APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 2002, p. 2576–2579 0099-2240/02/$04.00⫹0 DOI: 10.1128/AEM.68.5.2576–2579.2002 Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Vol. 68, No. 5
Efficacy of Common Laboratory Disinfectants on the Infectivity of Cryptosporidium parvum Oocysts in Cell Culture Susan C. Weir, Nicholas J. Pokorny, Ramon A. Carreno,† Jack T. Trevors,* and Hung Lee* Department of Environmental Biology, University of Guelph, Guelph, Ontario, Canada, N1G 2W1 Received 27 September 2001/Accepted 2 March 2002
Nine liquid disinfectants were tested for their ability to reduce infectivity of Cryptosporidium parvum oocysts in cell culture. A 4-min exposure to 6% hydrogen peroxide and a 13-min exposure to ammonium hydroxideamended windshield washer fluid reduced infectivity 1,000-fold. Other disinfectants tested (70% ethanol, 37% methanol, 6% sodium hypochlorite, 70% isopropanol, and three commercial disinfectants) did not reduce the infectivity after a 33-min exposure. The results indicate that hydrogen peroxide and windshield washer fluid or ammonium hydroxide disinfectant may be suitable laboratory disinfectants against C. parvum oocysts. Cryptosporidium parvum is a waterborne parasite that causes a diarrheal illness. While normally a self-limiting disease, infection in immunocompromised individuals is chronic and potentially fatal. There is no effective treatment for cryptosporidiosis. Furthermore, C. parvum oocysts are resistant to chlorine, which is normally used in water treatment. C. parvum oocysts are infectious for humans, with a 50% infective dose of 132 oocysts in human volunteer studies (5). Research on survival, persistence, disinfection, and treatment of C. parvum is necessary. However, safe handling of C. parvum oocysts in the laboratory requires appropriate measures for disinfection and inactivation of spills. Information on the efficacy of disinfectants on oocyst infectivity is essential for their evaluation and potential use in the laboratory, as effective disinfectants and decontamination procedures are required in laboratories working with infectious agents. Previous studies have shown that vital dye staining and excystation rates may overestimate the number of infectious oocysts present (2, 4, 8, 9). Infectivity assays using animals are expensive, and it is difficult to obtain quantitative results. However, in vitro infectivity assays are useful for quantitatively measuring antimicrobial effects of disinfectants against C. parvum oocysts (4, 9). Commonly used laboratory disinfectants include bleach (hypochlorite), ethanol (70%, vol/vol), and a variety of commercial preparations, such as Wescodyne. Useful disinfectants can be sprayed or poured onto laboratory surfaces and, after a brief period, cleaned up with paper towels for subsequent disposal. Laboratory disinfectants should kill target organisms in a short period of time. Incubations of disinfectants on spilled microorganisms longer than an hour may be impractical and unreasonable. The purpose of this research was to evaluate the efficacy of selected laboratory disinfectants against C. parvum oocysts after 4, 13, and 33 min of exposure. Cell cultures were grown and maintained as described previously (4). Briefly, human ileocecal adenocarcinoma (HCT-8)
cells (ATCC CCL-244) (American Type Culture Collection, Manassas, Va.) were grown in a maintenance medium: RPMI 1640 (Fisher Scientific, Mississauga, Ontario, Canada) supplemented with 5% (vol/vol) fetal bovine serum, 2 mM L-glutamine, 20 mM HEPES (ICN Biomedicals, Aurora, Ohio), 10% (vol/vol) Opti-MEM (Life Technologies, GIBCO BRL, Burlington, Ontario, Canada), 62.5 g of penicillin per ml, 100 g of streptomycin per ml, and 0.125 g of amphotericin per ml (all antibiotics were from Sigma-Aldrich Canada, Oakville, Ontario, Canada). Cells were maintained in 75-mm2 culture flasks at 37°C under a 5% CO2 atmosphere. Cells were passaged every 3 to 4 days by trypsinization with trypsin-EDTA (ICN Biomedicals Inc.). During infectivity experiments, trypsinized cells from 95 to 100% confluent cultures in 