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 SUSCEPTIBILITY OF PATHOGENIC FREE-LIVING AHEBAE TO CHEMOTHERAPEUTIC AGENTS 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 Jennifer Jane Donald 1979 ii ABSTRACT ======== The treatment of inf ect i ons caus ed by pathogenic fr ee- l iving amebae (PFLA) has, u n ti l o nly r ecent l y, b een far fr o m succe s s ful . Th e co n t inued scr een i n g of c hemot h e r apeu tic agent s agai n st amebae of the g e nera Naeg l er i a a nd Acan thamoeba i s the refor e of the ut1no st i mpor t a nc e. Se v e n c h emo t h erapeuti c agents, amphoter i ci n B, r i fampici n , tetracy c l i ne, po l y my xin B sulphate, 5 - fluo r o cytos i n e , miconazo l e a nd R41 ,400 were screen e d f or a ct i v ity agai ns t a non -pat hoge nic and p a t hog enic species of Naeg l e ri a a nd a non - pathogen i c a nd pathoge n ic species of Acan t hamoeb a in axenic c ul t ure. For th e Naeg l e ria spp . ampho t e ri c in B, mi co n a zole and R41, 400 were foun d to b e acti ve . Acan ­ t h amoebae spp. were foun d to b e susceptib l e only t o 5 -fluo r ocytos ine and R41 ,400 . The poss ibl e u se of c ombinat i ons of drugs agai n s t the amebae was al so i nve s t igat e d in axenic cu lture . Fo r Naeg leria fow l e r i ( MsT) a mphoter i c i n B wit h e ithe r tetra cycli n e or r ifampi c i n showed a s y n er ­ g i st i c e f fect . Polymyxin B sulphate and 5 - fluo rocy to s i n e showed syn erg i s tic ac t ivity a gainst Ac a nthamo eba cu l ber t s on i ( A-1 ) but wh e n po l ymyx:in B wa s comb i ned wi t h tetracyc line or ri famp i c in no s i g n if i cant additive e ff ec t wa s s een. Aft e r axen i c culture testing the suscept i b i lity of the path o­ gen ic s pec i e s,~. fowl e ri ( MsT) a nd A. c ul ber tson i (A- 1) , to the agen ts wh ich s howed ac tiv ity,was i nves t i gated i n a Ve r o ce ll c ul t u re s ystem . Fo r N. f ow l eri (MsT) t h e resu l t s of axeni c testi n g were con firmed with a mpho t eri ci n B, mic onazo l e a nd R41, 400 p r o t ecti n g t h e monol ayer f r om the d e s tru c tive e ff e ct s of t h e amebae . 5-Fluorocy tosine inh ibit e d th e formation of cy topa th i c e f fect (CPE) when the cell cultu r es we r e inoc ulated with A. culbe r t soni (A-1) but viable a mebae were still pre sent. R41,400 had no effect on A. culbe rtson i (A-1) at concentra­ tions at or above those whi ch were cy t otoxic to the Ve r o cells. The use of combinations of drugs was also investigated i n Vero cell culture. Amphotericin Band rifampicin showed an antagonistic rather than a synergistic effect when used against~- fowleri (MsT) in cell culture but amphote r icin B and tetracyc~ine showed synergistic activity. For A. culbertsoni (A-1) the synergistic activity of polymyx in B and 5-fluorocytosine was confirmed. The lack of an addi tiv e e ff e c t between polymyxin Band either tetracycline or rifampicin was also shown in cell culture. The new imidazole derivative R41,400, which showed promise against N. fowleri (MsT) in i n vitro tests was then tested in the in vivo situation. Mice experimenta lly infected with B- fowl eri (MsT) were treated once or twice daily intraperitoneally with different doses of R41,400 . At the higher dosage l evels tested the drug app eared to have a deieterious effect , the average time for death being less than that for the controls . iii ACKNOWLEDGEMENTS ---------------- I am indebted to the Department of Microbiology and Genetics, Massey University for providing the opportunity and facilities for this investigation . In particular I would like to thank: My supervisor, Dr. Tim Brown; Professor D.F. Bacon, Dr. Heather Brooks, Dr. Ray Cursons and other academic and technical staff of the Department of Microbiology and Genetics. Mrs. Elizabeth Keys for her help with the Vero cell cultures. Roche Products Pty. Ltd. for financial support and the supp ly of 5-fluorocytosine . and Ethnor Pty . Ltd. for financial support, supply of micon&zole and the opportunity to test R41,400. I would also like to thank: Ms Karen Walker for the excellant typing, Massey University Library staff for the numerous interloan requests, The Central Photographic Unit, Massey University and finally a special thanks to my parents for their help and encouragement. iv TABLE OF CONTENTS ABSTRACT ACKNOWLEDGEMENTS LIST OF TABLES LIST OF FIGURES LIST OF PLATES CHAPTER ONE : INTRODUCTION 1,1 . The History of Free -Living Amebae as Disease Agents 1. 2, Occurence and Distribution 1 . 3. Pa thogenicity 1 . 4. Immunity 1. 5 . Diagnosis 1. 6. Contro l Measures 1.7. PAM Cases and Thei r Treatment CHAPTER TWO : MATERI ALS 2.1. Arneba Cul t u res Used 2. 2. P l ate Media 2. 2.1. Arneba Sal i ne Agar 2. 2. 2 . Ameba 1% Saline Aga r 2.3. Axenic Media fo r Amebae 2.3.1. Page's Arneba Saline 2.3.2. CYM Medium 2.3.3. 4.0% Neff Medium 2.3.4. CGIN and CGHVS 2. 4. Cell Culture Media 2.5. Antibiotic Solutions 2.5.1. Amphotericin B 2.5.2. Rifampicin 2.5.3. Tetracycline hydrochloride 2.5.4. Polvmvxin B sulphate_ 2.5.5. 5-Fluorocytosine ii iv viii X x ii 1 1 2 3 4 5 6 7 16 16 20 20 20 21 21 21 22 22 24 25 25 25 25 25 26 2.5.6. ~.iiconazole 2.5.7. R41,400 2.6. Misce ll aneous Soluti ons 2 .7. Experimental Animals CHAPTER THREE : METHODS 3.1. Sterilization 3.2. Axenic Culture Techniques 3.2.1. Maintainence of stock axenic cultures 3.2.2 . Axenic drug testing 3.3. Cell Culture Techniques 3.3.1. Maintainence of stock Vero cell cultures and preparation of KIMAX tubes for drug testing 3.3.2. Cell culture drug testing 3.4. In Vivo Testing of R41,400 CHAPTER FOUR: RESULTS 4.1. In Vitro Axenic Drug Testing of Naegleria spp. 26 26 27 27 28 28 28 28 28 29 29 30 31 32 32 4.1.1. Amphotericin B 32 4.1.2. Tetracycline, Rifampicin and 5-Fluorocytosine 3 5 4.1.3. Polymyxin B 42 4.1 .4. Miconazole 42 4.1.5. R41,400 47 4.2. In Vitro Axenic Drug Testing of Acanthamoeba spp. 4. 