BackgroundThe aim of this study was to characterize the spread of carbapenemase-producing Klebsiella pneumoniae (CPKP) in a tertiary level hospital using ongoing active surveillance with rectal swab cultures. Furthermore, this study analyzed the presence of CPKP in the clinical samples (CS) of a single patient as well as the evolution of Colistin-sensitive strains (CoS) to Colistin-resistant strains (CoR).MethodsThis study was performed from January 1, 2012 to December 31, 2014. In 2012, a survey was conducted in the Intensive Care Department. In autumn 2013, active monitoring was extended to the Surgery Department, and since mid-2014, the surveillance has included the Medical Department as well. Only the first isolated strain from each patient was included. Antimicrobial susceptibility testing was performed on CPKP isolates: Klebsiella pneumoniae carbapenemase, oxacillinase-48, Verona integron-encoded metallo-β-lactamase and New Delhi metallo-β-lactamase were detected using a validated in-house PCR method, and multilocus sequence typing (MLST) was used to investigate the clonal transmission of strains.ResultsA total of 15,104 patients were included in the study, and 496 consecutive non-replicated strains of CPKP were collected: 149 strains were collected in 2012 (39 [26.2 %] from surveillance rectal swabs [SRS]), 133 strains were collected in 2013 (70 [52.6 %] from SRS) and 214 strains were collected in 2014 (164 [76.6 %] from SRS). We observed a significant increase in the percentage of positive SRS cases in 2014 relative to 2013 and 2012 (p = 0.0001 and p = 0.0172, respectively) and in the proportion of CPKP first isolated by SRS relative to those identified by CS (p < 0.0001). Among all available samples, the number of CoR isolated from SRS was higher in 2013 and 2014 compared with 2012 (p = 0.0019 and p = 0.008, respectively). ST-258 and ST-512 were more prevalent in the tested specimens, and a new single locus variant (SLV) of ST-512 (ST-745) was isolated.ConclusionsThe results of this 3-year study of 15,104 patients highlight the clinical relevance of antimicrobial resistance as well as the drug-selection pressure of colistin therapy. The active surveillance in the three different departments increased the level of CPKP cases isolated by SRS.

dFor pneumococcal disease surveillance, simple and cost-effective methods capable of determining all serotypes are needed. Combining a single-tube multiplex PCR with fluorescently labeled primers followed by amplicon analysis using automated fluorescent capillary electrophoresis, each serotype of 92 reference isolates and 297 recently collected clinical isolates was successfully determined. Streptococcus pneumoniae is a major human pathogen responsible for a wide variety of infections, from colonization and mild disease, such as sinusitis or otitis, to more-severe and lifethreatening infections, such as invasive pneumonia or meningitis. The capsular polysaccharide is considered a major virulence factor in pneumococcal disease, with the composition, order, and linkage of the monosaccharides that make up the capsule determining a specific antigenic response that classifies pneumococci in different serotypes (1). It has long been known that antibodies against capsular polysaccharides are serotype specific and protective (2), and pneumococcal conjugate vaccines (PCVs), which included the polysaccharides of a limited number of serotypes, were developed to prevent pneumococcal diseases in young children. The introduction of PCVs was accompanied by a decrease in the incidence of invasive pneumococcal diseases but also by a change in the distribution of circulating serotypes (3, 4).Knowing which S. pneumoniae serotypes cause infection is crucial in the surveillance of pneumococcal disease. Surveillance studies usually comprise many isolates, so techniques are needed that can type a large number of isolates simply and accurately. Among the developed techniques, and following the description of genes encoding the pneumococcal capsule (the cps gene cluster) by the Sanger Institute (http://www.sanger .ac.uk/Projects/S_pneumoniae/CPS/), are different PCR-based strategies. Those performed in a single PCR normally detect a limited number of serotypes (5-7), whereas those detecting more serotypes usually require 3 to 8 multiplex PCRs that are performed simultaneously (8) or sequentially (9-14), real-time multiplex PCR (15), or the combination of conventional and real-time PCR (16).By use of multiplex PCR combined with fragment analysis using automated fluorescent capillary electrophoresis (FAFmPCR), Lawrence et al. (17) detected 5 serotypes and 3 serogroups, whereas Selva et al. (18) identified 68 pneumococcal serotypes by multiplexing 40 pairs of primers in a unique reaction that was cost-effective in terms of reagent costs and labor time requirements. The primers used in the work of Selva et al. were those available on the CDC website (http://www.cdc.gov/streplab /downloads/pcr-oligonucleotide-primers.pdf).In the present work using a similar strategy, we designed a single-tube multiplex PCR with 55 fluorescently labeled pairs of primers to identify 92 capsular serotypes described up to 2010 (19) (see PCR mix preparation, amplification conditions, and readout in the supplemental material). The design of the primers for the...

Background: While surfactant protein composition of bronchoalveolar lavage (BAL) has been described in lung diseases of adults and premature infants, scanty data are available on surfactant protein composition beyond the neonatal period.

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