Design, Setting and Ethics
This retrospective cohort study was performed in The First Affiliated Hospital, College of Medicine, Zhejiang University, from July 2014 to August 2018. It is one of the largest hospitals in eastern China, having approximate 2500 beds and 131,000 annual admissions. The first report of KPC-kp in China came from here in 2007 . Since then, KPC-kp has been endemic in the hospital .
The present study was conducted according to the principles stated in the Declaration of Helsinki and approved by the ethical review board of The First Affiliated Hospital, College of Medicine, Zhejiang University.
The source population consisted of all patients admitted to the hospital during the study period, whose bacterial cultures were identified as CRKP. The clinical specimens mainly included blood, sputum/tracheal aspirate, pleural fluid and ascites. Further detailed collection of clinical information was performed for the patients whose isolates were verified as KPC-kp. Patients aged <18 years and those with missing or incomplete clinical records were excluded. Patients with polymicrobial infections and those with KPC-kp colonization were excluded as well. Infection or colonization was defined according to the criteria of the Center for Disease Control and Prevention of America . Only the first episode for each patient was included in our analysis.
Clinical Data Collection
For all eligible KPC-kp infection cases, chart review was performed to obtain the clinical data. Infectious disease specialists independently collected the following clinical information using the patients’ electronic charts and/or paper records: demographic conditions, unit and duration of hospitalization, underlying diseases, Charlson Comorbidity Index, abscess information and previous invasive procedures. Patients were defined as immunosuppressed if they had HIV or AIDS, were post-transplant, had received chemotherapy within 4 weeks, had received immunosuppressive agents for more than 2 weeks, or had neutropenia. Previous use of antibiotics was defined as antimicrobial therapy (intravenous or oral) within 4 weeks before infections. Invasive procedures included surgery and mechanical ventilation performed within 4 weeks prior to the infections. Appropriate empirical or definitive antimicrobial therapy was defined as previously described .
The primary outcome variables were all-cause mortality at day 7 and day 28. Other secondary outcome variables were infection-related mortality and in-hospital mortality. Septic shock and multiple organ dysfunction syndrome (MODS) at the onset of infections, as well as the length of stay (LOS) from culture to discharge, were also assessed.
Bacterial Identification and Antimicrobial Susceptibility Testing
Bacterial isolates identification and antimicrobial susceptibility testing were performed using Vitek 2 automated system (bioMérieux, France) and the results were interpreted according to Clinical and Laboratory Standards Institute (CLSI) 2019 criteria . For tigecycline, colistin and ceftazidime-avibactam (CAZ/AVI), minimum inhibitory concentrations (MICs) were determined using broth microdilution method. The susceptibility results were categorized in accordance with the CLSI criteria for CAZ/AVI, while European Committee on Antimicrobial Susceptibility Testing (version 9.0, http://www.eucast.org/clinical_breakpoints/) for colistin and tigecycline. The species identification was verified by MALDI-TOF MS (Bruker Daltonics Inc., Fremont, CA, USA). Pseudomonas aeruginosa ATCC27853 and Escherichia coli ATCC 25922 were used as the quality control strains of antimicrobial susceptibility testing.
All of the CRKP were subjected to detection of blaKPC by PCR. The isolates carrying blaKPC were further investigated for pLVPK-related genetic loci rmpA, rmpA2, iucA and iroN. The primers used for PCR amplification and sequencing were acquired from a previous study . Considering the primers of rmpA and rmpA2 target only the plasmid-borne genes, while iucA and iroN for both plasmid-borne and chromosomally-encoded genes, KPC-kp isolates of positive rmpA or rmpA2 were defined as pVir+-KPC-kp according to the definition previously described [11, 21-23].
Multilocus sequence typing (MLST) was carried out according to the protocol described on the K. pneumoniae MLST website ((https://bigsdb.pasteur.fr/klebsiella/klebsiella.html), and the results were analysed using the corresponding database.
Data were analysed using SPSS software (version 20; SPSS Inc., Chicago, IL, USA). Categorical variables were presented as absolute numbers and their relative frequencies, and analyzed by chi-square or Fisher exact test. For continuous variables, the Kolmogorov-Smirnov test was applied to distinguish between normal and non-normal distribution. Normally distributed data were expressed as mean and standard deviation (SD) and analysed using Student's t-test, while non-normally distributed data were present with median and interquartile rang (IQR) and compared using Mann-Whitney test. To identify independent predictors for pVir+-KPC-kp infections, we used logistic regression. All variables with P value <0.1 in univariate analysis as well as clinical importance were incorporated into multivariable regression model. P value <0.05 was considered statistically significant.