The effectiveness of hydrogen peroxide liquid or gas plasma on protozoan oocysts

To the Editor: Gastroendoscopy is increasingly in demand to diagnose and treat illnesses of the gastrointestinal tract. Because endoscopes may become contaminated with infectious agents from the intestine or rectum, they have to be reprocessed. The infectious agents that are most difficult to destroy (second to prions) are the oocysts of coccidian parasites such as Cryptosporidium and Cyclospora (1,2). No liquid disinfectants, including the much-studied aldehydes, have been shown to be entirely effective. We used hydrogen peroxide, a newer oxygenated product to the market, in both liquid and gaseous form, in a bioassay to test its effectiveness in inactivating oocysts. Because human coccidial parasites, such as Cryptosporidium, are hazardous to handle in the laboratory, we used a close alternative, Eimeria acervulina, as a surrogate (3). This parasite causes coccidiosis in chickens. In this bioassay, if oocysts are inactivated, they will not produce evidence of infection, lesions or replicate themselves to produce oocysts in chickens. Our studies confirmed that despite a 3 h soak in any of the hydrogen peroxide liquid disinfectants, some oocysts remained infectious (Table 1). In contrast to liquid disinfectants, a hydrogen peroxide gas plasma system, operating at less than 50°C, inactivated all E acervulina oocysts (Table 2) from the surface (aluminum foil) and from the inside of narrow tubing (inner diameter of 3 mm). None of the 40 chicks inoculated with gas plasma-treated oocysts produced lesions (n=15 chicks inoculated) or oocysts (n=25 chicks inoculated). Oocysts that were either airdried on aluminum foil or inside narrow tubing, but not treated with gas plasma, remained infectious for all chicks (n=25). Heattreated controls did not produce infection. These results were similar to the study by Vassal et al (4), in which 105 Cryptosporidium parvum oocysts treated in a 75 min cycle, hydrogen peroxide gas plasma system (STERRAD 100s [Advanced Sterilization Products, USA]), were found to be noninfectious to immunocompromised Sprague-Dawley rats. This similarity in behaviour between E acervulina oocysts and Cryptosporidum oocysts in their response to hydrogen peroxide also supports the usefulness of E acervulina as a surrogate for testing the sterilization of endoscopes. In addition, E acervulina oocysts are safe and economical to handle and provide verifiable and clear evidence of inactivation.


LETTER TO THE EDITOR The effectiveness of hydrogen peroxide liquid or gas plasma on protozoan oocysts
To the Editor: Gastroendoscopy is increasingly in demand to diagnose and treat illnesses of the gastrointestinal tract.Because endoscopes may become contaminated with infectious agents from the intestine or rectum, they have to be reprocessed.The infectious agents that are most difficult to destroy (second to prions) are the oocysts of coccidian parasites such as Cryptosporidium and Cyclospora (1,2).No liquid disinfectants, including the much-studied aldehydes, have been shown to be entirely effective.We used hydrogen peroxide, a newer oxygenated product to the market, in both liquid and gaseous form, in a bioassay to test its effectiveness in inactivating oocysts.Because human coccidial parasites, such as Cryptosporidium, are hazardous to handle in the laboratory, we used a close alternative, Eimeria acervulina, as a surrogate (3).This parasite causes coccidiosis in chickens.In this bioassay, if oocysts are inactivated, they will not produce evidence of infection, lesions or replicate themselves to produce oocysts in chickens.
Our studies confirmed that despite a 3 h soak in any of the hydrogen peroxide liquid disinfectants, some oocysts remained infectious (Table 1).In contrast to liquid disinfectants, a hydrogen peroxide gas plasma system, operating at less than 50°C, inactivated all E acervulina oocysts (Table 2) from the surface (aluminum foil) and from the inside of narrow tubing (inner diameter of 3 mm).None of the 40 chicks inoculated with gas plasma-treated oocysts produced lesions (n=15 chicks inoculated) or oocysts (n=25 chicks inoculated).Oocysts that were either airdried on aluminum foil or inside narrow tubing, but not treated with gas plasma, remained infectious for all chicks (n=25).Heattreated controls did not produce infection.
These results were similar to the study by Vassal et al (4), in which 10 5 Cryptosporidium parvum oocysts treated in a 75 min cycle, hydrogen peroxide gas plasma system (STERRAD 100s [Advanced Sterilization Products, USA]), were found to be noninfectious to immunocompromised Sprague-Dawley rats.This similarity in behaviour between E acervulina oocysts and Cryptosporidum oocysts in their response to hydrogen peroxide also supports the usefulness of E acervulina as a surrogate for testing the sterilization of endoscopes.In addition, E acervulina oocysts are safe and economical to handle and provide verifiable and clear evidence of inactivation.

ACKNOWLEDGEMENTS:
The authors gratefully acknowledge the contributions of Ms Wenrong Sun for assisting with the microbiological tests, Ms Alyssia Greig for laboratory technical assistance and Ms Vreni Lall for her help with the STERRAD 100S portion of the present study.
The present project was approved by the Animal Care Committee at Ryerson University (Toronto, Ontario).No funding was received from the manufacturers or distributors of any of the products used in the study.Heat-treated controls 0/3 0/2 0/5 0/5 0/15 *Fecal collections of three chicks before necropsy showed an abundance of oocysts.Five of five means that five birds produced evidence of infection, either lesions or oocysts, out of five birds that were inoculated.Likewise, zero of five means that no birds produced evidence of infection, neither lesions nor oocysts, out of five birds that were inoculated

TABLE 2
Infectivity of sporulated Eimeria acervulina oocysts treated by a hydrogen peroxide gas plasma system*.Inocula between 2000 and 20,000 oocysts were given orally to each chick on the first or second day of hatch ©2007 Pulsus Group Inc.All rights reserved *STERRAD 100s (Advanced Sterilization Products, USA).ND Not determined