Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only. The thesis may not be reproduced elsewhere without the permission of the Author. 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 Christopher John Elmsly i980 ABSTRACT 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 Na egleria and the genus Acanthamoeba, are the causative organisms of PAM and AM*respectively. PAM is a rapidly fatal disease affecting the central nervous s ystem (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 arnebae of the Naegleria spp. and Acantharnoeba spp.,is of great i mp ortance. AM is a lso essentially confined to the CNS althou gh it may take the form of chronic granulomata in the liver, spleen, uterus and kidneys (Martinez et al., 1977). ii Six chemotherapeutic agents: Ampho tericin B, 5-Fluorocytosine, Kanamycin, Oxy t et racycline, Tylosine and Levamisole were tested for activity a gainst 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 Band Oxytetracycline were found to be active and the Acanthamoeba spp. were found to be only susceptible to Levamisole. The s ynergistic combinations of drugs against the amebae were also investiga ted in axenic culture. In preliminary trials Kanamycin together with Oxy t etracycline showed promise against Naegleria fowleri (MsM) but thi s wa s later shown not to be the case. Amphotericin Bin combination wi th 5-Fluorocytosine was also shown not to be synergistic, however Amphotericin Bin 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 menin gitis caused by Acanthamoeba infections. Levamisole was also tested against N. gruberi (Pl200f) 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 usi ng aged amebae. It was found that the age of the stock culture bore no relation to the activity of the drug. iii After axenic culture testing, the susceptibility of the pathogenic N. fowleri (MsM) and A. culbertsoni (A-1) to the a gents 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 Band Oxytetracycline protecting the monolayer from the destructive effects of the amebae,both when used singly and at a greater efficiency when added t ogether as a synergistic combination. Levamisole, although effective to some extent a ga inst Acanthamoeba spp. in ax enic culture,failed to show any activity a gainst 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 i midazoles; 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. ACKNOWLEDGMENTS I would like to personally thank the following : The Department of Microbiology and Genetics, Massey University for providing the opportunity and facilities for this investigation. My supervisor , Dr Tim Brown for his supervision and guidance; and Dr Ray Cursons for his ro~ny suggestions . Professor D. F. Bacon and other academic and technical staff of the Department . Mrs Elizabeth Keys for her technical assistance and friendship throughout this study. Ethnor Pty. Ltd. , and Roche Product s Pty . Ltd . , for their financial support and supply of chemotherapeut ic a gents. Mrs Penny van Doorn for the excellen t typing . Ma ssey University Library staff for t he numerous interloan requests. The Central Photographic Unit , Massey University . The Small Animal Production Unit for the seemingly endless supply of mice , and all those others , without whom this study would not have been possib]e . TABLE OF CONTENTS ABSTRACT ACKNOWLEDGMENTS LIST OF TABLES LIST OF FIGURES LIST OF PLATES CHAPTER ONE: INTRODUCTION 1.1 The History of Free-Living Ameba as Disease Agents 1.2 Classification 1.3 Occurrence and Distribution 1.4 Pathogenicity 1.5 Immunity 1.6 Control Measures 1. 7 Diagnosis 1.8 PAM cases and Their Treatment CHAPTER TWO: MATERIALS 2 .1 Ameba Cultures Used 2.2 Plate Media 2.2.1 Ameba Saline Agar 2.2.2 Ameba 1% Saline Agar 2.2.3 Sabouraud Agar 2 .3 Axenic Media for Amebae 2 .3.1 Page's Ameba Saline (PAS) 2 .3.2 CYM Medium 2.3 .3 4.0% Neff Medium 2.3.4 CGHV and CGHVS Medium 2.4 Cell Culture Media 2.4.1 Vero Cell Culture 2.4.2 4.4% Bicarbonate Solution 2.4.3 lOx Trypsin/Versene Mixture 2.4.4 Antibiotics 2.4.5 Eagle's Growth Medium 2.4.6 Eagle's Maintenance Medium 2.5 Antibiotic Solutions 2.5.1 Amphotericin B 2.5.2 Tetracycline hydrochloride 2.5.3 Polymyxin B sulphate ii iv ix xi xii i 1 1 3 3 4 5 6 7 9 18 18 18 18 24 24 24 24 25 25 26 27 27 27 27 27 27 27 28 28 28 28 2.5.4 5-Fluorocytosine 2.5.5 Miconazole 2.5.6 R41,400 2.5.7 Levamisole HCl 2.5.8 Kanamycin 2.5.9 Oxytetracycline 2.5.10 Tylosine 2.6 Miscellaneous Solutions 2.6.1 Phosphate Buffered Saline 2.6.2 Physiological Saline 2.6.3 Hanks Solution 2.6.4 Erythrosine B CHAPTER THREE : METHODS 3.1 St erilization 3.2 Axenic culture techniques 3.2 .1 Maintenance of ~tock axenic cultures 3.2.2 Axenic drug testing 3.3 Cell Culture Techniques 3.3.1 Maintenance of stock Vero cell cultures and preparation of KLIMAX tubes for drug testing 3.3.2 Cell culture d rug testing 3.4 In Vivo Testing of Drugs 3.4.1 Intraperitoneal injection method 3.4.2 Intraveniri cu] ar injection method 3.5 Bioassay of Imidazole Levels in Mouse Serum 3.5.1 R41,400 3.5.2 Miconazole nitrate CHAPTER FOUR: RESULTS 4.1 In Vitro Axenic Drug Testing of Naegleria spp. 4.1.1 Kanamycin and Tylosine 4.1.2 Oxytetracycline 4.1.3 Levamisole 4.1.4 5-Fluorocvtosine 4.1.5 Amphotericin B 29 29 29 29 30 30 30 30 30 30 31 31 32 32 32 32 32 33 33 34 34 35 35 36 36 37 38 38 38 43 43 48 48 vi 4.2 In Vitro Axenic Drug Testing of Acanthamoeba spp . 4.2 .l Kanamycin , tylosine and oxytetracycline 4.2 . 2 Levamisole 4 . 3 . 3 5-Fluorocytosine 4.3 In Vitro Axenic Testing of Various Aged Cultures of N. gruberi (Pl200f) and A. castellanii (1501) Against 5 1 51 58 58 vii Leva mi sole 62 4.3 .1 N. gruberi (Pl200f) 4.3.2 A. castellanii (1501) 4.4 The Testing of Drug Combinations agains t Naegleria fowleri (MsM) in Axenic Culture 4 . 4 .1 Amphotericin Band 5-fluorocytosine 4.4.2 Amphotericin Band oxytetracycline 4.4 . 3 Oxytetracycline and tylosine 4 . 5 The Testing of Drug Combinations a gainst Acanthamoeba culbertsoni (A-1) in Axenic Culture 4 . 5 .1 Amphotericin Band 5-fluorocytosine 4 . 6 Cell Culture Drug Testing of Naegleria fowleri (MsM) 4 . 6 .l Amphotericin B 4 . 6 . 2 Oxytetracycline 4 . 6 . 3 Amphotericin Band Oxytetracycline 4 . 7 Cell Culture Drug Testing of Acanthamoeba culbertsoni (A-1) 4.7. 