Thie report contains the collective views of am international group of experts ond dost not necescarily represent the deck Mont or the stated policy of the World Health Organtzation WORLD HEALTH ORGANIZATION TECHNICAL REPORT SERIES No. 421 AMOEBIASIS Report of a WHO Expert Committee WORLD HEALTH ORGANIZATION GENEVA 1969(© World Health Organization 1969 Publications of the World Health Organization enjoy copyright protection in accord tance with the provisions of Protocol 2 of the Universal Copyright Convention. Mever- theless governmental agencies or learned) and professional societies may reproduce data for excerpts or ilustrations from them without requesting an authorization from the World Health Organization. For rights of reproduction or translation of WHO publications in foto, appiestion should be made to the Division of Editorial and Referenes Services, World Health Organization, Geneva, Switzerland. The World Health Organization’ welcomes such applications Tae designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the part of the Director- General of the World Health Organization concerning the legal status of any country Of territory or of its authorities, oF concerning the delimitation ofits frontiers. The mention of specific companies oF of certain manufactures" products does not Imply that they ate endorsed oF recommended by the World Health Organization in preference to others of a similar nature which are not mentioned. Errors and omissions excepted, the names of proprietary products are distinguished by initial capital leters.CONTENTS 1, Introduction 2. Definitions, classiication of disease conditions 24 Definitions... 2.2 Classification of disease conditions 43. The amoebae of man and animals 3.1 Amocbae parasitic in man 312 Related amoebae found in animals 313 Buamoeba hitoltica » 3.4 Entamoeba histolytica and E. histlyueaike amoebic. 4. Epidemiology 4e1 Transmission 4.2 Prevalence of infection 4° Onset of Bepatte amoctiasis 5, Laboratory diagnosis 5.1 Microscopic methods 5.2 Immunodiagnostic methods 6. Therapy. 6.1 Present drug situation 6.2 Drugs in practical use 6.3 Clinical tals and assessment of cures 6.4 Mass treatment and chemoprophslaxis 1. Control 1-1 Individual measures 7.2 Community measures 8, Research suggestions ‘The amoebae of man and animals | Epidemiology : Diagnosis Therapy 815 Control 8. Recommendations AcknowledgementWHO EXPERT COMMITTEE ON AMOEBIASIS Teheran, 27 Seplember 1968 Members :* Dr M. M. Brooke, Chief, Laboratory Consultation and, Development Section, ‘National Communicable Disease Center, Atlanta, Ga., USA (Rapporteur) Dr L. $. Diamond, Research Zoologist, Laboratory of Parasitic Diseases, National Institate of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Md., USA Dr Rawewan Jarumilinta, Parasitologist, Department of Medical Research, CIBA Ld, Basle, Switzerland Dr L. H. Lamy, Head, Protozootogy Department, Pasteur Institute, Paris, (ice: Chairman) Dr B, G. Macgeaith, Professor of Tropical Medicine, Dean, Liverpool Schoo! of Tropical Medicine, Liverpool, England (Chairman) Dr A. Neghme, Professor of Parasitology, School of Medicine, University of Chile, Sentiago, Chile Dr E, Nnochiri, Associate Professor of Medical Parasitology and Microbiology, Lagos Medical School, Nigeria Dr A, Zweibaum, Chargé de Recherche, National Institute of Health and Medical Research, Paris, France Secretariat Dr Ansati, Chief, Parasitic Diseases, WHO (Secretary) Dr P. C. Beaver, Professor of Tropical Diseases and Hygiene, Department of Tropical Medicine and Public Health, School of Medicine, Tulane University, New Orleuas, La, USA (Consultan Médecin-Genéral F. C. Blanc, Professor of Tropical Medicine, Tropical Diseases “Hospital, University of Ait-Marseilles, Marseilles, Franee (Consultant) Dr R. A. Neal, Head, Protozoology Section, Wellcome Laboratories of Tropical Medizine, Beckenham, Kent, England (Consultant) [Air Vice Macshal W, P. Stamm, Senior Consultant in Pathology and Tropical Medi- cine, RAF Institute of Pathology and Tropical Medicine, Halton, Aylesbury, Buckinghamshire, England (Consultant) * Unable to attend: Dr M. A. Sarkisyan, Laboratory of Protozootogy, Institute of Epidemiology and Hygiene, Mintsry of Health, Prevan, Armenian SSR, USSR.Wid Heh Org. techn, Rep. Ser. 1969, 421 AMOEBIASIS Report of a WHO Expert Committee A WHO Expert Committee on Amoebiasis met in Teheran from 2 to 7 September 1968, The meeting was opened by Dr N. Ansari, Chief, Parasitic Diseases, on behalf of the Director-General. Professor B. G. Maegraith was elected Chairman, Dr L. H. Lamy Vice-Chairman, and Dr M. M. Brooke Rapporteur. 1. INTRODUCTION Amoebiasis is known to occur in every part of the world, and is estimated to affect 10% of the world’s population, although its prevalence and severity may differ from area to area or increase in special circum stances. Thus,it is clearly a public health problem of international impor- tance. Following the Sixth International Congresses on Tropical Medicine and Malaria in 1958, which recommended an assessment of amoebiasis and of the parasite in vatious parts of the world, WHO undertook a prelimi- nary survey on the prevalence of the disease and on the diagnostic criteria adopted by experts. Two questionnaires were distributed to selected investigating bodies : one to laboratories and the other to hospitals. ‘The results were inconclusive because of the widely varying diagnostic criteria used by parasitologists and clinicians. In the last twenty years, views on the relation between amoebic infection and the symptoms attributed to the disease have been so fundamentally diverse as to prove irreconcilable. ‘The