Surface area matters: An evaluation of swabs and surface area for environmental surface sampling of healthcare pathogens.

Flocked and foam swabs were used to sample five healthcare pathogens from three sizes of steel and plastic coupons; 26 cm2, 323 cm2, and 645 cm2. As surface area increased, 1-2 log10 decrease in recovered organisms (P < .05) was observed. Sampling 26-cm2 yielded the optimal median percent of pathogens recovered.

The effect of disinfectants on the microbial community on environmental healthcare surfaces using next generation sequencing.

BACKGROUND: Healthcare-associated infections are a significant economic burden and cause of avoidable morbidity and mortality within healthcare systems. The contribution of environmental contamination to healthcare-associated infection transmission has been recognized, but the mechanisms by which transmission occurs are still being investigated. The objective of this study was to characterize the microbial communities of disinfected, non-critical healthcare surfaces using next generation sequencing technology. METHODS: Composite environmental surface samples were from high-touch surfaces in rooms of patients isolated for infections with multidrug-resistant organisms during their hospitalization. Information on the disinfectant product used and cleaning type (routine or terminal) was collected. 16S rRNA gene amplicon sequencing and analysis were performed. Community analysis was conducted to determine the bacterial composition and compare the detection of target pathogens by culture from 94 Contact Precaution rooms. RESULTS: Overall percent agreement between culture and sequence methods ranged from 52%-88%. A significant difference was observed in bacterial composition between rooms cleaned with bleach and those cleaned with a quaternary ammonium compound for composite 2 (overbed table, intravenous pole, and inner room door handle) (ANOSIM R = 0.66, P = .005) but not composite 1 (bed rails, television remote control unit, call buttons, and telephone). CONCLUSIONS: Surfaces in bleach-cleaned rooms contained a higher proportion of gram-positive microbiota, whereas rooms cleaned with quaternary ammonium compound contained a higher proportion of gram-negative microbiota, suggesting disinfectant products may impact the healthcare environment microbiome.

Development of a rapid-viability PCR method for detection of Clostridioides difficile spores from environmental samples.

Clostridioides difficile is a common pathogen that is well known to survive for extended periods of time on environmental healthcare surfaces from fecal contamination. During epidemiological investigations of healthcare-associated infections, it is important to be able to detect whether or not there are viable spores of C. difficile on surfaces. Current methods to detect C. difficile can take up to 7 days for culture and in the case of detection by PCR, viability of the spores cannot be ascertained. Prevention of C. difficile infection in healthcare settings includes adequate cleaning and disinfection of environmental surfaces which increases the likelihood of detecting dead organisms from an environmental sample during an investigation. In this study, we were able to adapt a rapid-viability PCR (RV-PCR) method, first developed for detection of viable Bacillus anthracis spores, for the detection of viable C. difficile spores. RV-PCR uses the change in cycle threshold after incubation to confirm the presence of live organisms. Using this modified method we were able to detect viable C. difficile after 22 h of anaerobic incubation in Cycloserine Cefoxitin Fructose Broth (CCFB). This method also used bead beating combined with the Maxwell 16 Casework kit for DNA extraction and purification and a real-time duplex PCR assay for toxin B and cdd3 genes to confirm the identity of the C. difficile spores. Spiked environmental sponge-wipes with and without added organic load were tested to determine the limit of detection (LOD). The LOD from spiked environmental sponge-wipe samples was 104 spores/mL but after incubation initial spore levels of 101 spores/mL were detected. Use of this method would greatly decrease the amount of time required to detect viable C. difficile spores; incubation of samples is only required for germination (22 h or less) instead of colony formation, which can take up to 7 days. In addition, PCR can then quickly confirm or deny the identity of the organism at the same time it would confirm viability. The presence of viable C. difficile spores could be detected at very low levels within 28 h total compared to the 2 to 10-day process that would be needed for culture, identification and toxin detection.

Elution efficiency of healthcare pathogens from environmental sampling tools.

Standardizing healthcare surface sampling requires the evaluation of sampling tools for organism adherence. Here, 7 sampling tools were evaluated to assess their elution efficiencies in the presence of 5 pathogens. Foam sponges (80.6%), microfiber wipes (80.5%), foam swabs (77.9%), and cellulose sponges (66.5%) yielded the highest median elution efficiencies.

Assessment of the Overall and Multidrug-Resistant Organism Bioburden on Environmental Surfaces in Healthcare Facilities.

