Nasal colonization with Staphylococcus aureus is a risk factor for ventricular assist device infection in the first year after implantation: A prospective, single-centre, cohort study

Journal Pre-proof

Nasal colonization with Staphylococcus aureus is a risk factor for ventricular assist device infection in the first year after implantation: a prospective, single-centre, cohort study ´ Dennis Nurjadi , Katharina Last , Sabrina Klein , Sebastien Boutin , Bastian Schmack , Florian Mueller , Klaus Heeg , Arjang Ruhparwar , Alexandra Heininger , Philipp Zanger PII: DOI: Reference:

S0163-4453(20)30097-9 https://doi.org/10.1016/j.jinf.2020.02.015 YJINF 4458

To appear in:

Journal of Infection

Accepted date:

20 February 2020

´ Please cite this article as: Dennis Nurjadi , Katharina Last , Sabrina Klein , Sebastien Boutin , Bastian Schmack , Florian Mueller , Klaus Heeg , Arjang Ruhparwar , Alexandra Heininger , Philipp Zanger , Nasal colonization with Staphylococcus aureus is a risk factor for ventricular assist device infection in the first year after implantation: a prospective, single-centre, cohort study, Journal of Infection (2020), doi: https://doi.org/10.1016/j.jinf.2020.02.015

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd on behalf of The British Infection Association.

Highlights 

S. aureus was the most common cause of Ventricular-Assist-Device (VAD) infections



Nasal colonization increased the risk of S. aureus-VAD-infection about 4-fold



All VAD-infections occurred at least 7 weeks after implantation



Genotyping showed that 75% of infecting S. aureus was from endogenous origin



Sustained interruption of endogenous transmission may half burden of VADinfections

1

Nasal colonization with Staphylococcus aureus is a risk factor for ventricular assist device infection in the first year after implantation: a prospective, single-centre, cohort study Dennis Nurjadi1, Katharina Last1, Sabrina Klein1, Sébastien Boutin1, Bastian Schmack2, Florian Mueller2, Klaus Heeg1, Arjang Ruhparwar2,3, Alexandra Heininger1,4*, Philipp Zanger1,5*

*contributed equally to the manuscript

1

Department of Infectious Diseases, Medical Microbiology and Hygiene, Heidelberg

University Hospital, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany 2

Department of Cardiac Surgery, Heidelberg University Hospital, Im Neuenheimer Feld 110,

69120 Heidelberg, Germany 3

Department of Cardiac Surgery, Essen University Hospital, Hufelandstraße 55, 45147 Essen,

Germany 4

Unit of Hospital Hygiene, Mannheim University Hospital, Theodor-Kutzer-Ufer 1-3, 68167

Mannheim 5

Heidelberg Institute of Global Health, Heidelberg University Hospital, Im Neuenheimer Feld

324, 69120 Heidelberg, Germany

Running title: S. aureus colonization in VAD-patients

3602 words

2

Correspondence to: Philipp Zanger, MD MSc, Heidelberg Institute of Global Health & Department of Infectious Diseases, Medical Microbiology and Hygiene, Heidelberg University Hospital, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany; Phone: +49 6221 56-4067, Email: [email protected]

Abstract (249 words) Objectives: To assess, whether S. aureus nasal colonization is a risk factor for infections in patients with durable ventricular assist device (VAD). Methods: Prospective, single-centre, cohort study (i) ascertaining S. aureus nasal colonization status of patients admitted for VAD-implantation and detecting time to first episode of VAD-specific or -related infection according to International Society for Heart and Lung Transplantation criteria during follow-up and (ii) comparing whole genomes of S. aureus from baseline colonization and later infection. Results: Among 49 patients (17 colonized, 32 non-colonized), S. aureus VAD-infections occurred with long latency after implantation (inter quartile range 76-217 days), but occurred earlier (log-rank test P=0.006) and were more common (9/17, 52.9% vs. 4/32, 12.5%, P=0.005; incidence rates 2.81 vs. 0.61/1000 patient days; incidence rate ratio 4.65, 95% confidence interval 1.30-20.65, P=0.009) and among those nasally colonized with S. aureus before implantation. We found a similar but less pronounced effect of colonization status when analysing its effect on all types of VAD-infections (10/17, 58.8% vs. 7/32, 21.9%, P=0.01). These findings remained robust when adjusting for potential confounders and restricting the analysis to ‘proven infections’. 75% (6/8) of paired S. aureus samples from colonization and VAD-infection showed concordant whole genomes. Conclusions: In patients with durable VAD, S. aureus nasal colonization is a source of endogenous infection, often occurring months after device-implantation and affecting mostly 3

the driveline. Hygiene measures interrupting the endogenous route of transmission in VADpatients colonized with S. aureus long-term may about half the burden of infections and require clinical scrutiny. Key words (MeSH): Heart-Assist Devices; Survival Analysis; Risk Factors; Infection Control; Heart Failure; Cardiovascular Surgical Procedures; Methicillin-Resistant Staphylococcus aureus; Microbiota; Cohort Studies; Whole Genome Sequencing; Graphical Abstract (high resolution figure uploaded separately)

4

Introduction The prevalence of heart failure is rising (1, 2) thus constantly widening the gap between donor hearts and patients listed for transplantation (3). For an increasing number of patients with advanced heart failure, durable ventricular assist devices (VAD) prolong survival and improve quality of life (4) by serving as bridge to transplant, or, for those not eligible for heart transplantation, as destination therapy, i.e. as a life-long support (4, 5). Their long-term use, however, is hampered by stroke, bleeding, and infection. The latter affect 14 to 58% of VAD-users (6-8) and account for a substantial burden of hospital-admissions (9), morbidity, and premature death (10-12). According to registry data, an infection of the tunnelled percutaneous driveline that connects the device with the external controller and power source, is the leading type of VAD-specific infection (8), pointing to the exit site as main port of entry for pathogens (13-15). In an estimated 56% of all episodes, Staphylococcus aureus is isolated from VAD-infections (11, 14, 16). This is not surprising, considering that S. aureus is also the leading cause of peritonitis in patients with peritoneal dialysis catheter (17). Although many VAD-specific infections can be managed (18), their role as risk factor for serious non-infectious complications such as pump thrombosis and stroke (15) further underlines the need for improved infection prevention in VAD-patients. Currently, our knowledge on modifiable risk factors for infectious complications of VAD use is limited. A recent review of published studies concluded that younger age and an increasing body mass index were the only risk factors that were reproducibly associated with an increased risk of infections in this population (15). In patients undergoing cardiac surgery other than VAD-implantation, S. aureus nasal carriage was shown to be an independent risk factor for S. aureus-SSI (19), with results from molecular typing supporting an endogenous route of infection (20). However, similar studies on the role of the commensal skin flora for infectious complications in patients newly receiving or already carrying a VAD system are 5

