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Clostridioides difficile contamination in the environment of a clinical microbiology laboratory and laboratory workers
Clinical Microbiology and Infection xxx (xxxx) xxx
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Original article
Clostridioides difficile contamination in the environment of a clinical microbiology laboratory and laboratory workers zquez-Cuesta 1, 3, R. Onori 1, 3, L. Villar-Go mara 1, 2, 3, L. Alcala 1, 3, 4, E. Reigadas 1, 2, 3, *, S. Va ~ oz 1, 2, 3, 4, E. Bouza 1, 2, 3, 4, * M. Marín 1, 2, 3, 4, A. Martin 1, P. Mun ~o n, Madrid, Spain Department of Clinical Microbiology and Infectious Diseases, Hospital General Universitario Gregorio Maran Medicine Department, School of Medicine, Universidad Complutense de Madrid (UCM), Madrid, Spain 3) n Sanitaria Gregorio Maran ~o n, Madrid, Spain Instituto de Investigacio 4) CIBER de Enfermedades Respiratorias (CIBERES CB06/06/0058), Madrid, Spain 1) 2)
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 April 2019 Received in revised form 20 June 2019 Accepted 22 June 2019 Available online xxx
Objectives: Clostridioides difficile infection has traditionally been considered to be transmitted predominantly within health-care settings. It is not recognized as a pathogen that presents a risk of laboratory acquisition. Data on laboratory contamination and acquisition by laboratory personnel are lacking. Our objective was to assess environmental contamination by C. difficile and its potential for transmission in a clinical microbiology laboratory. Methods: Laboratory surfaces were screened for C. difficile. Samples were taken in areas that handle C. difficile isolates (high-exposure (HE) areas), areas adjacent to HE areas or those processing faecal samples (medium-exposure (ME) areas), and areas that do not process faecal samples or C. difficile isolates (low-exposure (LE) areas). We examined C. difficile carriage (hands/rectal samples) of laboratory workers. Results: A total of 140 environmental samples were collected from two HE areas (n ¼ 56), two ME areas (n ¼ 56) and two LE areas (n ¼ 28). Overall, 37.8% (37/98) of surfaces were contaminated with C. difficile, and 17.3% (17/98) with toxigenic C. difficile (TCD). HE areas were significantly more contaminated with TCD than LE areas (38.1% (16/42) versus 0.0% (0/14), p 0.005) and ME areas (38.1% (16/42) versus 2.4% (1/ 42), p <0.001). Hands were colonized with TCD in 11.8% (4/34) of cases. We found no rectal carriage of C. difficile. Conclusions: We found a significant proportion of laboratory surfaces to be contaminated with toxigenic C. difficile, as well as hand colonization of laboratory personnel. We recommend specific control measures for high-risk areas and laboratory personnel working in these areas. E. Reigadas, Clin Microbiol Infect 2019;▪:1 © 2019 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Editor: F. Allerberger Keywords: Clostridioides difficile Environmental contamination Polywipes Surface sampling Transmission
Introduction Clostridioides difficile infection (CDI) is the leading cause of hospital-acquired diarrhoea in developed countries [1e4]. It has traditionally been considered to be transmitted predominantly
* Corresponding authors. E. Reigadas Ramírez, Servicio de Microbiología Clínica y ~o n”, C/ Enfermedades Infecciosas, Hospital General Universitario “Gregorio Maran n Dr. Esquerdo, 46, 28007 Madrid, Spain; S.E. Bouza, Instituto de Investigacio ~o n, C/ Dr. Esquerdo, 46 28007 Madrid, Spain. Sanitaria Gregorio Maran E-mail addresses: [email protected] (E. Reigadas), [email protected] (E. Bouza).
within health-care settings, although this concept has recently been challenged [5]. The hospital environment seems to play an important role in cross transmission [6,7], and strains can also be transferred via the hands of healthcare workers [8]. Contact precautions are therefore required when CDI is suspected [9]. Clostridioides difficile has occasionally been shown to be transmissible to both patient-care staff and laboratory technicians [10e12], although the role of the environment in such transmission has not been properly evaluated. Spores of C. difficile can survive in the environment for long periods of time, are naturally resistant to environmental stress, and can attach to surfaces and remain viable, suffering only a 10%
https://doi.org/10.1016/j.cmi.2019.06.027 1198-743X/© 2019 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Please cite this article as: Reigadas E et al., Clostridioides difficile contamination in the environment of a clinical microbiology laboratory and laboratory workers, Clinical Microbiology and Infection, https://doi.org/10.1016/j.cmi.2019.06.027
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E. Reigadas et al. / Clinical Microbiology and Infection xxx (xxxx) xxx
reduction in viability after 1 year [13]. Spores are particularly difficult to eradicate with traditional cleaning methods, as they are resistant to the disinfectants used in health-care settings, such as quaternary ammonium compounds and ethanol-based products [14]. Although microbiology laboratories seem to be exposed to high spore concentrations of C. difficile, data on laboratory contamination are lacking and no particular safety precautions are recommended. Our objective was to assess environmental contamination by C. difficile in a clinical microbiology laboratory and its potential for transmission. Methods Setting, design and study population Our institution is a large teaching hospital with 1350 beds, serving a population of approximately 715 000 inhabitants, situated in Madrid, Spain. The clinical microbiology laboratory receives samples from patients hospitalized at our centre and from all the outpatient institutions in our catchment area. Systematic testing for toxigenic C. difficile is performed on all diarrhoeic stool samples from patients aged >2 years. The microbiology laboratory receives approximately 20 662 stool samples annually (data from 2018); of these, 8960 (43.4%) were stools for toxigenic C. difficile testing, with a positivity rate of 8.5%. The average indoor temperature is 22 Ce24 C and the relative humidity is 30%e35%. The staff working directly in laboratory areas comprise 12 clinical microbiologists, 12 residents and 28 laboratory technicians. The laboratory works 24 hours per day, 7 days per week. Most of the staff work in the morning shift, which is when most of the samples are processed. Daily routine cleaning is usually performed during the evenings with disinfectant products containing sodium hypochlorite.
