10-19

35

20.2

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21.1

20-39

33

19.1

5

13.2

40 or more

23

13.3

4

10.5


121

69.9

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110

63.6

85

49.1


74

42.8

25

65.8

0.62

27

71.1

0.38

15

39.5

0.28

19

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0.42

18.4

0.81

7.9

0.41

18

47.4

0.90

63.0

23

60.5

0.78

20.8

12

31.6

0.15

102

59.0

18

47.4

0.19

105

60.7

19

50.0

0.23

51

29.5

7

18.4

0.17

Active fisting #

29

16.8

7

Passive fisting #

22

12.7

3

Had group sex §

80

46.2


109


36



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14

HIV infected *

59

34.3

21

58.3

0.007

Syphilis diagnosis

10

5.8

0

0

0.13


13

7.5

0

0

0.08

Chlamydia diagnosis

30

17.3

7

18.4

0.87


109

63.0

20

52.6

0.24


13

7.5

0

0

0.08


110

63.6

23

60.5

0.72


103

59.5

25

65.8

0.48

149

86.1

32

84.2

0.76


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165

95.4

38

100

0.18

Pubic hair

149

86.1

31

81.6

0.47

Body hair

110

63.6

20

52.6

0.21

45

26.0

11

29.0

0.71

14

8.1

3

7.9

0.97

9

5.2

4

10.5

0.22

23

13.5

8

21.6

0.21

Has pets




6

3.5

1

2.6

0.79

49

28.3

14

36.8

0.30

55

31.8

11

29.0

0.73

11

29.0

0.28

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20.8


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Author’s contribution IJ analysed the data and wrote the first manuscript draft. MvR collected the data and analysed the data, MSvdL supervised the epidemiological analysis and wrote the article, HdN performed the molecular strain typing and wrote the article, AvD performed the cultivation experiments, designed the study and wrote the aricle, HdV conceptualized and supervised the study and approved the final manuscript. Technical appendix, statistical code, and dataset available from the corresponding author at Dryad repository, who will provide a permanent, citable and open access home for the dataset.


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Community associated MRSA prevalentie en risicofactoren onder mannen die seks hebben met mannen Hoofdonderzoeker: dr. H. J. C. de Vries, dermatoloog, SOA polikliniek, cluster infectieziekten, GGD Amsterdam tevens afd. Dermatologie, AMC Amsterdam en Centrum Infectieziekten bestrijding, RIVM, Bilthoven. Overige onderzoekers: dr. M Schim van der Loeff, arts-epidemioloog, afdeling onderzoek, infectieziekten, GGD Amsterdam drs. P Gruteke, arts-microbioloog, streeklaboratorium, cluster infectieziekten, GGD Amsterdam, tevens afd. microbiologie, OLVG, Amsterdam dr. A van Dam, arts-microbioloog, streeklaboratorium, cluster infectieziekten, GGD Amsterdam, tevens afd. microbiologie, OLVG, Amsterdam mw. G.B. Linde, hoofdanalist, streeklaboratorium, cluster infectieziekten, GGD Amsterdam dr. A de Neeling, microbioloog, projectleider antimicrobiële resistentie, Centrum Infectieziekten bestrijding, RIVM, Bilthoven. dr. J.S.A. Fennema,. hoofd, cluster infectieziekten, GGD Amsterdam. research verpleegkundige, vacature

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Inhoud

1. Contact informatie ......................................................................................................... 4 2. Protocol ondertekeningpagina .................................................................................. 5 3. Samenvatting .................................................................................................................. 6 4. Achtergrond ..................................................................................................................... 7 5. Relevantie ......................................................................................................................... 8 7. Studie opzet...................................................................................................................... 9 8. Behandeling bij MRSA dragerschap/symptomatische MRSA infectie ....... 10 9. Sample-size berekening en statistiek................................................................... 11 9. Referenties ..................................................................................................................... 12

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Appendix I: Patientinformatie en toestemmingsformulier

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1. Contact informatie Hoofdonderzoeker dr. H.J.C. de Vries, SOA polikliniek, Weesperplein 1 1018 WZ Amsterdam Cluster Infectieziekten, GGD Amsterdam, Tel: +31-20-555 5429 Email: [email protected] en