extremely scarce (6). Only one retrospective study in 82 patients with VAD analysed the effect of colonization with any type of multi-resistant bacteria at multiple anatomic sites at time of implantation, but did not find a significant effect on VAD-specific/-related infections (4/28 in colonized vs. 3/54 in non-colonized, P=0.18) (21). Furthermore, VAD-infections frequently develop along the driveline, often months after the actual implantation procedure (6, 22). This underlines that associated risk factors are unlikely to be similar to those observed in association with SSI after thoracotomy and explains why the role of S. aureus nasal colonization as a risk factor for infection in the population of VAD-patients is still unclear. To this end, we conducted a single-centre prospective cohort study in patients with end-stage heart failure who underwent durable VAD-implantation and investigated, whether pre-operative S. aureus nasal colonization status has an effect on the later risk of experiencing VAD-infections, with a focus on infections caused by S. aureus. Given an association between S. aureus colonization and VAD related infection was demonstrated, decolonization therapy could be a promising approach, as previously discussed in other surgical (23-27) and non-surgical (28) patient populations.

6

Methods We conducted a prospective cohort study on nasal colonization with S. aureus as a risk factor for ‘VAD-infections’ in patients with newly implanted durable VAD. From January 2017 to August 2018, consecutive nasal swabs taken for mandatory screening of methicillinresistant S. aureus (MRSA) from patients admitted for VAD implantation were also analysed for growth of methicillin-sensitive S. aureus. Patients with one nasal swab result available up to 14 days or less before implantation were included. No other in- or exclusion criteria applied. Only patients colonized with MRSA underwent decolonization therapy, according to the institutional protocol: nasal ointment (mupirocin or octenidine) tid, body wash (octenidine) qd, and pharyngeal wash (octenidine) tid for 5 days, followed by swabs (nares, perianal, wound) for three days to confirm success or to prompt repetition of decolonization therapy. With the start of the implantation procedure, all patients received 1.5 grams of cefuroxime i.v. tid for 72 hours. No other prophylactic antimicrobial treatment was provided to the study patients. Results of the study-related screening for methicillin-sensitive S. aureus were not communicated. Beginning in January 2019, two independent infectious diseases specialists who were not directly involved in the routine care of VAD-patients and blinded for colonization status of study participants, retrospectively reviewed all clinical records, classified VAD-related and VAD-specific infections according to International Society of Heart and Lung Transplantation (ISHLT) criteria (29), and recorded alternative censoring events (death, heart transplantation, end of study at 31.12.2018), for a maximum follow-up period of 365 days post VADimplantation. A period between two physician contacts without documented evidence of infection or associated treatment was recorded as follow-up free of infections.

Laboratory methods

7

Immediate placement of the nasal swab system (Copan eSwab, Hain Life Science, Germany) in liquid Amies medium after sampling was shown to be highly sensitive in detecting MRSA nasal colonization (30) and is routinely used for colonization screening at Heidelberg University Hospital. To screen for S. aureus including MRSA, 10µl of swabmedium each was inoculated on one BD BBLTM CHROMagarTM Staph aureus/BBLTM CHROMagarTM MRSA II biplate (Becton Dickinson, Germany) and on one Columbia Blood Agar plate with 5% sheep’s blood to check for any growth as proof of valid sampling. Culture media were incubated at 35±1°C with 5% CO2 for 20 hours. Colonies with typical morphology were further analysed to the species level by MALDI-TOF (Bruker Diagnostics, Germany). Clinical samples from infection were processed by the routine diagnostic laboratory according to current microbiology standards. Nasal and clinical S. aureus isolates of subjects enrolled in the study were cryopreserved for further analysis. Nasal and clinical S. aureus isolates of colonized study subjects with later events that were classified as VAD-infection were retrieved from the cryostock, genomic DNA extracted using the DNeasy Mini Kit (Qiagen GmbH, Germany), the repeat region of protein A gene (spa-typing) sequenced by Sanger sequencing as described elsewhere (31) and the whole genome sequenced on a Illumina Miseq platform (Illumina, USA) as described elsewhere (32) with minor modifications. Assembly and core genome multilocus sequence genotyping (cgMLST) were performed using SeqSphere+ software version 5.0 (Ridom, Germany). For comparison, only genes present in all isolates were analysed, using a threshold of 95% of the defined cgMLST targets (1733 genes) and accessory genomes (203 genes). Allelic differences between nasal and clinical isolates of a given patient were plotted against each other to calculate genetic distance. Isolates with equal spa-type and less than 24 allelic differences in cgMLST were defined as identical and thus considered indicative of an endogenous infection

8

(33). Assembled sequences of all S. aureus isolates are publicly available at NCBI Genbank under the BioProject number PRJNA561696.

Data management and statistics As a primary outcome, we analysed differences in the incidence rate of S. aureus VAD-infections. Secondary outcome measures included time to S. aureus VAD-infections as well as incidence rate of and time to infection to VAD-infections by pathogens of any type. To this end, we considered (i) each patient starting with the day of VAD-implantation (baseline) ‘at risk’ of infection, (ii) S. aureus colonization status at implantation the main exposure, and (iii) VAD-infections either caused by S. aureus as primary and by any pathogen as secondary failure events. Both outcomes (S. aureus VAD-infections, any type of VADinfections) were assessed in separate analyses; on a single episode per participant basis (i.e. patients would not become at risk again after an infection and could thus only contribute one infection). VAD-infections were defined as the composite of ‘VAD-specific’ and ‘VAD-related’ infections according to ISHLT criteria (29). Incidence rates per 1000 days of observation and incidence rate ratios were calculated comparing the rate of infection in patients with and free of nasal colonization at baseline. We constructed Kaplan-Meier curves and compared the observed differences in survival functions using the log-rank test. We fitted Cox-regressions models and analysed for potential confounding by co-variates that were both, associated with colonization and risk factors for infection. We defined confounding as a difference of at least 10% when comparing crude and adjusted hazard ratios from Cox-models that were fitted using the same set of observations, with and without the putative confounder as additional explanatory variable. Before doing so, we checked for the proportional hazards assumption using the ‘stphplot’ and ‘estat phtest’

9

commands. The proportion of S. aureus-VAD-infection attributable to S. aureus nasal colonization in VAD-patients (preventable fraction) was estimated using adjusted hazard ratios and the ‘punafcc’ command (34). All statistical procedures were carried out in Stata 15 (Stata, USA).

