Impact of single-room contact precautions on acquisition and transmission of vancomycin-resistant enterococci on haematological and oncological wards, multicentre cohort-study, Germany, January−December 2016

Background Evidence supporting the effectiveness of single-room contact precautions (SCP) in preventing in-hospital acquisition of vancomycin-resistant enterococci (haVRE) is limited. Aim We assessed the impact of SCP on haVRE and their transmission. Methods We conducted a prospective, multicentre cohort study in German haematological/oncological departments during 2016. Two sites performed SCP for VRE patients and two did not (NCP). We defined a 5% haVRE-risk difference as non-inferiority margin, screened patients for VRE, and characterised isolates by whole genome sequencing and core genome MLST (cgMLST). Potential confounders were assessed by competing risk regression analysis. Results We included 1,397 patients at NCP and 1,531 patients at SCP sites. Not performing SCP was associated with a significantly higher proportion of haVRE; 12.2% (170/1,397) patients at NCP and 7.4% (113/1,531) patients at SCP sites (relative risk (RR) 1.74; 95% confidence interval (CI): 1.35–2.23). The difference (4.8%) was below the non-inferiority margin. Competing risk regression analysis indicated a stronger impact of antimicrobial exposure (subdistribution hazard ratio (SHR) 7.46; 95% CI: 4.59–12.12) and underlying disease (SHR for acute leukaemia 2.34; 95% CI: 1.46–3.75) on haVRE than NCP (SHR 1.60; 95% CI: 1.14–2.25). Based on cgMLST and patient movement data, we observed 131 patient-to-patient VRE transmissions at NCP and 85 at SCP sites (RR 1.76; 95% CI: 1.33–2.34). Conclusions We show a positive impact of SCP on haVRE in a high-risk population, although the observed difference was below the pre-specified non-inferiority margin. Importantly, other factors including antimicrobial exposure seem to be more influential.


Introduction
The prevalence of colonisation and infection by vancomycin-resistant enterococci (VRE) is increasing globally [1,2]. These gut commensals may cause infections mainly in individuals with immunosuppression, patients being treated at an intensive care unit (ICU), receiving haemodialysis or being extensively exposed to antimicrobials [3].
In order to reduce in-hospital acquisition and transmission of VRE, single-room contact precautions (SCP) including staff and visitors wearing gloves and gowns are recommended by some national infection prevention and control committees [4][5][6]. However, the underlying evidence is heterogeneous and contradictory [4]. The only two cluster-randomised studies did not observe any impact of targeted or universal wearing of gloves and gowns by staff and visitors on colonisation and infection rates with multidrug-resistant organisms including VRE in ICUs. However, they did not assess the impact of single-vs multiple-bed rooms [7,8]. Similarly, a meta-analysis including eight studies on discontinuation of contact precautions, but not necessarily including single-room accommodation, showed no effect on VRE infection rates [9]. In contrast, a time-series analysis published in 2019, showed a decrease in VRE colonisation and infection rates following a hospital relocation to a building with only single rooms [10]. Furthermore, all available studies lack high-resolution sequence-based typing data to determine transmission of VRE.
Single room contact precautions can have severe effects on patient health including psychological complications, as well as fewer contacts to healthcare providers resulting in a delay in diagnostic and therapeutic procedures [11,12]. Considering the unclear effectiveness and the potential harm associated with SCP, we aimed to assess the impact of SCP on in-hospital acquisition and transmission of VRE in haematological/oncological patients including molecular genetic analyses.

Study design and setting
A prospective 12-month cohort study was performed in 2016 at the haematology/oncology departments of the University hospitals in Cologne, Freiburg, Hamburg and Tübingen. The four sites are situated in different regions of Germany with ca 160 to 700km distance between sites. Inpatients were screened for intestinal colonisation with VRE within 72h of admission, once weekly and within 72h of discharge. As per their standard of care, two sites performed SCP for patients colonised or infected with VRE and two did not (NCP). SCP included accommodation in single rooms with en suite bathrooms and without cohorting, and wearing of gloves and gowns by staff and visitors. Patients at NCP sites and those at SCP sites without known VRE colonisation or infection were accommodated either in single or double rooms. Figure 1 and the Supplementary Table S1 further illustrates design and infrastructure.
To ensure comparability of hand hygiene, sites implemented a multimodal programme including compliance assessment with 150 observations at four different time points per site (Supplementary Text S1, Table  S2). The study was registered with clinicaltrials.gov (NCT02623413), the protocol is available at https:// www.clinicalsurveys.net/uc/statistic_analysis/1f5d/ images/CONTROL_protocol_2015-09_07_MV_signed. pdf .

