Effect of disinfectants on pathogenic free-living amoebae: in axenic conditions.

The amoebicidal properties of chlorine, chlorine dioxide, ozone, and deciquam 222 were examined in axenic conditions. Naegleria spp. were found to be more sensitive to chlorine and chlorine dioxide than Acanthamoeba spp. No marked difference in sensitivity to ozone or deciquam 222 could be detected between the pathogenic (A-1) and nonpathogenic (1501) strains of Acanthamoeba and the pathogenic (MsT) and nonpathogenic (P1200f) strains of Naegleria. Methods of disinfection are discussed with reference to suitability of the disinfectants to real conditions.     

Specimen housing unit for cinemicrographic studies in the vertical plane.

The destructive action of chlorine on the pathogenic Naegleria fowleri and Acanthamoeba culbertsoni, the nonpathogenic N. gruberi, and an avirulent Acanthamoeba isolate was investigated. N fowleri is somewhat more sensitive to chlorine than N. gruberi, whereas the two Acanthamoeba strains are very resistant. This study yields information needed for the destruction of amoebic cysts in drinking water and swimming pools. It also gives some explanation for the occurence of Acanthamoeba strains in these waters.     

Isolation and molecular identification of free-living amoebae of the genus Naegleria from Arctic and sub-Antarctic regions

Twenty-three freshwater samples with sediment taken from two regions in the Arctic, Spitzbergen and Greenland, and one region in sub-Antarctica, Ile de la Possession, were cultured for amoebae at 37 degrees C and room temperature (RT). Only two samples yielded amoebae at 37 degrees C and the two isolates were identified from their morphological features to belong to the genus Acanthamoeba. Vahlkampfiid amoebae were isolated from 11 samples at RT. Morphological analysis of the cysts identified all 11 isolates as belonging to the genus Naegleria, although only about half of them (45%) transformed into flagellates. Ribosomal DNA sequence analysis demonstrated that these isolates represent novel species and that N. antarctica, N. dobsoni and N. chilensis are their closest relatives. Not surprisingly, these three species also grow at lower temperatures (<37 degrees C) than the majority of described Naegleria spp. Two of the eight new species were found in both Arctic and sub-Antarctic regions, and other new species from the Arctic are closely related to new species from the sub-Antarctic. Therefore, it seems the Naegleria gene pool present in the polar regions is different from that found in temperate and tropical regions.     

Resistance of pathogenic Naegleria to some common physical and chemical agents

Resistance of pathogenic Naegleria to drying, low and high temperature, and two halogens was studied. Dying made trophozoites nonviable instantaneously and cysts nonviable in less than 5 min. Trophozoites degenerated in hours at temperatures below 10 degrees C and in minutes when frozen; cysts survived according to the equation th - t0/theta 1,440/1.122T (t0 is survival at 0 degrees C; Tis temperature between 0 and 10 degrees C), but 1.5 h at --10 degrees C to 1 h at --30 degrees C. At 51, 55, 58, 63, and 65 degrees C, trophozoites survived about 30, 10, 5, 1 and less than 0.5 min, respectively, cysts survived three to four times longer at 51 degrees C and six to seven times longer at 55 to 65 degrees C. Cyst destruction rates by heat indicated first-order kinetics with 25,400 cal/1 degree C for energy of activation. Cyst destruction rates by free chlorine and I2 also conformed to first-order kinetics. Concentration-contact time curves yielded concentration coefficient values of 1.05 for free chlorine and 1.4 for I2 and point to superchlorination as an effective means of destroying the cysts if free residuals are used as a guide and allowance is provided for low temperature and/or high pH waters.     

Resistance of pathogenic Naegleria to some common physical and chemical agents.