whereas the low-exposure areas with no such activity were sampled once. An additional, final sample was taken in all areas after thorough cleaning with a disinfectant containing a high amount of chlorine. The laboratory surfaces sampled in each area included computer keyboard and mouse, laboratory bench top, multi-use soap dispenser, alcohol gel multi-use dispenser, tap and drawer pulls. Laboratory bench-top surfaces were measured (cm2) to be able to ensure that the bench-top surfaces sampled from each of the different areas were equivalent for comparison (approximately 19 845 cm2). All other object surfaces were also measured for equivalence and an estimate for each object (cm2) was performed (keyboard 759e854.59 cm2, mouse 161 193.75 cm2, multi-use soap dispenser 29.90 cm2, alcohol gel multi-use dispenser 18.90 cm2, tap 132 cm2 and drawer pulls 16 cm2. Surface samples were taken using a Polywipe™ pre-moistened sponge device (Medical Wire & Equipment Co., Corsham, UK). The Polywipe sponge was passed over the surface under study and rieux, Basingstoke, UK) placed on a ChromID C. difficile plate (BioMe directly at the time of capture. The plates were then incubated for 48 hours at 35 Ce37 C under anaerobic conditions. After the incubation period, C. difficile colonies were counted. Identification of colonies suspected of being toxigenic C. difficile was confirmed using an immunochromatographic system (C Diff Quik-Chek Complete assay, TechLab, Blacksburg, VA, USA). All the toxigenic strains were characterized using PCRribotyping according to the procedure described by Stubbs et al. [15]. Phylogenetic analysis of ribotyping profiles was performed using the unweighted pair group method with arithmetic mean (UPGMA) and Dice coefficients (BIONUMERICS 5.0). The profiles of our isolates were compared with international ribotyping profile libraries. Ribotypes were named using the international designation.
Surface sampling procedure
Laboratory personnel sampling
Laboratory surfaces were screened for C. difficile, and sampling was performed in areas that were categorized into three groups depending on the expected level of exposure to C. difficile. We defined high-exposure (HE) areas as those that handled C. difficile isolates. The areas adjacent to high-exposure areas and areas where faecal samples were processed were considered medium-exposure (ME) areas. Laboratory areas where faecal samples or C. difficile isolates were not processed were considered low-exposure (LE) areas. Two different areas were sampled for each group: two highexposure areas consisting of a C. difficile diagnostic area (HE 1 area; activities in this area included C. difficile rapid test performance, toxigenic culture interpretation, and strain isolation and conservation) and a molecular biology section (HE 2 area), where C. difficile ribotyping was performed. The two medium-exposure areas sampled were the bacteriology section, which was adjacent to the C. difficile diagnostic area (ME 1 area), and the sample reception and processing area, where faecal samples were cultured and stool viral antigen tests were performed (ME 2 area). The lowexposure areas sampled consisted of areas where strains other than C. difficile were identified and susceptibility testing of bacteria other than C. difficile was performed (LE 1 area) and the virology section (LE 2 area). Each of the high- and medium-exposure areas was sampled three times (before the morning shift work started working with faecal samples/isolates, during the morning shift while working with faecal samples/isolates, and after the morning shift ended),
Samples from voluntary laboratory personnel working in high-, medium- and low-exposure areas were collected. We examined C. difficile carriage on the hands and in rectal samples of laboratory workers who had previously consented. Hands were sampled on the same day as the environmental sampling was performed and rectal swabs were collected during that week and the following one. Hand sampling was performed by rubbing the complete surface of the hand using a Polywipe sponge, which was placed on a rieux) directly at the time of ChromID C. difficile plate (BioMe sampling. Rectal swabs were obtained by self-sampling on the condition that swabs contained macroscopically visible faecal matter; if not the sample was rejected, and another acceptable sample was obtained. The swabs used were Copan FecalSwabs™ (Copan Italia, Brescia, Italy) with liquid CaryeBlair medium. Once received, they were cultured on a ChromID C. difficile plate (Biorieux, Basingstoke, UK). Me
Data analysis Data were analysed using PASW STATISTICS FOR WINDOWS, Version 18.0 (SPSS Inc, Chicago, IL, USA). Qualitative variables appear with their frequency distribution. Quantitative variables are expressed as the median and interquartile range. Groups were compared using the Fisher exact test for categorical variables and the ManneWhitney or t test for continuous variables. A p value <0.05 was considered significant.
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an 85.0% reduction in the medium-exposure areas (from 253 to 38 CFU). Thirty-four laboratory workers were screened (5 residents, 7 attendings and 22 laboratory technicians). Of these, we detected hand contamination in ten workers (five from high-exposure areas, three from medium-exposure areas and two from low-exposure areas). The median CFU per positive hand sample for C. difficile was 4 CFU. Most workers were laboratory technicians (80%; 8/10), and 20% (2/10) were attendings. Toxigenic C. difficile was found on the hands of four laboratory workers (11.8% (4/34); all laboratory technicians working in high-exposure areas), with a median of 16 CFU (range 2e130 CFU/hand). Two of them were colonized with ribotype 014, one was colonized with ribotype 001 and the remaining one with ribotype 054. We sampled a plastic thimble used for quicker sorting of paper notes by the worker with the highest rate of hand carriage and observed that it was heavily contaminated (58 CFU). We obtained rectal swab samples from 25 laboratory workers and found no rectal carriage of C. difficile.