Data capture
Inpatients with a minimum stay of one night and at least one screening sample were included. Documentation included demographics, hospitalisation period, and VRE detection. Bloodstream infections (BSI) due to VRE were noted including subsequent hospitalisations within the study period at any ward. Attributable mortality was defined as death within 7 days of onset of VRE BSI or of the last VRE-positive blood culture. Antimicrobial exposure by class was documented until detection of VRE or discharge, with emphasis on antimicrobials previously described as risk factors for VRE acquisition [13][14][15][16]. For VRE-positive patients, patient movement data including ward,

Figure 1
Study design for impact of single-room contact precautions on acquisition and transmission of VRE on haematological and oncological wards, Germany, January −December 2016 occupied rooms and occupation dates were documented. Data from multiple hospitalisations in the participating department during the study period (hospitalisation level) were cumulated to patient level. We monitored data completeness and assessed screening adherence, antimicrobial consumption and hand hygiene compliance (Supplementary Text S1).  Flow-chart of patient inclusion and isolate collection in multicentre cohort-study on impact of single-room contact precautions on acquisition and transmission of VRE on haematological and oncological wards, Germany, January − December 2016  Nucleotide Archive under the study accession number PRJEB25579 [19].

Endpoints and definitions
The primary endpoint was the rate of in-hospital acquisition of VRE (haVRE) in NCP vs SCP sites during 1 year. In-hospital acquisition was defined as a screening or clinical culture obtained > 72h after admission yielding VRE in a patient with a negative admission screening.
In order to test non-inferiority of NCP to SCP in preventing haVRE, a non-inferiority margin of 5% was defined in the statistical analysis plan. Colonisation with VRE does not constitute an immediate hazard to the patient and only few colonised patients develop subsequent infection [20,21]. Based on previously published VRE rates in a similar patient cohort [20], we decided that a difference in the rate of haVRE below 5% would not outweigh the adverse effects of SCP and would not be sufficient to be attributable to SCP. Secondary endpoints included rates of (i) VRE colonisation overall and at admission, (ii) VRE BSI, and (iii) patient-to-patient transmissions of VRE. Patient-topatient transmission was defined as the detection of closely related isolates from at least two patients in the same ward with overlapping hospitalisation periods and with at least one patient with haVRE for each transmission event. Close relatedness of isolates was defined as ≤ 10 allele differences in cgMLST, which is lower than the published threshold of 20 alleles to allow for a higher resolution [22]. The term "potential transmission event" was used to refer to pairs or clusters of closely related isolates regardless of confirmation by patient movement data.
Additionally, incidence densities for haVRE and VRE BSI per 1,000 patient days (pd) at risk were calculated counting days at risk from admission to discharge or the day of the respective event (haVRE or VRE BSI). Days of patients already colonised with VRE were censored for haVRE.

Sample size and statistical analysis
Assuming an overall rate of haVRE of 8% in both groups [20], a one-sided type I error rate of 2.5%, and a dropout rate of 30% due to missed screening, 475 patients per group were required to demonstrate non-inferiority regarding the margin of 5% with a power of 80% (calculated using https://www.sealedenvelope.com/power/ binary-noninferior/). To minimise seasonal influence, the study was conducted over 1 year without sample size limitation.
Supplementary Text S3 details the statistical analyses and sensitivity analyses performed. Briefly, distribution of data within groups, both at hospitalisation level (counting each hospitalisation separately) and patient level, was described as absolute numbers plus percentage, mean, median and interquartile range (IQR), and groups compared using Pearson's chi-squared test, Fisher's exact test or Mann-Whitney-U tests, as appropriate. Differences in the endpoints comparing NCP to SCP sites were presented as relative risk (RR) with 95% confidence intervals (CI). The effect of covariates and time-varying covariates on haVRE was estimated using the Fine-Gray subdistribution hazard regression model accounting for competing risks [23]. Results were displayed with subdistribution hazard ratio (SHR) and 95% CI. Multiple hospitalisations per patient were included, while hospitalisations of patients already colonised with VRE were excluded. A two-sided p value < 0.05 was considered significant.

Ethical statement
The study was conducted in line with the Declaration   Figure  2). Table 1 shows patient characteristics. Patients at NCP sites were younger, had a higher proportion of lymphoma as underlying disease and a longer cumulated length of stay. Furthermore, exposure to any antimicrobial was more frequent at NCP sites, while exposure to antimicrobials active against VRE and to fluoroquinolones was more frequent at SCP sites. Findings at hospitalisation level -with each hospital stay of all included patients counted separately -were similar to those at patient level (Supplementary Table  S3).  Table  S4). The Table 2 shows the rates of VRE endpoints. Median cumulative length of stay until haVRE was 16 days (interquartile range (IQR): 11-38) at NCP and 15 days (IQR: 9-35) at SCP sites (p = 0.475).