OBJECTIVE To determine the typical microbial bioburden (overall bacterial and multidrug-resistant organisms [MDROs]) on high-touch healthcare environmental surfaces after routine or terminal cleaning. DESIGN Prospective 2.5-year microbiological survey of large surface areas (>1,000 cm2). SETTING MDRO contact-precaution rooms from 9 acute-care hospitals and 2 long-term care facilities in 4 states. PARTICIPANTS Samples from 166 rooms (113 routine cleaned and 53 terminal cleaned rooms). METHODS Using a standard sponge-wipe sampling protocol, 2 composite samples were collected from each room; a third sample was collected from each Clostridium difficile room. Composite 1 included the TV remote, telephone, call button, and bed rails. Composite 2 included the room door handle, IV pole, and overbed table. Composite 3 included toileting surfaces. Total bacteria and MDROs (ie, methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci [VRE], Acinetobacter baumannii, Klebsiella pneumoniae, and C. difficile) were quantified, confirmed, and tested for drug resistance. RESULTS The mean microbial bioburden and range from routine cleaned room composites were higher (2,700 colony-forming units [CFU]/100 cm2; ≤1-130,000 CFU/100 cm2) than from terminal cleaned room composites (353 CFU/100 cm2; ≤1-4,300 CFU/100 cm2). MDROs were recovered from 34% of routine cleaned room composites (range ≤1-13,000 CFU/100 cm2) and 17% of terminal cleaned room composites (≤1-524 CFU/100 cm2). MDROs were recovered from 40% of rooms; VRE was the most common (19%). CONCLUSIONS This multicenter bioburden summary provides a first step to determining microbial bioburden on healthcare surfaces, which may help provide a basis for developing standards to evaluate cleaning and disinfection as well as a framework for studies using an evidentiary hierarchy for environmental infection control. Infect Control Hosp Epidemiol 2016;1426-1432.

Outbreak of Serratia marcescens bloodstream infections in patients receiving parenteral nutrition prepared by a compounding pharmacy.

BACKGROUND: Compounding pharmacies often prepare parenteral nutrition (PN) and must adhere to rigorous standards to avoid contamination of the sterile preparation. In March 2011, Serratia marcescens bloodstream infections (BSIs) were identified in 5 patients receiving PN from a single compounding pharmacy. An investigation was conducted to identify potential sources of contamination and prevent further infections. METHODS: Cases were defined as S. marcescens BSIs in patients receiving PN from the pharmacy between January and March 2011. We reviewed case patients' clinical records, evaluated pharmacy compounding practices, and obtained epidemiologically directed environmental cultures. Molecular relatedness of available Serratia isolates was determined by pulsed-field gel electrophoresis (PFGE). RESULTS: Nineteen case patients were identified; 9 died. The attack rate for patients receiving PN in March was 35%. No case patients were younger than 18 years. In October 2010, the pharmacy began compounding and filter-sterilizing amino acid solution for adult PN using nonsterile amino acids due to a national manufacturer shortage. Review of this process identified breaches in mixing, filtration, and sterility testing practices. S. marcescens was identified from a pharmacy water faucet, mixing container, and opened amino acid powder. These isolates were indistinguishable from the outbreak strain by PFGE. CONCLUSIONS: Compounding of nonsterile amino acid components of PN was initiated due to a manufacturer shortage. Failure to follow recommended compounding standards contributed to an outbreak of S. marcescens BSIs. Improved adherence to sterile compounding standards, critical examination of standards for sterile compounding from nonsterile ingredients, and more rigorous oversight of compounding pharmacies is needed to prevent future outbreaks.

Storage Effects on Sample Integrity of Environmental Surface Sampling Specimens with Bacillus anthracis Spores.

The effect of packaging, shipping temperatures and storage times on recovery of Bacillus anthracis. Sterne spores from swabs was investigated. Macrofoam swabs were pre-moistened, inoculated with Bacillus anthracis spores, and packaged in primary containment or secondary containment before storage at -15°C, 5°C, 21°C, or 35°C for 0-7 days. Swabs were processed according to validated Centers for Disease Control/Laboratory Response Network culture protocols, and the percent recovery relative to a reference sample (T0) was determined for each variable. No differences were observed in recovery between swabs held at -15° and 5°C, (p ≥ 0.23). These two temperatures provided significantly better recovery than swabs held at 21°C or 35°C (all 7 days pooled, p ≤ 0.04). The percent recovery at 5°C was not significantly different if processed on days 1, 2 or 4, but was significantly lower on day 7 (day 2 vs. 7, 5°C, 102, p=0.03). Secondary containment provided significantly better percent recovery than primary containment, regardless of storage time (5°C data, p ≤ 0.008). The integrity of environmental swab samples containing Bacillus anthracis spores shipped in secondary containment was maintained when stored at -15°C or 5°C and processed within 4 days to yield the optimum percent recovery of spores.

