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    Environmental factors affecting encystment of P.F.L.A. together with disinfection studies: a thesis is presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University, Palmerston North, New Zealand
    (Massey University, 1986) Harvey, Dawn
    ABSTRACT Free living amoebae from the genera Naegleria and Acanthamoeba have been implicated in fatal and several non-fatal infections of the human central nervous system, and other organs. They can be isolated from a worldwide range of environments. The common occurance of these organisms in nature may be attributed to the ability to form resistant cysts to withstand adverse environmental conditions. Research was performed to determine factors that will promote the encystment of amoebae, particularly Naegleria species. The parameters examined for the induction of encystment were: the type of substrate amoebae were growing on liquid or solid, the presence of bacteria, the cell concentration and nutrient availability and incubation at temperatures other than the optimal growth temperature. Higher percentages of amoebae encysted on solid surface environ­ments in comparison to the liquid media. In liquid media a greater percentage of trophozoites only formed a pre-encystment or roundform stage. The factor required for the complete encyst­ment of roundforms was not present. The encystment of Naegleria fowleri was not significantly influenced by the presence or absence of the bacterial species used (E. cloacae). Encystment of Naegleria spat different cell concentrations, using a nutrient media, a soil extract broth and a non-nutrient media was examined. Complete encystment of cells did not occur where nutrients were either high or absent, and the cell concentration was low. The ability of trophozoites of N. fowerli, N. gruberi, culbertsoni, and A. castellanii to encyst at a range of temperatures from 4°C - 44°C was studied. The trophozoites of Acanthamoeba sp could encyst over a wider temperature range in comparison to the trophozoites of Naegleria sp. The effect of disinfection using Baquacil was studied. Previously isolated strains of baquacil resistant N. fowleri, still had higher disinfection survival rates compared to sensitive strains. Resistance of trophozoites to Baquacil was not affected even after storage as a cyst. Baquacil resistant strains of N. fowleri were still sensitive to chlorination. Disinfection of amoebic cysts using chlorine and Baquacil was investigated. Amoebic cysts require higher levels of disinfection for inactivation in comparison to trophozoites. Acanthamoeba cysts have a greater tolerance to chlorination than Naegleria sp.
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    The susceptibility of pathogenic free-living amebae to chemotherapeutic agents : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University
    (Massey University, 1979) Donald, Jennifer Jane
    The treatment of infections caused by pathogenic free-living amebae (PFLA) has, until only recently, been far from successful. The continued screening of chemotherapeutic agents against amebae of the genera Naegleria and Acanthamoeba is therefore of the utmost importance. Seven chemotherapeutic agents, amphotericin B, rifampicin, tetracycline, polymyxin B sulphate, 5-fluorocytosine, miconazole and R41,400 were screened for activity against a non-pathogenic and pathogenic species of Naegleria and a non-pathogenic and pathogenic species of Acanthamoeba in axenic culture. For the Naegleria spp. amphotericin B, miconazole and R41,400 were found to be active. Acanthamoebae spp. were found to be susceptible only to 5-fluorocytosine and R41,400. The possible use of combinations of drugs against the amebae was also investigated in axenic culture. For Naegleria fowleri (MsT) amphotericin B with either tetracycline or rifampicin showed a synergistic effect. Polymyxin B sulphate and 5-fluorocytosine showed synergistic activity against Acanthamoeba culbertsoni (A-1) but when polymyxin B was combined with tetracycline or rifampicin no significant additive effect was seen. After axenic culture testing the susceptibility of the pathogenic species, N. fowleri (MsT) and A. culbertsoni (A-1), to the agents which showed activity, was investigated in a Vero cell culture system. For N. fowleri (MsT) the results of axenic testing were confirmed with amphotericin B, miconazole and R41,400 protecting the monolayer from the destructive effects of the amebae. 5-Fluorocytosine inhibited the formation of cytopathic effect (CPE) when the cell cultures were inoculated with A. culbertsoni (A-1) but viable amebae were still present. R41,400 had no effect on A. culbertsoni (A-1) at concentrations at or above those which were cytotoxic to the Vero cells. The use of combinations of drugs was also investigated in Vero cell culture. Amphotericin B and rifampicin showed an antagonistic rather than a synergistic effect when used against N. fowleri (MsT) in cell culture but amphotericin B and tetracycline showed synergistic activity. For A. culbertsoni (A-1) the synergistic activity of polymyxin B and 5-fluorocytosine was confirmed. The lack of an additive effect between polymyxin B and either tetracycline or rifampicin was also shown in cell culture. The new imidazole derivative R41,400, which showed promise against N. fowleri (MsT) in in vitro tests was then tested in the in vivo situation. Mice experimentally infected with N. fowleri (MsT) were treated once or twice daily intraperitoneally with different doses of R41,400. At the higher dosage levels tested the drug appeared to have a deleterious effect, the average time for death being less than that for the controls.