Ethical issues This study was approved by the Ethics Committee of Hospital ~o n. Informed consent was General Universitario Gregorio Maran obtained from laboratory personnel. Results A total of 140 environmental samples were collected from two high-exposure areas (n ¼ 56), two medium-exposure areas (n ¼ 56), and two low-exposure areas (n ¼ 28). Ninety-eight samples were collected with routine cleaning conditions at three different time-points (before (n ¼ 42), during (n ¼ 28) and after (n ¼ 28) working with faecal samples/isolates), and 42 samples were collected after an additional cleaning. Overall, 37.8% (37/98) of surfaces sampled with routine cleaning conditions were contaminated with C. difficile, and 17.3% (17/98) were contaminated with toxigenic C. difficile (Tables 1 and 2). High-exposure areas were significantly more contaminated with toxigenic C. difficile than low-exposure areas (38.1% (16/42) versus 0.0% (0/14), p 0.005) and medium-exposure areas (38.1% (16/ 42) versus 2.4% (1/42), p <0.001). The most contaminated highexposure area was the bench top for C. difficile DNA extraction, with a mean of 690 CFU/m2 (first morning sampling 1450 CFU/m2). Overall, we found six different toxigenic ribotypes in the 23 laboratory surfaces positive for toxigenic C. difficile, the most common ribotype was ribotype 014 (56.52% (13/23); all isolated from HE 2 area), followed by ribotype 106 (21.74% (5/23); all isolated from HE 1 area), ribotype 081 (8.69% (2/23); both from HE 2 area), ribotype 062 (4.35% (1/23) from HE 2 area), ribotype 001 (4.35% (1/23); from ME 1 area) and ribotype 056 (4.35% (1/23) from ME 1 area). Overall, all types of surfaces that were sampled were contaminated with C. difficile in any of the areas. The surfaces that were most frequently contaminated with C. difficile (toxigenic and nontoxigenic) were the taps (64.3% (9/14) of the samples from taps were positive for C. difficile), followed by the computer mouse (42.9%; 6/14), bench tops (42.9%; 6/14), and drawer pulls (35.70%; 5/14). The surfaces that were most frequently contaminated with toxigenic C. difficile were the bench tops (35.7%; 5/14), computer mouse (28.6%; 4/14), drawer pulls (21.4%; 3/14), and taps (14.3%; 2/ 14). The surfaces with the highest total CFU counts were the bench tops, followed by the soap dispensers. The most contaminated surface in terms of higher CFU/cm2 was the soap dispensers. After additional cleaning with a high-concentration of hypochlorite (35 g/L active chlorine), we observed a 93.3% decrease in the CFU count in high-exposure areas (from 2780 to 187 CFU) and
Discussion A high proportion of laboratory surfaces were contaminated with C. difficile after routine cleaning, and a considerable proportion were contaminated with toxigenic C. difficile corresponding mostly to ribotypes that are frequently encountered in our country [16]. We were able to detect several ‘grey zones’, which had an extremely high values for C. difficile CFU. After implementing additional cleaning with a high-chlorine solution, we observed a dramatic reduction in the CFU count in those areas. Few studies have assessed contamination by C. difficile outside the environment of the patient's room, that is, in other “at-risk units”. Torabi et al. evaluated the frequency of bacterial contamination in devices and staff from endoscopy and colonoscopy units and found C. difficile in 10.5% of imaging devices and 10.8% of environmental samples [17]; this percentage was lower than that observed in our study. We found that the percentage of surfaces contaminated with C. difficile was as high as that observed for the rooms of CDI patients. Environmental contamination of patients' surroundings is reported to be between 10% and 58%, with higher contamination rates in areas close to symptomatic patients than in those close to asymptomatic patients [18e22]. Few studies assess the degree of contamination per surface unit rather than the positivity rate [23,24]. In one, the degree of C. difficile contamination from the patients' environment ranged from of 0.013 to 2.56 CFU/cm2 [23]. Another study found that the highest concentration of C. difficile recovered from a room occupied by a CDI patient was 0.13 CFU/cm2 [24]. In our study, we found a
Table 1 Clostridioides difficile CFU counts in samples taken from high-exposure areas with routine cleaning conditions Surface sampled
High-exposure areas High-exposure Area 1 (culture and rapid tests) CFU before work
Bench top Keyboard Mouse Handles Soap dispenser Alcohol gel dispenser Tap a b c
0 0 4c 11c 146b 1b 3b
CFU during work b
2 0 4a 4b 31b 0 20b
High-exposure Area 2 (molecular biology) CFU after work c
3 0 5c 18b 18b 0 27b
CFU before work 1254 0 0 5a 0 0 0
a
CFU during work 593 0 0 0 0 0 2a
a
CFU after work 219a 28a 29a 35a 7a 8a 3a
Toxigenic C. difficile. non-toxigenic C. difficile. Both toxigenic and non-toxigenic C. difficile.
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Table 2 Clostridioides difficile CFU counts in samples taken from medium-exposure areas with routine cleaning conditions Surface sampled
Medium-exposure areas Medium-exposure Area 1
Bench top Keyboard Mouse Handles Soap dispenser Alcohol gel dispenser Tap
Medium-exposure Area 2
CFU before work
CFU during work
CFU after work
CFU before work
CFU during work
CFU after work
0 0 0 0 0 0 0
0 0 3b 0 0 0 1b
0 0 0 0 0 0 0
0 0 0 0 * * 0
0 0 2b 0 * * 0
1a 0 0 0 * * 0
*Items shared with high-exposure Area 1. a Toxigenic C. difficile. b non-toxigenic C. difficile.