Molecular epidemiology and patient-to-patient transmission
Surveillance isolates were missing in seven NCP and five SCP patients, from two NCP and one SCP patients two isolates each were included due to different resistance patterns. Overall, 287 isolates from 276 NCP patients and 218 isolates from 210 SCP patients were sequenced (Figure 1).  Figure S1 details the steps of confirmation of patient-to-patient transmission including information on previous room occupancy.

Relatedness of bloodstream infections and surveillance isolates
Of 17 VRE BSIs, one occurred in a patient without detected colonisation and in one patient the corresponding surveillance isolate was not available. Of the remaining 15 BSIs, bloodstream isolates were closely related to the corresponding surveillance isolate (Supplementary Figure S3).

Relatedness of isolates across sites
When merging the cgMLST datasets, close relatedness of isolates from different sites became apparent ( Figure 3). We identified nine large trans-regional clusters (TRC) comprising up to 99 isolates per cluster from all sites. In total, 297 of 494 sequenced isolates (60.1%) were part of a TRC. ST-117 was the most frequent ST (199/297 isolates; 67.0%). Supplementary  Tables S7 and S8 show details of TRC.  Table  S9f) showed the strongest association with haVRE and the respective SHRs increased with length of exposure (Tables S9d and S9g). Of note, in these four sensitivity models, hospitalisation at an NCP site was not an independent risk factor for haVRE.

Potential confounders at the patient level
In the analysis at hospitalisation level (Supplementary File 4, Tables S10a-j), acute leukaemia (SHR 1.64-2.84, depending on the model used), NCP site (SHR 1.36-1.87, depending on the model used) and antimicrobial exposure with different variations were, again, independent risk factors. In detail, cephalosporins, glycopeptides and other antimicrobials increased the risk, while antimicrobials active against VRE such as linezolid and daptomycin had a protective effect (Table  S10a). Of note, hand hygiene compliance was not confirmed as an independent risk factor in any of the models.
As an exploratory analysis, we applied the same set of variables to the endpoint confirmed patient-to-patient transmission with similar results (Supplementary Tables S11a-k, S12a-k).

Potential confounders at study site level
Screening adherence defined as complete admission screening, weekly screenings and one within 72h before discharge was achieved in 70.6% (1,719/2,435) of hospitalisations at NCP and 76.1% (2,299/3,023) at SCP sites. A complete admission screening and one within 7 days before discharge was performed in 88.7% (2,159/2,435) of hospitalisations at NCP and 87.7%% (2,652/3,023) at SCP sites (Supplementary Table S13).
Hand hygiene compliance was assessed less frequently than planned and results differed between sites (Supplementary Figure S4). The compliance at sites NCP2 and SCP1 decreased during the study with compliance as low as 48% at NCP2 during their third and last assessment (Supplementary Figure S3).
Antimicrobial consumption density assessed as defined daily doses per 100 pd differed considerably between sites and groups. SCP sites reported higher consumption of cephalosporins, carbapenems, fluoroquinolones and site SCP1 also of vancomycin. Linezolid and daptomycin consumption varied considerably between sites (Supplementary Tables S14 and S15, Figure S4).