Resistance of pathogenic Naegleria to drying, low and high temperature, and two halogens was studied. Dying made trophozoites nonviable instantaneously and cysts nonviable in less than 5 min. Trophozoites degenerated in hours at temperatures below 10 degrees C and in minutes when frozen; cysts survived according to the equation th - t0/theta 1,440/1.122T (t0 is survival at 0 degrees C; Tis temperature between 0 and 10 degrees C), but 1.5 h at --10 degrees C to 1 h at --30 degrees C. At 51, 55, 58, 63, and 65 degrees C, trophozoites survived about 30, 10, 5, 1 and less than 0.5 min, respectively, cysts survived three to four times longer at 51 degrees C and six to seven times longer at 55 to 65 degrees C. Cyst destruction rates by heat indicated first-order kinetics with 25,400 cal/1 degree C for energy of activation. Cyst destruction rates by free chlorine and I2 also conformed to first-order kinetics. Concentration-contact time curves yielded concentration coefficient values of 1.05 for free chlorine and 1.4 for I2 and point to superchlorination as an effective means of destroying the cysts if free residuals are used as a guide and allowance is provided for low temperature and/or high pH waters.     

Viability of pathogenic and nonpathogenic free-living amoebae in long-term storage at a range of temperatures.

The long-term storage of pathogenic and nonpathogenic strains of both Naegleria and Acanthamoeba spp. were tested on Page amoeba saline agar slopes for 24 months at room temperature and for 8 months at -10, 4, 10, and 15 degrees C. Acanthamoeba strains showed better survival potential than Naegleria strains, particularly when they were stored at temperatures equal to or lower than room temperature.     

Viability of pathogenic and nonpathogenic free-living amoebae in long-term storage at a range of temperatures

The long-term storage of pathogenic and nonpathogenic strains of both Naegleria and Acanthamoeba spp. were tested on Page amoeba saline agar slopes for 24 months at room temperature and for 8 months at -10, 4, 10, and 15 degrees C. Acanthamoeba strains showed better survival potential than Naegleria strains, particularly when they were stored at temperatures equal to or lower than room temperature.     

Propidium iodide as an indicator of Giardia cyst viability.

The use of propidium iodide, whose uptake indicates cell death or damage, was investigated to assess the viability of heat-inactivated and chemically inactivated Giardia muris cysts. This was done by comparing propidium iodide staining with excystation. We first determined that propidium iodide could be used with an immunofluorescence detection procedure by showing that the percentages of Giardia lamblia cysts stained with this dye before and after subjecting them to a fluorescence detection method were similar. G. muris cysts were then exposed to heat (56 degrees C), 0.5 to 4 mg of chlorine per liter (pH 7.0, 5 degrees C), 0.1 to 10 mg of a quaternary ammonium compound per liter, or 2 mg of preformed and forming monochloramine per liter (pH 7.2, 18 to 20 degrees C). A good positive correlation between percent propidium iodide-stained cysts and lack of excystation was demonstrated for G. muris cysts exposed either to heat or to the quaternary ammonium compound. However, no significant correlation between absence of excystation and propidium iodide staining was found for cysts exposed to chlorine or monochloramines. These results demonstrate that the propidium iodide staining procedure is not satisfactory for determining the viability of G. muris cysts exposed to these two commonly used drinking water disinfectants.     

Propidium iodide as an indicator of Giardia cyst viability.

The use of propidium iodide, whose uptake indicates cell death or damage, was investigated to assess the viability of heat-inactivated and chemically inactivated Giardia muris cysts. This was done by comparing propidium iodide staining with excystation. We first determined that propidium iodide could be used with an immunofluorescence detection procedure by showing that the percentages of Giardia lamblia cysts stained with this dye before and after subjecting them to a fluorescence detection method were similar. G. muris cysts were then exposed to heat (56 degrees C), 0.5 to 4 mg of chlorine per liter (pH 7.0, 5 degrees C), 0.1 to 10 mg of a quaternary ammonium compound per liter, or 2 mg of preformed and forming monochloramine per liter (pH 7.2, 18 to 20 degrees C). A good positive correlation between percent propidium iodide-stained cysts and lack of excystation was demonstrated for G. muris cysts exposed either to heat or to the quaternary ammonium compound. However, no significant correlation between absence of excystation and propidium iodide staining was found for cysts exposed to chlorine or monochloramines. These results demonstrate that the propidium iodide staining procedure is not satisfactory for determining the viability of G. muris cysts exposed to these two commonly used drinking water disinfectants.     