basi issue has revolved around the identification of the etiological agent and the assessment of its pathogen- icity to man, subjects on which there bas been wide disagreement among linicians and laboratory workers. Notwithstanding the spate of literature, the debate on “disease” versus “infection” remained unsettled. No comparison has been possible between morbidity surveys carried out by various investigators because the same phenomena were studied and inter- preted in different ways, as shown by the replies to the WHO question- Progress in the epidemiology of amoebiasis has not only suffered from the impossibility of assessing the public health significance of the disease in reliable quantitative terms, but has also been hampered by the lack of precise knowledge on some fundamental matters, such as the main modes —s—6 AMOEBIASIS of transmission of the disease, or the reasons for the differences in sus- ceptibility between hosts infected with the same strain. Finally, individual hygiene apart, prospects of controlling the disease ‘on a community basis were rather limited as there was no easily applied chemical or environmental method and no safe preventive or curative drug for use in mass treatment. In the circumstances, WHO was not in position to assist countries in assessing their amoebiasis problem or in controlling the disease. Now, however, WHO proposes to undertake a programme of activities on amoebiasis. There are several reasons why there is now more hope of success. First, there is a new trend in the pattern of study of intestinal diseases, whether of viral, bacterial, protozoal or helminthic origin. In countries well provided with laboratory facilities, etiological diagnosis is. widely practised, and consequently the identification of pathogenic and con- comitant agents has become more accurate. Of course, there arc many other countries where this is not done, particularly developing countries Where intestinal diseases are highly prevalent. Alongside the improve- ‘ments in parasitological methods, it seems that serological tests now being developed may further facilitate an etiological diagnosis by detecting cle- rments in the Serum that are indicative of tissue invasion by amoebae. ‘New techniques also offer great potential for the identification of species and strains of amoebae by their biochemical composition and ultrastruc- ture, as well as by their antigenic nature. The possibility of culturing amocbae in vitro in axenic media will encourage the study of the physiology and biochemistry ofthese organisms. Indeed, the multidisciplinary approach to the study of biological problems adopted by the young generation of rescarch workers is much more promising than traditional research within isolated disciplines. In the not too distant future it should be possible to separate pathogenic and non-pathogenic amoebae and to understand the pro- cess whereby apparently non-pathogenic amoebae may become pathogenic. Recent work has also brought new insights into the physiology of the colon epithelium. These may help in assessing the parts played in the pathogenesis of amoebic disease by direct action of the parasite on the host's tissues and by the host’s reaction. Furthermore, remarkable advances have been made in the chemo- therapy of amocbiasis and in the prevention of certain complications. This is of great significance, for treatment with toxic drugs was often instituted automatically upon discovery of a few cysts in faccal smears or ‘on presumptive clinical evidence, a practice that could lead to serious and sometimes irreversible accidents. It is now clear that inthe past the adminis- tration of a drug was often based on a wrong conception of its action, This is why drugs developed in recent years have aroused such interest compounds now available are less toxic and more effective.REPORT OF A WHO EXPERT COMMITTEE 7 As with other infectious diseases, a further circumstance encouraging WHO to participate more actively in amoebiasis work is the danger that incteasing movements of people will both contribute to the spread of amoebiasis and bring about changes in the present epidemiological patterns, so making it more difficult to interpret the findings and control the infection. ‘The Committee has not attempted an exhaustive survey of amoebia: In this report, it reviews recent advances in the parasitology, epidemiology, therapy, and control of amoebiasis, and expresses its opinion on some controversial problems raised by the discase. In addition to making recom- mendations on laboratory diagnostic procedures and on the methodology fot epidemiological and other investigations, the Committee suggests areas in which further research could achieve greater progress towards the con- trol of amoebiasis 2. DEFINITIONS, CLASSIFICATION OF DISEASE, CONDITIONS 2.1 Definitions ‘The Committee agreed upon the following definitions and concepts, which are based on the present state of knowledge of amoebic disease conditions. Amoebiasis. This denotes the, condition of harbouring Entamoeba histo- Iytica, with or without clinical manifestations. E, histolytica, With the recognition of E. hartmanni as a species, there is no further need to use the terms ‘small race” and “Targe race” E. histolytica. Minuta. This term refers to trophic forms of E. histolytica in the non- dysenterie stool. The definition is given to avoid possible confusion with E, hartmanni E, histolytica-like amoebae. ‘These are amoebae isolated from man that are morphologically indistinguishable from E. histolytica, but unlike the latter are capabie of indefinite multiplication in vitro at temperatures ranging from 10°C to 