Among antibiotic-resistant organisms, the Gram-negative bacteria are now the most important challenge because of the rapid worldwide spread of mechanisms conferring resistance to multiple drugs. The most recent and worrying problem is the emergence and spread of carbapenemases. Additionally, carbapenem-resistance is known to be very frequent among Acinetobacter baumannii isolates for many years. Overall, the therapeutic options available against carbapenem-resistant Enterobacteriaceae (CRE) and A. baumannii (CRAB) are very limited. The best available treatment (BAT) against CRE is unknown, which is a challenge for therapeutic decisions and also for the design of randomized trials with new drugs. The generic objectives of EURECA are to obtain high-quality observational data to inform the design of randomized controlled trials for complicated intraabdominal infections, pneumonia, complicated urinary tract infections and bloodstream infections due to Carbapenem-resistant Enterobacteriaceae (CRE) and carbapenem-resistant Acinetobater baumannii, and to provide cohort data that could eventually be used as historical controls for future comparisons with new drugs targeting CRE. This will be achieved by a prospective, multinational cohort study of patients with targeted infections due to CRE and CRAB, and by matched case-control-control studies.

HYPOTHESIS:

H1: 5 independent predictors for cure and mortality can be identified, including active empirical therapy, early targeted optimized therapy and early source management if needed.

H2: For pneumonia, cIAI (complicated intrabdominal infection) and BSI (bloodstream infection), combination therapy with two active drugs, one of them being (if available) an "active" beta-lactam (such as meropenem or imipenem if minimum inhibitory concentration [MIC] <16 mg/L, aztreonam if isolate is susceptible as in many metallo-beta-lactamase producers, or cephalosporin if isolate is susceptible as in some OXA-48 producers). For cUTI (complicated urinary tract infection), monotherapy with an "active" beta-lactam as above, colistin or an aminoglycoside (if active in vitro) is as effective as combination therapy.

H3. Clinical cure rate at test of cure (TOC) will be 50% with BAT. H4: Specific carbapenemase types do not independently influence cure rate or mortality.

H5: CRE infections caused by isolates showing a carbapenem MIC <16 mg/L are associated with higher probability of cure and lower mortality.

H6: five significant independent predictors (risk factors) for CRE infection can be found.

H7: the targeted infections due to CRE are significantly and independently associated with higher mortality, hospital stay and hospital costs than infections caused by carbapenem-susceptible Enterobacteriaceae (CSE) or than other diseases causing hospitalisation.

SPECIFIC OBJECTIVES. O1. To characterise the features, clinical management and outcomes of hospitalised patients with cIAI, pneumonia, cUTI and BSI caused by CRE and CRAB.

O1A. To provide cohorts of patients with the targeted infections caused by CRE and CRAB that would eventually be used as historical cohorts for comparison of efficacy and safety of newer drugs against these organisms.

O1B. To identify the outcome predictors of patients with cIAI, pneumonia, cUTI and BSI caused by CRE and CRAB, with identification of the best alternative therapy (BAT).

O1C. To exploratively investigate the importance of the specific carbapenemase and carbapenem-MIC in the outcome of CRE and CRAB infections.

O2. To identify the risk factors for target infections caused by CRE to inform a more efficient design of future randomized clinical trials for these infections.

O3. To assess the mortality, length of hospital stay and hospital costs associated with target infections caused by CRE.

DESIGN AND STUDIES To answer the above objectives, a prospective, multinational, multicentre, observational and analytic project including 3 studies was design.

Study 1. For the analysis of outcome predictors of CRE and CRAB infections (objective 1), a prospective cohort study of patients with the target infections due to CRE and CRAB will be performed.

Study 2. For the analysis of risk factors for target infections caused by CRE (objective 2), a nested case-control-control will be performed. The first group of controls will be formed by matched patients with CSE infections, and the second groups of controls will be formed by admitted patients non-infected patients by CRE or CSE.

Study 3. For the analysis of cost, outcome impact and length of stay associated to target infections caused by CRE (objective 3), a matched cohorts study will be performed. The cohorts will be formed by selected patients with infections due to CRE and the patients with infections due to CSE (identical to the CSE control group above). Additionally, a control group of admitted patients not infected by CRE or CSE will be studied (identical to the admitted control group above).

SETTING This study will be performed in 50 European hospitals from Spain, Italy, Greece, Turkey, Serbia, Croatia, Montenegro, Kosovo, Albania, Bulgaria and Romania.

STUDY PERIOD The recruitment period of the study is planned from February 2016 to June 2017.