1 Levamisole 62 62 67 67 69 72 74 74 76 77 77 80 82 83 4.8 In Vivo Drug Testing of Naegleria f owleri (MsT and }~M) 85 4.8 .1 R41 ,400 (Ketoconazole) 4.8 . 2 Miconazole nitrate 4 . 8 . 4 Amphotericin Band tetracycline 4. 8 . 5 Amphotericin Band oxytetracycline 85 88 90 95 4. 9 In Vivo Drug Testing of Acanthamoeba culbertsoni (A-1) 98 4.9 . 1 5-Fluorocytosine 98 4 . 9.2 Polymyxin B sulphate 4 . 9. 3 5-Fluorocytosine and polymyxin B sulphate 4.10 In Vivo Determination of Serum Drug Levels 4.10.1 Miconazole nitrate 4.10.2 R41,400 (Ketoconazole) 98 101 103 103 105 CHAPTER FIVE : DISCUSSION 5.1 Treatment of Naegleria Infections 5.2 Treatment of Acanthamoeba Infections 5.3 Drug Dosage an d Animal Size BIBLIOGRAPHY viii 110 110 128 134 137 ix LIST OF TABLES I. II. III. IV. V. VI. VII. VIII. IX. x. XI XII. XIII . XIV. xv . XVI. XVII. XVIII . XIX. xx. XXI. Cases of PAM Reported afte r 1974 Probable and Definite Survivors of PAM Ameba Cultures Used Effect of the Size of Inocula of N. fowleri (MsM) on Time Needed for Development of CPE The Effect of Amphotericin B on N. fowleri (MsM) in Cell Culture The Effect of Oxytetracycline on N. fowleri (MsM) in Cell Culture The Effect of Ampho teri cin Band Oxytetracycline Together on N. fowleri (MsM) in Cell Culture Effect of the Size of Inocula of A. culbertsoni (A- 1) on Time Needed for Development of CPE The Effect of Levamisole on A. culbertsoni (A-1) in Cell Culture The Treatment of Mice Inoculated with N . fowleri (MsM) Using Ketoconazole (R41,400)Given IP every 24 Hours The Treatmen t of Mice Inoculated wi th N. fowleri (MsM) Using Ketoconazole (R41,400) Given IP every 12 Hours The Treatment of Mice Intranasally Inoculated with N. fowleri (MsM) Using Ketoconazole (R4 l, 400) Administered by I . Vent. Injection The Treatment of Mice Inocu] ated with N . fowler i (MsM) 10 11 18 76 78 79 81 82 84 86 86 87 Using Miconazole Nitrate Administered by IP Injection 89 The Treatment of Mice Inoculated with N. fow ler i (MsM) Using Miconazole Nitrate Given by I.Vent Injection 89 Treatment of Mice Using Amphotericin B (Given I.Vent.) and Tetracycline (Given IP) 91 The Treatment of Mice with Arnphotericin Band Tetracycline . Arnphoteri cin B Was Administered Both I.Vent . and IP, with Tetracycline Given IP 92 The Treatment of Mice with Arnphotericin Band Te tracycline Alone and in Combination. Amphotericin B Was Administered I. Vent. and Tetracycline Given Both I. Vent. and IP 93 The Treatment of Mice Infected with N. fowler i (MsM) or (MsT) Using Amphotericin Band Tetracycline Given IP 94 The Treatment of Mice Infected with N. fowleri (MsM) Using Amphotericin B (I . Vent . ) and Oxytetracycline (IP) 96 The Treatment of Mice Infected with N. fowleri (MsM) or (MsT) Using Amphotericin _B and Oxytetracycline Given IP 97 The Treatment of Mice Intranasally Inoculated with A. culbertsoni (A-1) Using 5-Fluorocytosine Injected IP 99 XXII . XXIII . XXIV . XXV. Y.XVI . The Treatment of Mice Intranasally Infected with A. culbertsoni (A-1) Using 5-Fluorocytosine Injected I. Vent. The Treatment of Mice Infected with A. culbertsoni (A-1) Using -Polymyxin B Administered I.Vent. The Treatment of Mice Infected with A. culbertsoni (A-1) Using the Synergistic Combination of Polymyxin Band 5-Fluorocytosine given I.Vent . The Results of the Assay of Serum Samples Against Candida pseudotropicalis for Miconazole ':'he ~£sults of the Assay of Serum Samples Agains t Candida piropsilosis for Ketoconazole X 99 100 102 103 105 LIST OF FIGURES 1. 2. 3. 4. 5. Intraventricular Injection of Mice The Effect of Kanamycin on N; gruberi (Pl200f) The Effect of Kanamycin on N. fowleri (MsM) The Effect of Tylosine on N. gruberi (Pl200f) The Effect of Tylosine on N. fowleri (MsM) 6.The The Effect of Oxytetracycline on N. gruberi (P1200f) 7. 8. 9. 10. 11. 12. 13. 14 . 15. 16. 1 7. 18. 19. 20. 21. 22. 23. 24. 25 . 26. 27 . ?R. .,o . 31. The Effect of Oxytetracycline on N; fowleri (MsM) The Effect of Levamisole on N. gruberi (Pl200f) The Effect of Levamisole on N. fowleri (MsM) The Effect of 5-Fluorocytosine on N. fowleri (MsM) The Effect of Amphotericin B on N. fowleri (MsM) The Effect of Kanamycin on A. castellanii (1501) The Effect of Kanamycin on A. culbertsoni (A-1) The Effect of Tylosine on A. castellanii (1501) The Effect of Tylosine on A. culbertsoni (A-1) The Effect of Oxytetracycline on A. castellanii (1501) The Effect of Oxytetracycline on A. culbertsoni (A-1) The Effect of Levamisole on A. castellanii (1501) The Effect of Levamisole on A. culbertsoni (A-1) The Effect of 5-Fluorocytosine on A. culbertsoni (A-1) The Effect of Levamisole on a 48 Hour Culture of N. gruberi (Pl200f) The Effect of Levamisole on a 96 Hour Culture of N. gruberi (Pl200f) The Effect of Levamisole on a 48 Hour Culture of A. castellanii (1501) The Effect of Levamisole on a 96 Hour Culture of A. castellanii (1501) The Effect of Amphotericin Band 5-Fluorocytosine Alone and in Combination on N. fowleri (MsM) The Effect of Amphotericin . B and Oxytetracycline Alone and in Combination on N. fowleri (MsM) The Effect of Amphotericin Band Oxytetracycline Alone and in Combination on N. fowleri (MsM) The Effect of Oxytetracycline and Tylosine Alone and in Combination ort N. fowleri (MsM) The Effect of Amphotericin Band 5-Fluorocytosine Alone and in Combination on ·A. culbertsoni (A-1) Standard Curve Showing Zone Diameter Plotted Against Known Miconazole Concentrations Concentrations of Miconazole nitrate in the Serum of Mice After an Initial Injection of 60mg.kg- 1 IP 36 39 40 41 42 44 45 46 47 49 50 52 53 54 55 56 57 59 60 61 63 64 65 66 68 70 71 73 75 107 109 xi 32 . 33 . Standard Curve Showing Zone Diameter Plotted Against Known R41,400 Concentrations Concentrations of R41,400 in the Serum of Mice After an Initial Injection of 60mg.kg- 1 IP xii 108 109 LIST OF PLATES 1. 2. 3. 4 . 5. 6 . 7 . 8 . 9. 