Biocides and Protozoa

BIOCIDES AND PROTOZOA

The activity of biocides has been described in a number of amoebae, notably in Giardia spp. and Cryptosporidium spp., which are major waterborne pathogens, and Acanthamoeba spp., a pathogen mainly associated with contact lenses and contact lens solutions. Trophozoites (the actively-growing form) of Acanthamoeba spp. have been shown to be susceptible to low concentrations of chlorhexidine, PHMB, QACs, oxidizing agents (hydrogen peroxide, chlorine dioxide, peracetic acid, ozone), chlorine-releasing  agents  and isothiazolinones. Glutaraldehyde might possess only a poor trophocidal activity. In general higher concentrations and much longer contact times are needed to achieve a cysticidal activity (e.g. hydrogen peroxide 3% for 4 hours) and the concentration of biocide (e.g. PHMB, QAC) used for the disinfection of contact lenses might be ineffective against Acanthamoeba cysts. Other biocides such as iodine and bromine and the isothiazolinones have been shown to have no activity against Acanthamoeba cysts. It should be noted that differences in inactivation using the same biocides and parameters have been observed against cysts of different species.

Since most biocides have a poor cysticidal efficacy at a low concentration, the combination of biocides or the formulation excipients can become important. For example, a combination of chlorhexidine and thiomersal and/or EDTA has been shown to be cycticidal within 24 hours. QAC cysticidal activity can be improved when combined with tributyltin neodecanoate. A combination of hydrogen peroxide (3%) with catalase and potassium iodide (50 μM) was shown to enhance significantly cysticidal activity against A. polyphaga.

The mechanisms of action of biocides against trophozoites are similar to those observed on bacterial structures. For example, cationic biocides have been shown to damage the cytoplasmic membrane and to induce pentose leakage in Acanthamoeba spp.

Amoebal trophozoites undergo encystation when exposed to detrimental conditions, which include biocide (e.g. diamidines, chlorhexidine) exposure. Cysts are a dormant form which enable survival for many years in the environment. They are a dehydrated structure with a double wall composed of cellulose and relatively small numbers of proteins. The outer ectocyst wall is composed mainly of protein and lipid containing materials and the inner endocyst wall contains cellulose.

Encystation is a relatively rapid process that can be divided into three principal stages: induction, during which cellular components are degraded; immature cysts, during which the first cell wall is synthesized; and mature cysts, during which the second cell wall is formed.

The composition and morphological aspects of the cyst wall vary between species and depend upon the composition of the media used during encystation.

The double cyst wall represents a permeability barrier for biocides (e.g. chlorhexidine, PHMB, diamidines). In addition, the metabolically-dormant nature of the cyst might affect the cysticidal activity of biocides. It is thus not surprising that cysts represent a challenge for disinfection. This is now of particular concern following a report that protozoal cysts can protect intracellular bacterial pathogens from disinfection.