Ethical considerations The Ethics committee, Medical Faculty of Heidelberg University (S-187/2017), reviewed the protocol of this study. Individual informed consent was waived based on the study’s strict non-interference with routine patient care.

10

Results From January 2017 to August 2018, 49 patients were scheduled for VAD implantation, routinely screened for MRSA colonization, and thus consecutively enrolled in the study. Of these 49 patients with a newly implanted, durable VAD, 16 were nasally colonized with methicillin-sensitive and one with methicillin-resistant S. aureus (MRSA) prior to implantation. Table 1 gives an overview of patient characteristics at enrolment, stratified by colonization status at baseline. Nasal colonization and risk of infection During follow-up, 17 episodes of VAD-infections occurred. Of these, 12 were caused by methicillin-sensitive S. aureus, one by MRSA, and four by other pathogens (2 Pseudomonas aeruginosa, 1 Candida krusei, 1 Escherichia coli). Using infections caused by S. aureus and pathogens of any type as distinct failure events, the 49 members of the cohort contributed 9754 and 9349 patient days (pdys) at risk, respectively. The incidence rates of infection in the overall cohort were 1.33/1000 pdys (95% CI 0.77-2.30) for infections caused by S. aureus and 1.82/1000 pdys (95% confidence interval 1.13-2.93) for VAD-infections caused by any pathogen, respectively. The median time to first S. aureus infection was 157 days (interquartile range 76-217, minimum-maximum 44-333) (Figure 1) and to first infection of any type 161 days (IQR 106-285, min-max 44-365) (Supplementary Material, Figure). We found S. aureus infections to occur more often in patients colonized than in patients free of S. aureus colonization at baseline (9/17, 52.9% vs. 4/32, 12.5%, P=0.005) (Table 2). Taking difference in follow-up time into account, these findings translated into an incidence rate of S. aureus infection in colonized compared to non-colonized VAD-patients of 2.81 vs. 0.61/1000 pdys (incidence rate ratio 4.61, 95% CI 1.29-20.47, P=0.009) (Table 3). S. aureus-infection-free survival during the first 365 days post VAD-implantation was significantly longer among those free of S. aureus at baseline (log rank test P=0.006): after 11

114 and 285 days, 25% and 50% of carriers had already experienced one or more episodes of S. aureus infection. Non-carriers, in contrast, did not cross these thresholds during follow-up. Endogenous origin of S. aureus For eight of overall nine colonized patients with S. aureus infection, the isolate of nasal carriage at baseline could be re-cultured from the cryostock and was thus available for genotypic comparison. Six of these eight comparisons (75%) showed concordant spagenotypes and less or equal than 24 alleles difference in their core genome (Figure 2) - a value that is generally regarded as confirmatory for isolates being concordant (33). Death, transplantation, censoring, and loss to follow-up Overall, we recorded 14 deaths during follow-up, which were equally distributed over comparison groups (4/17, 23.5% vs. 10/32, 31.3%, P=0.7). However, deaths due to S. aureus infections showed a non-significant trend towards being more likely in patients colonized at baseline (2/17, 11.8% vs. 0/32, 0%, P=0.1). There was no difference in the proportion of patients undergoing heart transplantation, or being censored at 365 days or due to the end of study at 31st December 2018 (Table 2). None of the patients enrolled in the study was lost to follow-up. Robustness of findings Checking for the robustness of our initial findings, we first estimated the risk of VADinfections irrespective of type of causative pathogen and found a less pronounced, but still marked effect of S. aureus nasal colonization on VAD-infections overall (10/17, 58.8% vs. 7/32, 21.9%, P=0.01; IRs 3.14/1000 vs. 1.14/1000 pdys; IRR 2.76, 95% CI 0.95-8.55, P=0.04) when compared to the effect of colonization on S. aureus infections (Table 2). Similarly, infection-free survival during the first year post implantation differed significantly between colonized and non-colonized patients (log rank test P=0.04): 25% of carriers had 12

already experienced one or more episodes of VAD-infection after 114 days of follow-up while it took 266 days for non-carriers to reach this threshold (Supplementary material, Figure). In a second step, we then increased diagnostic specificity by restricting failures to ‘proven’ VAD-infections, according to ISHLT criteria (29). Using this approach, we found a stronger, but less precise effect estimate (IRR 5.59, 95% CI 1.00-56.67, P=0.03) for infections caused by S. aureus, indicating an even stronger underlying association of colonization with proven infection, but lower statistical power due to less failure events (Table 2). Finally, we fitted a Cox proportional hazards model to adjust for differences at baseline between comparison groups and to check for potential confounding. Using VADinfections caused by S. aureus as the outcome of interest, we detected some negative confounding by age (crude HR 4.23, 1.30-13.75, P=0.02; adjusted HR 4.92, 1.47-16.53, P=0.01), diabetes (adjusted HR 4.73, 1.44-15.61, P=0.01), and by days of mechanical ventilation (adjusted HR 5.14, 1.42-18.62, P=0.01). We did not detect confounding by the remaining baseline characteristics (days of intensive care, renal function, cause of heart failure, body mass index). In particular there was no evidence of positive confounding that may have led to a spurious association of colonization and infection in the first place. A multivariable model that simultaneously adjusted for all confounders from the analysis of single confounders (age, diabetes, days of mechanical ventilation) found, that carriers have 5.63-times the risk of non-carriers to acquire an S. aureus infection during the first year post implantation (95% CI 1.54-20.66, P=0.009). Similar negative confounding was observed for the Cox model that used VAD-infection caused by any type of pathogen as outcome of interest (crude HR 2.92, 95% CI 1.08-8.19, P=0.04) when simultaneously adjusting for diabetes, age and days of ventilation (adjusted HR 3.60. 95% CI 1.21-10.75, P=0.02). Measures of impact (preventable fractions) 13

Assuming a causal relationship and taking the prevalence of colonization observed in our cohort as population estimate, we calculated that 56.94% (95% CI 4.07-80.68%, P=0.04) of S. aureus VAD-infections can be attributed to S. aureus nasal colonization. In the presence of a fully effective intervention against colonization and assuming that initially colonized patients receiving the intervention adopt a risk of VAD-infection that is equal to those free of S. aureus in the first place, this would translate into 45.57% (95% CI -0.73-70.48%, P=0.05) of all types of VAD-infections being preventable.