35°C, and in hypotonic media. ‘These properties are also accompanied by antigenic and biochemical differences. ‘The first such amoeba to be recognized was the Laredo strain Strain of E. histolytica, This is a laboratory-established population of an isolate, the source of which is well documented. When maintained axenically or monoxenically, a strain has relatively stable biological properties. When a strain is maintained with an ill-definéd mixture of microbial associates, the biological properties of the amoeba-bacteria complex may vary greatly.8 AMOEBIASIS 2.2 Classification of disease conditions E. histolytica is the only species of intestinal amoeba known for certain to be pathogenic to man, and was the chief concern of the Committee. Clinical and serological evidence suggest that E. histolytica may sometimes invade tissues without producing any symptoms ; this view is supported by the frequent development of amoebic liver abscess without any his- tory of gut infection. ‘While infection without tissue invasion is recognized to be a common occurrence, it eannot yet be distinguished from infection with asymptomatic, tissue invasion. Consequently, in the classification recommended below, the term “carrier ” is not used in clinieal diagnosis and no distinction is made between asymptomatic amoebiasis with and without invasion. ‘Any classification of symptomatic intestinal amoebiasis (jc., condition in which clinieal manifestations are attributable to E. histolytica) is arbitrary, since there is bound to be some overlapping. However, the condition can conveniently be divided into 2 main clinical states: amoebic dysentery ‘and non-dysenteric amocbie colitis. The duration of both these conditions varies, so the terms “acute” and “chronic” are best avoided. ‘The more important complications and sequelae of amoebic colitis are listed. Attention is drawn to post-dysenteric colitis, which may follow treated amoebic dysentery. A functional form of post-dysentcric colitis, is one of the group of conditions forming the irritable colon syndrome. OF greater importance is ulcerative post-dysenteric colitis, a direct sequel to severe amoebie dysentery. Amoeboma is a localized form of intestinal amoebiasis that on clinical examination may be confused with a neoplasm. It is clasified separately in order to draw attention to its existence “Hepatic amoebiasis is by far the commonest form of extra-intestinal amoebic disease. Its two forms are clinically indistinguishable, except that in the acute non-suppurative form no abscess can be identified, whereas in amoebic liver abscess there is definite clinical evidence of the presence of an abscess. The classification of amoebiasis used by the Committee is as follows Asymptomatic Symptomatic Intestinal amoebiasis Diysentery® Non-lysenteric colitis ‘Amocboma ‘Amocbic appendicitis T Acute dysenteric symptoms are sometimes accompanied by a tender and enlarged liver; in this Condition there is thought ta be no actual invasion of the liver by amoebae, lind it is best referred to as nonspecific hepatomegaly, which does not involve any tssumption as to its exaet etiologyREPORT OF A WHO EXPERT COMMITTEE 9 Complications and sequelae of intestinal amoebiasis: perforation and peritonitis ; Thuemorrhage’; intussusception ; post-dysenteric colitis; stietare Extreintestinal amoebiasis Hepatio:* (2) Acute non-suppurative (@) Liver abscess Complications of liver abscess: rupture oF extension ; bacterial infection ; haemato= ‘enous spread to other organs Cutaneons Involvement of other organs, i., ling, brain, spleen, etc., without manifest liver involvement 3. THE AMOEBAE OF MAN AND ANIMALS. Parasitic amoebae are selective with regard to the part of the body they inhabit, and most species live in the colon. ‘The life eycle of an amoebic colony typically begins with a trophic amoeba (trophozoite), which grows and reproduces by binary fission; the daughter individuals spread and increase the size of the colony. Once the colony is well established, some of the trophozoites cease their vegetative function and secrete an enveloping membrane to form a cyst. Thus immobilized, the encysted amoeba is carried passively from the colony to the outside of the host organism, and to anew host. When it reaches the colon, the trophic amoeba emerges through the cyst membrane and the cycle is repeated. Seven species of amoeba are natural parasites of man: Entamoeba histolytica Schaudinn, 1903 : Entamoeba hartmanni von Prowazek, 1912 ; Entamoeba coli (Grassi, 1879) Hickson, 1909 ; Entamoeba gingivalis (Gros, 1849) Smith & Barrett, 1914; Endolimax nana (Wenyon & O'Connor, 1917) Brug, 1918; Jodamoeba buetschlii (von Prowazek, 1912); and Dientamoeba fragilis Jepps & Dobell, 1918. Entamoeba gingivalis is an inhabitant of the mouth ; the others live in the colon. Free-living species ‘of amoeba have been found as facultative parasites of man, 3.1.1 Amoebae of the colon OF the 6 species of colon-inbabiting amoeba, only Enramoeba histo- Iptica is recognized as pathogenic to man ; although in many individuals * Chronic diffuse amoebic hepatitis has been reported by some authors, but the existence of this condition has not yet been widely accepted.10 AMOEBIASIS it too may live as a harmless commensal. Dientamoeba fragilis is regarded as non-pathogenic, although some evidence of an association with diarthoea hhas been recorded. Iodamoeba buetschlii has been reported on 2 occa sions in autopsy materials, although itis now recognized that the organisms found were probably a species of Harrmannella or some other free-living amoeba (see section 3.1.2). Entamoeba histolytica is the organism always implied