2. 1. Amphotericin B 4.2.2. Tetracycline and Rifampicin 4.2.3. 5-Fluorocytosine· 4.2.4. Polymyxin B 4.2.5. Miconazole 4.2.6. R41,400 4.3. The Testing of Drug Combinations again$t Naegleria fowleri (MsT) in Axenic Cultures 4.3.1. Amphotericin Band Tetracycline 4.3.2. Amphotericin Band Rifampicin 50 50 53 58 61 61 66 69 69 72 vi 4.4. The Test ing of Drug Combinations agai nst Acanthamoeba culbertsoni (A-1) in Axenic Culture 4.4.1. Po l ymyxi n Band 5-Fluorocytosine 4.4.2. Po l ymyxi n Band Tetracyc line 4 .4.3. Po l ymyxi n Band Rifampicin 4 . 5. Cell Culture Drug Testing of Naegleria fowleri (MsT) 4 . 5 .1. Amphotericin B 4.5.2. Miconazo le 4.5.3. R41,400 4.5.4 . Tetracycline and Rifampicin 4 . 6 . Cel l Culture Drug Testing of Acanthamoeba culbertsoni (A-1) 4.6.1. 5-Fluorocytosine 4.6.2 . Po l ymyxin B 4.6 .3. R4 1, 400 4,6.4, Tetracy cline and Rifampicin 4.7. The Testing of Drug Combinations against Naeg l eria fowleri (MsT ) in Cell Culture 4.7.1. Amphotericin Band Tetracycline 4.7.2. Amphotericin Band Rifampicin 4.8. The Testing of Drug Combinations against Acanthamoeba culbertsoni (A-1) in Cell Culture 4.8.1. Polymyxin Band 5-Fluorocytosine 4.8.2. Polymyxin Band Tetracycline 4.8.3. Polymyxin Band Rifampicin 4.9. In Vivo Testing of R41,400 CHAPTER FIVE: DISCUSSION 5.1. Treatment of Naegleria Infections 5.2. Treatment of Acanthamoeba Infections BIBLICCRAPHY v ii 75 75 78 78 83 8Lj 84 8 7 89 9 2 93 95 95 98 101 101 101 104 104 106 106 109 112 112 127 135 LI ST OF TABLES -------------- I Cases of Primary Amebic Meningo- en c epha litis Report ed After 1974 II Proba ble and Definite Survivors of Primary Amebic Meningo- en cephalitis II I Treatment Protoco l Used in a Case of PAM IV Ameba Culture s Used V Eff e ct of Size of Inocula of Naeg leria fowl e ri (MsT) on Time Needed for Development of CPE VI The Effec t of Amphoter i c in Bon Na eg l eri a f owl e r i ( ~ sT ) in Cell Culture VII The Ef fec t of Mi conazole on Naeg leria fowleri (MsT) in Cell Cul t ure VIII The Effect of R41,~00 on Naeg leria fowleri (MsT) in Ce ll Culture I X The Effec t o f Tetracyc line on Naeg l eri a fow l e ri (MsT) in Cell Cu lture X The Effect of Rifampicin on Naegl e ria fowl eri viii 8 9 14 16 83 85 86 88 90 (MsT) in Cell Culture 91 XI Effect of Size of lnocula of Acanthamoeba - --- culbertsoni (A-1) on Time Ne eded for Development of CPE 92 XII The Effect of 5-Fluorocytosine on Acanthamoeba culbertsoni (A-1) in Cell Culture 94 XIII The Effect of Polymyxin B sulphate on Acanthamoeba culbertsoni (A-1) in Cell Culture 96 XIV The Effect of R41,400 on Acanthamoeba culbertsoni (A-1) in Cell Culture 97 XV The Effect of Tetracycline on Acanthamoeba culbertsoni (A-1) in Cell Culture 99 XVI The Effect of Rifampicin on Acanthamoeba culbertsoni (A-1) in Cell Culture 100 XVII The Effect of Amphotericin Band Tetracycline together on Naegleria fowleri (MsT) in Cell Culture 102 XVIII The Effect of Amphotericin Band Rifampicin together on Naegleria fowleri (MsT) in Cell Culture 103 XIX The Effect of Polymyxin B sulphate and 5-Fluorocytosine together on Acanthamoeba culbertsoni (A-1) in Cell Culture 105 XX The Effect of Polymyxin B sulphate and Tetracycline together on Acanthamoeba culbertsoni (A~l) in Cell Culture • 107 XXI The Effect of Polymyxin B sulphate and Rifampicin together on Acanthamoeba culbertsoni (A-1) in Cell Culture 108 XXI I Intranasa l Infection of 25 .0 ~ 3. 2 g Swiss-White Ma le Mice with Naegleria fowleri (MsT). R41,400 given i n doses indicated . XXI II Intranasal Infection of 22 .0 :t 4 .0 g Swiss-White Male Mice with Naegleria fow l er i (MsT). R41,400 given in doses indicated . XXIV Intranasal Infection of 27 .0 i 4.0 g Swi s s - White Male Mice with Naeg l eria fowleri ( MsT) . R41, 400 give n i n doses indicated . XXV The Effect of Miconazo l e and R41, 400 on Naeg l er ia f owleri (MsT ). ix 109 110 110 125 LIST OF FlGURES 1. The Effect of Amphotericin Bon Naegleria gruberi (Pl200f) 33 2 . The Effect of Amphotericin Bon Naegleria fowleri (MsT) 34 3. The Effect of Tetracycline on Naegleri~ ~~beri (Pl200f) 36 4. The Effect of Tetracycline on Naegleria fowleri (MsT) 37 5. The Effect of Rifa~pic in on Naegleria ~uberi (Pl200£) 38 6. The Effect of Rifarnpicin on Naegleria fowleri (MsT) 39 7. The Effect of 5-Fluorocytosine on Naegleria gruberi (Pl200f) 40 8. The Effect of 5-Fluorocytosine on Naegleria fowleri (MsT) 41 9 . The Effect of Polymyxin Bon Naegleria gruberi (Pl200f) 43 10. The Effect of Polymyxin B on Naegleria fowleri (M.sT) 44 11. The Effect of Miconazole on Naegleria gruberi (Pl200f) 45 12. The Effect of Miconazole on Naegleria fowleri (MsT) 46 13. The Effect of R41,400 on Naegleria gruberi (Pl200f) 48 14. The Effect of R41,400 on Naegleria fow l eri (MsT) 49 15. The Effect of Amphotericin Bon Acanthamoeba castellanii (1501) 51 16. The Effect of Amphotericin Bon Acanthamoeba culbertsoni (A-1) 52 17. The Effect of Tetracyc line on Acanthamoeba castellanii (1501) 54 18. The Effect of Tetracycline oa Acanthamoeba culbertsoni (A-1) 55 19. The Effect of Rifampicin on Acanthamoeba castellanii (1501) 56 20 . The Effect of Rifampicin on Acanthamoeba culbertsoni (A-1) 57 21. The Effect of 5-Fluorocytosine on Acanthamoeba castellanii (1501) 59 22 . The Effect of 5-Fluorocytosine on Acanthamoeba culbertsoni (A-1) 60 23. The Effect of Polymyxin Bon Acanthamoeba castellanii (1501) 62 24. The Effect of Polymyxin Bon Acanthamoeba culbertsoni (A-1) 63 25. The Effect of Miconazole on Acanthamoeba castellanii (1501) 64 26. The Effect of Miconazole on Acanthamoeba culbertsoni (A-1) 65 27. The Effect of R41,400 on Acan~hamoeba castellanii (1501) 67 28. The Effect of R41,400 on Acanthamoeba culbertsoni (A-1) 68 29. The Effect of Arnphotericin Band