14

Discussion This study provides strong evidence for S. aureus nasal colonization being a risk factor for VAD-infections. We show that among patients with end-stage heart failure that carry S. aureus in their nares, during the first year after VAD-implantation, the hazard of experiencing VAD-infections caused by S. aureus or by any pathogen is about 5.6- and 3.7-times that observed in patients free of S. aureus at baseline, respectively. Moreover, and in line with published research in other patient groups (35-37), the presented whole genome typing studies demonstrate that most S. aureus infections in colonized VAD-users are endogenous infections, thus supporting a causal link between S. aureus nasal carriage and subsequent VAD-infection. Our results provide strong evidence in favour of S. aureus-colonized nares being a promising

target

for

infection

prevention

in

VAD-patients.

Preoperative

nasal

decontamination (38) and bathing or showering with skin antiseptics prior to elective surgery (39) have been evaluated to reduce the perioperative risk of infection in S. aureus carriers. Based on the evidence produced, the World Health Organization recommends decontamination of colonized nares with mupirocin ointment prior to cardiothoracic and orthopaedic surgery and also suggests to consider this approach in other types of surgery (40). By contrast, recent Cochrane reviews conclude that current evidence supporting these interventions is inconclusive (38, 39). In VAD-patients, several studies have investigated risk factors of infection (13, 14), but none has studied the role of nasal colonization and/or the effect of decolonization on the risk of VAD-infection. However, other preventive approaches including variations of driveline tunnelling (41) and modifications of driveline dressing have been tested to reduce infection rates (42). Based on the available body of evidence, a consensus statement on the prevention and management of mechanical circulatory support infection has been published in 2017, focusing on systemic perioperative antibiotic

15

prophylaxis, skin antisepsis before skin incision, and postoperative wound care (18). Due to the lack of evidence pointed out above, this consensus-statement limits its recommendation to nasal mupirocin and chlorhexidine washings before surgery for patients colonized with methicillin-resistant S. aureus (MRSA). Based on the data presented, clinical studies evaluating the effectiveness of decolonization measures targeting methicillin-sensitive and resistant S. aureus for the prevention of infections in VAD-patients seem justified. Our findings suggest that interventions similar to those in cardiothoracic and orthopedic patients that focus on the perioperative period only will most likely fail to prevent many of the late-onset infections in VAD-patients. We observed that S. aureus VADinfections manifest after a median of 157 days, i.e. more than five months post implantation, which is in contrast to other patient groups in cardiac surgery, in whom surgical site infections during the perioperative period are the leading complication (43). Besides, and most strikingly, VAD-users remained free from S. aureus infection during the first seven weeks of follow-up. Only two other studies in the field also reported pathogen specific VAD-infections and found a median time of 177 (22) and 161 days (6) to first S. aureus infection, thus corroborating our findings. Taken together, these observations support colonization and biofilm formation of the percutaneous hardware by endogenous S. aureus as pathogenetic model underlying the majority of VAD-infections, as recently hypothesized by Joost et al. (6). Also in line with this concept, 83% of VAD-specific infections primarily involve the driveline (8). In this regard, infections in VAD-patients resemble more those developing alongside catheters of patients on continuous ambulatory peritoneal dialysis (CAPD). Indeed, and similar to our findings in VAD-users, CAPD patients colonized with S. aureus have been shown to have higher risk of exit site and tunnel infections than their non-colonized counterparts (37, 44-49). To date, however, adequately powered, high quality RCTs providing conclusive support in favour of oral, nasal, or topical antimicrobials at the exit site for

16

preventing infections in colonized peritoneal dialysis patients are similarly lacking (28). Against this background, clinical trials evaluating interventions against late-onset infections in colonized patients with persisting percutaneous devices such as catheters and VADdrivelines seem warranted. The potential usefulness of prolonged decolonization measures is further supported by one recent trial showing 30% lower risk of infection in a mixed patient population of patients colonized with MRSA that underwent post-discharge decolonization therapy during the first 6 months of a one year follow-up period (50). This study has strengths and limitations. The prospective cohort design, complete follow-up of all enrolled subjects, and blinding of observers to exposure status provides certainty about the timeliness of events while greatly reducing the risk of selection and information biases. Besides, our findings remained robust when increasing the specificity to ‘proven infections’ and adjusting for potential confounders. Together, these study characteristics and observations are reassuring in that the presented findings describe a true underlying effect of colonization on VAD-infection. However, our sample, although large enough to detect the obviously large underlying effect of S. aureus colonization on infection, is too small to provide precise estimates. Besides, due to retrospective ascertainment of outcome status, we cannot rule out the misclassification in few members of the cohort. However, the resulting bias, if at all, will be towards the null, since the outcome classification was done ignorant of exposure status and thus outcome allocation, including the involved imprecisions, non-differential. Inclusion of subjects colonized with MRSA and associated decolonization therapy according to institutional standard procedures could theoretically have influenced our study results. However, we are confident that this influence – if present at all – was negligible, since only one patient with MRSA colonization was included. Furthermore, decolonization – through differentially lowering the risk of infection in those colonized – will result in an attenuation of the expected effect, i.e. lead to bias towards the null, thus rather

17

leading to an underestimation of the effect and strengthening our findings than providing the explanation for a spurious association. Also, we acknowledge that a substantial proportion of VAD-infections occur independent from S. aureus nasal colonization. Further research will be needed to elucidate modifiable risk factors for VAD-infections by pathogens other than S. aureus. Finally, S. aureus colonization of the throat and other anatomic sites is strongly linked to nasal colonization (51, 52), explaining its use a proxy for a subject’s general colonization status in this study and similar research (27). Nevertheless, additional information on extra nasal S. aureus colonization in VAD-patients could be important when designing potential interventions. In patients with durable VAD, S. aureus nasal colonization is a source of endogenous infection (as shown by genotyping), often occurring months after device-implantation, affecting mostly the driveline, and accounting for about half of all infections in VAD-patients. Hygiene measures interrupting the endogenous rout of transmission in VAD-patients with proven S. aureus colonization may about half the burden of infections and require clinical scrutiny. These studies should acknowledge that patients with percutaneous devices are at long-term risk of infection and thus build on published research on the post-discharge prevention of MRSA-infections in a general patient population (27) to achieve a similarly sustained benefit in preventing S. aureus infections in VAD-patients colonized with S. aureus.