or referred to in connexion with such terms as dysentery amoeba, amoebic colitis, or amoebiasis, and is the species involved in extra-intestinal amoebiasis, ¢.g., amoebic invasion and colonization of the liver, lung, brain, and skin. ‘A commonly held view some years ago was that when £. histolytica could bbe demonstrated in the faeces, even if no symptoms were present, it could be assumed that it produced lesions in the mucosa of the colon. Nowa- days, however, the view more generally held is that, although E. histolytica is unquestionably highly pathogenic at times, it is often not pathogenic at all. There is widespread uncertainty about the pathogenicity of the different kinds of E. histolytica-like amoebae and their relationships to each other. ‘The different species of amoebae are distinguished by their size at the trophic and cystic stages, the number and structure of the nuclei, and the number and shape of the cytoplasmic inclusions in the trophozoite (vacuoles) and cyst (chromatoidals). The morphological features of the different species tend to overlap in range and to vary accord'ng to the conditions of collection and methods of preparation, and no species is sufficiently distinct from all others to permit immediate and reliable identification of an individual amoeba. Moreover, amoebae that morphologically are essen- tially indistinguishable may show marked, differences in physiological traits, associated with varying degrees of pathogenicity or in some instances with the presence or absence of pathogenicity. For example, E. histolytica- like amoebae that reproduce in vitro at relatively low temperatures arc ‘generally less pathogenic in experimental animals than B. histolytica, which Fequire a higher temperature in culture 3.1.2 Freesliving amoebae as facultative parasites of man Free-living amoebae occasionally reported in man include Acanth- amoeba, Hartmannella and Naegleria. It is not certain, however, that specimens found in man have been properly identified, since in most cases identification has been based only on the appearance of amoebae in histo- logical sections. The following review necessarily adheres to the nomen- clature used in the reports, but it is realized that further work is needed to establish precisely which of the free-living amoebae may parasitize man, ‘About ten years ago it was found that an amoeba earlier observed in cultures of trypsinized monkey-kidney cells was highly pathogenic toREPORT OF A WHO EXPERT COMMITTEE nl mice and monkeys when injected by the intracerebral, intraspinal, or intra- nasal route. It was identified as a species of Acanthamoeba, and later studies with mice confirmed the extreme invasiveness and pathogenicity of the amoeba when introduced into the nasal cavity, whence it rapidly invaded the meninges and brain. In 1965 Acanthamoeba infection was reported as causing acute, fatal meningitis in 3 children and an adult in South Australia. ‘The disease, markedly similar in onset and course to a fulminating bacterial meningitis, was rapidly fatal in the children, ‘There was lethargy and dulling of interest on the first day of illness, followed by fever, headache, sore throat, and blocked nose om the second day, vomiting and impaired consciousness on the third day, and deepening coma and death on the fourth day. In the adult, the acute phase was preceded by 2 weeks of headache and sore throat. Further fatal cases have been reported, with minor variations in course and symptoms: 3 from Florida in 1966, 7 from Virginia in 1968, and 16 from northern Bohemia in 1968. To date, reports have been received of 32 cases of acute meningo-encephalitis in which amoebae morpholog- ically resembling Hartmannella or Acanthamoeba were found in the meninges and brain. Also, it has been reported that a Hartmannella species, initially mistaken for a virus (Ryan virus) has been isolated in tissue cultures from the nasopharynx of persons examined in Great Britain, It is also interest- ing to note that chronic, granulomatous brain lesions have been produced experimentally in mice by new isolates of Hartmannella from soil. These isolates were of relatively low virulence. Alll the human cases referred to above were diagnosed on histological evidence. An amoeba recently isolated from the cerebrospinal fluid of a patient who died from meningo-encephalitis in Florida has been identified as Naegleria Tn the light of this new evidence, it appears likely that the amoeba reported as Zodamoeba buetschiit in 2 fatal cases where the brain was invaded was in fact a species of Hartmannella or some other facultative parasite. In one of the patients, a badly debilitated adult, the amoeba was found in the colon and other locations as well as in the brain, Identification of fodamoeba was based mainly on nuclear morphology, and was arrived at by the exclusion of other “human amoebae”. In the other patient, a 6-year-old girl, there was amoebic invasion of the meninges and brain following a fall. Apparently, the fall resulted in a traumatic lesion that, either at once or some time later, reached the meninges. In an interval of several months before death, a craniotomy was performed and a granulo- ‘matous mass was surgically removed from the dura mater. It was reported that the amoebae were probably Jodamoeba buetschli, but in illustrations accompanying the autopsy report the amoebae appeared to have the characteristic centrally located karyosome of Hartmannella, not the eccen- tric karyosome of Jodamoeba.12 AMOEBIASIS| If it is accepted that these 2 deaths were due to Hartmannella, the total now stands at 34. The cases have occurred in Australia, Europe, and the USA, in