Tetracycline Alone a nd in Combination on Naegleria fowleri (MsT) 70 30. The Effect of Amphotericin Band Tetracycline Alone and in Combination on Naegleria fowleri (MsT) 71 31. The Effect of Amphotericin Band Rifampicin Alone'and in Combination on Naegleria fowleri (MsT) 73 32. The Effect of Amphotericin Band Rifampicin Alone and in Combination on Naegleria fowleri (MsT) 74 33. The Effect of Polymyxin Band 5-Fluorocytosine Alone and in Combination on Acanthamoeba culbertsoni (A-1) 34. The Effect of Polymyxin Band 5-Fluorocytosine Alone and in Combination on Acanthamoeba culbertsoni (A-1) 35. The Effect of Po l ymyxin Band Tetracycline in Combination on Acanthamo eba culbe rtsoni 36. The Effect of Polymyxin Band Tetracycline in Combination on Acanthamoeba culbe rtsoni Alone (A- 1) Alo:1e (A-1) and and 37. The Effec t of Po l ymyxin B arid Rifampicin Alone and in Combina t ion on Acanthamoeba culbe rts~ni (A-1) 38 , The Effect of Polymyxin Band Rifampicin Alo~e and in Co!llbination on Acanthamoeba culbertsoni (A-1) 39. Structura l Formulae of lmidazole b a se, Miconazole and Clotrinazole xi 76 77 79 80 8 1 82 134 :xii LIST OF PLATES 1. Trophozoite stage of Naegleria RI~ Q. er i_ (P1200f) 17 2. Trophozoite stage of Naegleria fowleri (MsT) 17 3. Trop:1ozoi te stage of Acantha'1loe::ia castellanii (1501) 18 4. Cyst stage of Acanthamoeba castellanii 0501) 18 5. Trophozoite stage of Acanthamoeba culbertsoni (A-1) 19 6. Cyst stage of Acanthamoeba culbertsoni (A-1) 19 CHAPTER ONE : IN TRODUCTION Th e histo ry of pathogen i c fr ee - l i ving amebae (P FLA) o f t h e ge n e ra Acan t h amoe ba 2nd Naeg l e ri a has be e n ex tensive l y r evi ewe d e ls e wh e re (Cu l b e rtson , 1971; Du ma e t .§1 . , 1971; Ch a n g , 19 7 1 , 19 74a ; Ca r te r, 19 7 2 ; Cu r son s , 1974 ; Cur sons & Brown, 197 6 ). Th e commones t d iseas e c au s e d by P FLA i s k nown as P r imar y Ameb ic Me n i n go - encepha l i t i s (PAM ) and i n a n ext e ns ive wor ldwi d e survey , Will aer t ( 19 74 ) tabu l a t e d 84 cases f rom a ll cont i ne n t s wi th th e ex ­ c e p t i o n of Antarc tica . S i nce the n a t l ea s t te,1 additiona l case s have b e e n repor t e d. Pri or to 19 68 , mo st case s we re attribu t e d t o Acanth amo e ba spp .. Th is is pro bab l y a r e f l ection of the p i o n eering work o f Culbertson e t a l. (1 958 , 1959 , 1965 ) wh om , whi l st working on the production of po lio vacci n e , f ound a n ameba which co ntam i nat e d the c u l tures o f monkey kidney ce ll s . When the cul t ur e s we re ino cul ated intra c e r e bra l ly i n to mice a nd primates, a n ec r o t izing, h emor rhagic meni ngo - e nceph a lit i s wa s produced that k ill ed t h e a n ima ls in f our t o s even days . Th e res ­ po ns ibl e ameba wa s ide n ti f ied as a n Acanthamoeba a nd they p r e d i cte d , o n the b a sis of this f i nd i ng, that this ameba c ou ld b e capabl e of produc ing di sease in huma ns. Thi s ameba wa s previ ous l y co nsid e red to b e a harml e ss, fr e e-l i ving a meba. However, in 19 68, Bu t t e t ~1-, Ca rt e r a nd Culbertson e t al., showed that the i n c r imi nating spe ci e s of most r e po rte d human c ases belonged to the r e lat e d g enus Naegl e ria. In 1970, on the basis of mo rphological, cultural and pathogenicity diff e r ences, Carter n a med the pathogenic speci e s Nae gl eria fowl e ri d i stinguishing it from the non-pathogenic Naegler i a gruberi. The disease caused by PFLA can be divided into two types: i) a swimming - associated acute meningo-encephalitis (Primary Amebic Meningo- encephalitis (PAM) - Martinez et al.(1977) caused by N. fowleri. and ii) a non-swimming - associated chronic meningo-encephalitis (Amebic Meningo-encephalitis (AM) - being regarded as a second­ ary invasion of the central nervous system (CNS) having spread from other foci of infection (Martinez et al., 1977)) caused by a variety of pathogenic Acanthamoebae, notably Acanthamoeba culbertsoni, Acanthamoeba castellanii and Acanthamoeba polyphaga (Chang, 1974a). 2 Subsequently, Acanthamoeba spp. have also b een indicated in a number of chronic illnesse s such as respiratory infections (Martinez et~-, 1975 ), corneal ulceration of the eye resulting i n bl i ndness (Nagington et~-, 1974; Visvesvara et al., 1975) and togethe r with Naegleria spp. in humidifier f ever (M.R.C. Symposium, 1977). Henceforth in the text the nomenclature of Martinez et al . C1977), PAM for Naegleria inf ections and AM for Acanthamoeba meningo-encephalitis, will be adopted. The controversy regarding the classification of PFLA (Cursons & Brown, 1976) appears to be settled with the majority of authors preferring Chang's (1971) classification scheme. The identification of isolates involves the exploitation of specific cytological, morph­ ological, physiological, immunological, growth and pathogenicity characteristics in an ordered sequence readily usable by hospital and public health laboratory staff (Cursons & Brown, 1976). 1.2 Occurence and Distribution The summary of recorded isolations from a wide variety of environme ntal sources provided by Cursons (1978) emphasizes the truly ubiquitous distribution of PFLA. The ability of PFLA to form resistant cysts undoubtedly enables them not only to withstand unfavourable conditions, e.g., the isolation of pathogenic Acanthamoebae from 2°c (Brown & Cursons, 1977), but also to take advantage of the intermittent occurrence of favourable conditions. The distribution of the pathogenic species in relation to non-pathogenic ones is still unknown (Cursons, 1978). In general, non~pathogenic species are more prevalent at ambient temperatures in temperate zones. The repeated isolations of PFLA from water above ambient temperature, i.e.,~30°C (De Jonckheere et al., 1977j De Jonckheere & Van De Voorde, 1977a; Stevens et al., 1977; Wellings et al., 1977; Cursons et al., 1978b), combined with their higher optimum temperature of growth (Griffin, 1972) suggests that pathogenic amebae are environmentally selected over non-pathogenic amebae in waters above ambient t emperature. The source of pathogenic amebae in these waters is unknown but as Cursons et al. 0978b) and Wellings 3 et al. (1977) have succeeded in isolating PFLA from soil it is possible that soil acts as a reservoir of pathogens and contamination occurs via run-off after rain (Cursons, 1978). 