18

Funding German Research Foundation (DFG) funding to D.N. (grant no. NU-403/1-1).

Conflicts Florian Mueller reports to have received fees as speaker outside the presented work from Abbott and Medtronic. Bastian Schmack reports to have received personal fees as surgical consultant outside the presented work from Abbott. All other authors, no conflicts.

19

References 1. Writing Group M, Mozaffarian D, Benjamin EJ, Go AS, Arnett DK, Blaha MJ, et al. Executive Summary: Heart Disease and Stroke Statistics--2016 Update: A Report From the American Heart Association. Circulation. 2016 Jan 26;133(4):447-54. PubMed PMID: 26811276. Epub 2016/01/27. 2. Heidenreich PA, Albert NM, Allen LA, Bluemke DA, Butler J, Fonarow GC, et al. Forecasting the impact of heart failure in the United States: a policy statement from the American Heart Association. Circ Heart Fail. 2013 May;6(3):606-19. PubMed PMID: 23616602. Pubmed Central PMCID: PMC3908895. Epub 2013/04/26. 3. Cowger JA. Addressing the Growing U.S. Donor Heart Shortage: Waiting for Godot or a Transplant? J Am Coll Cardiol. 2017 Apr 4;69(13):1715-7. PubMed PMID: 28359518. Epub 2017/04/01. 4. Rose EA, Gelijns AC, Moskowitz AJ, Heitjan DF, Stevenson LW, Dembitsky W, et al. Longterm use of a left ventricular assist device for end-stage heart failure. N Engl J Med. 2001 Nov 15;345(20):1435-43. PubMed PMID: 11794191. Epub 2002/01/17. 5. Bielecka A, Wierzbicka M, Goch J. The ventricular assist device: a bridge to ventricular recovery, a bridge to heart transplantation or destination therapy? Cardiol J. 2007;14(1):14-23. PubMed PMID: 18651430. Epub 2008/07/25. 6. Joost I, Bothe W, Pausch C, Kaasch A, Lange B, Peyerl-Hoffmann G, et al. Staphylococcus aureus bloodstream infection in patients with ventricular assist devices-Management and outcome in a prospective bicenter cohort. J Infect. 2018 Jul;77(1):30-7. PubMed PMID: 29778631. 7. Gordon RJ, Quagliarello B, Lowy FD. Ventricular assist device-related infections. Lancet Infect Dis. 2006 Jul;6(7):426-37. PubMed PMID: 16790383. 8. Hannan MM, Xie R, Cowger J, Schueler S, de By T, Dipchand AI, et al. Epidemiology of infection in mechanical circulatory support: A global analysis from the ISHLT Mechanically Assisted Circulatory Support Registry. J Heart Lung Transplant. 2019 Apr;38(4):364-73. PubMed PMID: 30733158. Epub 2019/02/09. 9. Leuck AM. Left ventricular assist device driveline infections: recent advances and future goals. J Thorac Dis. 2015 Dec;7(12):2151-7. PubMed PMID: 26793335. Pubmed Central PMCID: PMC4703684. Epub 2016/01/23. 10. Trachtenberg BH, Cordero-Reyes A, Elias B, Loebe M. A review of infections in patients with left ventricular assist devices: prevention, diagnosis and management. Methodist Debakey Cardiovasc J. 2015 Jan-Mar;11(1):28-32. PubMed PMID: 25793027. Pubmed Central PMCID: PMC4362062. Epub 2015/03/21. 11. Topkara VK, Kondareddy S, Malik F, Wang IW, Mann DL, Ewald GA, et al. Infectious complications in patients with left ventricular assist device: etiology and outcomes in the continuousflow era. Ann Thorac Surg. 2010 Oct;90(4):1270-7. PubMed PMID: 20868826. Epub 2010/09/28. 12. Kirklin JK, Pagani FD, Kormos RL, Stevenson LW, Blume ED, Myers SL, et al. Eighth annual INTERMACS report: Special focus on framing the impact of adverse events. J Heart Lung Transplant. 2017 Oct;36(10):1080-6. PubMed PMID: 28942782. Epub 2017/09/26.