10 . Trophozoite Trophozoite Cyst stage Cyst stage Flagellate Flagellate Trophozoite Trophozoite Cyst stage Cys t stage stage of Nae~leria gruberi (Pl200f) stage of Naegleria fowleri (MsM) of Nae~leria gruberi (Pl200f) of Naegleria fowleri (MsM) stage of Naegleria gruberi (Pl200f) stage of Naegleria fowleri (MsM) stage of Acantharnoeba castellanii (1501) s t age of Acanthamoeba culbertsoni (A-1) of Acanthamoeba castellanii (1501) of Acantharnoeba culbertsoni (A-1) 11. Inhibition of a background lawn of Candida pseudotropicalis 19 19 20 20 21 21 22 22 23 23 by Miconazole nitrate standards 104 12. Inhibition of a background lawn of Candida pseudotropicalis by serum samples taken at various time intervals, containing unknown concentrations of Miconazole nitrate 104 13 . Inhibition of a background lawn of Candida pirapsilosis by R41,400 standards 106 14. Inhibition of a background lawn of Candida pirapsilosis by serum samples taken at various time intervals, containing unknown concen trations of R41,400 106 xiii CHAPTER ONE: INTRODUCTION 1.1 The History of the Free-living Amebae as Disease Agents The history of Pathogenic Free-living Arnebae (PFIA) of the genera Acanthamoeba and Naegleria has been extensively reviewed elsewhere (Culbertson, 1971; Duma et al., 1971; Chang, 1971, 1974a; Carter, 1972; Cursons, 1974; Cursons and Brown, 1976). The commonest disease caused by PFLA is Primary Amebic Meningo-encephalitis (PAM) caused by ameba quite different to those traditionally re garded a s parasitic in man,and are not ordinarily parasitic in lower animals. They are ubiquitous in the environment and are free-living in water, sewage, soil and other decaying organic rnatter(Carter, 1972). Willaert in 1974, tabulated 84 cases from all the continents with the exception of Antarctica. Since then at least 10 additional cases have been reported (Table 1 ) • Acanthamoeba spp. were the first agents implicated in this disease, but PAM is now known to be caused by a free-living arnebae of the genus Naegleria . This was due mainly to the first case prototype of this illness which was described by Fowler and Carter in 1965. In 1968 Carter, Culbertson et al., and Butt et al., showed that the incriminating species of most reported cases belonged to the genus Naegleria, and in 1970 on the basis of morphological, cultural and pathogenicity differences Carter renamed the pathogeni c species Nae gleria fowleri distinguishing it from the non-pathogenic Naegleria gruberi . Prior to 1968, all cases of PAM were attributed to the Acanthamoeba spp. which was probably due to the pioneering work of Culbertson et al., (1958, 1959, 1965) who, whilst working on the production of polio vaccine found an ameba which contaminated the cultures of monkey kidney cells. These cultures, when innoculated intracerebrally into mice, produced a necroting, hemorrhagic meningo­ encephalitis that killed mice in 4-7 days. The re s pon s ible amebae was identified as an Acanthamoeba and they predicted on the basis of the finding, that this amebae could be capable of producing disease in humans. This ameba was previously considered to be a harmless free­ living organism. 1 The disease caused by PFLA can be divided into two entities (Chang, 1974 (a) ) : a) a swimming-associated acute rneningo-encephalitis known as Primary Amebic Meningo-encephalitis (PAM) (Martinez et al., 1977). This is the most important of the two and is caused by Naegleria fowleri (non-pathogen - Naegleria gruberi) . Infection is thought to occur in two ways: i) Naegleria contaminated water may be introduced into the upper nasal passages; ii) it may be due to the washing of trophozoites residing in 2 the lower nasal passages of a carrier into the upper nasal area. A pathogenic strain of Naegleria was isolated from a normal healthy carrier (Visvesvara et al., 1974). b) a non-swimming-associated chronic meningo-encephalitis (Amebic Meningo-encephalitis (AM) caused by amebae of the Acanthamoeba/Hartmanella group . It is considered that the mos t probable species is A. rhysodes along with another pathogenic species A. culbertsoni. Amebic Meningo-encephalitis due to the involvement of the Central Nervous System (CNS), appears to be a secondary phenomenon representing metatastic spread from a primary focus in the skin, genitourinary tract or respiratory tract (Martinez et al., 1977). Cutaneous ulceration is a possible point of entry with hematogenous spread to the CNS and lower respiratory tract.Infections in experimental animals have been reported (Martinez et al.,) due to this spread. Subsequently Acanthamoeba spp. have been indicated in a number of chronic illnesses such as respiratory infections (Martinez et al., 1975), corneal ulceration of the eye leading to blindness (Nadington et al., 1974; Visvesvara et al., 1974) and together with Naegleria spp. in humidifier disease (M.R.C. Symposium, 1977). The reidentification of the etiological agents of the 1968 cases of PAM in New Zealand as N. fowleri species (Cursons and Brown, 1975, 1976) has dismissed the notion of slime moulds in the etiology of PAM (Mandel et al., 1970). Henceforth, in the text, the nomenclature of Martinez et al., 1977, of PAM for Naegleria infections and AM for Acanthamoeba meningo­ encephalitis will be adopted. 1.2 Classification Study of the basic classifi ca tion of the small free-living amebae ha s been stimulated by the di s covery of their role in human disease. Carter (1970) accepted the decision of Page to retain the family designation of Vahlkampfiidae (for Nae gleria spp.) in preference to the revision by Singh and Das (1970). Recent studies by Fulton (1970) and more recently by Schuster (1975) on the mitosis of Naegleria and related amebae displaying promitosis,have affirmed the lack of validity of details such as "interzonal body" and "polar caps" for use in establishing a higher taxa such as Schi zopyrenidae. These points and others are extensively reviewed elsewhere by others (Page, 1974; Chang, 1971; Schuster, 1975; Visvesvara et al., 1975). In reviewing Acanthamoeba spp. it was clear that they shoul d be moved from the Hartmanellidae and related limax ameba e. Page (1967) 3 for one, proposed the replacement of the Acanthamoeba e in the Mayorellidae. In a recent review by Cursons and Brown (1976) the controversy regarding the classification of the PFLA appeared to be settled with the majority preferring Chang's 1971 classification scheme. The identifi­ cation of isolates involves the exploitation of specific cytolo gica l, morphological, physiolo gical, immunological, growth and pathogenicity characteristics in an ordered sequence readily usab le by hospita l and public health laboratory staff (Cursons and Brown, 1976). 