Effects of ozone, chlorine dioxide, chlorine, and monochloramine on Cryptosporidium parvum oocyst viability

Purified Cryptosporidium parvum oocysts were exposed to ozone, chlorine dioxide, chlorine, and monochloramine. Excystation and mouse infectivity were comparatively evaluated to assess oocyst viability. Ozone and chlorine dioxide more effectively inactivated oocysts than chlorine and monochloramine did. Greater than 90% inactivation as measured by infectivity was achieved by treating oocysts with 1 ppm of ozone (1 mg/liter) for 5 min. Exposure to 1.3 ppm of chlorine dioxide yielded 90% inactivation after 1 h, while 80 ppm of chlorine and 80 ppm of monochloramine required approximately 90 min for 90% inactivation. The data indicate that C. parvum oocysts are 30 times more resistant to ozone and 14 times more resistant to chlorine dioxide than Giardia cysts exposed to these disinfectants under the same conditions. With the possible exception of ozone, the use of disinfectants alone should not be expected to inactivate C. parvum oocysts in drinking water.     

Effects of ozone, chlorine dioxide, chlorine, and monochloramine on Cryptosporidium parvum oocyst viability.

Purified Cryptosporidium parvum oocysts were exposed to ozone, chlorine dioxide, chlorine, and monochloramine. Excystation and mouse infectivity were comparatively evaluated to assess oocyst viability. Ozone and chlorine dioxide more effectively inactivated oocysts than chlorine and monochloramine did. Greater than 90% inactivation as measured by infectivity was achieved by treating oocysts with 1 ppm of ozone (1 mg/liter) for 5 min. Exposure to 1.3 ppm of chlorine dioxide yielded 90% inactivation after 1 h, while 80 ppm of chlorine and 80 ppm of monochloramine required approximately 90 min for 90% inactivation. The data indicate that C. parvum oocysts are 30 times more resistant to ozone and 14 times more resistant to chlorine dioxide than Giardia cysts exposed to these disinfectants under the same conditions. With the possible exception of ozone, the use of disinfectants alone should not be expected to inactivate C. parvum oocysts in drinking water.     

Occurrence of Naegleria and Acanthamoeba in aquaria

Samples from 24 aquaria were incubated at 28, 37, and 45 degrees C for the isolation of Naegleria and Acanthamoeba. Naegleria was the predominant genus (60.9%), whereas Acanthamoeba represented 15.5% of the isolates. No pathogenic N. fowleri was identified, although a high number of strains were closely related to this species. One isolate (Aq/9/1/45D) was compared with an aquarium isolate (PPMFB-6) from Australia. The Belgian isolate was found to be more related to N. fowleri, whereas the Australian isolate was closer to N. gruberi.     

Occurrence of Naegleria and Acanthamoeba in aquaria.

Samples from 24 aquaria were incubated at 28, 37, and 45 degrees C for the isolation of Naegleria and Acanthamoeba. Naegleria was the predominant genus (60.9%), whereas Acanthamoeba represented 15.5% of the isolates. No pathogenic N. fowleri was identified, although a high number of strains were closely related to this species. One isolate (Aq/9/1/45D) was compared with an aquarium isolate (PPMFB-6) from Australia. The Belgian isolate was found to be more related to N. fowleri, whereas the Australian isolate was closer to N. gruberi.     

Identification of Acanthamoeba at the generic and specific levels using the polymerase chain reaction.