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afdeling Dermatologie, Academisch Medisch Centum, Universiteit van Amsterdam Meibergdreef 9 1105 AZ Amsterdam Tel: +31-20-566 9111

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afdeling Dermatologie, Academisch Medisch Centum, Universiteit van Amsterdam

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Studielokaties SOA polikliniek, Cluster Infectieziekten, GGD Amsterdam,

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Laboratoria streeklaboratorium, cluster infectieziekten, GGD Amsterdam Centrum Infectieziekten bestrijding (CIb), RIVM, Bilthoven

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2. Protocol ondertekeningpagina Community associated MRSA prevalentie en risicofactoren onder mannelijke soa poli bezoekers die seks hebben met mannen Handtekening hoofd onderzoeker: H.J.C. de Vries, MD, PhD, SOA polikliniek, Cluster Infectieziekten, GGD Amsterdam

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________________________ Handtekening

________________________ Datum

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3. Samenvatting Methicilline Resistente Staphylococcus Aureus (MRSA) is een Ziekenhuis bacterie die tot grote behandelproblemen leidt. Sinds 1996 wordt een Community Associated of Acquired MRSA (CA-MRSA) beschreven die buiten ziekenhuizen in de gezonde populatie voorkomt. In Nederland wordt CA-MRSA regelmatig aangetroffen onder varkenshouders en in de Verenigde Staten ook onder mannen die seks hebben met mannen (MSM, homo- en biseksuele mannen). CA-MRSA veroorzaakt huid en weke delen infecties zoals abcessen, impetigo, en folliculitis. Begin 2008 is een nog resistenter en virulenter CA-MRSA kloon beschreven onder MSM, de USA 300 kloon, eerst in San Francisco en Boston, en recent in Duitsland. Ook op de SOA polikliniek van de GGD Amsterdam is het eerste geval van USA 300 CAMRSA onder een MSM bezoeker gevonden. In deze studie willen we de volgende vragen beantwoorden: 1) Wat is de prevalentie van CA-MRSA asymptomatisch dragerschap onder MSM SOA poli bezoekers? 2) Wat zijn risicofactoren voor CA-MRSA dragerschap en/of infecties onder MSM? 3) Wordt CA-MRSA onder MSM verspreid via seksuele netwerken? 4) Wat is de prevalentie van MRSA in MSM bezoekers van de GGD SOA poli en de AMC polikliniek dermatologie met afwijkingen passend bij bacteriële huidinfecties? Bij de GGD Soa poli zullen 125 MSM (voldoende power om 3% prevalentie mee aan te tonen) nagekeken worden op dragerschap (asymptomatische infecties) dmv afname van kweken van neusholte, perianaal en van de glans penis. Daarnaast zullen bij de GGD Soa poli en AMC dermatologie poli 75 MSM met aanwijzing van huidinfectie gerichte kweken plus kweken van de neusholte, perianaal en van de glans penis worden afgenomen. Bij alle deelnemers wordt een risico anamnese voor CA_MRSA afgenomen. De verwachting is dat in totaal 40 asymptomatische S. aureus infecties (20-30% algehele bevolking), en 40 symptomatische S. aureus infecties zullen worden opgespoord. Indien MRSA word gevonden volgt uitgebreid moleculair onderzoek: MLVA, PCR en DNA sequencing ism het CIb/RIVM, dmv Bayesiaanse Monte Carlo Markov chain (MCMC) fylogenetische analyse. Met de sequence gegevens wordt getracht een seksueel netwerk in kaart te brengen naast risicofactor analyse.