Discussion
In this large prospective, multicentre study, a significant difference regarding haVRE between NCP and SCP sites was observed. This difference, was below the predefined non-inferiority margin of 5% and thus, according to our estimation, would not outweigh the adverse effects of NCP. We also observed a higher rate of patient-to-patient transmissions determined by cgMLST and patient movement data at NCP sites. Still, with 10 and seven detected BSIs due to VRE these were rare events at NCP and SCP sites. Despite previously reported regional differences in VRE rates in Germany [24], the rate of VRE colonisation at admission differed by only 1.3% between NCP and SCP sites. The competing risk regression analysis confirmed hospitalisation at a NCP site as an independent risk factor for haVRE in most models. However, exposure to antimicrobials in different variations showed an even stronger and in the sensitivity models more consistent impact on haVRE. In particular, exposures to cephalosporins, fluoroquinolones and glycopeptides were identified as significant risk factors for haVRE as previously reported [15,16,25]. Of note, in the sensitivity models with exposure to any antimicrobial or the group of antimicrobials not active against VRE as risk factors, NCP site was no more significantly associated with haVRE suggesting a less important effect of SCP on the acquisition and/or transmission of VRE as compared with exposure to antimicrobials. Our analysis also revealed an important role of acute leukaemia as underlying disease. This disease is associated with extensive antimicrobial exposure and longer and more frequent hospitalisations compared with other underlying diseases in this cohort, possibly explaining this association.
Interestingly, we identified several TRCs with close relatedness of isolates from different sites. To our knowledge, the present study entails the largest highresolution sequence-based analysis of trans-regionally collected clinical VRE isolates, but smaller studies have already indicated inter-site relatedness of VRE and in particular the expansion of ST-117 [26,27]. Given the high number of inter-site relatedness and the large geographic distances of participating sites of up to 700 km, direct transfer of individual study patients between sites is not a plausible explanation for this observation. It seems more conceivable that certain VRE clones have a high environmental stability facilitating the observed spread within the healthcare system over long distances and longer time periods than the study period. Since environmental sampling was not included in our study, we unfortunately cannot substantiate this hypothesis further. Still, this assumption, questions our interpretation of direct patient-to-patient transmissions based on genomic relatedness: A certain proportion of the observed transmissions might have been acquisitions from reservoirs in the healthcare environment rather than from patient-to-patient contacts. This highlights the importance of improved environmental cleaning in reducing VRE spread [28]. Looking at details of room occupancy in our study, some transmission might have occurred between patients subsequently but not simultaneously occupying the same room. Further research to investigate the frequency of inter-site relatedness of VRE isolates and trans-regional transmission pathways is needed to determine the clinical relevance of our findings.
Our study has several limitations. Since cluster-randomisation was not feasible due to regulatory reasons, there were clear differences in patient and site characteristics between groups compromising the direct comparison of VRE rates. However, we performed regression models to differentiate the influence of contact precautions from variables with marked variations between groups, e.g. exposure to antimicrobials, underlying disease and hand hygiene compliance. Furthermore, despite rigorous assessment of patient level data and application of up-to-date molecular analysis to investigate epidemiological relatedness of isolates, using only one VRE colony per patient for WGS might have led to underestimation of patient-to-patient transmissions. Since VRE may colonise inanimate surfaces for weeks [29], indirect transmission between patients without simultaneous in-hospital stay may occur but was not assessed. Similarly, we did not record environmental cleaning strategies or perform environmental screening in the participating wards. The existing German recommendations regarding cleaning and disinfection of the hospital environment leave the decision on the frequency of cleaning measures and the exact implementation of quality control to the local hospital hygiene department [30]. Thus, cleaning standards and performance may have varied between the participating sites, influencing the effectiveness of SCP and in-hospital transmission dynamics of VRE. Finally, in a proportion of cases classified as haVRE, VRE might have been present at very low levels in the gut already on admission, only becoming detectable by conventional culture after antimicrobial exposure and successive increase in intestinal bacterial load through antimicrobial selection [31,32].
The findings of this large multicentre study confirm a protective effect of SCP in this specific high-risk population with significant differences in the rates of haVRE and of VRE transmissions. However, estimating the clinical appropriateness of performing SCP remains difficult because of several aspects. Firstly, the observed difference in haVRE rates was just within the predefined non-inferiority margin. We deemed that a difference below 5% would not outweigh the wellknown adverse effects of SCP, but surely this estimation is arguable. Secondly, only 0.6% of all patients and 3.4% of VRE colonised patients developed a VRE BSI with only one attributable death in this high-risk population, showing a minor clinical relevance of VRE in our setting [21]. Thirdly, our regression analysis indicates a more relevant and more consistent impact of other factors, particularly antimicrobial exposure, on haVRE. This highlights the importance of antimicrobial stewardship for reduction of VRE burden. Lastly, the role patients colonised with VRE below the cultural detection threshold, as well as inter-site relatedness of isolates and underlying transmission pathways need to be investigated in more detail to understand to which extent the observed relatedness of VRE reflects true transmissions within the hospital that can be prevented by SCP. Though not assessed directly, improved environmental cleaning possibly guided by environmental screening results should be included into infection control strategies for VRE [28].

Conclusions
The presented evidence suggests a certain protective effect of SCP for haVRE in patients hospitalised on haematological and oncological wards. More importantly, our study adds to the available evidence underlining the exposure to antimicrobials as an important and modifiable factor for the acquisition of VRE.

Access to data
The raw dataset underlying our analysis is available by contacting the corresponding author upon reasonable request.
The raw sequencing reads were submitted to the European Nt Archive (https://www.ebi.ac.uk/ena/), Study Accession number PRJEB25579.