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Sampling efficiency of Candida auris from healthcare surfaces: culture and nonculture detection methods

Published online by Cambridge University Press:  22 June 2021

William A. FurinOpen the ORCID record for William A. Furin[Opens in a new window],
Lisa H. Tran,
Monica Y. Chan,
Amanda K. Lyons,
Judith Noble-Wang and
Laura J. RoseOpen the ORCID record for Laura J. Rose[Opens in a new window]
William A. Furin
Affiliation:
Division of Healthcare Quality Promotion, Clinical and Environmental Microbiology Branch, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GeorgiaOak Ridge Institute for Science and Education (ORISE), Department of Energy, Oak Ridge, Tennessee
Lisa H. Tran
Affiliation:
Division of Healthcare Quality Promotion, Clinical and Environmental Microbiology Branch, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GeorgiaOak Ridge Institute for Science and Education (ORISE), Department of Energy, Oak Ridge, Tennessee
Monica Y. Chan
Affiliation:
Division of Healthcare Quality Promotion, Clinical and Environmental Microbiology Branch, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia
Amanda K. Lyons
Affiliation:
Division of Healthcare Quality Promotion, Clinical and Environmental Microbiology Branch, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia
Judith Noble-Wang*
Affiliation:
Division of Healthcare Quality Promotion, Clinical and Environmental Microbiology Branch, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia
Laura J. Rose
Affiliation:
Division of Healthcare Quality Promotion, Clinical and Environmental Microbiology Branch, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia
*
Author for correspondence: Judith Noble-Wang, E-mail: JNobleWang@cdc.gov
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Abstract

Sponges and swabs were evaluated for their ability to recover Candida auris dried 1 hour on steel and plastic surfaces. Culture recovery ranged from <0.1% (sponges) to 8.4% (swabs), and cells detected with an esterase activity assay revealed >50% recovery (swabs), indicating that cells may enter a viable but nonculturable state.

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Type
Concise Communication
Information
Infection Control & Hospital Epidemiology , Volume 43 , Issue 10 , October 2022, pp. 1492 - 1494
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Copyright
© The Author(s), 2021. Published by Cambridge University Press on behalf of The Society for Healthcare Epidemiology of America

Candida auris has emerged as an often-misdiagnosed multidrug-resistant organism causing mortality rates of 30%–60% in hospitalized patients. Reference Chowdhary, Sharma and Meis1–Reference Vallabhaneni, Kallen and Tsay3 It has been shown to persist on surfaces ≥2 weeks. Reference Welsh, Bentz and Shams4,Reference Piedrahita, Cadnum, Jencson, Shaikh, Ghannoum and Donskey5 Transmission via fomites within healthcare facilities has been reported Reference Chowdhary, Sharma and Meis1,Reference Adams, Quinn and Tsay2 but standard sampling methods are lacking. We evaluated the influence of sampling devices, mucin in organic matrix, and drying time on cell recovery efficiency of 2 C. auris strains from steel and plastic surfaces. We also compared the detection of recovered cells using culture (colony forming units, CFU) and an alternate viability assay.

Methods

Steel coupons (S-180 grade, T-304; Stewart Stainless Supply, Suwanee, GA) and plastic coupons (0.80 in. thickness, P1 haircell texture, Kydex-T, Bloomsburg, PA) (322 cm2) were cleaned with a nonantimicrobial detergent, rinsed with ultrapure water, then rised with 70% ethanol. Steel was autoclaved at 121°C for 20 minutes and plastic was sterilized with ultraviolet (UV) light for 1 hour.

We obtained 2 C. auris isolates from the Centers for Disease Control and Prevention (CDC); B11103 (clade I) and AR0385 (clade IV). Yeast colonies were cultured on Sabouraud dextrose agar (SDA) for 48 hours at 37°C then suspended in a body fluid simulant; artificial test soil (ATS), or ATS with mucin (ATS-M) (Healthmark Industries, Fraser, MI). Coupons were inoculated with 500 µL cell suspension (105 CFU/mL), spread, and dried for 1 hour at ambient temperature and humidity in a closed BSC (light and fan off). Evaluations were also conducted by sampling 0, 20, and 40 minutes after inoculation.

