Efficacy of active rapid molecular screening and IPC interventions to reduce carbapenem-resistant Enterobacteriaceae infections in emergency intensive care unit – a prospective, multi-stage study

Background: To investigate whether molecular rapid active screening and infection-prevention and controlIPCinterventions can reduce the colonization and infection of carbapenem-resistant Enterobacteriaceae (CRE) in a general emergency intensive care unit (EICU). Methods: The study was designed as a before-and-after quasi-experiment. It was conducted in 3 stages. During stage 1, April 2018, the environment was prepared and the staff in EICU was trained. Stage 2 was the main experimental stage from May 2018 to January 2019. The active screening was tested by semi-nested real-time fluorescent PCR (polymerase chain reaction) detection with rectal swabs from all the patients on admission to EICU and the results would be feedback in 1 hour and other IPC interventions were conducted in strict supervision in this stage. In the last stage (stage 3), February 2019-April 2019, only active screening was strictly executed. Other IPC interventions were carried out by health workers without supervision. In the meantime, the atient information and culture results from clinical laboratory from January 2017-April 2019 were collected. Results: In this 1-year study, a total of 217 patients were enrolled. There were 23.04% of the patients were initially colonized or infected with CRE as tested by active molecular screening. The clinical culture detection rate of CRE colonization/infection was 3.37% during the baseline stage (before this study was carried out), from January 2017-March 2018. The rate decreased significantly to 1.20% (p<0.05) during the main experimental period in which active screening and IPC intervention were executed strictly. However, the rate increased again to 6 .12% in stage 3 in which only active screening were carried out in supervision. Based on the clinical characteristics of the EICU patients, we found there's a higher probability that the patients had invasive devices or skin-barrier damage on admission or had antibiotic use before admission were colonized or infected with CRE Conclusions : Rapid active screening by molecular detection and other IPC interventions conducted in supervision showed a significant reduction of CRE in nosocomial infection. The key to reduce the spread of CRE in the EICU is that all medical staff and healthcare workers execute IPC interventions strictly. positive and negative groups using Mann-Whitney U-test. The association between the xpert tests and the outcomes (alive or dead at discharge) active rapid molecular screening and IPC interventions were modeled by binary logistic regression.


Introduction
Carbapenem-resistant Enterobacteriaceae (CRE) represent a serious therapeutic problem due to their pronounced multidrug resistance, with main pathogens being Klebsiella pneumoniae, Escherichia coli, and other Enterobacteriaceae. The worldwide spread of CRE is a global burden and has become one of the priorities of the Centers for Disease Control (CDC) [1]. In the last decades, along with the advent of Extended Spectrum Beta-Lactamase (ESBL) -producing enterobacteria, carbapenems have been used more frequently due to their broad antibacterial spectrum, thus contributed greatly to the drastic raise of the CRE problem. [2]. In Europe and South America, the situation reached alarming levels, causing about 140,000 cases of healthcare-associated infections annually. [3] In some parts of Asia, especially eastern China, CRE have become endemic and reached epidemic dimensions. [4].
Infections with carbapenem-resistant gram-negative bacilli (CR-GNB), such as CRE, carbapenem-resistant Acinetobacter baumannii (CRAB), carbapenem-resistant Pseudomonas aeruginosa (CRPsA), are predominantly associated with high morbidity, mortality, and heavy economic burden [5]. Despite a variety of preventive measures and implemented intervention programs to control nosocomial infections, the culture detection rate of CRE has increased continuously, reaching a total incidence rate in ICU from 2% to 7% and a mortality rate of 18% to 48%, reaching even 78% in the patients infected with carbapenem-resistant Klebsiella pneumoniae after transplantation in Germany, 2013. [6] [7] [8] [9]. In 2017, a CRE clinical and economics outcomes model was developed in the United States to evaluate the health and financial burden of CRE, reporting a doubling of costs and a 5.1 fold increase of incidence rates, causing higher expenses than many chronic diseases. [10] Recent studies showed that the majority of CRE infections in China are caused by carbapenem-resistant Klebsiella pneumoniae (CR-KP), mostly leading to serious infection in the ICUs [11]. The major mechanisms of carbapenem-resistance in these strains include production of carbapenemases, production of efflux pumps and porin mutation or loss. [12] From 2010 to 2014, the database showed that the average probability of CRE infection from 15 large teaching hospitals in 13 regions of China increased from 1.9% to 5%. [13] The resistance rate of Klebsiella pneumoniae to carbapenems has increased by 10% from 2005 to 2014. [14]This is the highest numbert among all Gram-negative bacteria [15]. In our institution, the Shanghai East Hospital, the detection rates of CR-KP recorded from 2015 to 2017, ranged between 41.3% to 45.5%. This value was much higher than the reportd average in Shanghai (27.3%, data collected by CHINET, China Antimicrobial Surveillance Network) and attributed mainly to CR-KP infections in the ICU ward of our hospital, where the antibiotic resistance rate reached 61.12% in 2017.
The WHO published guidelines for the infection prevention and control (IPC) of CRE, CR-AB and CRPsA, including hand hygiene, patient surveillance, contact precautions, patient isolation (single isolation or concentration), environmental disinfection, environment surveillance, monitoring auditing and feedback [16]. However, the translations of the guidelines into the clinic was rather ineffective in China. One of the reasons was that the available global literature mostly reported active screenings conducted by bacterial culture tests (time factor: at least 24h to feedback the results) and the data were collected in settings that are unusual for China, e.g. ICUs with advanced facilities such as 1 bed-room, isolation rooms etc. [17] In addition, in real-world, the IPC interventions implemented in China mostly covered hand hygiene and contact precautions, while infected or colonized patients are under isolation according to local conditions. [18] These are mostly forcing the hospitals to use curtains, one-time hygiene materials and daily utensils, and special signs for isolation were used and contact precautions were strictly applied when private rooms for isolation was not enough. Household items were also separately used and disposed when there were more than one positive patients in the ward.
We conducted a prospective, multi-stages long-term study in order to investigate whether a combination of measures can provide an effective and efficient, as well as persevering results. We aimed to establish whether infection-prevention and control IPC together with detection and monitoring of CRE colonizations/infections (active screen at admission) of EICU patients by a rapid, semi-nested real-time fluorescent PCR (polymerase chain reaction) are effective towards the reduction of infection number, mortality and morbidity.
At the same time, we provided effective clinical research data to develop China-specific multi-drug resistant prevention and control guidelines.

