Infections Caused by ESbL-Producing Enterobacteriaceae in Italy
Infections Caused by ESbL-Producing Enterobacteriaceae in Italy
To assess the molecular epidemiology, clinical impact, treatment outcome and risk factors for
 infections caused by Enterobacteriaceae producing ESBLs in Italy in a large multicenter
 observational survey.

 SPECIFIC OBJECTIVES

 1. To collect consecutive nonreplicate isolates of Enterobacteriaceae resistant to
 expanded-spectrum cephalosporins from clinical specimens from inpatients and
 outpatients.

 2. To characterize the isolates for resistance phenotypes and for β-lactam resistance
 mechanisms.

 3. To investigate the clonality of isolates.

 4. To analyse the epidemiology of various resistance mechanisms/resistant clones.

 5. To collect clinical and epidemiological data for patients with infections caused by the
 ESBL producers.

 6. To analyse the epidemiology, risk factors and outcome for infections caused by ESBL
 producers.
RESEARCH PLAN AND METHODOLOGY

 Population All patients with infections (i.e. bacteremia, pneumonia, abdomen infections, skin
 infections, and urinary tract infections) caused by ESBL-producing Enterobacteriaceae.

 Exclusion criteria Children < 16 years

 Trial design Multicenter prospective cohort study. Patients corresponding to the study
 definition will be detected by daily inspection of microbiological databases by one dedicated
 physician. Epidemiological variables will be detected for all patients at study admission. To
 identify the risk factors for infections (i.e. bacteremia, pneumonia, abdomen infections,
 skin infections, and urinary tract infections) caused by ESBL-producing Enterobacteriaceae a
 case-control study will be performed. A case patient will be defined as adult patient ( 16
 years of age) who has an infection caused by ESBL-producing Enterobacteriaceae according to
 the definitions of the Center for Diseases Control and Prevention in Atlanta (CDC). In the
 Clinical Microbiology Laboratories, definition of ESBL production by the Clinical
 Microbiology Laboratories will be according to the CLSI guidelines (3), or to the BSAC
 guidelines (http://www.bsac.org.uk) for species other than Klebsiella spp., E. coli and
 Proteus mirabilis. One control will be selected for each case. Control will be chosen among
 all adult patients admitted to the same hospital during the same period (within 30 days) and
 in whom ESBL-producing Enterobacteriaceae were not isolated during their hospital stay. If
 more than one control will be available per case, patients with the date and time of
 admission closest to the case will be chosen.

 To identify the risk factors for mortality a second case-control study will be performed
 comparing infected patients who died to infected patients who survived (evaluable in patients
 with bacteremia, wound infections, pneumonia, and meningitis, only). This analysis will be
 performed adjusting the results for inadequate empiric antimicrobial therapy and site of
 infection (see following definitions).

 Data collection Medical records of in-patient admissions and microbiology and pharmacy
 databases will be reviewed. Data collected at the study enrollment will include: patient
 demographics, transfer from another hospital, resident of a long term facility or nursing
 home, previous hospitalization within one year, ambulatory status, requirement for chronic
 hemodialysis, presence of a central venous catheter (CVC), and ICU stay and surgical
 procedures in the 30 days prior to study inclusion. A composite score of co-morbid illnesses
 will be derived using the Charlson score. The severity of illness at presentation will be
 quantified using the criteria of McCabe and Jackson. Antibiotics administered during a 30-day
 period prior to study enrollment and for at least 48 hours will be recorded. For the risk
 factors analysis, oral and intravenous antibiotic exposure will be analysed by individual
 antibiotics and by classes, and will include penicillins, vancomycin, cephalosporins,
 antibiotics with predominantly anaerobic activity (metronidazole and clindamycin),
 aminoglycosides, quinolones, and carbapenems. Length of stay (LOS) after infection diagnosis,
 management of infections, timing and type of antimicrobial therapy, results of follow-up
 clinical cultures (if any), hematogenous complications and death will be also extracted from
 medical records. Microbiology records will be also reviewed for recovery of vancomycin
 resistant enterococci (VRE) or methicillin-resistant S. aureus (MRSA) in the 12 months prior
 to the study enrollment. All patients with infections caused by ESBL-producing
 Enterobacteriaceae will be followed up to hospital discharge or death.

 Mortality will be defined as death occurring during the study hospitalization. Appropriate
 antimicrobial therapy will be defined as the initiation of therapy with activity against the
 ESBL-producing Enterobacteriaceae (according to the results of the antimicrobial
 susceptibility pattern of the isolate) from the day before to 2 days after the initial
 positive clinical culture result.

 Outcomes Primary outcomes

 1. Risk factors for the development of infections caused by ESBL producers bacteria;

 2. Risk factors for inadequate initial antimicrobial therapy;

 3. Overall mortality and 30-day mortality among patients receiving inadequate initial
 antimicrobial therapy (evaluable in patients with bacteremia, abdominal infections and
 pneumonia, only);

 4. Variability by site of infection of overall mortality and 30-day mortality among
 patients receiving inadequate initial antimicrobial therapy (evaluable in patients with
 bacteremia, abdominal infections and pneumonia, only).