Coupons were sampled across the entire surface with either a cellulose sponge (cellulose) (3M Sponge-stick, St Paul, MN) or a polyurethane sponge (polyurethane) (Hygiena, Camarillo, CA), both moistened with neutralizing buffer, using a standard method 6 modified to 322 cm2 sample area. Sponges were held 1 hour at ambient temperature to simulate transport time and were then transferred to a stomacher bag containing 45 mL PBS with 0.02% Tween 80 (PBST) for processing. Cells were eluted from sponges using a Stomacher 400 Circulator (Seward Ltd, Worthing, UK) at 260 rpm for 1 minute. The eluate was concentrated by centrifugation, and the final volume was recorded and then cultured on SDA (48–72 hours, 37°C).

To determine the portion of cells remaining on the coupons after sampling, and to investigate the presence of viable but nonculturable cells, smaller steel coupons (26 cm2) were inoculated as described above using 100 µL 5×106 CFU/mL inoculum in ATS-M and sampled with a foam swab (swab) (Puritan; Guilford, ME). The swab was vortexed in 5 mL PBST, the coupon was sonicated in 15 mL PBST, and both eluates were cultured on SDA. Using a solid phase cytometer (ScanRDI, bioMerieux, Durham, NC) and the instrument’s standard protocol, Reference Costanzo, Borazjani and McCormick7 1 mL of each swab and coupon eluate was analyzed for cells exhibiting metabolic esterase activity. Cells with esterase activity cleave a fluorophore creating fluorescent events that can be detected by the solid-phase cytometer and verified visually by fluorescent microscopy.

Percent recovery (%R) was calculated relative to inoculum CFU or cell count via ScanRDI. Significance was determined using the Student t test, with significance set at P < .05. Solid-phase cytometry fluorescent events were verified visually by fluorescent microscopy as viable cells per swab or coupon and were compared to CFU per swab or coupon.

Results

The mean %R CFU varied with sampling device, surface type, and inoculum matrix, although %R for C. auris B11103 was consistently <1% (Table 1), and for AR0385 was <3%. Using cellulose for sampling, the %R was significantly greater when C. auris was suspended in ATS-M compared to ATS (P < .01) from both surfaces (Table 1). All subsequent studies used the ATS-M formula. The sampling mean %R was significantly higher using polyurethane than cellulose, regardless of surface type. Combining data for steel and plastic using B11103 in ATS-M, the mean %R for polyurethane was 0.41% (SD, 0.32%) and for cellulose was 0.15% (SD, 0.12%). For directly inoculated cellulose and polyurethane (positive controls), the %R ranged between 46% and 82% with significantly higher %R from polyurethane (P < .01). The mean %R decreased with increased drying time from both surfaces. When sampled with cellulose at 0, 20, and 40 minutes, mean %R from steel and plastic dropped from ∼50% to <1%, representing a CFU decrease of 2.5–3 log10. Furthermore, the variability in %R was also significant (P < .01) between the 2 strains evaluated on plastic surfaces using cellulose (Table 1). When examining eluates from swabs using ScanRDI, 1 log10 more C. auris cells were detected than by culture (Table 2). Although the %R was higher for swabs than for sponges, the surface area sampled was much smaller (26 cm2) and the results were much more variable (higher SD).

Table 1.

Percent Recovery of C. auris From 322 cm2 Steel and Plastic Coupons as Determined by Culture (CFU) a

Note. SD, standard deviation; CS, cellulose sponge; PS, polyurethane sponge; ATS, artificial test soil; ATS-M, artificial test soil with mucin (both formulations contain hemoglobin, proteins, carbohydrates, albumin, lipids, and vitamins to simulate body fluids).

a Variables tested were isolate, dry time, sampling tools and suspension matrix. Percent recovery was calculated relative to the inoculum (∼5 log/coupon).

b Experiments conducted on 2 or 3 independent days.

Table 2.