Settings
The study was conducted at the University Hospital of Shanghai East Hospital, Tongji University School of Medicine, Shanghai. Patients were prospectively recruited from May 2018 through April 2019 in the EICU department, which treats approximately 200-280 critically ill patients annually. The ward consists of 8 beds, including 1 private room and 2 beds in a relatively isolated area.

Infection-control interventions and data collection
Initially, routine culture data were collected from January 2017 to March 2018. This time period was called the baseline period. In this period, hardly any isolation measures or patient surveillance were done for the patients with CRE. Most of the nurses and doctors did hand hygiene and contact precautions strictly. However, the interventions including hand hygiene, contact precautions and environmental disinfection conducted by the health care workers didn't meet the standard according to our investigation. Clinical specimens were collected for culture only from the patients with infectious clinical symptoms. The data was used as baseline for comparisons to those from stage 3. The study was performed in three stages. In stage 1 (April 2018-May 2018), practical and theoretical EICU staff training on IPC model plan including: hand hygiene, patient surveillance, contact precautions, patient isolation (single isolation or concentration), environmental disinfection, environment surveillance, monitoring auditing and feedback, as well as of the goals, objectives, significance and exact methodology of the project. The whole ward was emptied and the health care workers disinfected the environment with sodium hypochlorite disinfectant, ultraviolet-light radiation and Clinell Universal Wipes which is a combination of benzalkonium chloride, didecyl dimethyl ammonium chloride and polyhexamethylene biguanide (PHMB). Such process of disinfection was also conducted in the area around the bed when a patient left the ward. Hygiene control group examined and confirmed that the ward has been contamination-free prior to the study onset. An additional hand hygiene inspection for all workers was conducted, followed by an intensive training of the study guidelines for sampling and transportation [19]. There isolation guidelines were constructed according to the spatial conditions of the ward in which there are 1 isolation room and 2 semi-isolated rooms(2 beds in one room and 6 beds in another room with bedside screen and curtain respectively). In case of multiple patients with infectious diseases, bedside screens, separations via curtains and a respective signalization for isolation, with instructions and precautions (clothing, hand disinfection etc.) were placed. Therapeutic supplies and household items were used separately for each patient and disposed after use. Daily operations of the health care workers were strictly directed and supervised, as were the measures of daily disinfection and final disinfection after patients' discharging from the ward [20]. then underwent testing with Gene Xpert, a semi-nested real-time fluorescent PCR method, by a trained lab technician. The Gene Xpert results were returned within one hour(the detection and results feedback were done by emergency lab technician during the nights and at weekends), while a culture sampling was set at the same time. During the hospitalization, various type of specimen from the patients (the patients with CRE at admission or clinical symptoms of infection) were sent to laboratory for culture like how was done in baseline period. In the 8 months period of Stage 2, environmental checks and disinfection measures were performed at least twice a day and the environment surveillance twice a month. In addition, sink monitoring and dust avoiding in the process of preparing beds were implemented [21]. The patients' isolation was arranged as described above, according to the results of Gene Xpert [22]