 Secondary outcomes

 1. Lenght of hospitalization;

 2. Costs of inadequate initial therapy;

 3. Days of defervescence.

 Statistical analysis At the end of the study two different analysis will be performed: the
 first one will consider patients with bacteremia, abdominal infections and pneumonia, and it
 will be focused on mortality; the second analysis will consider all infected patients and it
 will be focused on risk factor analysis and secondary outcomes.

 Sample size justification Assumptions

 Patients with bacteremia, pneumonia, abdomen infections, skin infections and urinary tract
 infections (first analysis):

 Mortality among patients with inadequate initial therapy: 25% Mortality among patients with
 appropriate initial therapy: 13%

 All included patients:

 Percentage of bacteremia: 20% Percentage of pneumonia: 10% Percentage of abdomen infections:
 15% Percentage of skin infections: 3% Percentage of urinary tract infections: 52%

 Primary outcomes Risk factors for mortality Difference of mortality between controls
 (inappropriate therapy) and cases (appropriate therapy): we anticipated a reduction from 25%
 to 13%.

 Secondary outcomes Difference of length of hospitalization between cases (appropriate
 therapy) and controls (inappropriate therapy): we anticipated a decrease from 80% (length of
 hospitalization < 15 days) to 40%.

 Difference of defervescence between cases (appropriate therapy) and controls (inappropriate
 therapy): we anticipated a decrease from 85% (defervescence < 3 days) to 30%.

 Assuming that p1=p2, where p1 is the proportion in population 1 and p2 is the proportion in
 population 2 and that alpha=0.05 (two-sided), power=0.80.

 Required sample size for analysis of risk factors for mortality: 366 patients with
 bacteremia, wound infections, pneumonia, and meningitis. To include 366 patients with the
 above reported infections the total sample size is likely to be 813 patients infected with
 ESBL-producing Enterobacteriaceae.

 Microbiological analysis of bacterial strains

 All ESBL-producing isolates obtained from patients included in the study at the Clinical
 Microbiology Laboratories of each Clinical Center will be collected and subjected to further
 investigation including:

 -  confirmation of the identification at the species level;

 -  determination of the antimicrobial resistance profile against a standard set of drugs;

 -  analysis of the ESBL determinants;

 -  analysis of clonal relationship between bacterial isolates of the same species. Results
 of microbiological analysis will be used for correlation with clinical data to assess
 the epidemiological and clinical impact of the ESBL-producing strains.

 Experimental methodology

 Confirmation of identification of the bacterial isolates at the species level will be carried
 out by standard phenotypic methods (10). In case of ambiguity of results, identification will
 be confirmed by 16S rDNA sequencing (16).

 Antimicrobial susceptibility testing will be carried out by broth microdilution as
 recommended by the CLSI (3), and categorical assignment will be carried out using the CLSI
 breakpoints (3). The following agents will be included: ertapenem, imipenem, meropenem,
 cefepime, ceftazidime, cefotaxime, piperacillin/tazobactam, amoxicillin/clavulanate,
 ciprofloxacin, levofloxacin, gentamicin, amikacin.

 The nature of the ESBL determinants will be investigated by molecular analysis using PCR and
 sequencing, as described previously (9, 15). The presence of ESBL determinants of the TEM-,
 SHV, CTX-M- and PER-type (i. e. those known to be circulating in Italy (9, 15)) will be
 investigated. In case of negative results with the available probes, the ESBL phenotype will
 be reconfirmed and, if positive, the nature of -lactamase deerminants will be studied by
 means of: (i) analytical isoelectric focusing (IEF) on crude extracts using previously
 described methods (14); (ii) DNA microarray technology, using a microarray for β-lactamase
 genes that has been developed in our laboratory, which includes probes for uncommon ESBL
 genes such as VEB, GES, BES, SFO, and TLA enzymes. In case of new ESBL genes undetected by
 the above methods (an unlikely occurrence), In the case of production of ESBL but unaccounted
 for by the presence of known resistance determinants, the resistance determinants will be
 studied by means of a shotgun-cloning approach followed by mapping and sequencing.

 Clonal relatedness among isolates of the same species will be investigated by high-throughput
 genotyping techniques such as RAPD (Random Amplified Polymorphic DNA) and/or by AFLP
 (Amplified Fragment Length Polymorphism) analysis and/or REP PCR (19-21). In selected
 isolates, the clonal relationships will be performed by macrorestriction analysis of genomic
 DNA by pulsed-field gel electrophoresis (PFGE).

 Plasmid analysis and investigation of resistance determinants to non β-lactam agents (by
 molecular techniques) will be considered in selected cases, should these information be
 relevant for correlation with clinical and epidemiological data.
Inclusion Criteria:

 -  All patients with infections (i.e. bacteremia, pneumonia, abdomen infections, skin
 infections, and urinary tract infections) caused by ESBL-producing Enterobacteriaceae

 Exclusion Criteria:

 -  Children < 16 years