1.3 Occurrence and Distribution The ability of PFLA to form resistant cysts undoubtedly enables them not only to withstand unfavourable conditions, e.g. the isolation of Acanthamoeba spp. from 2°c (Brown and Cursons, 1977) and from Antarctic soils (Brown et al., pers. comm.), but also to take advantage of the intermittent occurrence of favourable condition s . The PFLA appear to be truly ubiquitous organisms, as isolations have been recorded from a variety of environmental sources such as a ir (Kingston and ~arhurst , 1968), humidifier systems (M.R.C. Symposi um, 1977), freshwater, brackish and ocean systems (De Jonckheere et a l ., 1975; Brown and Cursons, 1977; Stevens et al., 1977a; Wellings c t al., 1977), chlorinated swimming and domestic waters (Cerva, 1971a; Anderson and Jamieson, 1977; Cerva and Huldt, 1974), from bottled drinking water (Desmet-Faix, 1974), from a home dialysis unit (Casemore, 1977), from soil (Anderson and Jamieson, 1972; Cursons et al., 1978b), and from sewage (Singh and Das, 1972; Chang, 1974a) Isolations have also been recorded from cell cultures (Jahnes e t al., 1957; Stevens and O'Dell , 1973a ; Willaert e t al ., 1978~ throat and nasa l cavities (Elridge and Tobin, 1967; Cerva et al., 1973; Chang e t al ., 1975), eye inf e c tions (Nagington et al., 1974; Visvesvara et al. , 1975) , gastrointestinal washings (Hoeff l er and Rubel, 1974) , cold­ blooded vertebrates (Frank, 1974 ) , snails (Kingston a nd Taylor, 19 76) , and fish (Taylor, 1977). Temperature and pH a re equally t olerated over a wide range wi th in vitro growth r epor ted up to 45°c (Griffin, 1972) and a pH range of 4.6 to 9.5 (Carter, 1970). The dis tribution of the pa thogenic species in relation t o non-pathogenic ones is s till unknown (Cursons, 1978) though i n general, non-pa t hogenic species are more prevalent at ambient temperature in tempera te zones . The r epeated i so l ations of PFLA from waters above ambient temperatur e , i .e. grea ter or equal to 30°c (De Jonckheere et al., 1975 ; Stevens et al ., 1977a; Welli ngs et al ., 1977 ; Cursons et a l., 1978b) , combined with their higher optimum temperature of growth (Griffith, 1972 ) suggests that the pathogenic amebae are environmentally selected over non-pathogenic amebae in waters above ambien t temperature . The source of the pathogenic arnebae in these waters is unknown though the fac t that Cursons e t al. (1978b), and Wellings et a l. (1977) have isolated PFLA from the soil , which is the preferred habitat of small free-l i ving arnebae (Singh, 1978) , makes it possib l e that the soil ac t s as a ~0 servoir of pathogens in t he same way as it does fo r Cryptococcus , and t ha t contamination occurs via run off af ter rain (Cursons, 19 78) . 1.4 Pathogenic ity The invasion of or gans and tissues b y PFLA is now well documen ted (Culbertson e t al ., 1959, 1968, 1972; Carter, 1968 , 1970, 19 72; Callicot e t al. , 1968; Chang , 1971, 1974a & b, 1976; Cul'bc- t son, 1971; Martinez et al. , 1973, 1975, 1977 ; Visvesvar a and Bal a~u t h, 1975; Wong et al. , 1975 a & b). The CNS i nvasion by Na e~lPria spp . occurs primarily via the nasal mucosal epithelium, ma i~l ) due to the pathological condition of the cribriform plate and subjacent nasal passages (Culbertson, 1971; Carter, 1972) and this has been verified experimentally by Martinez et al. (1973). Using mice they s howed that amebic invasion occurs through the disruption of the olfactory mucosa , penetration into the submucosal plexus , probably by phagocytosis by the ameba~ of the s ustentacular cells of the olfactory 4 5 neuroepithelium,and finally through the cribriform plate to the CNS. In cases of Acanthamoeb a meningo-encephalitis,the involvement of the CNS appears to be a secondary phenomenon representing metatastic spread from a primary focus in the skin, geni tourinary or respiratory tract (Martinez et al., 1975, 1977; Culbert son, 1971). Martinez et al. (1975) reported lower respiratory tract infections in experimental animals. AM . due to Acanthamoeba spp.,appears to be due to an opportunistic infection of the CNS. AM occurs in patients who are chronically ill, debilitated or in those whose cell mediated immune responses have been impaired as a result of either underlying sys temi c disease or its treatment by i mmunosuppressive me thods (Kernohan et al., 1960; Jager and Stamm, 1972; Robert and Rorke , 1973; Bhagwandeen et al., 1975). Acanthamoeba infections of sites with reduced accessibility to the immune s ystem (e.g. the e ye) also demonstrates the opportunistic nature of these infections . Isolations of Acanthamoeba from the cornea of the eye ( Nagington et al., 1974) were shown to be of low virulence and infection only resulted after damage to the cornea (Visvesvara et al., 1975). Once CNS invasion has occurred, destruction of the surroundin g tissue is thought to be brou ght about by a combination of phagocytosis and pinocytosis of host tissu e by N. fowleri,and solely by pinocytosis in the case of A. culbertsoni (Visvesvara and Callaway, 1974; Maitra et al., 1974, 1976). The extensive reports of the possession of lysosomal and hydrolytic enzymes is reported elsewhere (Bowers and Korn, 1973; Martinez et al., 1975; Chang, 1976; Maitra et al., 1976; Cursons and Brown, 1976), and it is also speculated that the levels of the cytopathic enzymes produced may explain the degrees of virulence amon g the Acanthamoeba and the ·N: fowleri isolates (Cursons, 1978; Culbertson, 1971; De Jonckheere and van de Voorde, 1977b). 