1.3. Pathogenicity The invasion of organs and tissues by PFLA is now well document ed (Culbertson et al., 1959 , 1968, 1972; Cart er, 1968, 1970, 1972; Call i ­ cott et _tl., 1968 ; Chang, 1971, 1974a & b, 1976; Culbertson, 1971; Martinez et~ - , 1973, 1975 , 1977; Vi svesvara & Balamuth, 1975; Wong et E-l•, 1975a & b; Hoffman et al., 1978). It has been es tabl i shed experimentally that the portal of entry into the CNS in N. fowleri infection is via d i sruption of the olfactory mucosa, penetration of the organisms into the submucosal nervous pl exus, probably by phagocytosis of the amebas by the sustentacular cells of the olfactory neuroepithel­ ium a nd passage through the cribiform plate to the subara chnoid space ( Martinez et al., 1973). Ho\vever, in cases of Acanthamoeba meningo-encephalitis the involvement of the CNS appears to be a secondary phenomenon representi ng metastatic spread from a primary focus in the skin, genitourinary or respiratory tract (Martinez et al ., 1977). Cutaneous ulceration as a possible point of entrance with hematogenous spread to the CNS was reported by Bhagwandeen et al. (1975) and Martinez et al. (1977) report involvement of the genitourinary tract. Lower respiratory tract infection in experimental animals have been reported (Martinez et al., 1975). AM due to Acanthamoeba spp. appears to be an opportunistic infection of the CNS. AM occurs in patients who are chronically ill, debilitated or those whose cell-mediated immune responses have been impaired as a result of either underlying systemic disease or its treatment by immunosuppresive methods (Kernohan et al., 1960; Jager & Stamm, 1972; Robert & Rorke, 1973; Sotelo-Avila et al., 1974; Bhagwan­ deen et al., 1975). Acanthamoeba infections of sites with reduced accessibility to the immune system e.g., the eye also demonstrate the opportunistic nature of these infections. Isolates from the cases reported by Nagington et al. (1974) were shown by Visvesvara et al. (1975) to be of low virul ence and infection resulted after damage to the cornea . Once invasion of the brain has been established i 1, PAM and AM, destruction of surrounding brain tissue is thought to be brought about by a combi nati on of phagocytosis and pinocytosis of host tissue by N. fowleri and solely by pinocytosis i n the case of A. culbertsoni (Vi svesvara & Callaway , 1974; Mai tra et ~ - , 1974, 1976) . The examination of sections from brains i nfected with either 4 N. fowleri or A. culbertsoni revea ls areas of extensive demye lination leaving the trophozoites surrounded by a clear halo (Martinez et a l., 1975; Chang, 1976 ; Maitra e t a l., 1976 ). Many authors have speculated on the possibil ity that enzymes secreted by the amebae are responsible for thi s. Exper i ment s have shown that both cytotoxi c enzymes a nd phos­ pholipases are produced by PFLA (Elson et al ., 1970; Chang, 1971, 1974a, 1976; Hax et al., 1974; Visvesvara & Callaway, 1974; Vi c toria & Korn , 1975; Visvesvara & Ballamu t h, 1975; Cursons & Brown, 1976, 1978; Cursons e t .§:.1.•, 1978c ; Maitra et al . , 1976 ). The l eve l of production of such cytopathic enzymes may explai n the diffe r ences in virulence reported amongst Acanthamoeba and N- fowl eri isol a tes (Culber tson, 1971; De J onckheere & Van De Voorde , 19 77b). l . 4. 1~!:!!}.!!Y -------- The lm·J incidence of PAM and AM in the human population ha s puzzled many authors in view of the ease a nd frequ ency of isolation of virulent PFLA from the environment (Anderson & J amieson, 1972; Cursons et al., 1976b, 1977; John et al., 1977; Wellings et al., 1977; Haggerty & John, 1978). This has led to speculation on the existence of host related susceptibility factors and the demonstration of specific antibodies to free-living amebae in human sera has been reported (Chang & Owens, 1964; Edwards et al., 1976; Cursons et al., 1977; M.R.C. Symposium, 1977). Observations that previous exposure of mice to live N• gruberi significantly protected them against a subsequent lethal challenge with ~- fowleri (John et al., 1977) supports the idea that unwitting ex­ posure to the ubiquitous~ - gruberi may immunize against~- fowleri. A similar immunization may also occur with Acanthamoeba . Cell-mediated immunity (CMI) also appears to play an important part in protection against PFLA (Diffley et al., 1976; Cursons et al., 1977). 1 .5. !2i§g!:!Q§i§ --------- Successful treatment of this rapidly progressive disease is whol l y dependent on prompt and definitive diagnosis . The survival of a nine year old female in Torrance, California (Seidel et a l. , pers . comm., 1978) and a fourteen year old male in Australia (Anderson & Jamieson, 1972) i s attributed to early diagnosis and prompt treatment . 