Strengths and limitations The main strength of our study was that the relocation of the hospital provided us the opportunity to determine the difference in risk on acquisition of ESBL-E between multipleoccupancy rooms and single-occupancy rooms for patients from different departments and specializations. Additionally, performing WGS analyses provided us additional insights in ESBL-E colonization compared to only microbiological culture methods. However, our study also has some limitations. The most important limitation was the low prevalence of ESBL-E, and the low incidence of ESBL-E acquisition. As a result, we did not have enough statistical power to perform multivariate analyses and were thus unable to correct for possible confounding factors, such as differences in hand hygiene compliance and cleaning protocols. Since we did not perform a sample-size calculation before the start of the study, it is possible that our study is underpowered. Additionally, we used perianal samples instead of rectal samples. While perianal swabs are less invasive then rectal swabs and might increase participation, it is known that the sensitivity of perianal swabs is lower compared to rectal swabs (36). By using selective broths and culture methods, we aimed to minimize the risk of false-negative results, but it is likely that ESBL-E carriers and ESBL-E acquisitions were missed. Additionally, repeated sampling throughout the hospitalization period would also have decreased the chance for false-negative samples. A final sampling limitation is that patients missed at discharge were asked to sample at home, which meant a delay in sampling. Therefore, not all discharge samples might be representative of the situation at discharge. Furthermore, we have introduced selection bias as a consequence of our inclusion criteria, and by the fact that the proportion of patients admitted to the different specializations was different in the old building compared to the new hospital building. An explanation for this is the fact that after the relocation of hematology patients from the Cancer Institute to the new hospital building, it was easier to approach and hence include these patients. Finally, we did not include all patients admitted to the participating departments. Therefore, we were unable to determine the exact dynamics of ESBL-E within and between departments. Conclusion Due to the design of the study, a significant decrease in ESBL-E acquisition after relocating to the new hospital could not be shown, but the transition to a hospital with 100% singleoccupancy rooms was associated with a significant decrease in intra-hospital patient transfers and, hence, a significant decrease in exposure to square meters. By determining that transferred patients had higher odds on ESBL-E acquisition, we showed that the transition to 100% single-occupancy rooms can indirectly impact ESBL-E acquisition. Additionally, the large proportion of ESBL-E carriers that remains unidentified by clinical samples highlights the need for an improved risk-assessment screening at admission. Future research is needed to determine the impact of 100% single occupancy rooms on factors that could impact ESBL-E and HRMO acquisition, such as exposure to square meters as a measure for exposure to the hospital environment, and to develop an effective risk-assessment screening. 40 Chapter 2.1
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