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4. Achtergrond Methicilline Resistente Staphylococcus Aureus (MRSA) is een bacterie die clinici voor grote behandelproblemen plaatst doordat er voor de eerste keuze antibiotische opties veelal resistentie bestaat. In ziekenhuizen kan MRSA moeilijk behandelbare infecties veroorzaken onder ernstig zieke patiënten. Omdat transmissie verloopt via huid-huid contact is strenge isolatie vervolgens geboden om transmissie te voorkomen. Om diezelfde reden worden met enige regelmaat afdelingen gesloten om uitbraken van MRSA te kunnen elimineren (zo is december j.l. in het Boven IJ-ziekenhuis nog voor zes van de elf afdelingen een opnamestop afgekondigd). Sinds 1996 wordt melding gemaakt van MRSA infecties onder ogenschijnlijk gezonde individuen die buiten het ziekenhuis, in de gemeenschap zijn opgelopen. Deze infecties werden het eerst gesignaleerd onder veehouders (met name varkenshouders), “Native Americans” in de VS en worden Community Associated of Acquired MRSA (CA-MRSA) genoemd. CA-MRSA veroorzaakt meestal huid en weke delen infecties zoals abcessen, impetigo (krentenbaard), folliculitis (haarzakontstekingen) maar daarnaast ook longontstekingen. Daar veel eerste keuze antibiotica onwerkzaam zijn kan vertraging in de behandeling optreden waardoor de infectie kans ziet zich verder te verspreiden en aanleiding kan geven tot systemische infecties zoals sepsis, hartklepontstekingen en andere orgaanproblemen. Van later datum zijn meldingen van uitbraken van CA-MRSA onder gevangenen, sporters die intensief huid-huid contact hebben (worstelaars) en sinds 2003 ook onder mannen die seks hebben met mannen (MSM).[1] Binnen de groep van CA-MRSA zijn nog gevaarlijker varianten aanwijsbaar zoals de multi-resistente USA300 kloon die resistent is tegen een nog groter deel van de eerste keuze preparaten voor huid en wekedelen infecties zoals clindamycine, tetracycline, co-trimoxazol, en beta-lactam preparaten. Voortschrijdende resistentie van de USA300 kloon is gemeld doordat extra resistentie genen via incorporatie van het plasmide pUSA03 heeft plaats gevonden. Hierdoor bestaat er tevens resistentie tegen macroliden, clindamycine en mupirocine.[2] pUSA03 positieve USA300 CA_MRSA wordt gerapporteerd onder MSM in San Francisco en Boston. Naast eerdere MRSA infectie, clindamycine gebruik, vormt man-man seks de sterkste risicofactor voor een CA-MRSA infectie met deze USA300 kloon. Overigens is hiv status in deze studie niet geassocieerd met CA-MRSA infectie. Ook in Europa is de USA300 positieve CA-MRSA gemeld in een HIV positieve Duitse MSM.[3] In maart j.l. is ook op de SOA polikliniek een HIV positieve MSM gediagnosticeerd met een huidinfectie op basis van MRSA die viel binnen de US300 groep (SPA 8). Verder is gerapporteerd dat sommige USA300 klonen virulentiegenen meedragen die coderen voor het cytotoxine Panton-Valentine Leukocidin (PVL) en fenol-oplosbare peptiden die er voor zorgen dat deze stammen aanleiding kunnen geven tot meer invasieve en ernstiger infecties, zoals de potentieel lethale necrotiserende fasciitis.[4] PVL positieve CA-MRSA wordt gerapporteerd onder 2 HIV positieve MSM in Amsterdam.

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De meeste meldingen van CA-MRSA in MSM betreffen symptomatische infecties, veelal van de huid en wekedelen in de genitaal streek, de billen en het perineum.[5] Daarnaast zijn CA-MRSA infecties onder HIV positieve mannen geassocieerd met seksueel risicogedrag.[6] Op basis van deze bevinding is seksuele overdracht van CA-MRSA voorstelbaar. Er heeft echter nog geen goed gestructureerd onderzoek plaats gevonden om deze aanname verder te onderbouwen. Daarnaast is er nog geen zicht op asymptomatisch dragerschap van MRSA stammen onder MSM en de relevantie van dragerschap voor de verdere verspreiding van de infectie binnen de gemeenschap. In een recente Rotterdamse studie naar de prevalentie van S. aureus positieve neus swabs onder 443 HIV patiënten kwam en prevalentie van 0,45% MRSA positieve patiënten aan het licht waarbij geen PVL positieve stammen werden gevonden.[7] Dit betreft de Rotterdamse situatie en daarnaast is het onderzoek beperkt tot neus uitstrijkjes waardoor geen uitspraak gedaan kan worden over dragerschap in de anogenitale regio.