median C. difficile concentration of 0.07 CFU/cm2 (range 0.0002e4.88 CFU/cm2), and the most contaminated laboratory site was almost twice as contaminated as the highest reported for clinical settings. Therefore, in theory, the risk of acquiring C. difficile in the laboratory is similar to or even higher than the risk run by other healthcare workers who come into direct contact with the patient. The highest contamination rates observed in the areas were C. difficile culture and ribotyping were performed suggest that laboratories in which C. difficile isolates are handled are at higher risk. Indeed, the four laboratory workers whose hands were colonized with toxigenic C. difficile worked in those areas. A recent review revealed colonization by C. difficile of the hands of health-care workers who provide direct care to patients with CDI (0%e59% of workers) [8], with a mean reported frequency of hand contamination of 11%, which is consistent with our results (11.8%). The frequency of hand contamination among health-care workers caring for CDI patients has been reported to correlate with the intensity of environmental contamination by C. difficile. Hence, no hand contamination was observed with environmental contamination of 0%e25%; the frequency of hand contamination was 8% when these levels were 26%e50% and 36% when they were >50% [19]. We were unable to find studies evaluating carriage of C. difficile among microbiology laboratory personnel. In our study, that the ribotypes in three out of the four laboratory workers whose hands were colonized with toxigenic ribotypes, matched with the ribotypes encountered in the areas in which they worked. In clinical settings, frequently touched surfaces with high degrees of contamination include toilet seats, sinks, call buttons, bed rails and telephones [14]. Within the laboratory, we also found that frequently touched surfaces, such as bench tops and soap dispensers, were more frequently contaminated. Interestingly, we found alcohol dispensers to be colonized with C. difficile. It is well known that C. difficile is not eradicated by alcohol, and the role of alcohol dispensers as potential fomites was recently studied. However, C. difficile was not recovered in studies sampling 30e120 alcohol dispensers [25,26]. We recovered C. difficile from three laboratory alcohol dispensers. We had previously recorded a similar finding, in alcohol dispensers from patients' rooms during a ribotype 027 outbreak that occurred in our hospital in 2014e2015. Environmental cleaning is essential for preventing hospital infections, although few data have been reported on practices for hospital environments not directly related to patients. Environmental decontamination of the rooms of patients with CDI using hypochlorite (diluted 1/10) or a sporicidal product is recommended [9]. We observed a reduction in the C. difficile CFU count of as high as 93% after intensive cleaning with a more concentrated
hypochlorite. Our data provide evidence that the regular routine cleaning of the laboratory is not enough and that targeted cleaning in the laboratory can reduce C. difficile contamination. In this sense, cleaning should be reinforced to avoid cross-contamination of samples and for protection of laboratory personnel, especially in laboratories where C. difficile culture and ribotyping are performed. These areas should be cleaned with the highest chlorine proportion, and should be cleaned at least once per working shift. The term ‘grey zones’ applied to cleaning is used to refer to those areas or surfaces where cleaning is not performed or where it is suboptimal. Grey zones have been identified in hospital environments where there is contact with patients [27]. Here, we identified grey zones in the microbiology laboratory. Surfaces in close proximity to equipment make cleaning more difficult, as do items that do not specifically fall under the responsibility of housekeeping staff or in which cleaning responsibilities rely on different staff/ departments, so often leading to a cleaning gap. Our study is limited by the fact that it is a single-centre study with a limited number of samples from laboratory workers, additional multi-centre studies are needed. As for rectal carriage of C. difficile, samples were obtained using swabs that were diluted in liquid medium; the dilution process and the lack of an enrichment step before culture could have led to an underestimation of the carriage rate. Also, ribotyping of the patients' isolates handled at the time of the environmental sampling was not performed. However, to the best of our knowledge, our study is the first to address contamination of microbiology laboratory surfaces and laboratory personnel by C. difficile. In conclusion, we found a significant proportion of laboratory surfaces to be contaminated with toxigenic C. difficile, as well as hand colonization of laboratory personnel managing this microorganism. We recommend specific control measures for high-risk areas and for laboratory personnel working in high-risk areas. These include disposable gloves and gowns and strict disinfection of hands with water and soap. Materials used in the diagnosis of C. difficile and frequently touched surfaces should be routinely decontaminated with disinfectants containing a high proportion of chlorine. Transparency declarations The authors declare no conflicts of interest. Funding source This study was financed by Fondo de Investigaciones Sanitarias (FIS), Research Project number PI13/00687 and PI16/00490, and by the European Regional Development Fund (FEDER) ‘A way of
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making Europe’. Laura Villar holds a grant from the Complutense University of Madrid (Beca UCM, CT27/16eCT28/16). Acknowledgements We thank Thomas O'Boyle for his help in the preparation of the manuscript. We are particularly grateful to the laboratory workers who agreed to participate in the study. Some of the results of this study were presented in poster form at the 28th European Congress of Clinical Microbiology and Infectious Diseases (21e24 April, 2018, Madrid, Spain). References [1] Dubberke ER, Olsen MA. Burden of Clostridium difficile on the healthcare system. Clin Infect Dis 2012;55:S88e92. [2] Wiegand PN, Nathwani D, Wilcox MH, Stephens J, Shelbaya A, Haider S. Clinical and economic burden of Clostridium difficile infection in Europe: a systematic review of healthcare-facility-acquired infection. J Hosp Infect 2012;81:1e14. [3] Asensio A, Bouza E, Grau S, Rubio-Rodriguez D, Rubio-Terres C. Cost of Clostridium difficile associated diarrhea in Spain. Rev Esp Salud Publica 2013;87: 25e33. [4] Miller MA, Hyland M, Ofner-Agostini M, Gourdeau M, Ishak M. Morbidity, mortality, and healthcare burden of nosocomial Clostridium difficile-associated diarrhea in Canadian hospitals. Infect Control Hosp Epidemiol 2002;23: 137e40. [5] Durovic A, Widmer AF, Tschudin-Sutter S. New insights into transmission of Clostridium difficile infection-narrative review. Clin Microbiol Infect 2018;24: 483e92. [6] Barbut F, Menuet D, Verachten M, Girou E. Comparison of the efficacy of a hydrogen peroxide dry-mist disinfection system and sodium hypochlorite solution for eradication of Clostridium difficile spores. Infect Control Hosp Epidemiol 2009;30:507e14. [7] Weber DJ, Anderson DJ, Sexton DJ, Rutala WA. Role of the environment in the transmission of Clostridium difficile in health care facilities. Am J Infect Control 2013;41:S105e10. [8] Jullian-Desayes I, Landelle C, Mallaret MR, Brun-Buisson C, Barbut F. Clostridium difficile contamination of health care workers' hands and its potential contribution to the spread of infection: review of the literature. Am J Infect Control 2017;45:51e8. [9] Vonberg RP, Kuijper EJ, Wilcox MH, Barbut F, Tull P, Gastmeier P, et al. Infection control measures to limit the spread of Clostridium difficile. Clin Microbiol Infect 2008;14:2e20. [10] Kaplan N, Davies A, Davies P. Clostridium difficile in a healthcare worker. J Hosp Infect 1996;32:322.