children and young adults (with 1 exception) between the ages of 6 and 28, and death iisually took place 1-7 days after the onset of symptoms and signs that were no different from those of acute menin- gitis. More than half the patients had swum in freshwater lakes or pools @ few days before the onset of illness. ‘These observations support the conclusion drawn from experimental evidence that the brain is invaded directly from the nasopharynx, along the olfactory nerves. In some in- stances, invasion may occur through open lesions of the skin or buccal 3.2. Related amoebae found in animals Monkeys in captivity harbour amoebae indistinguishable from the 4 common amoebae of man (Entamoeba coli, E. histolytica, lodamoeba buetschlii and Endolimax nana). Whether these differ physiologically from the forms found in man has not been determined. It is possible to infect Macaca mulatta with E. histolytica from man, and the behaviour and pathogenicity of E. histolytica appears to be the same in the chimpanzee E. histolytica has occasionally been reported in the domestic pig, but the records are of doubtful reliability. On the other hand, there are a number of well-documented instances of infection of man by E. polecki, a common amoeba of pigs. ‘Amoebae resembling E. coli live in the caecum of rats, mice, and many ‘other species of rodent, In the common laboratory rat and mouse, these amoebae are called E, muris; in the guinea-pig they are known as E. cobayae, and in the rabbit as E. cuniculi. A species of amoeba indistinguishable from E. muris is found in the Mongolian gerbil (Meriones unguiculatus). An Endolimax species resembling End. nana of man, though somewhat smaller, ‘occurs in the guinea-pig. . histolytica has occasionally been reported in the rat, but it has not been established that it is ever enzootic in non- primates, Occasionally dogs and cats are found to harbour amocbae that resemble, and are assumed to be, E. histolytica. Several species of amoebae have been found in various reptiles, but only 2 are of special interest : . invadens and E. terrapinae. As name implies, £. invadens is highly pathogenic. However, its pathogenicity differs greatly from host to host, for it is lethal in snakes but not in herbivorous turtles, and is invasive in snakes at high temperatures only. E, terrapinae, on the other hand, is not pathogenic at any temperature, Both species resemble E. histolytica in morphology and motility, and are useful in experimental studies on the biology of parasitic amoebae[REPORT OF A WHO FXPERT COMMITEE 13 3.3. Entamoeba histolytica 3.3.1 Ultrastructure Electron microscopy has revealed differences in subcellular organization between £. histolytica and metazoan cells or the larger free-living amoebae. Neither mitochondria of the type seen in other cells nor the Golgi complex hhas been observed in £. histolytica or in other species of Entamoeba that hhave been investigated. On the other hand, histochemical studies of E. histolytica have revealed small granuies that appear to have a mitochondrial function. The endoplasmic reticulum seems to be poorly developed. The cytoplasmic membrane is in the form of a typical unit membrane consisting, of 3 layers. The limiting membrane of the food vacuoles has the same structure as the cytoplasmic membrane. E. histolytica, like the other amoebae with limax-type movement, has a differentiated tail region or uroid. This organelle is the site of protein contraction, and produces the flow of eytoplasm that enables the organism to move. It also has excretory functions. ‘The arrangement in the nucleus of peripheral and karyosomal (endo- somal) material has essentially the same appearance, whether observed by electron microscopy or by conventional light microscopy. The nuclear membrane is 2layered, and presents a discontinuous appearance as if perforated by a series of pores. Various numbers of helical bodies representing an array of ribosomes have been observed. Polycrystalline bodies representing a mass of ribosomes, hhave been seen in amoebae of the related species . invadens, and the chromatoidals of the cyst appeared as a much larger polycrystalline mass of ribosomes. Whorllike structures have been seen and interpreted either as viruses or as membranes remaining after the disappearance of food vacuoles. ‘The modifications of the typical subcellular organization that are found in Entamoeba may reflect changes due to the mode of locomotion, and may also be related to adaptation to the parasitic environment.” The electron microscope observations will be better understood when the localization of enzymes and other biochemical or physiological informa- tion can be related to subcellular organization, Of particular interest in connexion with tissue invasion is the localization of enzymes in the cyto- plasmic membrane. 