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5. Relevantie CA-MRSA vormt in de VS een toenemende bedreiging voor risico populaties zoals mensen die leven in instituties bijvoorbeeld bewoners van zorginstellingen en gevangenen, immuun gecompromitteerden, en verder ook minderheden en MSM.[8] Naast de resistentie en samenhangende behandelproblematiek zijn er aanwijzingen dat CA-MRSA ook meer virulente varianten omvat die aanleiding kunnen geven tot aanzienlijke morbiditeit zoals longontstekingen en ernstige wekendelen infecties. Er bestaat op dit moment geen inzicht in de verspreiding van CA-MRSA binnen de Amsterdamse populatie. Het ligt voor de hand eerst te onderzoeken of CAMRSA in Amsterdam onder een van de reeds geïdentificeerde risicogroepen (MSM) voorkomt. Daarnaast is het vanuit de preventie belangrijk te weten hoe CA-MRSA zich binnen een populatie verspreidt en dienen netwerken en risicofactoren te worden blootgelegd. Als blijkt dat CA-MRSA transmissie onder MSM binnen seksuele netwerken plaatsvindt, dan zal de SOA polikliniek een belangrijke taak hebben in verder onderzoek, behandeling, voorlichting en preventie. Daarnaast is het uit oogpunt van de medewerkers van Soa poliklinieken van belang te weten in hoeverre zij kans lopen op besmetting met MRSA. Dit heeft directe implicaties voor de uitoefening van hun werk aangezien MRSA dragers in de gezondheidzorg niet mogen werken totdat dragerschap is geëlimineerd.

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6. Doelstellingen 1. Wat is de prevalentie van community associated MRSA asymptomatisch dragerschap onder mannelijke SOA poli bezoekers die seks hebben met mannen? 2. Wat zijn risicofactoren voor community associated MRSA dragerschap en/of infecties onder mannen die seks hebben met mannen? 3. Wordt community associated MRSA onder mannen die seks hebben met mannen verspreid via seksuele netwerken? 4. Wat is de prevalentie van community associated MRSA veroorzaakte huidinfecties onder MSM bezoekers van de GGD SOA poli en de AMC polikliniek dermatologie?

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7. Studie opzet Tijdens het hoogrisico MSM spreekuur op de soa polikliniek (waarbij op afspraak MSM worden gezien met meerdere bewezen SOA in de voorgeschiedenis), en tijdens de reguliere spreekuren zullen 125 MSM gevraagd worden deel te nemen aan de studie. De huid zal worden nagekeken (mn genitaal, perianaal en de nates) op tekenen van huidinfecties (folliculitis, cellulitis, carbunkels, impetiginisatie, abcessen en fistels) en indien aanwezig zullen hiervan gericht kweken worden afgenomen. Standaard worden banale en MRSA specifieke kweken afgenomen uit de neusholte, de keelholte, perianaal en van de glans penis. Daarnaast zullen bij 1) de GGD SOA poli, 2) de AMC polikliek dermatologie bij 75 MSM bezoekers met afwijkingen passend bij een bacteriële huidinfectie een lesionale banale kweek worden afgenomen, en standaard kweken perianaal, van de glans penis en van de neusholte. Bij alle deelnemers zal een vragenlijst afgenomen worden gericht op het achterhalen van risicofactoren samenhangend met dragerschap of symptomatische infecties met S. aureus Naar verwachting zal bij 40 deelnemers asymptomatisch dragerschap worden gevonden (dit komt voor bij 20-30% van de algemene bevolking). In de groep met huidafwijkingen komen naar verwachting 40 positieve S. aureus kweken aan het licht. De volgende risicofactoren zullen worden onderzocht: reisgedrag, (seksuele)partner selectie, seksuele technieken, hygiëne technieken (o.m. klysma gebruik, nagelbijten, scheergedrag, gebruik en delen van huidproducten), huidinfecties bij personen in de directe omgeving, contact met dieren, druggebruik, bezoek aan en behandeling in zorginstellingen, bezoek/verblijf in openbare gelegenheden en penitentiaire inrichtingen, beroep, hobbies, sportuitoefening, saunabezoek, massage, antibioticagebruik, eerdere (MRSA) infecties en hiv status (indien positief eveneens laatste CD4 getal en viral load). Voorafgaande aan de studie zal getracht worden dmv enkele kwalitatieve interviews overige relevante risicofactoren voor transmissie van huidinfecties te achterhalen. Indien een kweek positief is voor S. aureus volgt uitgebreid resistentieonderzoek. Indien MRSA wordt aangetoond volgt ism het CIb/RIVM (dr. A de Neeling) moleculair onderzoek bestaande uit: MultipleLocus Variable-Number Tandem Repeat Analysis (MLVA), polymerase chain