Recovery of C. auris From 26 cm2 Steel Coupona

Note. CFU, colony-forming units; SD, standard deviation; NA, not available.

a Cells detected by culture (CFU) and esterase activity (ScanRDI) from the inoculum suspension, from swabs after sampling the coupon, or from the coupon after sampling. Data represents mean of n=18 replicates.

b ScanRDI indicates the cells determined as viable by metabolic esterase activity.

c %R ScanRDI indicates the percent of viable cells determined by ScanRDI, relative to the ScanRDI inoculum.

d Titer of inoculum placed on coupons: 100 µL of 4.52 × 106/mL suspension.

Discussion

Candida auris has been found on surfaces in healthcare facilities Reference Chowdhary, Sharma and Meis1,Reference Vallabhaneni, Kallen and Tsay3,Reference Piedrahita, Cadnum, Jencson, Shaikh, Ghannoum and Donskey5 and has shown persistence extending ≥2 weeks. Reference Welsh, Bentz and Shams4,Reference Piedrahita, Cadnum, Jencson, Shaikh, Ghannoum and Donskey5 We found recovery by traditional culture methods to be low after only 40 minutes drying time, yet patient shedding and/or fomite contamination can occur throughout the day Reference Adams, Quinn and Tsay2 and can continue drying on surfaces for much longer than 1 hour. Our findings suggest that the actual contamination on surfaces may be 1–2 log10 greater than what is reflected by CFU counts. The mean %R by CFU from directly inoculated sponges was >60%, indicating that neither sampling device nor processing method influenced CFU or induced viable but nonculturable cells in C. auris. We also noted that the directly inoculated polyurethane yielded a higher mean %R than cellulose, indicating less cellular adherence to polyurethane, also noted by West-Deadwyler et al. Reference West-Deadwyler, Moulton-Meissner, Rose and Noble-Wang8 Furthermore, %R improved slightly from both steel and plastic surfaces using the polyurethane sponge, indicating better recovery of C. auris. The CDC recommends using swabs for sampling surface areas <26 cm2 and sponges for 645 cm2. 6

Although %R varied between C. auris strains, the low %R for both strains was unanticipated because the same methods applied to antimicrobial-resistant bacterial pathogens yielded higher %R, ranging from 7.7% (SD, 5.2%) for carbapenemase-producing Klebsiella pneumoniae to 58.9% (SD, 12.7%) for Clostridium difficile. Reference Houston, Rose, West-Deadwyler and Noble-Wang9

Background particle fluorescence from sponge materials prevented ScanRDI assay comparison to culture, but from swabs, we observed almost 50% cell recovery by esterase activity and only 9% by culture. Thus, although many cells are recovered, they are nonculturable. These results support previous findings that yeasts may become viable but nonculturable, Reference Welsh, Bentz and Shams4,Reference Mills, Johannsen and Cocolin10 though surprisingly, this occurred after only 1 hour of drying. If this holds true for other C. auris isolates, contamination on environmental surfaces could be 1 log10 higher than detected by culture and culture could misrepresent actual contamination by 2–3 log10. The question of cell resuscitation potential and subsequent transmission from healthcare surfaces is worth investigating. We are not aware of published evidence supporting C. auris resuscitation, though it has been reported in other yeasts. Reference Mills, Johannsen and Cocolin10

This study was limited to 2 C. auris strains, 2 simulated body fluids, and 3 sampling devices, and each of these can influence recovery efficiency. However, the data presented here contribute to creating a standard method for C. auris surface sampling and to interpreting results obtained in the context of an outbreak investigation or in evaluating environmental infection control interventions.

Acknowledgments

We acknowledge our colleagues Anastasia Litvintseva, Joseph Sexton, and Rory Welsh of the Mycotic Diseases Branch, Centers for Disease Control and Prevention, Atlanta, Georgia.

References herein to any specific commercial product, process, or service by trade name, trademark, manufacturer, or otherwise does not necessarily constitute or imply its endorsement, recommendation, or favoring by the US government.

Financial support

This project was supported in part by an appointment to the Science Education Programs at National Institutes of Health (NIH), administered by ORAU through the US Department of Energy Oak Ridge Institute for Science and Education.

Conflicts of interest

All authors report no conflict of interests relevant to this article.

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Figure 0

Table 1.Percent Recovery of C. auris From 322 cm2 Steel and Plastic Coupons as Determined by Culture (CFU)a

Figure 1

Table 2.Recovery of C. auris From 26 cm2 Steel Coupona