We have adapted the polymerase chain reaction to identify strains of Acanthamoeba. Using computer-assisted analysis, primers were designed from an anonymous repetitive sequence and from published sequences of 18S and 5S ribosomal RNA genes of A. castellanii. Amplification of a short ribosomal DNA target (272 base pairs) at restrictive annealing conditions (greater than 50 degrees C) resulted in a single band that was unique for the genus and distinguished Acanthamoeba from Naegleria. This assay functioned with fresh and formalin-fixed cells as starting material. Amplification of longer targets (400-700 base pairs) at less restrictive annealing conditions (less than 47 degrees C) led to more than one band. This multiple banding pattern could reproducibly classify Acanthamoeba at the strain level and was, in certain cases, diagnostic for known pathogenic strains. However, these assays need to be further refined to make them relevant for clinical purposes.     

In-vitro activity of miltefosine and voriconazole on clinical isolates of free-living amebas: Balamuthia mandrillaris, Acanthamoeba spp., and Naegleria fowleri

The anticancer agent miltefosine and the antifungal drug voriconazole were tested in vitro against Balamuthia mandrillaris, Acanthamoeba spp., and Naegleria fowleri. All three amebas are etiologic agents of chronic (Balamuthia, Acanthamoeba) or fulminant (Naegleria) encephalitides in humans and animals and, in the case of Acanthamoeba, amebic keratitis. Balamuthia exposed to <40 microm concentrations of miltefosine survived, while concentrations of >or=40 microM were generally amebacidal, with variation in sensitivity between strains. At amebastatic drug concentrations, recovery from drug effects could take as long as 2 weeks. Acanthamoeba spp. recovered from exposure to 40 microM, but not 80 microM miltefosin. Attempts to define more narrowly the minimal inhibitory (MIC) and minimal amebacidal concentrations (MAC) for Balamuthia and Acanthamoeba were difficult due to persistence of non-proliferating trophic amebas in the medium. For N. fowleri, 40 and 55 microM were the MIC and MAC, respectively, with no trophic amebas seen at the MAC. Voriconazole had little or no inhibitory effect on Balamuthia at concentrations up to 40 microg/ml, but had a strong inhibitory effect upon Acanthamoeba spp. and N. fowleri at all drug concentrations through 40 microg/ml. Following transfer to drug-free medium, Acanthamoeba polyphaga recovered within a period of 2 weeks; N. fowleri amebas recovered from exposure to 1 microg/ml, but not from higher concentrations. All testing was done on trophic amebas; drug sensitivities of cysts were not examined. Miltefosine and voriconazole are potentially useful drugs for treatment of free-living amebic infections, though sensitivities differ between genera, species, and strains.     

Amoebae from antarctic soil and water.

Samples of soil and water were taken from the McMurdo Sound-Dry Valley region of Antarctica. Of the 70 samples cultured, 22 yielded amoebae capable of clonal growth at 30 degrees C. None of the isolates was pathogenic for mice. Acanthamoeba isolates appeared to show better survival potential than Naegleria isolates.     

Cellular response of the amoeba Acanthamoeba castellanii to chlorine, chlorine dioxide, and monochloramine treatments

Acanthamoeba castellanii is a free-living amoebae commonly found in water systems. Free-living amoebae might be pathogenic but are also known to bear phagocytosis-resistant bacteria, protecting these bacteria from water treatments. The mode of action of these treatments is poorly understood, particularly on amoebae. It is important to examine the action of these treatments on amoebae in order to improve them. The cellular response to chlorine, chlorine dioxide, and monochloramine was tested on A. castellanii trophozoites. Doses of disinfectants leading to up to a 3-log reduction were compared by flow cytometry and electron microscopy. Chlorine treatment led to size reduction, permeabilization, and retraction of pseudopods. In addition, treatment with chlorine dioxide led to a vacuolization of the cytoplasm. Monochloramine had a dose-dependent effect. At the highest doses monochloramine treatment resulted in almost no changes in cell size and permeability, as shown by flow cytometry, but the cell surface became smooth and dense, as seen by electron microscopy. We show that these disinfectants globally induced size reduction, membrane permeabilization, and morphological modifications but that they have a different mode of action on A. castellanii.     