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Please cite this article as: Reigadas E et al., Clostridioides difficile contamination in the environment of a clinical microbiology laboratory and laboratory workers, Clinical Microbiology and Infection, https://doi.org/10.1016/j.cmi.2019.06.027
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Original article
Clostridioides difficile contamination in the environment of a clinical microbiology laboratory and laboratory workers zquez-Cuesta 1, 3, R. Onori 1, 3, L. Villar-Go mara 1, 2, 3, L. Alcala 1, 3, 4, E. Reigadas 1, 2, 3, *, S. Va ~ oz 1, 2, 3, 4, E. Bouza 1, 2, 3, 4, * M. Marín 1, 2, 3, 4, A. Martin 1, P. Mun ~o n, Madrid, Spain Department of Clinical Microbiology and Infectious Diseases, Hospital General Universitario Gregorio Maran Medicine Department, School of Medicine, Universidad Complutense de Madrid (UCM), Madrid, Spain 3) n Sanitaria Gregorio Maran ~o n, Madrid, Spain Instituto de Investigacio 4) CIBER de Enfermedades Respiratorias (CIBERES CB06/06/0058), Madrid, Spain 1) 2)
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 April 2019 Received in revised form 20 June 2019 Accepted 22 June 2019 Available online xxx
Objectives: Clostridioides difficile infection has traditionally been considered to be transmitted predominantly within health-care settings. It is not recognized as a pathogen that presents a risk of laboratory acquisition. Data on laboratory contamination and acquisition by laboratory personnel are lacking. Our objective was to assess environmental contamination by C. difficile and its potential for transmission in a clinical microbiology laboratory. Methods: Laboratory surfaces were screened for C. difficile. Samples were taken in areas that handle C. difficile isolates (high-exposure (HE) areas), areas adjacent to HE areas or those processing faecal samples (medium-exposure (ME) areas), and areas that do not process faecal samples or C. difficile isolates (low-exposure (LE) areas). We examined C. difficile carriage (hands/rectal samples) of laboratory workers. Results: A total of 140 environmental samples were collected from two HE areas (n ¼ 56), two ME areas (n ¼ 56) and two LE areas (n ¼ 28). Overall, 37.8% (37/98) of surfaces were contaminated with C. difficile, and 17.3% (17/98) with toxigenic C. difficile (TCD). HE areas were significantly more contaminated with TCD than LE areas (38.1% (16/42) versus 0.0% (0/14), p 0.005) and ME areas (38.1% (16/42) versus 2.4% (1/ 42), p <0.001). Hands were colonized with TCD in 11.8% (4/34) of cases. We found no rectal carriage of C. difficile. Conclusions: We found a significant proportion of laboratory surfaces to be contaminated with toxigenic C. difficile, as well as hand colonization of laboratory personnel. We recommend specific control measures for high-risk areas and laboratory personnel working in these areas. E. Reigadas, Clin Microbiol Infect 2019;▪:1 © 2019 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Editor: F. Allerberger Keywords: Clostridioides difficile Environmental contamination Polywipes Surface sampling Transmission
Introduction Clostridioides difficile infection (CDI) is the leading cause of hospital-acquired diarrhoea in developed countries [1e4]. It has traditionally been considered to be transmitted predominantly
* Corresponding authors. E. Reigadas Ramírez, Servicio de Microbiología Clínica y ~o n”, C/ Enfermedades Infecciosas, Hospital General Universitario “Gregorio Maran n Dr. Esquerdo, 46, 28007 Madrid, Spain; S.E. Bouza, Instituto de Investigacio ~o n, C/ Dr. Esquerdo, 46 28007 Madrid, Spain. Sanitaria Gregorio Maran E-mail addresses: [email protected] (E. Reigadas), [email protected] (E. Bouza).
within health-care settings, although this concept has recently been challenged [5]. The hospital environment seems to play an important role in cross transmission [6,7], and strains can also be transferred via the hands of healthcare workers [8]. Contact precautions are therefore required when CDI is suspected [9]. Clostridioides difficile has occasionally been shown to be transmissible to both patient-care staff and laboratory technicians [10e12], although the role of the environment in such transmission has not been properly evaluated. Spores of C. difficile can survive in the environment for long periods of time, are naturally resistant to environmental stress, and can attach to surfaces and remain viable, suffering only a 10%
https://doi.org/10.1016/j.cmi.2019.06.027 1198-743X/© 2019 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Please cite this article as: Reigadas E et al., Clostridioides difficile contamination in the environment of a clinical microbiology laboratory and laboratory workers, Clinical Microbiology and Infection, https://doi.org/10.1016/j.cmi.2019.06.027
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reduction in viability after 1 year [13]. Spores are particularly difficult to eradicate with traditional cleaning methods, as they are resistant to the disinfectants used in health-care settings, such as quaternary ammonium compounds and ethanol-based products [14]. Although microbiology laboratories seem to be exposed to high spore concentrations of C. difficile, data on laboratory contamination are lacking and no particular safety precautions are recommended. Our objective was to assess environmental contamination by C. difficile in a clinical microbiology laboratory and its potential for transmission. Methods Setting, design and study population Our institution is a large teaching hospital with 1350 beds, serving a population of approximately 715 000 inhabitants, situated in Madrid, Spain. The clinical microbiology laboratory receives samples from patients hospitalized at our centre and from all the outpatient institutions in our catchment area. Systematic testing for toxigenic C. difficile is performed on all diarrhoeic stool samples from patients aged >2 years. The microbiology laboratory receives approximately 20 662 stool samples annually (data from 2018); of these, 8960 (43.4%) were stools for toxigenic C. difficile testing, with a positivity rate of 8.5%. The average indoor temperature is 22 Ce24 C and the relative humidity is 30%e35%. The staff working directly in laboratory areas comprise 12 clinical microbiologists, 12 residents and 28 laboratory technicians. The laboratory works 24 hours per day, 7 days per week. Most of the staff work in the morning shift, which is when most of the samples are processed. Daily routine cleaning is usually performed during the evenings with disinfectant products containing sodium hypochlorite.