3.3.2 Cultivation in vitro and nutritional requirements Conventional techniques of cultivating E. histolytica involve growing the amoebae in association with one or more species of micro-organism. Media have been prepared from a variety of natural materials, but eggs and serum are chiefly used. Synthetic media have also been described. Whatever medium is selected, it must have the ability to support the growth of suitable bacteria for several days, and rice starch must be added. The4 AMOEBIASIS: growth with bacteria is complex, for each of the 3 components— microbial associates, amoebae, and medium—interacts with each of the others. Starch is not essential for the growth of the amoebae, but when present in the medium it speeds up the growth rate by providing carbo- hydrate ina form readily assimilated by the amoebae. Amoebae are usually particulate feeders, but in axenic media they ingest by pinocytosis. In many strains of E. histolytica the composition of the flora is unknown. ‘There are various procedures whereby the unknown flora can be replaced by one or more known species of bacteria favourable to growth, or by other organisms such as Trypanosoma cruzi Axenic cultures provide the investigator with a source of amoebae free of the influences of the microbial associates, a prerequisite for many bio- chemical, nutritional, and immunological studies. ‘The medium for axenic cultivation of E. histolytica was developed in stages. The amoebae, origi- nally cultivated in a complex diphasic medium containing a cell-free extract, of chick embryo, can now be cultivated in a clear liquid medium without the extract. Moreover, this liquid medium can be used for mass cultivation. Inocula of 10000 amoebae per ml of medium result in 10-15-fold increases after cultivation for 72 hours at 35°C, Under these conditions, the average yield of protein antigen is 0.87 mg per million amoebae. Horse serum, or some satisfactory substitute, is an essential and critical component of the medium. The optimum concentration appears to be 10%. Because of variations in growth-promoting ability, each batch of scrum has to be pretested. A standard of serum acceptibility and # technique for determining compliance with this standard have been developed. Both bovine and rabbit sera appear to be good replacements for horse serum, but lamb serum, although it will support growth, is relatively ineffective. Samples of human serum from two individuals failed to support serial growth, Panmede, an ox-liver digest, is also an essential ingredient of the ‘medium. Unsuccessful attempts have been made to replace it with the following liver preparations : solubilized aqueous liver paste; liver frac- tion 1 (NF); liver fraction L; dehydrated liver infusion ; and liver con- centrate (NF #) 1:20. ‘The source of all amoebae for axenic cultures has been monoxenic, amocba-trypanosomatid fiagellate cultures. There is evidence that the axenization of a given strain of amoeba is dependent, at least in part, upon the composition of the medium used for axenic growth, and on the par- ticular trypanosomatid associate employed in the monoxenic source cul- tures. 2 Committee on National Formulary of the American Pharmaceutical Association (1960) The natonel formulary, Washington, 11th e&REPORT OF A WHO EXPERT COMMITTEE 15 Antigens prepared from axenically cultivated E. histolytica (4 strains) and from an E. histolytica-like amoeba (Laredo strain) have been studied by immunoelectrophoresis and evaluated in the indirect haeniagelutination, test, These studies showed the 4 strains of E. histolytica to be essentially similar, but significantly different from the E. histolyticaslike amoeba. In the haemagelutination test, a concéntration of 30-40 jug protein antigen per ml was found adequate for the sensitization of sheep erythrocytes. When the £. histolytica antigens are injected subcutaneously into guinea- pigs sensitized with homologous antigen, they induce a typical delayed- type skin reaction, which suggests they might be acceptable for studies in human subjects. Ribosomal fractions prepared from axenically cultivated E. histolytica and ribosomes from rat liver showed similar ability to promote incorpora- tion of “C-labelled amino acid into an acid-precipitable product. Tetra- cycline was found to depress polypeptide formation by the ribosomes of amoebic origin, suggesting that this antibiotic has a direct effect upon the amoebae. Axenically cultivated E, histolytica is non-infective to guinea-pigs. How: ever, infectivity and a degree of virulence are restored when the amoebae are reassociated with a mixed bacterial flora, 3.3.3 Bneystment Eneystment in vivo is a response not to unfavourable conditions, but rather to special conditions that are at present ill-defined. Cysts are rarely observed in the stools of patients with amoebic dysentery. Encystment appears to require normal intestinal transit In addition to cysts, a variable number of trophozoites may be found in infected individuals. ‘Their presence does not necessarily imply colonic or rectal ulceration, but may merely be due to failure to encyst. Strains isolated and maintained in vitro soon lose their ability to encyst. However, some strains with suitable bacterial associates form large num bers of cysts when maintained by a special technique. In such strains the production of cysts is regular and predictable, ‘The conditions for encystment have not been defined, but can be sum- marized as the transfer of poorly growing amoebae to a richer medium where their rate of division is increased. Cysts form when the right bacterial flora is present. Strains that will not encyst, e.g., E. histolytica strains isolated from patients with amoebic dysentery, can be induced to do so by transferring the bacterial flora from encysting strains. 