5 A Naegleria brain infection should be suspected when th ere is a history of swimming about seven day s prior to the abrupt onset of fever, headache, sore - throa t, nausea and vomiting ( Carter, 1972; Chang, 1974a) . The most important laboratory procedure for the diagnos i s of PAM is the microsc opic examination of cerebra-spinal fluid (CSF) . Overall, the CSF is indistinguishable from t hat obtained from pat i ents with bacterial meningitis and diagnosi s re l ies upon amebae being seen i n t he fl ui d a nd the culture of these for complete diagnosis . Species identification can th en be achieved by the method outlined by Cursons & Brown ( 1976) . In post- mortem diagnosis, a degree of encephaliti s is invar iably present . The brain shows swel l ing and redness with the purulent exudat~ more extensive on the ventral surface of the cerebrum or cerebe llum and over t he bra in stem ( Car ter , 197 2) , The gr ey mat t er of t he cer ebra l h emispheres a nd cerebe llum sho ws var iabl e si zed l es i ons wh ich tend to be hemo rrhagic and qui t e sof t wh en they are l arge (Cul ber t son, 1971 ) . The existence of r edness a nd destruction of t he ol f ac t or y nerve occur s only i n PAM and could s e r ve to distingui sh i t from bac t er i a l mening i tis (Ca r ter, 19 72 ). Th e immunof luorescent an tibody (IFAB) technique applied to h isto logic ·bra i n s ection s taken pos t- mor tem , i s a valuabl e t ool in i dent i fi cation of amebae i n brains of pat i ents who d ied from men i ngo­ e ncephal i tis. Anti sera can be pr oduc ed in rabbits and can be made spec ies-spec i fic by sui tabl e abso r pt ion. In additi on t o their value in clinical diagnosis IFAB provide rapid screening methods for the detect­ ion of PFLA in swi mmi ng pool, tap and other domest i c and recreational water supplies. Immunoperoxi dase methods have been used by Culbertson (1975) and Cursons et al. (1976) to demonstrate both Naegleria and Acanthamoeba in the brain sections of patients who died from PAM and AM respectively. This method may be shown in the future to be more valuable than the immunofluorescent technique. It has certain advantages over IFAB, e.g., permanent preparations can be made, no specialized equipment is necessary and cl ear , definitive staining of the tis sue elements r esults (Culbertson, 1975). 6 Acanthamoeba brain infections are difficult to diagnose even in advanced cases due to lack of specific symptoms and signs and the absence of amebae in the CSF (Chang, 1974a) . Nasa l and throat swabs may provide more information. Post - mo r tem diagnosis relies on the presence of c onf i ned, superficial l esions in the grey mat ter with a minimum inflammatory r eaction, and the find i ng of double-walled wrinkled cysts i n appar ­ ent l y normal tissue borderi ng the lesion (Chang, 1974a) . In all the reported cases, except that reported by Bhagwandeen et~ - (1 97 5) , there was l ack of evidence of the i nvolvement of the olfactory bu l b and the absence of inflammatory reactions i n the surrounding grey a nd white matter. These observations may help in dist i ngu i shing between PAM and AM . Positive d iagnosis was possible in the cases of eye infections reported by Nagington e t al. (1 974) and J on e s e t a l. (1975) by iso­ lation , and subs equent identification of Acanthamoebae spp . taken from corneal scrapings . 1. 6 . Control Measure s ---------------- The necess ity for an effective disinfectant can be judged by the increasing number of i so lations of free-living amebae from potable and tr eated and untreated recreational water s (Cerva, 1971 a ; Chang, 1971; Anderson & J amieson, 1972 ; Cerva & Huldt, 1974; Ma let et al., 1976; Lyons & Kapur, 1977). The majority of amebae isolated i n these studies belonged to the genus Acanthamoeba indicating its greater r e s i stance to chlorine than Naegleria spp .. In reviewing sixteen fatal cases of PAM from an indoor chlorinated swimming pool Cerva (1971a) stated that, " it appears that the constant presence of numerous populations of a mebae of the limax group cannot be prevented even under the strictest observations of all routine safety measures applied to potable waters." However, Lyons & Kapur (1977) in a survey of 30 halogenated public swimming pools concluded that the low amebic densities (~ 1.1- 1 ) in the majority of the pools illustrated that these organisms could be adequately controlled by proper pool maintain­ ence. The possession of resistant cysts, however, constantly compli- 7 cates the disinfection process. In a s tudy of alternative disinfectants, Cursons et~- (1978b) found that deciquam 222, chlorine, chlorine dioxide and ozone all possessed potential disinfecting properties for PFLA , but at higher leve ls than those for disinfecting bacteria . Of the four disinfectants exami ned, deciquam 222 proved to be the most ef f ective amebicide followed by chlor ine, chlorine d ioxide and ozone . The final choice of a particular di sinfec t ant must however, remain ti ed to the physical and chemica l properties of the water to be disinf ected. 1.7. PAM Cases and Their Treatment In 1974, Willaert provided an extensive worldwi de survey of cases of PAM . Since then at l east ten additi onal cases have been reported (Table I). Conceivably, the actual number of cases may be hi gher since the symptoms of PAM parallel those of aseptic meningi t is . Retrospective studies have disclos ed a possible case dating back to 1909 (Symrne r s, 1969 ) and fluor esc~nt antibody staining has conf irmed that t h e 1948 case reported by Derrick , originally thought to be due to Iodamoeba butschl ii was in fact caused by~- f owleri (McMillan, 1977). The reid ent i fication of the etiological agents of the 19 68 cases of PAM in New Zealand as N. fowle r i (Cursons & Brown, 1975 ; Cursons et al., 1976a ) has dismissed the notion of slime moulds being involved in the etiology of PAM (Mandal et al., 1970). The r esu lts of treatment of PAM have been far from encouraging. Willaert's summary (1974) provides information on ten possible survivors of PAM and the California n case of Seide l et al. (pers. comm., 1978) makes the world total eleven survivors (Table II). Such a result is hardly surprising in the earlier cases, where the amebic nature of the disease had not been suspected, and treatment