whereas the low-exposure areas with no such activity were sampled once. An additional, final sample was taken in all areas after thorough cleaning with a disinfectant containing a high amount of chlorine. The laboratory surfaces sampled in each area included computer keyboard and mouse, laboratory bench top, multi-use soap dispenser, alcohol gel multi-use dispenser, tap and drawer pulls. Laboratory bench-top surfaces were measured (cm2) to be able to ensure that the bench-top surfaces sampled from each of the different areas were equivalent for comparison (approximately 19 845 cm2). All other object surfaces were also measured for equivalence and an estimate for each object (cm2) was performed (keyboard 759e854.59 cm2, mouse 161 193.75 cm2, multi-use soap dispenser 29.90 cm2, alcohol gel multi-use dispenser 18.90 cm2, tap 132 cm2 and drawer pulls 16 cm2. Surface samples were taken using a Polywipe™ pre-moistened sponge device (Medical Wire & Equipment Co., Corsham, UK). The Polywipe sponge was passed over the surface under study and rieux, Basingstoke, UK) placed on a ChromID C. difficile plate (BioMe directly at the time of capture. The plates were then incubated for 48 hours at 35 Ce37 C under anaerobic conditions. After the incubation period, C. difficile colonies were counted. Identification of colonies suspected of being toxigenic C. difficile was confirmed using an immunochromatographic system (C Diff Quik-Chek Complete assay, TechLab, Blacksburg, VA, USA). All the toxigenic strains were characterized using PCRribotyping according to the procedure described by Stubbs et al. [15]. Phylogenetic analysis of ribotyping profiles was performed using the unweighted pair group method with arithmetic mean (UPGMA) and Dice coefficients (BIONUMERICS 5.0). The profiles of our isolates were compared with international ribotyping profile libraries. Ribotypes were named using the international designation.
Surface sampling procedure
Laboratory personnel sampling
Laboratory surfaces were screened for C. difficile, and sampling was performed in areas that were categorized into three groups depending on the expected level of exposure to C. difficile. We defined high-exposure (HE) areas as those that handled C. difficile isolates. The areas adjacent to high-exposure areas and areas where faecal samples were processed were considered medium-exposure (ME) areas. Laboratory areas where faecal samples or C. difficile isolates were not processed were considered low-exposure (LE) areas. Two different areas were sampled for each group: two highexposure areas consisting of a C. difficile diagnostic area (HE 1 area; activities in this area included C. difficile rapid test performance, toxigenic culture interpretation, and strain isolation and conservation) and a molecular biology section (HE 2 area), where C. difficile ribotyping was performed. The two medium-exposure areas sampled were the bacteriology section, which was adjacent to the C. difficile diagnostic area (ME 1 area), and the sample reception and processing area, where faecal samples were cultured and stool viral antigen tests were performed (ME 2 area). The lowexposure areas sampled consisted of areas where strains other than C. difficile were identified and susceptibility testing of bacteria other than C. difficile was performed (LE 1 area) and the virology section (LE 2 area). Each of the high- and medium-exposure areas was sampled three times (before the morning shift work started working with faecal samples/isolates, during the morning shift while working with faecal samples/isolates, and after the morning shift ended),
Samples from voluntary laboratory personnel working in high-, medium- and low-exposure areas were collected. We examined C. difficile carriage on the hands and in rectal samples of laboratory workers who had previously consented. Hands were sampled on the same day as the environmental sampling was performed and rectal swabs were collected during that week and the following one. Hand sampling was performed by rubbing the complete surface of the hand using a Polywipe sponge, which was placed on a rieux) directly at the time of ChromID C. difficile plate (BioMe sampling. Rectal swabs were obtained by self-sampling on the condition that swabs contained macroscopically visible faecal matter; if not the sample was rejected, and another acceptable sample was obtained. The swabs used were Copan FecalSwabs™ (Copan Italia, Brescia, Italy) with liquid CaryeBlair medium. Once received, they were cultured on a ChromID C. difficile plate (Biorieux, Basingstoke, UK). Me
Data analysis Data were analysed using PASW STATISTICS FOR WINDOWS, Version 18.0 (SPSS Inc, Chicago, IL, USA). Qualitative variables appear with their frequency distribution. Quantitative variables are expressed as the median and interquartile range. Groups were compared using the Fisher exact test for categorical variables and the ManneWhitney or t test for continuous variables. A p value <0.05 was considered significant.
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an 85.0% reduction in the medium-exposure areas (from 253 to 38 CFU). Thirty-four laboratory workers were screened (5 residents, 7 attendings and 22 laboratory technicians). Of these, we detected hand contamination in ten workers (five from high-exposure areas, three from medium-exposure areas and two from low-exposure areas). The median CFU per positive hand sample for C. difficile was 4 CFU. Most workers were laboratory technicians (80%; 8/10), and 20% (2/10) were attendings. Toxigenic C. difficile was found on the hands of four laboratory workers (11.8% (4/34); all laboratory technicians working in high-exposure areas), with a median of 16 CFU (range 2e130 CFU/hand). Two of them were colonized with ribotype 014, one was colonized with ribotype 001 and the remaining one with ribotype 054. We sampled a plastic thimble used for quicker sorting of paper notes by the worker with the highest rate of hand carriage and observed that it was heavily contaminated (58 CFU). We obtained rectal swab samples from 25 laboratory workers and found no rectal carriage of C. difficile.