Active rapid molecular screening by GeneXpert Carba-R Assay
A rectal swab was obtained from the patient by a paired-swab at admission to the EICU.
One swab would be placed into the sample reagent vial and vortexed at high speed for 10 seconds. The prepared sample would be aspirated using the transfer pipette provided and transferred into the Xpert Carba-R Assay cartridge and then it would be put into the GeneXpert instrument. The results are given in 45 minutes. Rapid active screenings by molecular detection were carried out immediately after the sampling by GeneXpert Carba-R Assay, simultaneously detecting five carbapenemase genes including blaKPC, blaIMP blaNDM blaVIM and blaOXA-48.
Surveillance cultures were sampled simultaneously with another unused rectal swab.
Further routine surveillance cultures were obtained, including sputum, endotracheal aspirate, urinary tract, blood, and infection sites (individually selected according to patient's symptoms/differential diagnosis). The culture sampling has been repeated weekly or more often, when apposite (e.g. in case of fever, elevation of inflammatory markers etc.) Thus, we were able to monitor the incidence of CRE colonization or infection once a week or anytime if the patient got a fever or developed other symptoms of infections. All isolates were identified by the MALDI-TOF MS(Autof ms1000) and routine antibiotic susceptibility tests were performed by the VITEK2 compact system (bioMérieux, France) as to determine carbapenem resistance. The control strain was Escherichia coli ATCC 25922. Susceptibility breakpoints were interpreted as per the Clinical and Laboratory Standards Institute guidelines (CLSI M100-S28) [24].

Multiplex PCR detect Carbapenemase genes
Carbapenemase genes of the isolates collected from the rectal swabs culture of the patients on admission in EICU were verified by multiplex PCR with primers as shown below. (Table 2) [25]. In addition, a retrospective analysis of genetic correlations between the isolates from both rectal swabs on admission and the clinical culture during the hospitalization was performed by pulsed-field gel electrophoresis (PFGE). If the strains from different patients shared highly similar PFGE patterns, we suggested it might be a nosocomial infection.  c. Stains collected from clinical culture during the hospitalization.   IPC intervention reduces nosocomial CRE infection. However, we observed a raise of the rate increased to 6.12% in stage 3 ( Table 5), suggesting that rapid active screening by molecular detection and supervised IPC interventions lead to an effective decrease in CRE infection, while routine checks and unsupervised interventions fail to maintain this effect.
In order to exclude effect of any change in in-hospital patients in stage 2 and stage 3. The main diagnoses in both stages were listed in table 6 from most frequent to least frequent.
There was no significant difference in diseases between stage 2 and stage 3. We compared the duration of total in-hospital and in-EICU stay in stage 2 and stage 3. (Fig 5) It showed no significant difference both in total in-hospital and in-EICU stay between stage 2 and stage 3 which meant the increase of detection rate might not be caused by a longer duration.   Fig 5 The duration of total inhospital and inEICU stay in stage 2 and stage 3. The duration showed no significant difference both in total inhospital and inEICU stay between stage 2 and stage 3.

Discussion
Carbapenem antibiotics are extensively used worldwide due to their strong antibacterial activity and broad spectrum [26]. CRE infections are increasingly challenging due to a growing antimicrobial resistance and thus causative for a high mortality. Increased participation during the education phase of the intervention program was reported as an important factor to decrease the incidence of carbapenem-resistant pathogen infections. [29]. Feedback from screening services, rapid turnaround time and efficient communication were all positively correlated to overall success in outbreak control [29]. Based on this study, we designed and designated one entire month for educating and training of medical professionals and health care workers, familiarizing them with the study-specific IPC intervention model, screening measures, general hygiene. These educational approaches resulted in higher compliance and better adherence to study protocols, as well as in further reduction of the infection rates. Our findings suggest that environmental surveillance is of high importance in IPC interventions. The "environment" is defined as all areas of the ward, involved hand colonization and hygiene, clothing of medical staff and medical staff themselves. [31] Environmental monitoring was helpful in providing a clear feedback to health care providers about disinfection processes. In our study, environmental monitoring was conducted twice a month. If case of unsatisfactory outcomes, the ward underwent a total disinfection process with an additional surveillance thereafter. Such approach was efficient and effective in CRE reduction and positive in terms of individual hygiene awareness of the health care providers.
In the third phase of the study, the incidence of CRE increased to 6.12%, which was

Consent for publication
Not applicable

Availability of data and materials
All data analysed during this study are provided in the attached file(Additional file 1).
Leaders in Medical Disciplines in Shanghai (grant number 2017BR032).  The dendrogram of PFGE results of CRE strains. The PFGE patterns were various among the isolates collected from 16 patients.  The duration of total inhospital and inEICU stay in stage 2 and stage 3. The duration showed no significant difference both in total inhospital and inEICU stay between stage 2 and stage 3.

Supplementary Files
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