consisted only of antibacterial agents such as sulpha-drugs, penicillin, strep­ tomycin, tetracyclines and chloromphenicol (Fowler & Carter, 1965; Butt et al., 1968; Cerva & Novak, 1968; Dos Santos, 1970; Van den Driessche et al., 1973). However, even in later cases where the anti­ protozoal drugs emetine, chloroquine and metronidazole were often used, the course of the disease was not affected in the slightest (Carter, 1968, 1970, 1972; Duma et al., 1971), except in the unproved case of Grundy and Blowers (1970) in which survival was attributed to chloroquine. In this case, amebae, believed to be Naegleria, were NUMBER CAUSATIVE COUNTRY YEAR OF CASES ORGANISM DIAGNOSIS TREATMENT OUTCOME REFERENCE 1974 1 N. fowleri (MsT) isolation Penicillin di ed Cursons et ~-, 1976b from CSF Ampicillin NEW Amphotericin B ZEALAND 1978 1 ~- fowleri (MsM) isolation Amphotericin B died Cursons e t al., pers. from CSF comm., 1978 1974 1 N. fowleri (Lovell) isolation Unknown died De Jonckheere, 1977 from CSF 1974 1 Acanthamoeba sp. IFAB Steroids died Martinez et ~-, 1977 U.S.A. Penicillin 1975 1 Acanthamoeba sp. IFAB Unknown died Hoffman et al., 1978 post-mortem 1978 1 Naegleria sp. isolation Amphotericin B survived Seidel et al ., pers, from CSF Miconazole comm ., 1978 Rifampi n VENEZUELA 1 !:. . culbertsoni IFAB Steroids died Marti nez et al., 1977 1 A. castellanii IFAB Steroids died Martinez et al ., 1977 PERU - Antibiotics 1972 1 Acanthamoeba sp. post - mor tem Antibiotics died Bhagwandeen et al., ZAMBIA Amphoter icin B 1975 1958 1 Acanthamoeba sp. post- mor t em Penicillin d ied Ri ngst ed et ~., 1975 KOREA Strep tomyci n Chloramphenicol Table I: Cases of Primary Amebic Meningo- enc ephali t is Reported After 1974 (modified from Cursons, 1978) C'. NUMBER CAUSATIVE COUNTRY YEAR OF CASES ORGANISM TREATMENT REFERENCE 1968 1 Naegleria Metronidazole - Grundy & Blowers, UGANDA Emetine - Penicillin 1970 Chloroquine. 1967 1 A. astronyxis Ampici llin Callicott et al., Penicilli n - G 1968 U.S.A. 1978 1 Naegleria Amphotericin B Seidel~ ..el_., pers. Miconazole comm. 1978 Rifampin 1970 2 Naegleria Streptomycin Pan & Ghosh, 1971 Isonicoteinhydrosine INDIA Sulphadexanathosone Amphoteri cin B 1973 3 N. fowleri Unk:nown S . R. Das, pers. comm . to Willaert(l974) 1969 2 Naegleria Antibiotics Apley et al., 1970 ENGLAND Sulphadiazine Amphotericin B 1971 1 N. fowleri Amphotericin B Anderson & Jamieson, AUSTRALIA - Sulphadiazine 1972 Table II: Probable and Definite Survivors of Primary Arnebic Meningo-encephalitis \0 s een in the CSF and survived f or awhi l e i n cu l ture but were not po s it ively i d enti f ied, The patient a l so presented atypical c l inica l features . Treatment consisted of metronidazo l e , emeti ne, penicillin , sulphane and chl or oquine . 10 The ineffectiveness of antibacterial agents against pathogenic Naegl eria has been confirmed in vitro by Carter (1969) and Mandal et §1_ . (1970) . Of the antiprotozoal agents, emetine HCl is effective agains t !:i , fowleri i n vitro . Carter (19 69 ) r eported a minimum immobil­ izing l eve l of 12 . 5 ;-< g . cm- 3 a nd Krishna Prasad (1972) and Das (1975) r eport minimum amebicida l concentrations of 16 and 15.l" g.cm- 3 respect ­ ive l y , but, it does not protec t anima l s from the dis ease (Culbertson et al., 1968) probably because it is unable to pass the blood-brain barrier (Parmer & Cottril l, 1949) . The ineffectiveness of chloroquine a nd metroni dazo l e has a l so been co nf i r med by i n vit r o t e st s and a nima l protection studies (Carter, 1969; Manda l et ~-, 1970; Duma et a l. , 1971) . The only drug to appear promisi ng i n the ear l y 1970' s wa s t h e a nti f unga l agen t amphotericin Band as can be seen from Tabl e II i t was used i n the treatment of all survivors (except the unproved case of Grundy & Bl owers, 1970 a nd Callicot t e t .§.1_. , 1968) . Amphoteric in B is a po l yene a nt ibio tic a nd ha s been shown to be h ighly amebici da l to pathogen ic Naeg l e ri a i n v i tro (Ca r ter, 1969; Handa l e t al., 1970; Duma e t al. , 1971 ; Schuster & Rechthand, 1975 ; Visvesvara & Balamu t h , 1975; Duma & Fi nl ey , 1976 ; De Jonckheer e & Van De Voorde , 1977; Dona ld e t al., 1979 ) and t o protect mi ce fr om the d i s ease ( Culber tson e t a l., 19 68 ; Ca rt er, 1969; Das, 1971). In 1969 , Carte r suggested t ha t a mphote ricin B be tri ed i n the t reatment of PAM by s i multaneous intravenous (IV) and intraventricular (IVent) administration; the doses r ecommended were - 0.25 mg.kg-l IV and 1.0 mg into the cerebral ve nt r i cles in the first 24 hours . Carter (1972) also suggested sulphadiazine should always be used as well as amphotericin Bin case the amebae should occasionally prove to be Acanthamoebae . These amebae have been shown to be resistant to both drugs in vitro (Casemore, 1970; Chang, 1971; Visve svara & Balamuth, 1975; Duma & Finley, 1976; Nagington & Richards, 1976; Donald et al . , 1979), but there is good evidence that they are affected by sulpha­ diazi ne in vivo (Culbertson et al., 1965). Subsequently, such treatment was tr i ed on two patients in the U.S.A. (Duma et al., 1971) who were in the early stages of the disease 11 and should ha ve r e spond e d . I n th e f i rst of the s e , (Pati e nt 3, Duma e t al ., 1971) a ri ght v entr i cu l ar tap was performed a nd aft e r r emova l of flu i d (containing many motile a mebae) 1. 5 mg of amphot e r i ci n B di ssolved i n 5 cm3 of 5% dextrose inj ect i on solut i on (D 5 W) was s l owly i n jec t ed into the vent ri c l e . 10 mg of a mphot e ricin Band 10 mg d exame thason e we r e administ e r ed IV . A nasoga stric tube was inserted, t h r ough wh ich 400 mg o f metronidazol e was gi ven f our t imes dai ly . Th e pati e nt a lso r e c e ived chloro quine ba se, 200 mg an d dexamethason e , 4 mg, intramuscu l a rl y ( IM ) every six ho u r s. Sixteen hours after a dmission the ventricu l a r tap was r epeated a nd 1. 