Ethical issues This study was approved by the Ethics Committee of Hospital ~o n. Informed consent was General Universitario Gregorio Maran obtained from laboratory personnel. Results A total of 140 environmental samples were collected from two high-exposure areas (n ¼ 56), two medium-exposure areas (n ¼ 56), and two low-exposure areas (n ¼ 28). Ninety-eight samples were collected with routine cleaning conditions at three different time-points (before (n ¼ 42), during (n ¼ 28) and after (n ¼ 28) working with faecal samples/isolates), and 42 samples were collected after an additional cleaning. Overall, 37.8% (37/98) of surfaces sampled with routine cleaning conditions were contaminated with C. difficile, and 17.3% (17/98) were contaminated with toxigenic C. difficile (Tables 1 and 2). High-exposure areas were significantly more contaminated with toxigenic C. difficile than low-exposure areas (38.1% (16/42) versus 0.0% (0/14), p 0.005) and medium-exposure areas (38.1% (16/ 42) versus 2.4% (1/42), p <0.001). The most contaminated highexposure area was the bench top for C. difficile DNA extraction, with a mean of 690 CFU/m2 (first morning sampling 1450 CFU/m2). Overall, we found six different toxigenic ribotypes in the 23 laboratory surfaces positive for toxigenic C. difficile, the most common ribotype was ribotype 014 (56.52% (13/23); all isolated from HE 2 area), followed by ribotype 106 (21.74% (5/23); all isolated from HE 1 area), ribotype 081 (8.69% (2/23); both from HE 2 area), ribotype 062 (4.35% (1/23) from HE 2 area), ribotype 001 (4.35% (1/23); from ME 1 area) and ribotype 056 (4.35% (1/23) from ME 1 area). Overall, all types of surfaces that were sampled were contaminated with C. difficile in any of the areas. The surfaces that were most frequently contaminated with C. difficile (toxigenic and nontoxigenic) were the taps (64.3% (9/14) of the samples from taps were positive for C. difficile), followed by the computer mouse (42.9%; 6/14), bench tops (42.9%; 6/14), and drawer pulls (35.70%; 5/14). The surfaces that were most frequently contaminated with toxigenic C. difficile were the bench tops (35.7%; 5/14), computer mouse (28.6%; 4/14), drawer pulls (21.4%; 3/14), and taps (14.3%; 2/ 14). The surfaces with the highest total CFU counts were the bench tops, followed by the soap dispensers. The most contaminated surface in terms of higher CFU/cm2 was the soap dispensers. After additional cleaning with a high-concentration of hypochlorite (35 g/L active chlorine), we observed a 93.3% decrease in the CFU count in high-exposure areas (from 2780 to 187 CFU) and
Discussion A high proportion of laboratory surfaces were contaminated with C. difficile after routine cleaning, and a considerable proportion were contaminated with toxigenic C. difficile corresponding mostly to ribotypes that are frequently encountered in our country [16]. We were able to detect several ‘grey zones’, which had an extremely high values for C. difficile CFU. After implementing additional cleaning with a high-chlorine solution, we observed a dramatic reduction in the CFU count in those areas. Few studies have assessed contamination by C. difficile outside the environment of the patient's room, that is, in other “at-risk units”. Torabi et al. evaluated the frequency of bacterial contamination in devices and staff from endoscopy and colonoscopy units and found C. difficile in 10.5% of imaging devices and 10.8% of environmental samples [17]; this percentage was lower than that observed in our study. We found that the percentage of surfaces contaminated with C. difficile was as high as that observed for the rooms of CDI patients. Environmental contamination of patients' surroundings is reported to be between 10% and 58%, with higher contamination rates in areas close to symptomatic patients than in those close to asymptomatic patients [18e22]. Few studies assess the degree of contamination per surface unit rather than the positivity rate [23,24]. In one, the degree of C. difficile contamination from the patients' environment ranged from of 0.013 to 2.56 CFU/cm2 [23]. Another study found that the highest concentration of C. difficile recovered from a room occupied by a CDI patient was 0.13 CFU/cm2 [24]. In our study, we found a
Table 1 Clostridioides difficile CFU counts in samples taken from high-exposure areas with routine cleaning conditions Surface sampled
High-exposure areas High-exposure Area 1 (culture and rapid tests) CFU before work
Bench top Keyboard Mouse Handles Soap dispenser Alcohol gel dispenser Tap a b c
0 0 4c 11c 146b 1b 3b
CFU during work b
2 0 4a 4b 31b 0 20b
High-exposure Area 2 (molecular biology) CFU after work c
3 0 5c 18b 18b 0 27b
CFU before work 1254 0 0 5a 0 0 0
a
CFU during work 593 0 0 0 0 0 2a
a
CFU after work 219a 28a 29a 35a 7a 8a 3a
Toxigenic C. difficile. non-toxigenic C. difficile. Both toxigenic and non-toxigenic C. difficile.
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Table 2 Clostridioides difficile CFU counts in samples taken from medium-exposure areas with routine cleaning conditions Surface sampled
Medium-exposure areas Medium-exposure Area 1
Bench top Keyboard Mouse Handles Soap dispenser Alcohol gel dispenser Tap
Medium-exposure Area 2
CFU before work
CFU during work
CFU after work
CFU before work
CFU during work
CFU after work
0 0 0 0 0 0 0
0 0 3b 0 0 0 1b
0 0 0 0 0 0 0
0 0 0 0 * * 0
0 0 2b 0 * * 0
1a 0 0 0 * * 0
*Items shared with high-exposure Area 1. a Toxigenic C. difficile. b non-toxigenic C. difficile.