75-mm2 flasks were washed and suspended in 5 ml of maintenance medium. Cells were diluted 20-fold in maintenance medium, and 750-l aliquots were added to each chamber in an eightwell chamber slide (Falcon culture slides; Becton Dickinson, Franklin Lakes, N.J.). Slides were incubated at 37°C under an atmosphere of 5% CO2. After 24 h, the medium was removed and replaced with 650 l of growth medium, which was maintenance medium with the concentration of fetal bovine serum increased to 10% (vol/vol). Purified oocysts of C. parvum (GCH1 isolate) were obtained from the AIDS Research and Reference Reagent Program, Division of Acquired Immunodeficiency Syndrome, National Institute of Allergy and Infectious Diseases, National Institutes of Health, through McKesson HBOC BioServices, Rockville, Md. All experiments were performed with oocysts between 5 and 6 months old. The oocysts were stored in 2.5% (wt/vol) potassium dichromate at 4°C throughout the experimentation period. For each disinfectant, three replicates of 3.6 ⫻ 106 oocysts were each washed twice with sterile phosphate-buffered saline (PBS) (pH 7.2) in sterile 1.5-ml microcentrifuge tubes. Oocysts were centrifuged at 11,750 ⫻ g at 22 to 24°C, and the supernatant was completely removed. Oocysts were suspended in 1 ml of freshly prepared chemical disinfectant solution. Nine liquid chemical disinfectants were tested for their ability to reduce infectivity of C. parvum oocysts. The active ingredients of the disinfectants tested were as follows: bleach, 6.0% (wt/vol) sodium hypochlorite (Dutch Chemicals Inc., Montréal, Quebec, Canada); ethanol, 70% (vol/vol) eth-
* Corresponding author. Mailing address: Department of Environmental Biology, University of Guelph, Guelph, Ontario, N1G 2W1, Canada. Phone: (519) 824-4120, ext. 3367 (J.T.T.) or 3828 (H.L.). Fax: (519) 837-0442. E-mail:
[email protected] or
[email protected]. † Present address: Department of Nematology, University of California, Davis, Davis, CA 95616-8668. 2576
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anol (Sigma-Aldrich Canada Ltd.); isopropanol, 70% (vol/vol) isopropanol (Sigma-Aldrich); methanol, 37% (vol/vol) methanol (Sigma-Aldrich); hydrogen peroxide, 6% hydrogen peroxide (Fisher Scientific); in-house disinfectant, 2.5% (vol/vol) ammonium hydroxide (Sigma-Aldrich) in windshield washer fluid (Motomaster, Guelph, Ontario, Canada); Clinicide, 4.61% didecyl dimethyl ammonium chloride plus 3% n-alkyl dimethyl benzyl ammonium chloride, diluted 128-fold (MTC Pharmaceuticals, Cambridge, Ontario, Canada); Omega, 6% n-alkyl dimethyl benzyl ammonium chloride plus 6% n-alkyl dimethyl ethyl benzyl ammonium chloride, diluted 250-fold (Ecolab Professional Products, Mississauga, Ontario, Canada); Wescodyne, 9.1% polyethoxypolypropoxypolyethoxyethanoliodine complex plus 8.74% nonylphenoxypoly(ethylenoxy)ethanol-iodine complex, diluted 110-fold (West Chemical Products of Canada, Montréal, Quebec, Canada). As a positive control, oocysts were suspended in 1 ml of sterile PBS. Oocysts were exposed to disinfectants as described previously, with some modifications (11). Oocysts were incubated in the presence of disinfectants for 1, 10, and 30 min prior to being centrifuged for 3 min at 11,750 ⫻ g at room temperature. Supernatants containing disinfectants were removed, and oocysts were washed twice with 1 ml of sterile PBS. Neutralizing agents could not be applied easily and consistently to all of the various disinfectants tested, and therefore, they were not used. For this study, exposure time was considered the time oocysts were in suspension plus the first centrifugation step (3 min), excluding subsequent washing steps. After the washing, oocysts were suspended in 900 l of sterile PBS. Excystation was induced by adding 100 l of bleach (5.25% [wt/wt] sodium hypochlorite; Javex; Colgate-Palmolive Canada Inc., Toronto, Ontario, Canada), and oocysts were incubated on ice for 8 min. Oocysts were centrifuged for 3 min at 11,750 ⫻ g and washed twice with 1 ml of sterile PBS to remove bleach. Oocysts were suspended in 300 l of growth medium that