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    The in vitro and in vivo testing of chemotherapeutic agents against pathogenic free living amebae : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University, Palmerston North, New Zealand.
    (Massey University, 1980) Elmsly, Christopher John
    During the last ten years, there has been an increasing awareness of sporadic cases of Primary Amoebic Meningo-encephalitis (PAM) affecting primarily younger age groups and appearing in an acute fulminant form. The earliest positive case (Willaert, 1974) may have been in England in 1909 which shows that the disease has been with us for a long time. The pathogenic free-living amebae (PFLA), which comprises the genus Naegleria and the genus Acanthamoeba, are the causative organisms of PAM and AM*respectively. PAM is a rapidly fatal disease affecting the central nervous system (CNS),the treatment of which to date has been successful in only a small number of cases, and therefore the continual screening of suitable chemotherapeutic agents against amebae of the Naegleria spp. and Acanthamoeba spp., is of great importance. AM is also essentially confined to the CNS although it may take the form of chronic granulomata in the liver, spleen, uterus and kidneys (Martinez et al., 1977). Six chemotherapeutic agents: Amphotericin B, 5-Fluorocytosine, Kanamycin, Oxytetracycline, Tylosine and Levamisole were tested for activity against a non-pathogenic and a pathogenic species of Naegleria and a non-pathogenic and a pathogenic species of Acanthamoeba in axenic culture. For the Naegleria spp., Amphotericin B and Oxytetracycline were found to be active and the Acanthamoeba spp. were found to be only susceptible to Levamisole. The synergistic combinations of drugs against the amebae were also investigated in axenic culture. In preliminary trials Kanamycin together with Oxytetracycline showed promise against Naegleria fowleri (MsM) but this was later shown not to be the case. Amphotericin B in combination with 5-Fluorocytosine was also shown not to be synergistic, however Amphotericin B in combination with Oxytetracycline proved to be effective against N. fowleri. Amphotericin B was combined with 5-Fluorocytosine against A. culbertsoni (A-1) but was not found to be synergistically active. * Amebic meningitis caused by Acanthamoeba infections. Levamisole was also tested against N. gruberi (P1200f) and A. castellanii (0.1) at various stages in growth of the amebae (i.e. 24, 48 and 72 hour stock cultures) to determine the effect of using aged amebae. It was found that the age of the stock culture bore no relation to the activity of the drug. After axenic culture testing, the susceptibility of the pathogenic N. fowleri (MsM) and A. culbertsoni (A-1) to the agents which showed activity, was investigated in a vero cell culture system. For N. fowleri (MsM) the results of axenic culture testing were confirmed, with Amphotericin B and Oxytetracycline protecting the monolayer from the destructive effects of the amebae, both when used singly and at a greater efficiency when added together as a synergistic combination. Levamisole, although effective to some extent against Acanthamoeba spp. in axenic culture, failed to show any activity against the amebae in vero cell culture testing. In vivo animal protection studies were then performed using drugs that had been shown either in this or other studies to be effective against either Naegleria or Acanthamoeba spp. Chemotherapeutic agents tested on N. fowleri (MsM) included two imidazoles; Miconazole nitrate and Ketoconazole (previously known as R41,400), as well as Amphotericin B. The synergistic combination of Amphotericin B with either Tetracycline or Oxytetracycline was also investigated. For A. culbertsoni (A-1), 5-Fluorocytosine, and Polymyxin B were tried both singly and in combination. These drugs were injected by intraperitoneal (I.P.) and intraventricular (I.vent.) routes. The results were not promising, with none of the drugs offering significant protection even whilst using Amphotericin B which is considered the drug of choice. The question of adequate drug levels reaching the brain was tested out with two imidazoles, Ketoconazole and Miconazole. Serum samples were assayed against Candida pirapsilosis and C. pseudotropicalis respectively at various time intervals after innoculation with the drug, and a gradual increase and breakdown of the drug in the animal system could then be shown. These results showed that based on in vitro results, the levels of the imidazoles obtained in the serum after the first eight hours after injection, should have been sufficiently high to prevent amebic multiplication.
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    Disinfection and environmental studies on pathogenic free-living amoebae : a thesis presented in partial fulfillment of the requirements for the degree of Master of Science in Microbiology at Massey University
    (Massey University, 1982) Dawson, Mark William
    Over the last fifteen years, there has been an increasing awareness of sporadic cases of Primary Amoebic Meningoencephalitis (PAM), affecting primarily younger age groups and appearing in an acute fulminant form. The earliest positive case known, may have been in England in 1909. The pathogenic free-living amoebae (PFLA), which comprises the genera Naegleria and Acanthamoeba, are the causative organisms of PAM and Granulomatous Amoebic Encephalitis (GAE) respectively. PAM is a rapidly fatal disease affecting the central nervous system (CNS), and GAE although essentially confined to the CNS, may also take the form of granulomata in the liver, spleen, uterus and kidneys. A study on the disinfecting potential of Baquacil in axenic conditions, for comparison with the disinfecting potential of the chlorine, chlorine dioxide, ozone and Deciquam 222, showed that the order of effectiveness as amoebicides was Baquacil, chlorine, chlorine dioxide, ozone and Deciquam 222 in hard water. In soft water the order is Deciquam 222, Baquacil, chlorine, chlorine dioxide and ozone. Further study on the effect of Baquacil, chlorine and chlorine dioxide on amoebae, in conditions involving the use of a known Biochemical Oxygen Demand (BOD), a known bacteria concentration, and a combination of BOD and bacteria, confirmed Baquacil as a more effective amoebicide than chlorine, which in turn was more effective than chlorine dioxide. The concentrations of each disinfectant required were increased by the presence of a BOD, and of bacteria. The bacteria were preferentially destroyed over the amoebae with all three disinfectants. Baquacil resistant clones of Naegleria fowleri were isolated, although it is not known whether this resistance is due to genetic or physiological variation. Axenically and Monoxenically cultured amoebae were used, the latter to increase the resemblance of the amoebae to those found in the environment. Differences in survival rates were observed, the monoxenically cultured amoebae invariably having higher survival rates. Competition studies were done with Naegleria spp. and T.pyriformis on three bacteria species, after preliminary studies on the ability of the two protozoa to grow on eight species of bacteria. Of the three bacteria used in the competiton studies, Escherichia coli and Enterobacter cloacae were shown to support both Naegleria spp. and T.pyriformis, with the ciliate increasing in numbers by up to 3 fold over the controls, but the amoebae were affected only slightly, with a small decrease in numbers compared to the controls. A synergistic relationship was evident on the third bacteria species, Pseudomonas fluorescens, between Naegleria spp. and T.pyriformis, where as in the controls, this bacterium was not a good growth support bacteria for either protozoan.