910
the Cleveland VA hospital.2,6,7,15–17 Carriers with high burden of C. difficile in stool were more likely to have skin and/or environ- mental shedding, suggesting that this subset of carriers might pose a relatively high risk for transmission.15 Here, we tested the hypothesis that LTCF residents with CDI or asymptomatic carriage of toxigenic strains are an important source of transmission. Given the high frequency of interfacility transfer between LTCFs and hospitals, we examined transmission both in the LTCF and in the hospital during acute-care admissions.
Methods Setting
The Louis Stokes Cleveland VA Medical Center includes a 215-bed hospital and an adjacent 150-bed LTCF. The affiliated LTCF receives approximately half of the hospitalized patients that are discharged to long-term care, with the remainder being transferred to community LTCFs. The incidences of healthcare facility-associated CDI in the hospital and LTCF during the study were 8 and 2 cases per 10,000 patient days, respectively. A commercial polymerase chain reaction (PCR) assay (Xpert C. difficile,Cepheid,Sunnyvale, CA) wasusedfor CDI diagnostic testing. Infection control measures for CDI for the hospital and LTCF included contact precautions for patients with orders for CDI testing, continuation of contact precautions until at least 2 days after completion of CDI treatment, and use of bleach for daily and postdischarge CDI room disinfection. A fluorescentmarker method was used in both facilities to monitor and provide feedback on thoroughness of daily and postdischarge cleaning of CDI rooms.18
Participants and procedures
Between March 1, 2012, and August 31, 2012, we conducted a prospective cohort study to determine the role of LTCF residents with CDI or asymptomatic carriage in transmission of toxigenic C. difficile strains. Beginning February 16, 2012, current residents and new admissions to the LTCF with no diarrhea were screened to detect asymptomatic carriage of toxigenic C. difficile. The partici- pants were primarily from3 LTCF wards, including 2 wards with a mix of residential and postacute residents and a spinal cord injury rehabilitation ward. A smaller group of residents was enrolled from a dementia ward; residents with advanced dementia were excluded. Consenting subjects had perirectal, groin, skin (chest and abdo- men), and environment (bed rail, bedside table, call button, tele- phone) cultures collected within 24 hours of admission or upon enrollment, every 2 weeks for the first month, and then monthly during their LTCF stay. Because detection of C. difficile on a single occasion can represent transient “pass through” of spores rather than true colonization,19 we defined patients with detection of the same restriction endonuclease analysis (REA) type of toxigenic C. difficile on 2 or more occasions as persistent carriers. All stool specimens of LTCF residents and hospitalized
patients diagnosed with CDI between March 1, 2012, and August 31, 2012, were collected from the Microbiology Laboratory and cultured for toxigenic C. difficile. Healthcare-associated CDI was defined as the presence of diarrhea (≥3 unformed stools in 24 hours) and a positive PCR assay (Xpert C. difficile, Cepheid, Sunnyvale, CA) on stool specimens collected more than 3 days after admission or within 4 weeks of discharge to the community. Healthcare-associated CDI cases were classified as LTCF asso- ciated or hospital associated if the diagnosis was based on stool specimens collected more than 3 days after admission to the
Curtis J. Donskey et al
LTCF or hospital, respectively. Medical record review was con- ducted to obtain information on demographics, medical condi- tions, medications, prior CDI, and ward location. The research protocol was approved by the Louis Stokes Cleveland VA Medical Center Institutional Review Board.
Microbiology and molecular typing
Perirectal swabs and stool specimens were cultured as described previously.18 The number of C. difficile colonies per swab was counted. Restriction endonuclease analysis was performed for all isolates.20 For a subset of isolates that were linked based on REA typing with or without ward exposure, whole-genome sequencing (WGS) was performed. DNA was extracted using the Zymo Fun- gal/Bacteria DNA MicroPrep kit (Zymo Research, Irvine, CA). Libraries were prepared using the NEBNext Ultra DNA library prep kit for Illumina (New England BioLabs, Ipswich, MA), and paired-end reads (2×250 bp) were generated using the Illumina MiSeq reagent kit version 2 and MiSeq instrument (Illumina, San Diego, CA). Dynamic trimming was performed with SolexaQA++ version 3.1.2 software on all reads to meet or exceed 99.9% probability that the nucleotides were properly identified.21 Reads were assembled with an interative de Bruijn algorithm (IDBA version 1.1.2 software).22 The core sequences among these isolates were extracted from an alignment made using progressiveMauve version 2.4.0 software and the results were filtered using Clonal- FrameML version 1.21 software before a PhyML version 3.0 tree was constructed.23–25 Data were archived at theNational Center for Biotechnology Information under project number PRJNA296517.
Epidemiologic and statistical analysis
Epidemiologic relationships were determined based on WGS analysis and ward- or facility-level contact identified through tracking of patient movement. Donor and recipient isolates that differed by ≤2 single-nucleotide polymorphisms (SNPs) were considered genetically related.25 Donor and recipient isolates differing by 3–4 SNPs were considered possibly genetically rela- ted. Ward location was used to categorize possible transmission routes: (1) ward transmission (ie, recipient shared ward exposure with a putative donor either simultaneously or within 30 days of discharge of the putative donor); (2) nonward transmission (ie, recipient and donor in facility simultaneously or within 30 days of discharge of the putative donor but with no shared time on a ward). To be considered a donor, asymptomatic carriage or CDI must have been documented prior to the onset of CDI in the recipient. If multiple potential donors were identified for a reci- pient case, only 1 transmission event was recorded and the potential donor with the least SNP differences (eg, 0 SNP dif- ferences chosen over 2 SNP differences) and the greatest ward contact was selected as the most likely donor. The primary outcome was healthcare-associated CDI linked to transmission of toxigenic C. difficile based on WGS and ward- or facility-level contact. We determined the proportions of CDI cases linked to LTCF asymptomatic carriers, LTCF-associated CDI cases, and hospital-associated CDI cases. Data were analyzed using SPSS version 10.0 software (SPSS, Chicago, IL).
Results
A total of 201 LTCF residents were screened for asymptomatic carriage of toxigenic C. difficile. Those screened included 69 of 98
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