had been prewarmed to 37°C. Serial dilutions (10-fold) were prepared in prewarmed growth medium, and 100 l from each dilution was added to a corresponding well of a chamber slide containing a confluent monolayer of HCT-8 cells. From the initial (undiluted) suspension, oocysts were enumerated with a hemocytometer. Chamber slides were incubated at 37°C under 5% CO2. After 24 h, growth medium was removed, monolayers were washed with 1 ml of sterile PBS, and 750 l of fresh growth medium was added to each well. Chamber slides were incubated a further 24 h at 37°C under a 5% CO2 atmosphere. After incubation, the growth medium was removed, and HCT-8 monolayers were washed with 1 ml of sterile PBS and fixed in methanol for 20 min. The methanol was removed, chamber walls were removed from the slides, and the slides were air dried for 30 min. Fixed and dried cell monolayers on each slide were covered in antibody dilution-blocking buffer (PBS [pH 7.4], 1% bovine serum albumin, 10% normal goat serum, 0.02% sodium azide; Waterborne Inc., New Orleans, La.) for 30 min. The buffer was removed and replaced with fluorescein-labeled rat immunoglobulin G against sporozoites of C. parvum (Sporo-Glo [Waterborne Inc.]; 1:20 dilution in dilution-blocking buffer from 20⫻ stock antibody solution). The slides were placed in a lightproof box and incubated at 22°C for 1 h. The antibody solution was removed, and the slides were rinsed four times in sterile PBS. Coverslips were
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placed on the slides with a 2% DABCO mounting medium [2% 1,4-diazabicyclo(2,2,2)octane (Sigma-Aldrich) in 90% glycerol–10% PBS], and each chamber was viewed under epifluorescence at a ⫻100 magnification with a Nikon Eclipse E600 microscope. Foci of infection appeared as bright green clusters on monolayers at an excitation wavelength of 460 to 500 nm and an emission wavelength of 510 to 560 nm. Wells containing 10 to 100 foci of infection were enumerated, and the total number of foci were calculated according to the dilution of oocysts applied to each well. Where indicated, statistically significant differences between mean values of the control and disinfectant treatment were assessed by Student’s t tests using SigmaStat for Windows version 3.02 (SSPS Science, Chicago, Ill.). The numbers of foci of infection observed after exposure to various disinfectants are shown in Fig. 1. Oocyst infectivity was reduced 1,000-fold after 4 min of exposure to 6% hydrogen peroxide. However, no foci of infection were observed after exposure to 6% hydrogen peroxide for 13 or 33 min. These results agree with previous studies demonstrating the effect of hydrogen peroxide on C. parvum infectivity in MDCK cells (1). Barbee et al. reported a 500-fold reduction of infectivity after a 10-min exposure to 6% hydrogen peroxide and a ⬎1,000-fold reduction of infectivity after 20 min (1). Hydrogen peroxide also reduced excystation rates of C. parvum oocysts, and oocysts treated with hydrogen peroxide failed to infect neonatal mice at a dose of 105 treated oocysts per mouse (3). Hydrogen peroxide is readily available and nonhazardous at the 6% concentration used here. Treatment of oocysts with in-house Cryptosporidium disinfectant prepared from windshield washer fluid and ammonium hydroxide resulted in about a 104-fold reduction in infectivity after 13 min of exposure and 135-fold reduction after 33 min relative to the control values. The numbers of foci after 13 and 33 min of exposure to in-house disinfectant were significantly different from the control values (P ⫽ 0.003 and P ⬍ 0.001, respectively). Jenkins et al. found that 0.05 M ammonia inactivated more than 75% of C. parvum oocysts after 24 h exposure, as measured by viability dye staining and by excystation rates (7). They estimated that 99.999% of oocysts would be inactivated within 1 day by 5.8 M ammonia. In our study, the concentration of ammonia in the Cryptosporidium disinfectant was approximately 0.2 M. However, additional components of windshield washer fluid, such as detergents and methanol, may have contributed