Polymerase chain reaction detection of potentially pathogenic free-living amoebae in dental units

Several genera of amoebae can be found in water from dental units and on the inner surface of waterlines. The presence of bacterial biofilms on these surfaces is thought to favor the proliferation of amoebae. Potentially pathogenic Acanthamoeba and Naegleria spp. may be an infection risk for patients through contact with open surgical sites or aerosolization. A polymerase chain reaction of DNA extracted from pelleted samples showed that Acanthamoeba spp. and Naegleria spp. were present in water from dental units, suction lines, and suction filters at the dental clinic of the Université de Montréal. Acanthamoeba spp. were detected in 24.2% of 66 samples and Naegleria spp. in 3.0%. We discuss the infection risk associated with these results.     

Epidemiology of free-living ameba infections

Small free-living amebas belonging to the genera Acanthamoeba and Naegleria occur world-wide. They have been isolated from a variety of habitats including fresh water, thermal discharges of power plants, soil, sewage and also from the nose and throats of patients with respiratory illness as well as healthy persons. Although the true incidence of human infections with these amebas is not known, it is believed that as many as 200 cases of central nervous system infections due to these amebas have occurred worldwide. A majority (144) of these cases have been due to Naegleria fowleri which causes an acute, fulminating disease, primary amebic meningoencephalitis. The remaining 56 cases have been reported as due either to Acanthamoeba or some other free-living ameba which causes a subacute and/or chronic infection called granulomatous amebic encephalitis (GAE). Acanthamoeba, in addition to causing GAE, also causes nonfatal, but nevertheless painful, vision-threatening infections of the human cornea, Acanthamoeba keratitis. Infections due to Acanthamoeba have also been reported in a variety of animals. These observations, together with the fact that Acanthamoeba spp., Naegleria fowleri, and Hartmannella sp. can harbor pathogenic microorganisms such as Legionella and or mycobacteria indicate the public health importance of these amebas.     

Pathogenic free-living amoebae in Korea

Acanthamoeba and Naegleria are widely distributed in fresh water, soil and dust throughout the world, and cause meningoencephalitis or keratoconjunctivitis in humans and other mammals. Korean isolates, namely, Naegleria sp. YM-1 and Acanthamoeba sp. YM-2, YM-3, YM-4, YM-5, YM-6 and YM-7, were collected from sewage, water puddles, a storage reservoir, the gills of a fresh water fish, and by corneal washing. These isolates were categorized into three groups based on the mortalities of infected mice namely, highly virulent (YM-4), moderately virulent (YM-2, YM-5 and YM-7) and nonpathogenic (YM-3). In addition, a new species of Acanthamoeba was isolated from a freshwater fish in Korea and tentatively named Korean isolate YM-4. The morphologic characters of its cysts were similar to those of A. culbertsoni and A. royreba, which were previously designated as Acanthamoeba group III. Based on experimentally infected mouse mortality, Acanthamoeba YM-4 was highly virulent. The isoenzymes profile of Acanthamoeba YM-4 was similar to that of A. royreba. Moreover, an anti-Acanthamoeba YM-4 monoclonal antibody reacted only with Acanthamoeba YM-4, and not with A. culbertsoni. Random amplified polymorphic DNA marker analysis and RFLP analysis of mitochondrial DNA and of a 18S small subunit ribosomal RNA, placed Acanthamoeba YM-4 in a separate cluster based on phylogenic distances. Thus Acanthamoeba YM-4 was identified as a new species, and assigned Acanthamoeba sohi. Up to the year 2002 in Korea, two clinical cases were found to be infected with Acanthamoeba spp. These patients died of meningoencephalitis. In addition, one case of Acanthamoeba pneumonia with an immunodeficient status was reported and Acanthamoeba was detected in several cases of chronic relapsing corneal ulcer, chronic conjunctivitis, and keratitis.