Outbreak of Mycobacterium chelonae infection associated with tattoo ink.

BACKGROUND: In January 2012, on the basis of an initial report from a dermatologist, we began to investigate an outbreak of tattoo-associated Mycobacterium chelonae skin and soft-tissue infections in Rochester, New York. The main goals were to identify the extent, cause, and form of transmission of the outbreak and to prevent further cases of infection. METHODS: We analyzed data from structured interviews with the patients, histopathological testing of skin-biopsy specimens, acid-fast bacilli smears, and microbial cultures and antimicrobial susceptibility testing. We also performed DNA sequencing, pulsed-field gel electrophoresis (PFGE), cultures of the ink and ingredients used in the preparation and packaging of the ink, assessment of source water and faucets at tattoo parlors, and investigation of the ink manufacturer. RESULTS: Between October and December 2011, a persistent, raised, erythematous rash in the tattoo area developed in 19 persons (13 men and 6 women) within 3 weeks after they received a tattoo from a single artist who used premixed gray ink; the highest occurrence of tattooing and rash onset was in November (accounting for 15 and 12 patients, respectively). The average age of the patients was 35 years (range, 18 to 48). Skin-biopsy specimens, obtained from 17 patients, showed abnormalities in all 17, with M. chelonae isolated from 14 and confirmed by means of DNA sequencing. PFGE analysis showed indistinguishable patterns in 11 clinical isolates and one of three unopened bottles of premixed ink. Eighteen of the 19 patients were treated with appropriate antibiotics, and their condition improved. CONCLUSIONS: The premixed ink was the common source of infection in this outbreak. These findings led to a recall by the manufacturer.

Contaminated product water as the source of Phialemonium curvatum bloodstream infection among patients undergoing hemodialysis.

OBJECTIVE: We investigated a cluster of cases of bloodstream infection (BSI) due to the mold Phialemonium at a hemodialysis center in Illinois and conducted a cohort study to identify risk factors. DESIGN: Environmental assessment and cohort study. SETTING: A hemodialysis center in a tertiary care hospital. METHODS: A case patient was defined as a person who underwent dialysis at the center and had a blood sample that tested positive for Phialemonium curvatum on culture. We reviewed microbiology and medical records and tested water, surface, and dialysate samples by culture. Molds isolated from environmental and clinical specimens were identified by their morphological features and confirmed by sequencing DNA. RESULTS: We identified 2 case patients with BSI due to P. curvatum. Both became febrile and hypotensive while undergoing dialysis on the same machine at the same treatment station, although on different days. Dialysis machines were equipped with waste handling option ports that are used to discard dialyzer priming fluid. We isolated P. curvatum from the product water (ie, water used for dialysis purposes) at 2 of 19 treatment stations, one of which was the implicated station. CONCLUSION: The source of P. curvatum was likely the water distribution system. To our knowledge, this is the first report of patients acquiring a mold BSI from contaminated product water. The route of exposure in these cases of BSI due to P. curvatum may be related to the malfunction and improper maintenance of the waste handling option ports. Waste handling option ports have been previously implicated as the source of bacterial BSI due to the backflow of waste fluid into a patient's blood line. No additional cases of infection were noted after remediation of the water distribution system and after discontinuing use of waste handling option ports at the facility.

A multistate outbreak of Serratia marcescens bloodstream infection associated with contaminated intravenous magnesium sulfate from a compounding pharmacy.