1.5 IIIIlilunity Many authors have pondered over the low incidence of PAM a ~d AM cases with regards the ease and frequency of isolation of pathoge nic PFLA from the environment (Anderson and Jamieson, 1971; Cursons et al., 1977b, 1977; John ·et al., 1977; Wellings et al., · 1977; Haggerty and John, 1978). This has led many to speculate on the existence of probable host related susceptibility factors and the demonstration of specific antibodies to free-living amebae in human sera has been reported (Chang a nd Owens, 1964; Edwards et a l . , 1976; Cursons et al., 1977; MRC Symposi um , 1977). Adams et al., (1976) reported that mice surviving a primary intravenous injection of N. fowleri we re subsequent l y resistant to further challenge by the same route with a dose of amebae that produced a unifo r mly fa t al disease in untreated con trol mice . It was fur ther demonstrated by this group tha t mice i mmunized with live or formalized N. fowleri or live N. gruberi either subcut aneously , intraperitoneally, intravenously o r intramuscularly were significantl y pr o tected agains t a subsequent challenge with N. fowleri (John et al . , 1977) . The role of cell mediated immunity (CMI) in r es i stance to infec t ion by N. fowleri was reported by Diffley et al. (19 76) , who demonstrated tha t guinea-pigs surviving a normally fata l chall enge with N. fowleri, exhibited a delayed hypersensitivity when t ested intradermall y with a soluble fraction derived from N. fowler i (Cursons et al . , 1977) . Thong (1978) s ta ted tha t protective i mmuni ty to PAM could be transfer red t o syngeneic mice by immune sera but no t by i mmune spleen cells . The i mmunity may be related to agglutinating antibodies demonstrated in immune sera or an t itoxic antibodies in immune sera may be the active principle . These all tend to support the hypo t hesi s tha t unwitting exposure to the more ubiquitous non-pathogenic N. gruberi may i mmunize against N. fowleri and the same may also occur with Acanthamoeba spp . The fact tha t some underlying immunity exists was dernor.strated by Wong et al. (1975 a & b) who demonstrated 6 tha t prima t es ~e re apparently immune t o intranasal or intravenous innoculations of N. fowler i or A. culbertsoni unless on i mmunosuppressive drugs. However intrathecal innoculations were shown t o cause arnebic meningo- encephalitis. Culbertson has shown tha t mice immunized with Acantharnoeba spp . are resistant to intranasal challenge with A. culbertsoni but was unable to show the same with Naegleria spp . 1, 6 Control Measures Free- l iving amebae are widely dispersed in the environment a nd the fact that t hey can be isolated from chlorinated domestic and swimming waters (Cerv2 , ~971a; Anderson and J amieson, 1972; Cerva and Huldt, 1974; De Jonckheere and van de Voorde , 1976), as well as untreated recreationa l waters has led to an expression of concern by public health authorities over the possible contraction of PAM or AM via t hese sources . Cerva (1971a), afte r reviewing 16 fatal cases of PAM from an indoor chlorinated swimming pool stated that, there will always be the cons t ant presence of limax amebae even under the strict observations of all routine safety measures applied to swinnning pools and water systems. This was supported by a reported case of PAM in South Australia by Anderson and Jamieson (1972), in which the victim contracted the dis ease from domestic bath water, and that super chlorination to lOmg.1-l failed to eradicate Naegleria from the contaminated pool. However, Lyons and Kapur (1977) in a survey of 30 halogenated public swimming pools concluded that the low amebic densities (less than one per litre), in the majority of pools illustrated that these amebae could be adequately controlled by proper pool maintenance. The possession of resistant cysts however complicates the disinfection process. Derreumaux et al. (1974) demonstrated that O.Smg.1-l of H0Cl, the active disinfecting component of chlorine disinfection was able to eradicate both Naegleria and Acanthamoeba spp. De Jonckheere and van de Voorde (1976), showed that an initial concentration of chlorine 7 between 0.5 - l.Omg.1-l was cysticidal for Naegleria spp. but that Acanthamoeba culbertsoni cysts were not inactivated by levels up to 40mg.1-l. I In a study of alternative disinfectants by Cursons et al., (1978b) it was shown that deciquam 222, chlorine, chlorine dioxide and ozone a l l possessed potential disinfecting properties for PFLA, but at higher levels than those for disinfecting bacteria. Deciquam 222 was found to be the most effective followed by chlorine, chlorine dioxide and ozone, but the final choice of disinfectant mus t depend on the physical and chemical properties of the water to be treated. 1 . 7 Dia gnosis Early diagnosis and treatment along with careful intensive care treatment therapy is extremely important in the treatment of infections due to PFLA; more in those caused by Naegleria spp. The survival of a nine year old female in Torrance, California (Siedel et al., pers. connn . 1978) and that of a fourteen year old male in Australia (Anderson and Jamieson, 1972) could be attributed to this. Fluid restriction, management of cerebral edema and other complications of amebic meningo-encephalitis are all important in the care of these patients (Siedel et al., pers. comm. 1978) . Infections due to Naegleria spp. are usually characterized by a previous history of swiunning in freshwater some 7-14 days before expressing typical meningitis symptoms (Cursons et al.,1977; Carter, 1972; Chang, 1974a). The symptoms include severe headache (usually frontal), sore throat, nausea, vomiting, fever (39-41°C) accompanied by a stiff neck. Clinical isolation of amebae can be routinely done by cultivation of Cerebral Sninal Fluid (CSF), brain tissue or nasal discharge on Page 's Ameba Saline Agar spread with live E. coli or E. cloacae; by a xenic CYM culture ; or by passaging of suspected material through cell culture, at 37-45°C (Cur son s et al., 1978). The examination of CSF is still probably the mos t routine method of diagnosing general meningitis . The differences between amebic and bacterial meningitis are sli ght and although in positive amebic cases there tend to be a predominance of neutrophils in the CSF, a hi gh protein concentration and low sugar levels, complete dia gnosis relies on finding amebae in the fluid and the further cultivation of these for complete dia gnos is. Species identification can then be achieved by a method outlined by Cursons and Brown (1976). In post-mortem dia gnosis, a degree of encephalitis is invariably present. Severe brain swelling and redness, combined with purulent and haemorrhagic exudate containing numerous amebae is more extensive on the ventral surface of the cerebrum or cerebellum and over the brain stem. Amebae are also numerous in the olfactory nerve bundles which are virtually destroyed by purulent inflamma tion (Carter, 1969, 1972). The grey ma tter of the cerebral hemispheres and cerebellum shows variable sized lesions which tend to be haemorrhagic and quite soft when they are large (Culbertson, 1971). Puralent meningitis