20

13. Bejko J, Toto F, Gregori D, Gerosa G, Bottio T. Left ventricle assist devices and driveline's infection incidence: a single-centre experience. J Artif Organs. 2018 Mar;21(1):52-60. PubMed PMID: 28988400. Epub 2017/10/11. 14. Simeon S, Flecher E, Revest M, Niculescu M, Roussel JC, Michel M, et al. Left ventricular assist device-related infections: a multicentric study. Clin Microbiol Infect. 2017 Oct;23(10):748-51. PubMed PMID: 28323195. Epub 2017/03/23. 15. Pavlovic NV, Randell T, Madeira T, Hsu S, Zinoviev R, Abshire M. Risk of left ventricular assist device driveline infection: A systematic literature review. Heart Lung. 2019 Mar - Apr;48(2):90104. PubMed PMID: 30573195. Epub 2018/12/24. 16. Nienaber JJ, Kusne S, Riaz T, Walker RC, Baddour LM, Wright AJ, et al. Clinical manifestations and management of left ventricular assist device-associated infections. Clin Infect Dis. 2013 Nov;57(10):1438-48. PubMed PMID: 23943820. Pubmed Central PMCID: PMC3805171. Epub 2013/08/15. 17. Akoh JA. Peritoneal dialysis associated infections: An update on diagnosis and management. World J Nephrol. 2012 Aug 6;1(4):106-22. PubMed PMID: 24175248. Pubmed Central PMCID: PMC3782204. Epub 2012/08/06. 18. Kusne S, Mooney M, Danziger-Isakov L, Kaan A, Lund LH, Lyster H, et al. An ISHLT consensus document for prevention and management strategies for mechanical circulatory support infection. J Heart Lung Transplant. 2017 Oct;36(10):1137-53. PubMed PMID: 28781010. Epub 2017/08/07. 19. Munoz P, Hortal J, Giannella M, Barrio JM, Rodriguez-Creixems M, Perez MJ, et al. Nasal carriage of S. aureus increases the risk of surgical site infection after major heart surgery. J Hosp Infect. 2008 Jan;68(1):25-31. PubMed PMID: 17945393. Epub 2007/10/20. 20. Jakob HG, Borneff-Lipp M, Bach A, von Puckler S, Windeler J, Sonntag H, et al. The endogenous pathway is a major route for deep sternal wound infection. Eur J Cardiothorac Surg. 2000 Feb;17(2):154-60. PubMed PMID: 10731651. Epub 2000/03/25. 21. Papathanasiou M, Pohl J, Janosi RA, Pizanis N, Kamler M, Rassaf T, et al. Colonization With Multiresistant Bacteria: Impact on Ventricular Assist Device Patients. Ann Thorac Surg. 2018 Feb;105(2):557-63. PubMed PMID: 29174784. 22. Gordon RJ, Weinberg AD, Pagani FD, Slaughter MS, Pappas PS, Naka Y, et al. Prospective, multicenter study of ventricular assist device infections. Circulation. 2013 Feb 12;127(6):691-702. PubMed PMID: 23315371. Pubmed Central PMCID: PMC3695607. Epub 2013/01/15. 23. Schweizer M, Perencevich E, McDanel J, Carson J, Formanek M, Hafner J, et al. Effectiveness of a bundled intervention of decolonization and prophylaxis to decrease Gram positive surgical site infections after cardiac or orthopedic surgery: systematic review and meta-analysis. BMJ. 2013 Jun 13;346:f2743. PubMed PMID: 23766464. Pubmed Central PMCID: PMC3681273. Epub 2013/06/15. 24. Chen AF, Wessel CB, Rao N. Staphylococcus aureus screening and decolonization in orthopaedic surgery and reduction of surgical site infections. Clin Orthop Relat Res. 2013 Jul;471(7):2383-99. PubMed PMID: 23463284. Pubmed Central PMCID: PMC3676622. Epub 2013/03/07.

21

25. Urias DS, Varghese M, Simunich T, Morrissey S, Dumire R. Preoperative decolonization to reduce infections in urgent lower extremity repairs. Eur J Trauma Emerg Surg. 2018 Oct;44(5):78793. PubMed PMID: 29306970. Epub 2018/01/08. 26. Troeman DPR, Van Hout D, Kluytmans J. Antimicrobial approaches in the prevention of Staphylococcus aureus infections: a review. J Antimicrob Chemother. 2019 Feb 1;74(2):281-94. PubMed PMID: 30376041. Pubmed Central PMCID: PMC6337897. Epub 2018/10/31. 27. Bode LG, Kluytmans JA, Wertheim HF, Bogaers D, Vandenbroucke-Grauls CM, Roosendaal R, et al. Preventing surgical-site infections in nasal carriers of Staphylococcus aureus. N Engl J Med. 2010 Jan 7;362(1):9-17. PubMed PMID: 20054045. Epub 2010/01/08. 28. Campbell D, Mudge DW, Craig JC, Johnson DW, Tong A, Strippoli GF. Antimicrobial agents for preventing peritonitis in peritoneal dialysis patients. Cochrane Database Syst Rev. 2017 Apr 8;4:CD004679. PubMed PMID: 28390069. Pubmed Central PMCID: PMC6478113. Epub 2017/04/09. 29. Hannan MM, Husain S, Mattner F, Danziger-Isakov L, Drew RJ, Corey GR, et al. Working formulation for the standardization of definitions of infections in patients using ventricular assist devices. J Heart Lung Transplant. 2011 Apr;30(4):375-84. PubMed PMID: 21419995. 30. Saegeman V, Flamaing J, Muller J, Peetermans WE, Stuyck J, Verhaegen J. Clinical evaluation of the Copan ESwab for methicillin-resistant Staphylococcus aureus detection and culture of wounds. Eur J Clin Microbiol Infect Dis. 2011 Aug;30(8):943-9. PubMed PMID: 21298460. Epub 2011/02/08. 31. Harmsen D, Claus H, Witte W, Rothganger J, Claus H, Turnwald D, 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 Dec;41(12):5442-8. PubMed PMID: 14662923. Pubmed Central PMCID: PMC309029. Epub 2003/12/10. 32. Nurjadi D, Boutin S, Dalpke A, Heeg K, Zanger P. Draft Genome Sequence of Staphylococcus aureus Strain HD1410, Isolated from a Persistent Nasal Carrier. Genome Announc. 2018 May 10;6(19). PubMed PMID: 29748411. Pubmed Central PMCID: PMC5946038. Epub 2018/05/12. 33. Schurch AC, Arredondo-Alonso S, Willems RJL, Goering RV. Whole genome sequencing options for bacterial strain typing and epidemiologic analysis based on single nucleotide polymorphism versus gene-by-gene-based approaches. Clin Microbiol Infect. 2018 Apr;24(4):350-4. PubMed PMID: 29309930. Epub 2018/01/09. 34. Newson RB. Attributable and unattributable risks and fractions and other scenario comparisons. Stata Journal. 2013;13(4):672-98. 35. Skramm I, Fossum Moen AE, Aroen A, Bukholm G. Surgical Site Infections in Orthopaedic Surgery Demonstrate Clones Similar to Those in Orthopaedic Staphylococcus aureus Nasal Carriers. J Bone Joint Surg Am. 2014 Jun 4;96(11):882-8. PubMed PMID: 24897735. Epub 2014/06/05. 36. Kluytmans JA, Mouton JW, Ijzerman EP, Vandenbroucke-Grauls CM, Maat AW, Wagenvoort JH, et al. Nasal carriage of Staphylococcus aureus as a major risk factor for wound infections after cardiac surgery. J Infect Dis. 1995 Jan;171(1):216-9. PubMed PMID: 7798667. Epub 1995/01/01.