5 mg o f amphotericin B was agai n i nj ected into the v entric l e . Sev enty - two hours after admi s s i o n h e b ecame s h ock- l i k e , res p i rat i ons c eas e d and h e died. Th e second cas e (Patien t 4 , Duma et a l. , 1971 ) receiv ed , within 3 two hours o f admission , 1.5 mg amphoteri c i n B d i l uted in 5 c m D 5 W i nt rac i sterna lly (IC i st) and 10 mg a mphoter i c in B a nd 5 0 mg hy drocort ­ ison e IV over the next f our hou r s . In a ddition, chloro qui n e su l pha t e , 200 mg I M; me t r on i dazo l e , 50 0 mg by nas o gastr i c tube ev e r y s ix hours; and d i phe ny l hydanto i n (D i lant i n), 100 mg I M every eigh t hours, wa s g iv e n . E~ghteen h ours a ft e r admi ss io n, the pat ient aga in r eceived 1. 5 mg amphoter i cin B ICi s t and the IV a mphoter i cin B i n c r e as e d to 20 mg . The pati ent d ied 66 hours a f ter admi ssion . Car t er ( 1972) r e ports simi lar fi nd i n gs t o Duma et al . ( 197 1 ) in two pa t ients (7th a nd 9th , Tabl e III , Cart e r, 19 7 2) who h ad b een treated in t h e same way . Ap l ey e t a l.(1 9 70) d e scr i be d t h ree c ases of PAM in Grea t Br itain, t wo of wh i ch wer e d iagno s e d presumptive l y becau se of association with t h e f ata l proven ca s e . They had t h e same early symptoms but nei ther actua lly develo ped convincing s i gns o f men i ngitis. Naegl eria was cultured from the CSF o f the child who di e d and from one o f the others but the amebae prove d to be~- g r uberi. In the fatal infe ction, amphotericin B treatment was begun two days after admission, when amebae had been seen in the CSF. Amphoter- -1 icin B, 0.25 mg.kg in one daily dose over three to four hours IV increasing over a week to 1 mg.kg- 1 , was administered till the patient died on the sixteenth day after admi ssion. Sulphadiaz i ne, 160 mg IV every six hours was also given. On the s eventh day after admi ssion, 650 amebae.mm- 3 were seen in the CSF, but many appeared to be dead. The -3 concentration of amphotericin Bin the CSF was 0.184/'g.cm . On the eleventh day after admission, the CSF contai ned no amebae and the 12 -3 amphoteri cin B concentration was O. 224/< g . cm . Al though the d iagnosis i n this case was made relatively ear l y and treatment with amphotericin B started promptly the patient died after being tr eated for thirteen days. The second case reported by Apley et a l. (1970) was the brother of case one and was admitted to hospita l two days later. On the morning of admission he c omplained of headache and in the evening developed a sore throat and neck pains. A CSF sample was taken and it was cl ear and no amebae were seen. The clinical picture was that of an uppe ~ r e spirato ry tract infect ion but in view of case one treatment with amphotericin Band sulphadiazine was begun. By the seventh day he was symptom-f ree . On the eighth day he again c omplained of sore th r oa t with head and neck pains. No amebae were seen in the CSF but some were grown a nd appeared similar in morphology to those isolated fr om the CSF of case one . By the twelfth day he was afebrile and had no signs of meningitis but i n view of growth of amebae from the CSF taken four days ear l ier, amphotericin B treatment was started again. 0.25 mg amphot er- -1 -1 icin B.kg IV dai l y over four hours increasing to 0 . 75 mg.kg after four days fo r a total of ten days was given . CSF take n on the twelfth and eighteenth days a ppeared norma l and no amebae were grown. The patient was discharged, s ymptom-f ree . Ten days l ater a CSF sample was taken and again no amebae were grown. The th ird case was admitted to hosp ita l s i x days after case one. On the morning of admi ss ion he c ompla i ned of sore throat and headache, vomiting and abdominal pain. The CSF was normal and no treatment was given. On the third day, the temperature had become norma l but the h eadache continued and there was slight neck stiffness. No amebae were seen in the CSF but in view of slight lymphocytosis treatment was -1 started with sulphadia zine and amphotericin B (0.25 mg.kg daily in one dose over four hours IV). On the fourth day signs of drug toxicity were noted and treatment was stopped. On the eighth day, growth of amebae from case two was reported and although the patient was well, -1 -1 daily amphotericin B, 0.25 mg.kg increasing to 0.75 mg.kg was given IV for ten days. CSF specimens on the 8th, 14th and 24th days were normal. No amebae were isolated at any time from this case. He was qischarged on the fourteenth day, symptom-free (Apley et al., 1970). Apley et al. (1970) do not believe that isolation of amebae from the CSF of case two was due to laboratory cross-infection "but case three must be considered to have been only doubtfully infected with amebae. 11 Griffin (1976), has d isputed the diagnosis of Naegleria meningo-encephaliti s i n cases one and two . He contends that an Acanthamoeba was involved and that sulphadiazine, rather than ampho­ tericin B, which was given on admission, was responsible for the prolonged survival in the f i rst case and survival in the second . 