is usually inconspicuous and confined to the antero-basal aspects of the brain, and it is only rarely tha t one can find inflammation or amebic invasion in the posterior cerebral hemispheres, brain stem or cerebellum, and never in the spinal cord (Carter, 1969, 1972). The Indirect Immuno Fluorescent Antibody (IFAB) technique applied to hydrosoluble protein extracts of either Naegleria or Acanthamoeba spp. is a valuable tool in the identification of species. It can also be app lied to identify amebae in brain sections of suspected or proven patients,though is a time consuming process and is not recommended for r outine laboratory practice. Antisera can be produced in rabbits and can be made species specific by suitable absorption methods. IFAB me thoc s can also be used to provide rapid screening methods for dete ction of PFLA in swimming pools, tap and other domestic and recreational ~a ter supplies. Immunoperoxidase methods have been used to demonstrate both Naegleria and Acanthamoeba spp. in brain sections of patients who have died from PAM and AM respectively by Culbertson (1975) and Cursons et al., (1976). This is a method that may be shown to be more valuable 8 in the future than immunofluorescence techniques. It has certain advantages over IFAB in that permanent preparations can be made, no specialized equipment is necessary and clear definitive staining of tissue elements results (Culbertson, 1975). Acanthamoeba meningitis infections are difficult to diagnose even in advanced cases due to the lack of specific s ymptoms and the apparent lack of amebae in the CSF (Chang, 1974a). There is usually a history of poor health and immunological incompetence with few patients giving a past history of swimming. The onset is slow (>10 days) and insidious,with the lung, brain and kidneys being infected (Martinez et al., 1976). Acanthamoeba infections may initially produce a severe bronchopneumonia, the organisms then dessimina ting and reaching the CNS via the bloodstream (Marino, 1975). Post-mortem diagnosis relies on the presence of superficial lesions in the grey matter with granulomatosis inflamma tio~ and the presence of trophozoites and double walled wrinkled cysts in apparently normal tissue bordering the lesion (Chang, 1974a; Carter, 1972; Culbertson, 1971; Hoffmann et al., 1978). Many authors re gard this as diagnostic of Acanthamoeba infections. In the case of eye infections reported by Nagin g ton et al. (1974) and Jones et al. (1975), positive diagnosis was possible b y taking corneal scrapings, with subsequent isolation and identification of Acanthamoeba spp. 1. 8 PAM Cases and Their 'Treatment Since Willaert published the extensive review of world-wide cases due to PAM in 1974, there have been at least ten additional cases reported (Table I). Symmers (1969) reports a possible earliest case dating back to 1909. A later case reported by Derrick et al. (1948) 9 was originally thought to be due to Iodamoeba butschlii, but was later proven by fluorescent antibody staining to have been caused by N. fowleri (McMillan, 1977). The confusion in this case arose through the patient ha ~rj i!g ,,,widespread alimentary and systemic invasion as well as the typical pattern of cerebral invasion by morphologically identical amebae, thought to be caused by starvation of the patient, perhaps by reducing his gastric activity, bile secretion and amebicidal serum factor (Carter, 1970). The reidentification of the etiological agents of the 1968 cases of PAM in New Zealand as ·N~ fowleri (Cursons and Brown, 1975; Cursons · ·et ·al., 196 7a) has dismissed the notion of slime l'l'Odds being involved in ; NUMBER C.'\USATIVE COUNTRY YEAR OF CASES ORGANISM DI AGNOSIS TREATMENT OUTCOME REFERENCE . 1974 1 N. fowleri (MsT ) isolation Peni ci llin died Cur sons ~ ~-, 1976b NEW from CSF Ampicillin ZEALAND Amphotericin B 1978 l Ii. fowleri (MsM) isolation Amphotericin B died Cursons e t ~., pers. from CSF comm ., 1978 1974 l k!. fowleri (Lovell) isolation .Unknown died De Jonckheere, 1977 from CSF 1974 l Acanthamoeba sp, IFAB Steroids died Marti nez et .§.1., 1977 U,S,A, Penicillin 1975 l Acanthamoeba sp, IFAB Unknown died Hoffman ~ al. , 1978 post- mortem 1978 l Naegleria sp, isolation Amphote r ici n B survived Seidel g ,tl., pers, from CSF Miconazole co mm ,, 1978 Rif ampi n VENEZUELA l !:. . cul berts oni IFAB Steroids died Martinez g ~., 1977 1 A. cas tellanii IF.I\B Steroids died Marti nez ~ ~-, 19 77 PERU - Antibiotics 1972 l Acanthamoeba sp. post-mortem Antibiotics died Bhagwand een ~ ~., ZAMBIA Amphotericin B 1975 1958 l Acanthamoeba sp. post - mortem Penicillin died Ringsted ~ ~-, 1975 KOREA Streptomycin Chloramphenicol Table I: Cases of Primary Amebic Meningo-encephalitis Reported After 1974 (modified from Cursons, 1978) _. 0 NUMBER CAU SATIVE COUNTRY YEAR OF CASES ORGANISM TREATMENT 1968 l Naegleria Me tronidazole - UGANDA Emeti ne - Penicillin Chloroquine , - 1967 1 A, ast ron::txis Ampicillin Peni cillin - G U.S.A. 1978 1 Naegl eria Amphotericin B ~licona zo le Rifampin 1970 2 Naegler ia Streptomycin Isonicoteinhydrosine INDIA Sulphadexanathosone Ampho t e ricin B 1973 3 N. fowleri Unk.nown 1969 2 Naeg leria Antibi otics ENGLAND Sulphadiazine Amphotericin B 1971 1 !i . fowleri Amphotericin B AUSTRALIA Sulphadiazine Table II: Probable and Definite Survivors of Primar::t Arnebic Meningo-encephalitis REFERENCE Grundy & Blowers, 1970 Callicott~ iU:.