3.3.4 Enzymes and the pathogenicity of E. histolytica Despite numerous extensive studies with animal hosts, the factors governing the pathogenic activities of E. histolytica are stllIargely unknown,16 AMOEBIASIS, ‘The mechanism by which E. histolytica gains entry into the host tissues is still not fully understood. It is generally believed that it penetrates the gut epithelium of human beings and animals by virtue of histolytic enzymes that it secretes. ‘The evidence for the existence of such enzymes is based on observation of the initial contact with the gut mucosa, and on the histological studies of lytic necrosis in the tissue at later stages, when the amoebae are visible and are often surrounded by a clear area (ytic area) separating them from the adjacent host tissue. The earliest superficial changes in the mucosa seen in patients with amoebic ulceration are the typical bottle-neck ulcers and the honeycombing destruction of the sub- mucosa, although it is generally agreed that mechanical action may aid the amoebae in their penetration and migration. It has repeatedly been shown that E. histolytica possesses proteolytic activity in vitro towards various substrates. The proteolytic enzyme content of E. histolytica has been studied in detail in living organisms and in extracts of trophozoites, using both protein and synthetic substrates. The investigations have covered E histolytica strains that were isolated from human patients with amoebic dysentery and were pathogenic to rats and guinea-pigs, and strains isolated from human asymptomatic patients and not pathogenic to these animals. ‘The enzymes of Acanthamoeba sp. maintained in axenic culture have also been studied, but the pathogenicity of the organism to animals has not been tested. The living organisms and extracts ofall strains of E. histolytica examined were capable of hydrolysing gelatin, casein, fibrin, haemoglobin, and guinea-pig gut epithelium. Acanthamoeba sp. lysed gelatin and casein, but not haemoglobin, fibrin or epithelium. Hydrolysis of the gut epi- thelium by all the strains of £. histolytica and by trypsin produced similar amino acid chromatographic patterns. The use of various specific syn- thetic substrates for proteinases (endopeptidases and exopeptidases) showed that E. histolytica, whether pathogenic or not, has tryptic and peptic activity but no chymotryptic activity. Non-pathogenic strains of . histolytica contained carboxypeptidase, aminopeptidase, and dipeptidase, but carboxy- peptidase was not present in pathogenic strains. Acanthamoeba sp. con tained only pepsin, aminopeptidase, and carboxypeptidase. All strains of E. histolytica studied contain trypsin, therefore, but Acanthamoeba sp. does not. The use of synthetic substrates has shown that both E. histolytica and ‘Acanthamoeba sp. contain pepsin, but in experiments using casein optimum activity at an acid pH was not observed. Digestion of the epithelium by pepsin is regarded as unlikely, because pepsin works within a pH range Where the parasites are inactive in vitro. ‘The presence of trypsin, which is capable of digesting guinea-pig caccal epithelium, in all strains of E. histolytica, has prevented any distinction between the pathogenic and non-pathogenic strains. A distinction could be drawn between the potential tryptic activity of £. histolytica and theREPORT OF A WHO EXPERT COMMITTEE a7 absence of this potential in Acanthamoeba sp. In the non-pathogenic strains of E. histolytica it scems that the activity of the trypsin is in some way controlled or limited. The significance of the presence of carboxypeptidase in non-pathogenic strains of E. histolytica and Acanthamoeba sp. (and in 1 strain of E. coli) and its absence from the pathogenic strains is not understood. It is pos- sible that some residual peptide moiety survives the final digestion of protein by the pathogenic strains of . histolyrica and plays a part in con- trolling the parasite’s over-all proteolytic activity Afier E. histolytica has penetrated the gut epithelium (either by proteo- lytic enzymes, induction of autolysis in the epithelial cells, mechanical penetration, or through an area of epithelial damage) and reached the submucous tissue, the spread of the organism into the deeper tissue may well depend on the activity of enzymes of the “ spreading factor ” type including hyaluronidase, which has been shown to be « common component of the ground substance of connective tissue. Hyaluronidase activity has been studied in trophozoites and extracts of strains of E. histolytica that were isolated ftom patients with amoebic dysentery and were pathogenic to rats and guinea-pigs, as well as in those that were isolated from asymptomatic subjects and were non-pathogenic to the animals. Of 10 pathogenic strains examined, 8 contained hyaluronidase and 2 did not. ‘There was no hyaluronidase in any of the non-pathogenic strains or in the Acanthamoeba. Among the strains containing hyaluronidase, there was no correlation between the degree of hyaluronidase activity and the average grade of the lesions produced in guinea-pigs. A correlation has not been established, therefore, between the presence of this enzyme and the pathogenicity of the parasite. It is noteworthy, however, that all strains examined that contained hyaluronidase were in fact pathogenic to hhuman and animal hosts, although the absence of this enzyme did not necessarily indicate non-pathogenicity ‘The conclusion that has been drawn is that the study of the pattern of enzymes in an amoeba is not in itself sulficient to determine whether the parasite will be pathogenic when brought into relation with the host tissue. It has proved possible. admittedly, to differentiate between E. histolytica as a group and Acanthamoeba, which indicates that the genetic enzyme patterns of E. histolytica as a group (both pathogenic and non-pathogenic strains) differ from those of known non-pathogenic free-living organisms. For the present, however, pathogenicity must continue to be established only in terms of host-parasite relationships. Other enzymes observed in cultures of E. histolytica are amylase, phosphomonoesterase, glutaminase, maltase, esterase, and succinic dehy- drogenase, but their significance with regard to the amoeba’s pathogenicity has not yet been studied.18 AMOERIASIS| Trophozoites of various strains