BACKGROUND: In contrast to pharmaceutical manufacturers, compounding pharmacies adhere to different quality-control standards, which may increase the likelihood of undetected outbreaks. In 2005, the Centers for Disease Control and Prevention received reports of cases of Serratia marcescens bloodstream infection occurring in patients who underwent cardiac surgical procedures in Los Angeles, California, and in New Jersey. An investigation was initiated to determine whether there was a common underlying cause. METHODS: A matched case-control study was conducted in Los Angeles. Case record review and environmental testing were conducted in New Jersey. The Centers for Disease Control and Prevention performed a multistate case-finding investigation; isolates were compared using pulsed-field gel electrophoresis analysis. RESULTS: Nationally distributed magnesium sulfate solution (MgSO(4)) from compounding pharmacy X was the only significant risk factor for S. marcescens bloodstream infection (odds ratio, 6.4; 95% confidence interval, 1.1-38.3) among 6 Los Angeles case patients and 18 control subjects. Five New Jersey case patients received MgSO(4) from a single lot produced by compounding pharmacy X; culture of samples from open and unopened 50-mL bags in this lot yielded S. marcescens. Seven additional case patients from 3 different states were identified. Isolates from all 18 case patients and from samples of MgSO(4) demonstrated indistinguishable pulsed-field gel electrophoresis patterns. Compounding pharmacy X voluntarily recalled the product. Neither the pharmacy nor the US Food and Drug Administration could identify a source of contamination in their investigations of compounding pharmacy X. CONCLUSIONS: A multistate outbreak of S. marcescens bloodstream infection was linked to contaminated MgSO(4) distributed nationally by a compounding pharmacy. Health care personnel should take into account the different quality standards and regulation of compounded parenteral medications distributed in large quantities during investigations of outbreaks of bloodstream infection.

Growth and survival of Fusarium solani-F. oxysporum complex on stressed multipurpose contact lens care solution films on plastic surfaces in situ and in vitro.

PURPOSE: To analyze factors implicating the association of ReNu with MoistureLoc (ReNu ML) multipurpose contact lens solution (MPS) with the increased incidence of Fusarium keratitis. METHODS: Used contact lens cases with and without contact lenses and MPS containers were collected from patients with confirmed or possible Fusarium keratitis. Direct microscopy including transparent adhesive tape preparations and swab cultures were used to determine fungal colonization. Survival and growth of selected isolates of Fusarium spp. in drying MPS on plastic surfaces were determined by microscopy and recoverable colony counts on enriched agar. RESULTS: Discrete regions of fungal colonization, including occasional microcycle conidiation and chlamydospore formation, were observed on the surfaces of contact lens cases and, less often, on solution containers that had been used by patients with Fusarium keratitis associated with the use of ReNu ML. Isolates provisionally grouped with the F. solani-F. oxysporum complex were inhibited by fresh MPS in original solution containers and contact lens cases, but survived in stressed (drying) films of MPS, particularly ReNu ML. These in vitro test results were similar to the direct in situ observations of the materials from patients. CONCLUSIONS: Selective, rapid growth and survival of cells of the F. solani-F. oxysporum complex on plastic surfaces, particularly of contact lens cases with stressed ReNu ML films, may explain, in part, the recent Fusarium keratitis outbreak.

Viability of amoebae, fungal conidia, and yeasts: rapid assessment by flow cytometry.

Conventional methods for the evaluation of antimicrobials and disinfecting solutions with microorganisms involve culture-based techniques, which are time-consuming and underestimate the number of viable organisms. Rapid detection and viability measurements of microorganisms in homogenous and heterogenous microbial populations have been greatly enhanced by recent advances in the use of fluorescent stains in flow cytometry (FCM). FCM has been applied to enumerate, differentiate, and identify microorganisms, determine protein and DNA content of cells, analyze the physiological state of individual cells, and analyze the interaction of drugs, antibiotics, and antimicrobials with microbial cells. Four physiological states of cells can be distinguished by FCM: (1) reproductively viable, (2) metabolically active, (3) intact, and (4) permeabilized.FCM permits a rapid and quantitative measurement of the optical characteristics of cells as they pass through, in a single file, a focused beam of light. As cells are carried within a fast-flowing fluid stream and through the focus of exciting light, three parameters are measured: forward angle light scatter, side angle light scatter, and fluorescence emitted by dyes that have specific interaction with intracellular components of individual cells. FCM data that are presented in histogram and dot plots can be generated to give information on a variety of properties of interest among cells in the population as a whole.FCM offers major advantages in multiparameter data acquisition and multivariate data analysis, high-speed analysis, and cell-sorting capabilities. Disadvantages may be associated with the cost, which is usually over 100,000 (US Dollars) for a typical laser-based flow cytometer with just analyzing capabilities. Another disadvantage is that skilled personnel are usually required to operate these complex instruments so as to get optimum performance. A schematic overview of flow cytometry is presented in Fig. 1.