ENDPOINTS (MAIN OUTCOME VARIABLES) See below

STUDY VARIABLES AND DEFINITIONS

• CRE: any isolate identified as an Enterobacteriaceae showing a minimum inhibitory concentration (MIC) ≥1 mg/L if using any dilution method and/or ≤22 mm if using a disc-diffusion method for imipenem or meropenem (10 µg disks); all others will be considered carbapenem susceptible (CSE), but meropenem and imipenem susceptible isolates showing resistance to ertapenem will be excluded.
• CRAB: Any isolate identified as Acinetobacter baumannii showing a minimum inhibitory concentration (MIC) ≥16 mg/L for imipenem or meropenem if using any dilution method and/or ≤17 mm for meropenem and/or ≤15 for imipenem if using a disc-diffusion method.
• Independent variables for Study 1: demographics, comorbidities in adults (Charlson's index), comorbidities in children (ARPEC PPS definitions), type of acquisition, systemic inflammatory response syndrome severity in adults, sepsis criteria in children, Pitt score, SOFA score, APACHE-II score, PIM2, invasive procedures, neutropenia, microbiological variables (poly or monomicrobial infection, carbapenemase producer, carbapenemases type, susceptibility profile, carbapenem MIC), clinical management (source control and support therapy), antimicrobial therapy.
• Independent variables for Study 2: demographics, length of hospital stay, epidemiological variables (travels, previous contact with persons colonised by CRE, previous hospitalization, nursing home or other long term-care facility residency, previous colonisation by CRE), comorbidities, acquisition type, SIRS severity in adults, sepsis in children, Pitt score, SOFA score, APACHE-II score, PIM2, invasive procedures, neutropenia, type of infection, microbiological variables, antibiotics received in the last 3 months.
• Independent variables for Study 3: all those included in Study 2.

DATA COLLECTION AND FOLLOW-UP Data will be collected by trained local investigators. Patients will be followed for 30 days from day 0. Data prior to study entry will be collected by reviewing medical records or interviewing the patient, his/her family or the attending healthcare staff. After that, the patients will be followed prospectively. If the patients had been discharged before assessement, outcome must be assessed by an outpatient visit or phone call according to a pre-design questionnaire.

MICROBIOLOGICAL STUDIES. All procedures will be performed locally using accepted, standard microbiological protocols. Isolates identified as CRE or CRAB according to above criteria will be locally studied for carbapenemase production using the CARBA-NP test. Susceptibility tests to key antimicrobial agents will be collected. Isolates preservation CRE and CRAB isolates will be preserved locally at least at -20ºC.

SAFETY ASSESMENT No investigation drugs will be used in this study. Adverse event will be collected during follow-up as one variable of interest for analysis purposes.

SAMPLE SIZE :

• Study 1 The sample size for the CRE and CRAB cohorts were calculated so that the cohorts may serve as 'historical' cohorts for future comparison with new drugs for CRE and CRAB. To do so, and because the estimates for the outcome variable of the new drug is unknown, we seek to estimate the clinical cure rate of BAT with 95% confidence interval and 8% precision. For an estimated cure rate of 50% based on data from previous studies, 151 patients for each of the five types of infection are needed (due to CRE: cUTI, pneumonia, cIAI, and BSI; due to CRAB: BSI). However, because around 25% of patients will not receive BAT, we will need 201 patients per type of infection except for BSI due to A. baumannii, for which we will need 221 because some blood isolates identified as Acinetobacter spp in sites not specifying the species will not be A. baumannii (total, 1025 patients). Such sample size will be enough also to investigate the best available therapy with the merged CRE cohorts.
• Study 2: The CRE case group will be formed by 248 patients with CRE infections selected from the study 1 cohort. Per each CRE case, one CSE control patient and 3 non-infected controls will be selected. Because of possible heterogeneity due to the different infection types, we have chosen to over sample by a ratio of 248/201*100%. The number of matched controls has been chosen to be four which is well known to well approximate the power of full cohort data.
• Study 3 The CRE, CSE and non-infected cohorts will be formed by the same patients as in Study 2. Therefore, the sample size rationale is as for Study 2, however with the refinement that the nested case control matching is now viewed as an exposure density sampling.

STATISTICAL ANALYSIS

• Study 1. The outcomes associated with exposure to different variables will be compared; the targeted exposures will be empirical active antimicrobial therapy, early targeted optimized therapy, and early source control. Antimicrobial regimens will be analysed as empirical (administered before the susceptibility testing is available) and targeted (thereafter) therapy. The primary endpoint "mortality from any cause until day 30" will be analysed using survival methods (Kaplan-Meier, Cox regression). The other primary endpoint "clinical response at TOC" will be analysed as a dichotomous outcome, regression analyses will use the logistic regression model. The analysis of the secondary endpoints will be analogous. Microbiological response at TOC is a polychotomous outcome and will be analysed using multinomial logistic regression. Goodness of fit will be assessed throughout. Variable selection will be based on Akaike's information criterion.
• Study 2. Exposure to potential risk factors of patients will be compared between CRE cases and CSE controls, and between CRE cases and admitted controls. A stratified and weighted Cox analysis will be performed. Multilevel hospital data (local rate of CRE, antimicrobial consumption, infection control measures) will also be considered in the analyses above.
• Study 3. The impact of CRE infection on mortality, length of stay, cost, length of hospital and ICU stay, and length of mechanical ventilation of patients will be assessed by comparison with those of CSE infection and admitted patients. All time-to-event outcomes (for costs and extra hospital/ICU days see below) will be compared using survival techniques. Because the outcome infection due to CRE is subject to the competing risks of death in hospital w/o CRE infection and discharged alive from hospital with or without CRE infection, the analyses shall be supplemented by those of the competing outcomes. Goodness of fit will be assessed throughout. Extra hospital/ICU days will be estimated using the multistate approach of Beyersmann et al. Variable selection will be based on Akaike's information criterion. Goodness of fit will be assessed throughout.