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reaction (PCR) en DNA sequencing. Hiermee zullen genen betrokken bij antibiotica resistentie (o.m. USA300, pUSA03, ermC, mupA, msrA,en mphB) en virulentie (o.m. PVL en fenol oplosbare peptiden) in kaart worden gebracht. Indien bij een deelnemer MRSA wordt aangetoond dan wordt hij hiervan op de hoogte gesteld. Tevens moeten alle hulpverleners die tijdens het consult in aanraking zijn gekomen (dwz. huid/huid contact hebben gehad) met deze deelnemer worden nagekeken op MRSA dragerschap door middel van het afnemen van een neus, keel en perineumkweek conform de richtlijnen van commissie ziekenhuishygiëne en infectiepreventie van het AMC of de microbiologen van het streeklaboratorium van de GGD Amsterdam, naar gelang waar de deelnemer is onderzocht.

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8. Behandeling bij MRSA dragerschap/symptomatische MRSA infectie Indien bij een deelnemer of een hulpverlener die betrokken is bij het onderzoek MRSA wordt aangetoond dan worden de aanbevelingen ten aanzien van MRSA dragerschapbehandeling, zoals beschreven in de richtlijn “Optimaliseren van het antibioticabeleid in Nederland XI SWAB richtlijn Behandeling MRSA dragers”van de Stichting Werkgroep Antibioticabeleid (SWAB), Maart 2007 in acht genomen.[9] De aanbevelingen zijn verschillend voor ongecompliceerd en gecompliceerd MRSA dragerschap. Men spreekt van ongecompliceerd MRSA-dragerschap , wanneer dit voldoet aan onderstaande punten: individu zonder actieve infectie met MRSA en MRSA is in vitro gevoelig voor de toe te passen antibiotica en er zijn geen actieve huidlaesies en er is geen lichaamsvreemd materiaal dat een verbinding vormt tussen milieu interieur en milieu exterieur (bijvoorbeeld urine catheter, fixateur externa en dragerschap is in de neus gelokaliseerd. Men spreekt van compliceerde MRSA-dragerschap , wanneer dit voldoet aan minstens één van onderstaande punten: er zijn actieve huidlaesies en/of er is lichaamsvreemd materiaal dat een verbinding vormt tussen milieu interieur en milieu exterieur en/of MRSA is in vitro ongevoelig voor mupirocine, en/of eerdere behandelingen volgens de adviezen bij ongecompliceerd dragerschap hebben gefaald en/of dragerschap bevindt zich uitsluitend op andere plaatsen dan de neus, zoals keel, perineum, of huidlesies. Ongecompliceerd dragerschap: Mupirocine neuszalf 3 dd gedurende 5 dagen. Gedurende de behandeling worden huid en haren dagelijks met een desinfecterende zeep (Chloorhexidine zeep oplossing 40 mg/ml of betadine shampoo 75 mg/ml) gewassen, bij voorkeur onder de douche (niet in bad). Dagelijks schoon ondergoed, schone kleding, schone washandjes en handdoeken gebruiken. Op dag 1, 2 en 5 van de kuur beddengoed volledig verschonen. Bij het naar bed gaan dient tevens gedurende de behandeling schoon ondergoed dan wel pyjama te worden aangetrokken. Bij therapie falen: Nagaan of er sprake is van een reservoir in de thuissituatie (mens of dier). Als er een reservoir in de thuis situatie aanwezig is, dan dient deze gelijktijdig te worden meebehandeld.