median C. difficile concentration of 0.07 CFU/cm2 (range 0.0002e4.88 CFU/cm2), and the most contaminated laboratory site was almost twice as contaminated as the highest reported for clinical settings. Therefore, in theory, the risk of acquiring C. difficile in the laboratory is similar to or even higher than the risk run by other healthcare workers who come into direct contact with the patient. The highest contamination rates observed in the areas were C. difficile culture and ribotyping were performed suggest that laboratories in which C. difficile isolates are handled are at higher risk. Indeed, the four laboratory workers whose hands were colonized with toxigenic C. difficile worked in those areas. A recent review revealed colonization by C. difficile of the hands of health-care workers who provide direct care to patients with CDI (0%e59% of workers) [8], with a mean reported frequency of hand contamination of 11%, which is consistent with our results (11.8%). The frequency of hand contamination among health-care workers caring for CDI patients has been reported to correlate with the intensity of environmental contamination by C. difficile. Hence, no hand contamination was observed with environmental contamination of 0%e25%; the frequency of hand contamination was 8% when these levels were 26%e50% and 36% when they were >50% [19]. We were unable to find studies evaluating carriage of C. difficile among microbiology laboratory personnel. In our study, that the ribotypes in three out of the four laboratory workers whose hands were colonized with toxigenic ribotypes, matched with the ribotypes encountered in the areas in which they worked. In clinical settings, frequently touched surfaces with high degrees of contamination include toilet seats, sinks, call buttons, bed rails and telephones [14]. Within the laboratory, we also found that frequently touched surfaces, such as bench tops and soap dispensers, were more frequently contaminated. Interestingly, we found alcohol dispensers to be colonized with C. difficile. It is well known that C. difficile is not eradicated by alcohol, and the role of alcohol dispensers as potential fomites was recently studied. However, C. difficile was not recovered in studies sampling 30e120 alcohol dispensers [25,26]. We recovered C. difficile from three laboratory alcohol dispensers. We had previously recorded a similar finding, in alcohol dispensers from patients' rooms during a ribotype 027 outbreak that occurred in our hospital in 2014e2015. Environmental cleaning is essential for preventing hospital infections, although few data have been reported on practices for hospital environments not directly related to patients. Environmental decontamination of the rooms of patients with CDI using hypochlorite (diluted 1/10) or a sporicidal product is recommended [9]. We observed a reduction in the C. difficile CFU count of as high as 93% after intensive cleaning with a more concentrated
hypochlorite. Our data provide evidence that the regular routine cleaning of the laboratory is not enough and that targeted cleaning in the laboratory can reduce C. difficile contamination. In this sense, cleaning should be reinforced to avoid cross-contamination of samples and for protection of laboratory personnel, especially in laboratories where C. difficile culture and ribotyping are performed. These areas should be cleaned with the highest chlorine proportion, and should be cleaned at least once per working shift. The term ‘grey zones’ applied to cleaning is used to refer to those areas or surfaces where cleaning is not performed or where it is suboptimal. Grey zones have been identified in hospital environments where there is contact with patients [27]. Here, we identified grey zones in the microbiology laboratory. Surfaces in close proximity to equipment make cleaning more difficult, as do items that do not specifically fall under the responsibility of housekeeping staff or in which cleaning responsibilities rely on different staff/ departments, so often leading to a cleaning gap. Our study is limited by the fact that it is a single-centre study with a limited number of samples from laboratory workers, additional multi-centre studies are needed. As for rectal carriage of C. difficile, samples were obtained using swabs that were diluted in liquid medium; the dilution process and the lack of an enrichment step before culture could have led to an underestimation of the carriage rate. Also, ribotyping of the patients' isolates handled at the time of the environmental sampling was not performed. However, to the best of our knowledge, our study is the first to address contamination of microbiology laboratory surfaces and laboratory personnel by C. difficile. In conclusion, we found a significant proportion of laboratory surfaces to be contaminated with toxigenic C. difficile, as well as hand colonization of laboratory personnel managing this microorganism. We recommend specific control measures for high-risk areas and for laboratory personnel working in high-risk areas. These include disposable gloves and gowns and strict disinfection of hands with water and soap. Materials used in the diagnosis of C. difficile and frequently touched surfaces should be routinely decontaminated with disinfectants containing a high proportion of chlorine. Transparency declarations The authors declare no conflicts of interest. Funding source This study was financed by Fondo de Investigaciones Sanitarias (FIS), Research Project number PI13/00687 and PI16/00490, and by the European Regional Development Fund (FEDER) ‘A way of
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making Europe’. Laura Villar holds a grant from the Complutense University of Madrid (Beca UCM, CT27/16eCT28/16). Acknowledgements We thank Thomas O'Boyle for his help in the preparation of the manuscript. We are particularly grateful to the laboratory workers who agreed to participate in the study. Some of the results of this study were presented in poster form at the 28th European Congress of Clinical Microbiology and Infectious Diseases (21e24 April, 2018, Madrid, Spain). References [1] Dubberke ER, Olsen MA. Burden of Clostridium difficile on the healthcare system. Clin Infect Dis 2012;55:S88e92. [2] Wiegand PN, Nathwani D, Wilcox MH, Stephens J, Shelbaya A, Haider S. Clinical and economic burden of Clostridium difficile infection in Europe: a systematic review of healthcare-facility-acquired infection. J Hosp Infect 2012;81:1e14. [3] Asensio A, Bouza E, Grau S, Rubio-Rodriguez D, Rubio-Terres C. Cost of Clostridium difficile associated diarrhea in Spain. Rev Esp Salud Publica 2013;87: 25e33. [4] Miller MA, Hyland M, Ofner-Agostini M, Gourdeau M, Ishak M. Morbidity, mortality, and healthcare burden of nosocomial Clostridium difficile-associated diarrhea in Canadian hospitals. Infect Control Hosp Epidemiol 2002;23: 137e40. [5] Durovic A, Widmer AF, Tschudin-Sutter S. New insights into transmission of Clostridium difficile infection-narrative review. Clin Microbiol Infect 2018;24: 483e92. [6] Barbut F, Menuet D, Verachten M, Girou E. Comparison of the efficacy of a hydrogen peroxide dry-mist disinfection system and sodium hypochlorite solution for eradication of Clostridium difficile spores. Infect Control Hosp Epidemiol 2009;30:507e14. [7] Weber DJ, Anderson DJ, Sexton DJ, Rutala WA. Role of the environment in the transmission of Clostridium difficile in health care facilities. Am J Infect Control 2013;41:S105e10. [8] Jullian-Desayes I, Landelle C, Mallaret MR, Brun-Buisson C, Barbut F. Clostridium difficile contamination of health care workers' hands and its potential contribution to the spread of infection: review of the literature. Am J Infect Control 2017;45:51e8. [9] Vonberg RP, Kuijper EJ, Wilcox MH, Barbut F, Tull P, Gastmeier P, et al. Infection control measures to limit the spread of Clostridium difficile. Clin Microbiol Infect 2008;14:2e20. [10] Kaplan N, Davies A, Davies P. Clostridium difficile in a healthcare worker. J Hosp Infect 1996;32:322.
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Please cite this article as: Reigadas E et al., Clostridioides difficile contamination in the environment of a clinical microbiology laboratory and laboratory workers, Clinical Microbiology and Infection, https://doi.org/10.1016/j.cmi.2019.06.027