to oocyst inactivation. In our study, 37% methanol, a component of windshield washer fluid, significantly reduced oocyst infectivity by over half after 13 min of exposure (P ⫽ 0.006). Exposure times of less than 1 h are more practical for routine laboratory cleaning and disinfection of minor spills. Other common laboratory disinfectants tested included 70% ethanol, 6% sodium hypochlorite, and 70% isopropanol. None of these reduced the infectivity of C. parvum oocysts in cell culture after 33 min of exposure. Similar experiments with 5.25% sodium hypochlorite showed that oocysts were still infective for mice after 120 min of exposure (6). Three commercial disinfectants used in our study, Wescodyne, Clinicide, and Omega, also failed to reduce the infectivity of C. parvum oocysts after 33 min of exposure and actually resulted in an increase in focus development in cell culture. While it is un-
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FIG. 1. Number of foci observed after infection of HCT-8 cells with C. parvum oocysts exposed to various disinfectants for 4, 13, and 33 min. Control samples were oocysts suspended in sterile PBS. Values are the means ⫾ standard deviations for three independent replicates. ⴱ, No foci of infection observed.
certain why this occurred, it is known that pretreatment with dilute bleach induces excystation, the first step toward infection. Perhaps the lower concentration of disinfectant in these commercial preparations had a similar excystation-enhancing effect on C. parvum oocysts. Clinicide contains the alkaline detergent didecyldimethylammonium chloride as well as another quaternary ammonium compound. Vassal et al. showed that this detergent in combination with other germicidal agents, such as glutaraldehyde and chlorohexidine gluconate, did not reduce infectivity of C. parvum oocysts in rats (10). Omega also contains quaternary ammonium compounds and was ineffective at reducing oocyst infectivity. Treatment with Wescodyne, an iodine-based disinfectant, slightly increased the number of foci developed in cell culture. Wilson and Margolin reported that povidone-iodinebased disinfectant reduced excystation, especially after increased contact times (11). However, oocysts treated with povidone-iodine for up to 10 h still infected Caco-2 cells (11). Suitable disinfectants need to be available for work with infectious microorganisms such as C. parvum. Procedures for spill containment and inactivation are necessary for safe handling of C. parvum oocysts. All disinfectants used in this study were tested at room temperature (22 to 24°C) for short periods (4, 13, and 33 min) that would be reasonable for disinfecting small-scale spills in the laboratory. We found that common laboratory disinfectants, such as sodium hypochlorite and ethanol, were ineffective at reducing infectivity of C. parvum oocysts. The results indicate that hydrogen peroxide and the in-house Cryptosporidium disinfectant may be more suitable
laboratory disinfectants against C. parvum oocysts. These reagents are inexpensive and can be applied to laboratory bench tops, floors, and stainless steel surfaces. Inclusion of these disinfectants in laboratory spill kits and in routine laboratory disinfection will help prevent exposure of laboratory workers to C. parvum oocysts. This research was supported by grants from the Natural Sciences and Engineering Research Council (NSERC), Group Strategic Project program, and Ontario Ministry of Agriculture, Food and Rural Affairs (OMAFRA) Resources Management and Environment Program. J.T.T. and H.L. were also supported by individual NSERC Research grants. Some of the equipment used in this research was provided under the CFI/OCF funding programs. We thank the AIDS Research and Reference Reagent Program, DAIDS, NIAID, NIH, for providing us with the C. parvum GCH1 isolate that was originally made available by Saul Tzipori. We thank Shu Chen, Laboratory Services Division, University of Guelph, for providing technical and logistical assistance for this study.
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