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Bij tweede keer falen is er sprake van een gecompliceerd MRSA dragerschap: Indien er sprak is van actieve huidlaesies, eerst de huidlaesies behandelen, zonodig in overleg met dermatoloog. Is er na afloop van de behandeling sprake van ongecompliceerd dragerschap dan kan de behandeling die daar is aangegeven worden ingesteld. Controle kweken worden afgenomen en verder bewerkt volgens de richtlijnen van de Nederlandse Vereniging voor Medische Microbiologie (www.nvmm.nl). De eerste kweken ter beoordeling van de effectiviteit van de behandeling worden tenminste 48 uur na het beëindigen van de behandeling afgenomen. De frequentie van verdere kweekafname is onder andere afhankelijk van de gevolgen voor het betrokken individu. In de richtlijnen van de WIP zijn deze gevolgen vastgelegd (www.wip.nl).

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9. Sample-size berekening en statistiek De prevalentie MRSA dragerschap in de algemene bevolking wordt geschat op 0,03%.7 De hypothese luidt dat de MRSA prevalentie onder MSM bezoekers van de SOA polikliniek ten minste 3% bedraagt. Om de null hypothese (0,03% prevalentie onder de onderzochte groep) te verwerpen moeten ten minste 113 mannen worden geïncludeerd om een valide uitspraak met een power van 95% en een significantie level van 0.05 te kunnen doen. Met behulp van Bayesiaanse Monte Carlo Markov chain (MCMC) fylogenetische analyse van MRSA genen zoals SPA zullen clusters en associaties worden geanalyseerd. In combinatie met de uitgevraagde risicofactoren zal getracht worden inzicht te krijgen in de bijdrage van seksuele netwerken in de transmissie van MRSA onder MSM.

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9. Referenties (1) Outbreaks of community-associated methicillin-resistant Staphylococcus aureus skin infections--Los Angeles County, California, 2002-2003. MMWR Morb Mortal Wkly Rep 2003 Feb 7; 52(5):88. (2) Diep BA, Chambers HF, Graber CJ, et al. Emergence of multidrugresistant, community-associated, methicillin-resistant Staphylococcus aureus clone USA300 in men who have sex with men. Ann Intern Med 2008 Feb 19; 148(4):249-57.

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(3) Witte W, Braulke C, Strommenger B. Community-associated methicillinresistant Staphylococcus aureus ST8 ("USA300") in an HIV-positive patient in Cologne, Germany, February 2008. Euro Surveill 2008; 13(13). (4) Tiemersma E, Wannet W, op de Coul E, de Neeling AJ, van de Laar M. Introduction of surveillance of infections with Staphylococcus aureus containing the Panton-Valentine Leukocidin gene in the Netherlands. Euro Surveill 2003; 4(16).

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(5) Diep BA, Chambers HF, Graber CJ, et al. Emergence of multidrugresistant, community-associated, methicillin-resistant Staphylococcus aureus clone USA300 in men who have sex with men. Ann Intern Med 2008 Feb 19; 148(4):249-57.

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(6) Lee NE, Taylor MM, Bancroft E, et al. Risk factors for communityassociated methicillin-resistant Staphylococcus aureus skin infections among HIV-positive men who have sex with men. Clin Infect Dis 2005 May 15; 40(10):1529-34.

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(7) Melles DC, Pauw E, van den BL, et al. Host-microbe interplay in persistent Staphylococcus aureus nasal carriage in HIV patients. Microbes Infect 2008 Feb; 10(2):151-8.

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(8) Outbreaks of community-associated methicillin-resistant Staphylococcus aureus skin infections--Los Angeles County, California, 2002-2003. MMWR Morb Mortal Wkly Rep 2003 Feb 7; 52(5):88.

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(9) Stichting Werkgroep Antibioticabeleid (SWAB). Optimaliseren van het antibioticabeleid in Nederland XI SWAB richtlijn Behandeling MRSA dragers. 2007.