ETHICAL CONSIDERATIONS. Prior to initiation of a study site, approval will be sought from the appropriate regulatory agency and local Ethics Committees of Research or IRBs to conduct the study in accordance with regulatory requirements. This is an observational study and therefore no intervention is performed on behalf of the investigation. Management of all patients including all antibiotic regimens prescribed will be decided by the physician doctor/team in charge without any interference. The processing of the patients' personal data collected in this study shall comply with the Data Protection Act 1998 and with the European Directive on the Privacy of Data.

MONITORING The study will be monitored for quality and consistency of data.

Selection criteria for CRE GROUPS and CRAB GROUP:

Inclusion criteria (all must be fulfilled):

• Isolation of CRE or CRAB from a clinical sample (e.g., a sample obtained in the work-up of a patient with suspicion of infection; therefore, screening samples are not considered).
• The patient meets the criteria for any of the following infections (see definitions below): complicated urinary tract infection, pneumonia, intraabdominal infection or bloodstream infection (if the source of infection is any of the above, the patient will be included in both groups).
• Patient or his/her representative sign the inform consent if requested by the local Institutional Review Board (IRB).

Patients in these groups will be included until the needed sample sizes are reached.

Exclusion Criteria:

• The infection is considered to be polymicrobial according to standard microbiological interpretation of culture results (except for cIAI, in which polymicrobial infections are allowed).
• The patient was participating in a clinical trial that involved active treatment for the infections.
• The patient was previously included in the same cohort of this study for the same organism. A single episode of CRE or CRAB per patient can be included. Patients who suffer a CRE infection could later be included in the CRAB cohort if developing a CRAB infection and vice versa.
• Patients with do not resuscitate orders or with a life expectancy of <30 days. Selection criteria for CSE GROUP Inclusion criteria (all must be fulfilled)
• Isolation of CSE from a clinical sample (e.g., a sample obtained in the work-up of a patient with suspicion of infection; therefore, screening samples are not considered).
• The patient meets the criteria for any of the following infections (see definitions below): complicated urinary tract infection, pneumonia, intraabdominal infection or bloodstream infection (if the source of infection is any of the above, the patient will be included in both groups).
• The infection is the same as that of the index case; in case of BSI, the source of bacteraemia must be the same as the index case classified as follows: UTI, pneumonia, intraabdominal infection or any other.
• The type of acquisition is the same as for the index CRE case (nosocomial or community).
• The previous length of hospitalization before the infection onset is minus 1 up to minus 3 days the previous length of hospitalization before the CRE infection date in the CRE correspondent (up to minus 7 days if the CRE case occurred after 14 days of previous stay).
• The patient was admitted to the same type of service as the index case (medical, surgical, ICU, neonatal Unit, paediatric ICU, general paediatric wards).
• Patient or his/her representative sign the inform consent (if requested by local IRB).

Patients in this group will be included until the needed sample size is reached.

Exclusion criteria

• The infection is considered to be polymicrobial according to standard microbiological interpretation of culture results (except for cIAI, in which polymicrobial infections are allowed).
• Patient is participating in a clinical trial that involved active treatment for the infections at assessment.
• Patients with do not resuscitate orders or with a life expectancy of <30 days. The first patient found with all inclusion criteria and no exclusion criteria will be included.

Selection criteria for ADMITTED CONTROL GROUP Inclusion criteria (all must be fulfilled)

• Patient is admitted in the same hospital ward where was admitted the index CRE.
• The previous length of hospitalization is at least one day less than the previous duration of hospitalisation of the correspondent CRE case when the CRE infection occurred.
• Patient or his/her representative sign the inform consent (if requested by local IRB).

Patients in this group will be included until the needed sample size is reached.

Exclusion criteria

• Patient was participating in a clinical trial that involved active treatment for the infections at assessment.
• Patients with do not resuscitate orders or with a life expectancy of <30 days.

Because the search for CSE controls is more difficult, the search for admitted control patients can be started once a CSE control has been included; the first 3 patients with the above inclusion criteria and no exclusion criteria will be included.

The base-population for the studies are:

• Study 1: all patients with the targeted infections due to CRE or CRAB.
• Study 2 and study 3: all admitted patients with the targeted infections due to CRE or CSE, and all admitted patients.

Within COMBACTE consortium