22

37. Nouwen JL, Fieren MW, Snijders S, Verbrugh HA, van Belkum A. Persistent (not intermittent) nasal carriage of Staphylococcus aureus is the determinant of CPD-related infections. Kidney Int. 2005 Mar;67(3):1084-92. PubMed PMID: 15698449. Epub 2005/02/09. 38. Liu Z, Norman G, Iheozor-Ejiofor Z, Wong JK, Crosbie EJ, Wilson P. Nasal decontamination for the prevention of surgical site infection in Staphylococcus aureus carriers. Cochrane Database Syst Rev. 2017 May 18;5:CD012462. PubMed PMID: 28516472. Pubmed Central PMCID: PMC6481881. Epub 2017/05/19. 39. Webster J, Osborne S. Preoperative bathing or showering with skin antiseptics to prevent surgical site infection. Cochrane Database Syst Rev. 2015 Feb 20(2):CD004985. PubMed PMID: 25927093. Epub 2015/05/01. 40. Global guidelines for the prevention of surgical site infections. 2nd ed. Geneva: World Health Organization; 2018. 41. Wert L, Hanke JS, Dogan G, Ricklefs M, Fleissner F, Chatterjee A, et al. Reduction of driveline infections through doubled driveline tunneling of left ventricular assist devices-5-year follow-up. J Thorac Dis. 2018 Jun;10(Suppl 15):S1703-S10. PubMed PMID: 30034842. Pubmed Central PMCID: PMC6035948. Epub 2018/07/24. 42. Wus L, Manning M, Entwistle JW, 3rd. Left ventricular assist device driveline infection and the frequency of dressing change in hospitalized patients. Heart Lung. 2015 May-Jun;44(3):225-9. PubMed PMID: 25746483. Epub 2015/03/10. 43. Lepelletier D, Bourigault C, Roussel JC, Lasserre C, Leclere B, Corvec S, et al. Epidemiology and prevention of surgical site infections after cardiac surgery. Med Mal Infect. 2013 Oct;43(10):4039. PubMed PMID: 23988675. Epub 2013/08/31. 44. Luzar MA, Coles GA, Faller B, Slingeneyer A, Dah GD, Briat C, et al. Staphylococcus aureus nasal carriage and infection in patients on continuous ambulatory peritoneal dialysis. N Engl J Med. 1990 Feb 22;322(8):505-9. PubMed PMID: 2300122. Epub 1990/02/22. 45. Davies SJ, Ogg CS, Cameron JS, Poston S, Noble WC. Staphylococcus aureus nasal carriage, exit-site infection and catheter loss in patients treated with continuous ambulatory peritoneal dialysis (CAPD). Perit Dial Int. 1989;9(1):61-4. PubMed PMID: 2488184. Epub 1989/01/01. 46. Sesso R, Draibe S, Castelo A, Sato I, Leme I, Barbosa D, et al. Staphylococcus aureus skin carriage and development of peritonitis in patients on continuous ambulatory peritoneal dialysis. Clin Nephrol. 1989 May;31(5):264-8. PubMed PMID: 2736815. Epub 1989/05/01. 47. Lye WC, Leong SO, van der Straaten J, Lee EJ. Staphylococcus aureus CAPD-related infections are associated with nasal carriage. Adv Perit Dial. 1994;10:163-5. PubMed PMID: 7999818. Epub 1994/01/01. 48. Wanten GJ, van Oost P, Schneeberger PM, Koolen MI. Nasal carriage and peritonitis by Staphylococcus aureus in patients on continuous ambulatory peritoneal dialysis: a prospective study. Perit Dial Int. 1996 Jul-Aug;16(4):352-6. PubMed PMID: 8863325. Epub 1996/07/01. 49. Zimakoff J, Bangsgaard Pedersen F, Bergen L, Baago-Nielsen J, Daldorph B, Espersen F, et al. Staphylococcus aureus carriage and infections among patients in four haemo- and peritonealdialysis centres in Denmark. The Danish Study Group of Peritonitis in Dialysis (DASPID). J Hosp Infect. 1996 Aug;33(4):289-300. PubMed PMID: 8864941. Epub 1996/08/01.

23

50. Huang SS, Singh R, McKinnell JA, Park S, Gombosev A, Eells SJ, et al. Decolonization to Reduce Postdischarge Infection Risk among MRSA Carriers. N Engl J Med. 2019 Feb 14;380(7):63850. PubMed PMID: 30763195. Pubmed Central PMCID: PMC6475519. Epub 2019/02/15. 51. Wertheim HF, Melles DC, Vos MC, van Leeuwen W, van Belkum A, Verbrugh HA, et al. The role of nasal carriage in Staphylococcus aureus infections. Lancet Infect Dis. 2005 Dec;5(12):751-62. PubMed PMID: 16310147. Epub 2005/11/29. 52. Nurjadi D, Lependu J, Kremsner PG, Zanger P. Staphylococcus aureus throat carriage is associated with ABO-/secretor status. J Infect. 2012 Oct;65(4):310-7. PubMed PMID: 22664149. Epub 2012/06/06.

Table 1. Subject characteristics in a cohort of ventricular assist device carriers, stratified by Staphylococcus aureus colonization status at time of device implantation S. aureus nasal colonization at time of device implantation Yes, N=17 No, N=32 n % n % Baseline Male sex Age in years (median, IQR) Body mass indexc (median, IQR) Screening to implantation (median days, IQR) Cause of heart failurec dilated cardiomyopathy ischemic cardiomyopathy other/unclassified Type of ventricular assist device Left ventricular assist device Biventricular assist device Diabetes mellitus Renal functionc,d Normal to stage 2 Stage 3 to stage 4 Stage 5 Intensive care p.i. (median days, IQR) Mechanical ventilation p.i. (median days, IQR)

pa

16 55.0 27.1 6

94.1 49.6-61.4 25.1-30.7 2-26

26 51.8 27.5 7.5

81.3 44.9-58.5 22.5-30.1 1-22

0.4 0.2b 0.3b 0.8b

9 8 0

52.9 47.1 0.0

21 10 1

65.6 31.3 3.1

0.6

14 3 6

82.4 17.7 35.3

26 6 12

81.3 18.8 37.5

1.0 1.0

10 2 5 7 2

58.8 11.8 29.4 3-14 1-4

10 6 15 18 7

32.3 19.4 48.4 7-38.5 1-30

0.2 0.02b 0.1b

Data are number of observations and column %, if not indicated otherwise. IQR=interquartile range; ICU=intensive care unit; p.i.=post implantation; n.a.=not applicable. a; from Fisher’s exact test, if not indicated otherwise b; from Wilcoxon rank-sum test c; information of one non-carrier individual missing