•, 1968 Seidel g fil., pers. comm. 1978 Pan & Ghosh, 1971 S. R. Das, pers. comm, to Willaert (l974) Apley~~., 1970 Anderson & Jamieson, 19 72 .... .... the etiology of PAM (Mandel e t al ., 1970). The drug treatment of PAM has been very discouraging with ' Willaer t ' s summar y (1974) providing information of onl y ten possible survivors of the disease. The Californian case of Seidel et al . (1978) brings the world total to eleven cases (Table II). In the earlier cases , where the amebic nature of the disease had no t been suspec t ed, treatment consisted only of antibacteria l agents such as sulpha-drugs, penicillin, st r ep t omycin , tetracyclines and chloramphenicol (Fowler and Carter , 1965; But t et al ., 1968; Cerva and Novak , 1968 ; Dos Santos , 1970 ; Van den Driessche e t a l., 197 3) . However, even in later cases where the antiprotozoal drugs emetine , chloroquine and metronidazole were often used, the course of the disease was not affected in the slightes t (Car ter, 1968, 1970, 1972; Duma et al. , 1971) except in the unproven case of Grundy and Blowers (1970) in which surviva l was attributed to chl oroquine . Naegleria were supposedly isola t ed from the CSF bu t fai l ed to survive for any length of time in cul t ure and subsequently were no t positively identified . The patient a lso presented atypical symptoms and tr eatment consisted of me tronidazole, emetine, penicillin su]phane and chloroquine . The in vitro activity of antibacterial agents agains t pathogenic Naegleria has been extensively reviewed by many (Carter, 1969; Ma ndel et al . , 1970; Prasad, 1972 ; Thong et al ., 1977 ; Lee e t al. , 1979 , Donald e t al ., 1979) . Of the antiprotozoal drugs , emetine hydrochl or ide was shown t o be effec tive in vitro against N. fowleri (Carter , 1969 ; Prasad , 1972 ; Das , 1975) although it does not protect animals from the disease (Culbertson et al . , 1968) , probably due to its inability t o pass the blood-brain barrier (Parmer and Cottrill, 1949) . Chloroquine and metronidazole have a lso been shown to be ineffec tive both in i n vitro and in vivo studies (Carter, 1969 ; Mandel et alt , 1970; Duma et al . , 1971). 12 Amphotericin B was the only drug to appear promising in the early 70's, and as can be seen in Table II, it was used in the trea t ment of all survivors except the unproven case of Grundy and Blowers (1976) and Callicott et al;, (1968) . Amphotericin Bis an antifungal polyene antibiotic and in vitro t es ts have shown it to be very effective against Naegleria spp. (Carter, 1969 ; Mandel et al., 1970; Duma et al . , 1971; Schuster and Rechthand, 1975 ; Visvesvara and Balamuth, 1975; Duma and Finley, 1976; De Jonckheere a nd van de Voorde, 1977 ; Donald et al., 1979) and t o show i n vivo promise (Culbertson et al . , 1968; Carter, 1969; Das 197 1; Thong, 1978 , 1979) . Carter (1969) suggested that amphotericin B be tried in the treatment of PAM by simultaneous intravenous and intraventricular administration . The doses recommended were: 0.25mg.kg- 1 1V and 1.0mg into the cerebral ventricles (I.vent.)in the first 24 hours which were as high as he dared propose due to the highly toxic nature of the drug. Carter (1972) also suggested using sulphadiazine as well as amphotericin B initially,in case the amebae should prove to be Acanthamoeba. These amebae have shown to be resistant to both these drugs in vitro (Casemore, 1970; Chang, 1971; Visvesvara and Balamuth, 1975; Duma and Finley, 1976; Nagington and Richards, 1976; Donald et al ., 1979), but there is evidence to show that they are affected by sulphadiazine in vivo (Culbertson et al., 1965). Subsequently the treatment was tried on two patients in the U.S.A. (Duma et al., 1971) who were in the early stages 13 of the disease and should have responded. The first patient (patient 3, Duma et al., 1971) was given 1.5mg of amphotericin B through a ventricular tap which was repeated 16 hours later. 10mg amphotericin B was also administered I.vent. together with 10mg dexamethasone. The patient was also given 400mg metronidazole (orally); 200mg chloroquine base and 4mg dexamethazone intramuscularly (IM) every 6 hours. However, 72 hours after admission he became shocklike, respiration ceased and he died . The second patient (patient 4, Duma et al., 1971) received similar treatment though he died 66 hours after admission. Carter (1972) reported similar findings to Duma et al (1971) in two patients that had been treated in the same way (seventh and ninth patients Table III, Carter, 1972). There was also the added difficulty in getting the drug into the swollen brains by the intra­ ventricular method. Apley et al (1970) described three cases of PAM in Britain, two of which were diagnosed presumptively because of association with the fatal proven case. They had the same early symptoms, however, neither actually developed the convincing signs of meningitis . ·N; gruberi was cultured from the CSF of the child who died and from only one of the others. Amphotericin B was administered to the fatal case after finding amebae in the CSF. It was given IV in one daily dose of 0.25mg.kg-l given over three to four hours. This was increased to lmg.kg-l over one week, but the patient died on the sixteenth day after admission. It is interesting to note that on the seventh day after admission amebae were seen in the CSF though many appeared to be dead. By the eleventh day after admission, the CSF contained no amebae. It is also interesting that the drug was given py the intravenous route only, and yet produced high levels in the CSF, apparently destroying most of the amebae in the CNS. The patient's survival had been notably prolonged and Carter 14 (1972) postulated that maybe IV treatment on its own, but at a higher dosage rate, may be successful in further cases. This was in fact proved in a later case (Anderson and Jamieson, 1972). The second British case, a brother of case one,was admitted to hospital two days after case one, complaining of a headache and sore throat with neck pains. In view of patient one, amphotericin Band sulphadiazine treatment was begun although the CSF was clear . By day seven he was symptom clear though on day eight they returned and although the CSF was clear, some amebae were cultured which appeared to be similar to those from case one. Amphotericin B was given, 0.25mg.kg-l IV over four hours increasing to 0.75mg .kg-1 after four days for a total of 10 days after which the CSF was clear and no amebae were cultured. He was discharged symptom free. The third case was admi_tted to hospital six days after case one. He complained of sore throat, headache, vomiting and abdominal pain although CSF appeared normal . He was given amphotericin Band sulphadiazine though signs of drug toxicity were noted after four days and the treatment was stopped. It was on day eight that the growth of amebae from case two was reported and although the patient was well, amphotericin B treatment was recorrnnended at 0.25mg . kg- 1 . He was dischar ged after fourteen days, symptom free with no amebae having been isolated at any time (Apley et 2. l., 1970). "Case three must be considered to be only doubtful ly infected