13 The two cases reported by Pan and Ghosh ( 1971) were similarly inconclusive i n the nature of the etiological agent involved and the effective agent in treatment. Their report deals with two Indian children, aged six months and three years, with CNS infections of slow onset (3-5 months). CSF sampl e s s howed " mo til e amebae with thin ps eud­ opods ". No strains were isolated and both patients survived . They were treated with amphotericin B, sulphadiazine and intrathecal steroids . Anderson and Jamieson (1972) reported the case of a fourteen year ol d boy from Queensland who had typical acute symptoms and was already in the fourth day of illness and comatose by the time amphoter ­ icin B treatment was begun . The diagnosis was confirmed by finding 12,000 white cells . mm- 3 and numerous amebae in the CSF; the amebae were cultured and shown to be N. fowleri. Amphotericin B was given in -1 a dose of 1 mg . kg per day IV and penicillin, ampici ll in and sulphadia - zi ne, he had been having for three days previously, were continued. Within two days he became afebr i l e and was talking rationally. After -3 fiv e days the CSF white cell count h a d fallen t o 15.mm but many atypical amebae were still present. Amphote r ic in B wa s therefore given IT and later !Ve nt in small do ses (0.1 mg o n alterna t e day s) and the fluid gradually cleared. He was disc harged from ho sp i tal without any n eu rological deficit. Thi s case represents the first surviva l where t here is definite proof that~- fowleri was involved and survival c an be attributed to amphoteric i n B. Seidel et al. (pers. comm., 1978) report a more recent case in Torrance, California. The patient, a nine year old female, presented with typical symptoms of meningo-encephalitis three days before ad­ mission to hospital. Lumbar puncture revealed a purulent CSF and motile amebae were seen. Amphotericin B (1 mg.kg- 1 ), sulphadiazine . -1 -1 5 (50 mg.kg ), chloramphenicol (25 mg.kg ) and penicillin G (3.4 x 10 units) were all administered IV immediately on admission. 1.5 mg amphotericin B was also given IT. The patient was then transferred to Harbour General Hospital and was in a coma on admission but responsive to pain and tactile stimulation. On arrival the treatment outlined in Table III was inst i tuted. Table III: Treatment Protocol Used in a Case of PAM (Seidel et al., pers. comm., 19 78 ) DRUG ROUTE DOSAGE Amphoterici n B IV 1.5 -1 day + bid x 3 days mg .kg per • -1 day qd X 6 days 1.0 mg .kg pe r Amphoterici n B IT 1.5 mg per day X 2 days • 1mg QOD X 8 days Miconazole IV 350 - 2 day..:... tid x 9 days mg . m per Miconazole IT 10 mg X 2 days • 10 mg QOD x 8 days Rifampi n Ora l 10 mg .kg -1 per day tid x 9 days bid = twice da i ly tid = three times daily qd = every day QOD = every other day Sulphadiazine (4 g per day IV) was continued for three days 14 until studies confirmed the diagnosis of Naegleria meningo-encephalitis. Penicillin and chloramphenicol were continued f or three days until bacterial CSF cultures were negative. Decadron (dexamethasone) and Dilantin (diphenylhydantoin) were given for increased intracranial pressure and sei zure activity, respectively. The patient stabilized clinically over the first 48 hours. Gradually over the next month of hospitalization her mental status improved. No significant neurological deficits were noted at discharge. In a few of the other cases of PAM, where proof that N. fowleri was the etiological agent involved and amphotericin B given at effec­ tive doses the course of the disease was often too advanced to see any effect (Van Den Driessche et al., 1973; Cursons et al., 1976, pers. comm. 1979). Callicott et al. (1968), isolated an ameba identified as A. astronyxis from a spinal fluid sample from a patient with a purulent meningitis that remitted spontaneously . The authors were unable to provi de evidence that the organism caused the illness and was not just a cultural contaminant . 1 5 Kenney (1971), reported the case of a patient hospitalized for acute gastritis of unknown origin . Complement fixation tests revealed no antibodies to Entamoeba histolytica but did reveal antibodies to~­ culbertsoni . Over the next two months a rising titer to A. culbertsoni antigen was reported . Clinical investigation by a physician did not reveal any symptomatology suggestive of cerebromeningeal involvement. The patient refused a s pinal tap . The gastro-intestina l symptoms continued and a stool examinat ion revealed amebae which were called Iodamoeba butschlii. Because of the titre to Acanthamoeba antigen, the patient wa s put on anti a mebic th e rapy consisting o f Dehydro-Emetine (IM) and ch loroquin e . Complement f ixa tion tests two months later demonstrated that the serum titer had decreased. This case appears to demonstrate a form of disease between the symptom­ less carrier state an d fulminating meningo-encephalitis which may be found to b e more common than at present beli eved. The only human Acanthamoeba infections positively diagnosed during li fe under circumstances where chemotherapy cou ld have been tried were those in the eye . Nagington e t a l. (1 974) repeatedly iso ­ lat ed Acanth amoeba from t wo En g l ish patients with corneal ulcers. Warhus t a nd Thomas (1975) identified the amebae as~- castellanii a nd ~- polyphaga. One of the infections was in a 32 year old woman who had a mi ld unil ateral keratoconjunctivitis and uveitis which did not r e spond to t reatment which included chloramphenicol, idoxuridine, 3-fluorothymidine, gentamicin, methici llin and later on sulphadiazine (500 mg, six hourly). Six months after treatment, because of corneal ulceration, pain and loss of vision, a corneal graft was performed but the graft was rejected. The other infection described was in a 59 year old farmer with an identical clinical condition which required enucleation of the eye after one year. Treatment in this case included chloramphenicol, acetylcysteine, 3-fluorothymidine and clotrimazole eye drops. Jones et al. (1975) cultiva ted~- polyphaga from corneal ulcers of two patients in Houston, Texas. They reported suppression of the amebae with paromomycin. Griffin (1978) reported seeing similar material at the Armed Forces Institute of Pathology, Washington D.O. and it seems likely that Acanthamoeba in the eye will not prove to be strikingly rare or unusual.