24

d; according to classification proposed by National Kidney Foundation, New York, U.S. (www.kidney.org)

25

Table 2. Events during one-year follow-up in a cohort of ventricular assist device carriers, stratified by Staphylococcus aureus colonization status at time of device implantation S. aureus nasal colonization at time of device implantation Yes, N=17 No, N=32 n % n % Follow-up time (median days, IQR)b VAD-infection by any pathogen Subjects with ≥ one S. aureus VAD-infection d,e Proven infection Driveline infection superficial deep Blood stream infection Endogenous infectionsk VAD-infection by pathogen other than S.aureus First VAD-infection by other pathogen Proven deep driveline infection Blood stream infection Deaths S. aureus infections with fatal outcome Death not related to S. aureus infection Heart transplantation before other eventp Censored without event at 365 days p.i. (end of maximum follow-up) < 365 days p.i. (end of study period)

161.0 10 9 6 8 2f 6h 1i 6l 1n 1 1 0 4 2 2 1

74.0-333.0 58.8 52.9 35.3 47.1 11.8 35.3 5.9 75.0m 5.9 5.9 5.9 0.0 23.5 11.8 11.8 5.9

196.5 7 4 2 4 2g 2g 0 n.a. 3o 3 2 1 10 0 10 3

91.0-365.0 21.9 12.5 6.3 12.5 6.3 6.3 0 n.a. 9.4 9.4 6.3 3.1 31.3 0.0 31.3 9.4

4 0

23.5 0.0

7 5

21.9 15.6

pa 0.6c 0.01 0.005 0.02 0.01 0.6 0.02 0.3 n.a. 1.0 1.0 1.0 1.0 0.7 0.1 0.2 1.0 1.0 0.2

Data are number of observations and column %, if not indicated otherwise. IQR=interquartile range; ICU=intensive care unit; p.i.=post implantation; n.a.=not applicable. a; from Fisher’s exact test, if not indicated otherwise b; using first S. aureus infection as outcome of interest c; from Wilcoxon rank-sum test d; as classified by two independent infectious diseases clinicians, according to (29). e; includes three nasally colonized subjects with more than one episode of S. aureus infection: two episodes of S. aureus blood stream infections (BSI) occurred in two subjects that were previously diagnosed with probable (n=1) and proven (n=1) deep driveline infections, and one

26

episode of presumed S. aureus endocarditis occurred in one patient with previously diagnosed, proven deep driveline infection and previously diagnosed, proven BSI. f; both classified as “probable VAD-specific”, according to (29) g; classified as “proven” (n=1) and “probable” (n=1) “VAD-specific”, according to (29) h; classified as “proven” (n=5) and “probable” (n=1) “VAD-specific”, according to (29) i; classified as “presumed VAD-related”, according to (29) k; as supported by i.) identical spa-genotype and ii.) less or equal than 24 alleles difference in core genome MLST in S. aureus isolated from the nose at baseline and from clinical material during follow up (33) l; spa-types were t002, t005, t012, t1451, t2313, t18874 m; n=8, because one nasal S. aureus isolate of one colonized patient with later S. aureus infection could not be recultured, and was thus not available for genotyping n; Pseudomonas aeruginosa in one subject with no prior episode of VAD-infection o; Pseudomonas aeruginosa (n=1); Escherichia coli (n=1), Candida krusei (n=1); occurred in subjects with no prior episodes of VAD-infection p; i.e. before infection, death, or end of follow-up

27

Table 3. Incidence rate of infectionsa in a cohort of ventricular assist device carriers after 365 days of follow-up, stratified by nasal colonization status at time of device implantation

S. aureus nasal colonization at baseline Type of event

Any infectiona Any S. aureus infectiona Proven S. aureus infectionc

No. of events 10 9 6

Yes, N=17 rate days at /1000 risk days 3188 3201 3666

3.14 2.81 1.64

No, N=32 rate /1000 days

CI

No. of events

days at risk

1.69-5.83 1.46-5.40 0.74-3.64

7 4 2

6161 6553 6836

1.14 0.61 0.29

IRR

CI

Pb

CI 0.54-2.34 0.23-1.63 0.07-1.17

2.76 4.61 5.59

0.95-8.55 1.29-20.47 1.00-56.67

0.04 0.009 0.03

Data are number of events, person time at risk, 95% confidence intervals (CI) and incidence rates per 1000 days of follow-up from single event per subject analyses. a; includes ventricular assist device (VAD)-specific and VAD-related infections, according to (29) b; exact p-value c; includes proven ventricular assist device (VAD)-specific and VAD-related infections, according to (29)

28

Figure 1. Survival free of VAD-infection caused by S. aureus in 49 patients, by nasal carrier

0.00

0.25

0.50

0.75

1.00

status at time of device implantation

0

60

120

180

240

300

360

13 6

10 5

7 4

Days Number at risk Non-Carrier 32 Carrier 17

26 14

22 10

17 8

Non-carrier

Carrier

VAD-infection refers to VAD-specific and VAD-related infections, according to definitions proposed by the International Society of Heart and Lung Transplantation (29); log-rank test comparing survival curves gives P=0.006 Figure 2. Clonal relationship between S. aureus isolates from colonization and infection in patients with ventricular assist device

29

A: phylogenetic dendrogram based on unweighted pair group method with arithmetic mean based on 1733 core and 203 accessory genes as used by core genome multi locus sequence typing (Ridom SeqSphere+, Germany). Classic multi locus sequence typing (MLST) based on 7 housekeeping genes was assigned from the assembled draft genome, spa type was determined by Sanger sequencing. Isolates belonging to the same clone are indicated by the same background colour. Isolates from P9 were methicillin-resistant S. aureus (red font), all other isolates were methicillin-sensitive (black font). B: allelic difference between nasal and infection isolates for 9 colonized patients with S. aureus infection. Less than or equal to 24 allelic differences between isolate from nasal colonization and infection (dotted line) were defined as endogenous, following published guidance (33). Nasal isolate from P3 could not be recultured (missing data).

30