with Amebae" (Apley et al., 1970). Griffin (1976) has disputed the dia gnosis of Naegleria meningo­ encephalitis in cases one and two and believes that Acanthamoeba were in fact the ameba involved,and that sulphadiazine was responsible for the treatment of case two and the prolonged survival in case one. He also considers that the leve l of sulphadiazine in the CSF prevented the growth of amebae in culture. Pan and Ghosh (19 71) r eported the survival of two children (aged 6 months and 3 years) with CNS infections of slow onset (3-5 months). CSF samples showed "motile amebae with thin pseudopods" and, although no strains were isolated, treatment was with amphotericin B, sulphadiazine and intrathecal steroids . These two cases are considered inconclusive in the nature of the etiological agent involved and the effective agent in their treatment (Donald, 1979). The first successful treatment of N. fowleri PAM was that reported by Anderson and Jamieson (1972). A fourteen year old boy from Queensland was in the fourth day of illness and comatose by the time treatment was be gun. N. fowleri was cultured from the CSF, in which they could be plainly seen. Amphotericin B was given at a dose of lmg,kg-l per day IV, as well as penicillin,-ampicillin and sulphadiazine which he had been having for three days previously. He was afebrile and talking rationally within two days. After five days the CSF white cell count had dropped but amebae were still seen, therefore amphotericin B was given intrathecally (IT) and later I.vent. in small doses (0.1mg on alternate days). The fluid gradually cleared and he was discharged from hospital without any neurological defects. The second successful treatment of a N. fowleri PAM case 15 is that of a nine year old female from Torrance, California who showed typical symptoms of meningo-encepha litis three days before admission to hospital (Seidel et al., pers. comm,). Routine CSF cell count procedures revealed organisms with ameboid movements and the following medications were given: amphotericin B - 1.5mg IT and lmg.kg-l IV; sulphadiazine 50mg.kg-l IV; chloramphenicol 25mg.kg-l IV and penicillin 3.4 x 105 units IV, The patient was then transferred to Harbor General Hospital where she was in a coma on admission but responsive to pain and tactile stimulation. The following treatment was administered: (i) Amphotericin B was given IV at a dose of 1.Smg .kg-1 .day-l given in two doses daily for three days after which it was decreased to lmg.kg- 1 .day-l given in a single daily dose for six days. (ii) Amphotericin B was also given IT at l.Smg.day-l for two days after which it was decreased to 1,0mg every other day for eight days. This was administered through a lumbar intrathecal catheter. (iii) Miconazole was given IV at a dose of 350mg,m-2 ,day-l given thrice daily for nine days. (iv) Miconazole IT at lOmg.day- 1 for two days then 10mg every other day for eight days • . (v) Rifampin was given orally at a dose of lOmg.kg- 1 .day-l thrice daily for nine days. Sulphadiazine (IV - 4gms.day-1) was continued for three days until studies confirmed the diagnosis of Naegleria meningo-encephalitis. Penicillin and chloramphenicol were continued for three days until CSF cultures were shown to be negative for bacteria. Dexamethasone and diphenylhydantoin were given for increased intracranial pressure and seizure activity respectively. The· patient stabilized clinically over 16 the first forty eight hours. Gradually over the next month of hospitalization her mental status improved and no significent neurological deficits were noted at discharge (Seidel et al., -pers. comm). In other reported cases of PAM where there was proof of N. fowleri infection, and where amphotericin B was given as a treatment, the course of the disease was often too advanced to see any effect (Van Den Driessche et al., 1973; Donald, 1979). Amebic meningitis due to Acanthamoeba are a lot less common than those of Naegleria probably due to the need for some predisposing factor (Martinez et al., 1977; Kernohan et al., 1960; Jager and Stamm, 1972; Bhagwandeen et al. ,1975). Callicott et al. (1968) reported a survival due to A. astronyxis which was isolated from the CSF although the authors were unable to provide evidence as to whether the disease was in fact due to Aca nthamoeba. Several cases of Acanthamoeba infection have been reported though only after post-mortem examination where the brain sections were stained by indirect immunofluorescent antibody techniques (Ringsted et al., 1976; Martinez et al., 1977; Hof fmann et al., 1978; Willaert, 1978). A possible case was reported b y Kenney (1971) in a patient hospitalized for acute gastritis of unknown origin. Compliment fixation tests revealed no antibodies to Entamoeba histolytica though did reveal some to A. culbertsoni which rose over the next two months. Clinical examination failed to reveal any symptoms of cerebral involvement and the patient refused a spinal tap. The patient was put onto antiamebic treatment consisting of dehydro-emetine and chloroquine (IM). Compliment fixation tests two months later showed that the serum titre had decreased. The only human Acanthamoeba i n f ections positively diagnosed during life were those in the eye. Nagi ngton et al. (1974) repeatedly isolated Acanthamoeba from two English pa tients with corneal ulcers. Warhurst and Thomas (1975) identified the amebae as A. castellanii and A. polyphaga. In one case, chloramphenicol, iodoxuridine, 3-fluor­ thymidine, methicillin, gentamicin and later sulphadiazine were tried without any effect. After six months, because of corneal ulceration, pain and loss of vision, a corneal graft was performed which was rejected. The other infection was in a 59 year old farmer with an identical condition which required enucleation of the eye after one year. Treatment was in this case, chloramphenicol, acetylcysteine, 3-fluoro­ thymidine and clotrimazole. At the same time as the above eye infections, Jones et al. (1975) cultivated A. polyphaga from corneal ulcers of two patients in Houston, Texas. They reported suppression of the ameba e with paromomycin. It seems that these infections may not be so rare, and in cases of chronic corneal ulceration, amebic infection should always be considered (Nagington et al., 1974). 17