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STROBE 2007 (v4) checklist of items to be included in reports of observational studies in epidemiology*Checklist for cohort, case-control, and cross-sectional studies (combined) Section/Topic Title and abstract

Introduction Background/rationale Objectives

Item # 1

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Methods Study design

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Setting

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Participants

Recommendation (a) Indicate the study’s design with a commonly used term in the title or the abstract (b) Provide in the abstract an informative and balanced summary of what was done and what was found Explain the scientific background and rationale for the investigation being reported State specific objectives, including any prespecified hypotheses Present key elements of study design early in the paper Describe the setting, locations, and relevant dates, including periods of recruitment, exposure, followup, and data collection (a) Cohort study—Give the eligibility criteria, and the sources and methods of selection of participants. Describe methods of follow-up Case-control study—Give the eligibility criteria, and the sources and methods of case ascertainment and control selection. Give the rationale for the choice of cases and controls Cross-sectional study—Give the eligibility criteria, and the sources and methods of selection of participants (b) Cohort study—For matched studies, give matching criteria and number of exposed and unexposed Case-control study—For matched studies, give matching criteria and the number of controls per case Clearly define all outcomes, exposures, predictors, potential confounders, and effect modifiers. Give diagnostic criteria, if applicable For each variable of interest, give sources of data and details of methods of assessment (measurement). Describe comparability of assessment methods if there is more than one group Describe any efforts to address potential sources of bias Explain how the study size was arrived at Explain how quantitative variables were handled in the analyses. If applicable, describe which groupings were chosen and why (a) Describe all statistical methods, including those used to control for confounding (b) Describe any methods used to examine

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Study size Quantitative variables

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Statistical methods

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subgroups and interactions (c) Explain how missing data were addressed (d) Cohort study—If applicable, explain how loss to follow-up was addressed Case-control study—If applicable, explain how matching of cases and controls was addressed Cross-sectional study—If applicable, describe analytical methods taking account of sampling strategy (e) Describe any sensitivity analyses Results Participants

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Descriptive data

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(a) Report numbers of individuals at each stage of study—eg numbers potentially eligible, examined for eligibility, confirmed eligible, included in the study, completing follow-up, and analysed (b) Give reasons for non-participation at each stage (c) Consider use of a flow diagram (a) Give characteristics of study participants (eg demographic, clinical, social) and information on exposures and potential confounders (b) Indicate number of participants with missing data for each variable of interest (c) Cohort study—Summarise follow-up time (eg, average and total amount) Cohort study—Report numbers of outcome events or summary measures over time Case-control study—Report numbers in each exposure category, or summary measures of exposure Cross-sectional study—Report numbers of outcome events or summary measures (a) Give unadjusted estimates and, if applicable, confounder-adjusted estimates and their precision (eg, 95% confidence interval). Make clear which confounders were adjusted for and why they were included (b) Report category boundaries when continuous variables were categorized (c) If relevant, consider translating estimates of relative risk into absolute risk for a meaningful time period Report other analyses done—eg analyses of subgroups and interactions, and sensitivity analyses

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Discussion Key results

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Limitations

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Interpretation

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Summarise key results with reference to study objectives Discuss limitations of the study, taking into account sources of potential bias or imprecision. Discuss both direction and magnitude of any potential bias Give a cautious overall interpretation of results considering objectives, limitations, multiplicity of analyses, results from similar studies, and other

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Generalisability

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Other information Funding

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relevant evidence Discuss the generalisability (external validity) of the study results

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Give the source of funding and the role of the 3 funders for the present study and, if applicable, for the original study on which the present article is based *Give information separately for cases and controls in case-control studies and, if applicable, for exposed and unexposed groups in cohort and cross-sectional studies. Note: An Explanation and Elaboration article discusses each checklist item and gives methodological background and published examples of transparent reporting. The STROBE checklist is best used in conjunction with this article (freely available on the Web sites of PLoS Medicine at http://www.plosmedicine.org/, Annals of Internal Medicine at http://www.annals.org/, and Epidemiology at http://www.epidem.com/). Information on the STROBE Initiative is available at www.strobe-statement.org.

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BMJ Open. For peer review only - Journal: BMJ Open. Manuscript ID: bmjopen

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