of E. histolytica, pathogenic and non- pathogenic, have recently been found to have a cytotoxic effect on leucocytes originating from man, sheep, rabbits, chickens, guinea-pigs, hamsters, rats, and mice. £. coli, Acanthamoeba, and an unidentified non-pathogenic amoeba did not exhibit this activity. Once again, although the leucocyto- toxie action of £. histolytica made it possible to distinguish this parasite from other amoebae, the question of pathogenicity remains unsolved, for there is no apparent distinction between the strains known to be patho- genic to man and animals and those that are non-pathogenic. Since E. histolytica has been shown to display a variety of enzymatic activities, the Parasite might be assumed to act enzymatically upon the leucocyte. How- ever, trypsin at a high concentration showed no Ieucocytotoxic activity under the same experimental conditions, and it was concluded that this enzyme is not the causative agent of leucocytotoxic reaction. ‘As the process by which leucocytes in contact with £. histolytica are damaged is marked by rapid and extensive lysis of cytoplasmic granules, it seems reasonable to assume that the noxa of £. histolytica may disrupt the lysosomes in the leucocytes, thereby releasing hydrolytic enzymes that cause other structural elements in the leucocytes to be digested and finally result in generalized cell damage and death. 3.3.5. Histochemistry Although mitochondria have not been observed by electron microscopy, hhistochemical tests have revealed small granules that give evidence of oxidative-reductive function. Polysaccharides, mainly glycogen, are uni- formly distributed as gramules in the cytoplasm and as a hyaline mass in the cyst. Alkaline phosphatase is seen mainly in the nuclei of the trophic forms of E, histolytica obtained from fresh sigmoidoscopic or other faecal ma- terial in acute cases of dysentery, or from 48-hour-old cultures. In general, the intensity of reaction is greater in specimens obtained from nature than, in those cultured in vitro, The ectoplasm shows a bilaminar distribution of alkaline phosphatase. In the cyst it is the mucleus that shows a marked. reaction ; here the enzyme appears as a series of granules along the inner surface of the nuclear membrane, while the endosome reacts strongly when tested for the enzyme. The nucleoplasm may at times show a diffuse reaction, In the endoplasm, the enzyme is located in fine granules. The periphery of the food vacuoles always reacts positively to tests for alkaline phosphatase. It is possible that the layer of phosphatase on the outer border of the ectoplasm of the amoeba is concerned with the transfer of soluble food material, and 3 possible functions have been attributed to nuclear phosphatases : (4) protecting genes against a number of vigorous phosphorylating agents, such as adenosine triphosphate and acetylphos-REPORT OF A WHO EXPERT COMMITTEE 19 phate, (6) mediating in the final stage of gene formation, and (c) nucleic acid synthesis. ‘Acid phosphatase has been demonstrated in the mucleus and nucleo- plasm. A study of the chromatoidals revealed a core of RNA and an outer coat of DNA, but this has not: been confirmed by subsequent work. Granules giving the staining reactions of DNA and RNA have been observed in the cytoplasm of amoebae. In the nucleus of trophic forms of £. histolytica, Feulgen-positive ‘granules are arranged along the entire inner surface of the nuclear mem- brane, or sometimes in crescent form. The endosome (karyosome) appears, as a hollow sphere, and Feulgen-positive material is confined to the peri- phery. Some minute Feulgen-positive granules are also seen in the nucleo- plasm around the endosome. When the pyronine-methyl green technique 4s used, the areas stained by methyl green correspond to those that are Feulgen-positive and show the presence of alkaline phosphatase. Some- what similar results are obtained with specimens fixed in Schaudinn's fluid and stained with Heidenhain’s iron-alum haematoxylin stain. The tetranucleate cyst also gives similar findi Clearly, the available information about the cytochemical make-up of E. histolytica is very sketchy. Up to now, moreover, there has been no standard cytochemical analysis of various strains of Entamoeba from dif- forent parts of the world to provide an over-all picture of their basic similarities or dissimilarities, particularly as regards the degree of patho genicity or virulence. 3.3.6 Experimental methods for the study of pathogenicity (1) Inducing intestinal amoebiasis in animals Selection of host. Experimental intestinal infection with E. histolytica hhas been successfully induced in many host species, including monkeys, dogs, cats, rabbits, guinea-pigs, rats, and hamsters, either by injection cof amoebae or by administration of cysts. The host selected depends on the purpose of the study. If all that is required is to produce amoebic Ulceration of the gut, one of the more susceptible hosts would be suitable, -. kittens, dogs, or guinea-pigs. Dogs are also suitable when patent infection of the gut for prolonged periods is desired, since they do not die as a result. The guinea-pig has been extensively used for studies of the virulence of E. histolvtica strains because of its high susceptibility, but is not used for chemotherapeutic studies since it responds poorly to drugs that are effective in man. Rabbits are probably most suitable for immuno- logical studies. Young rats are most commonly used at present for virulence and chemotherapy studies, since they react in the same manner as man : E. histolytica strains causing symptoms in man are virulent for rats, whereas,20 AMOEBIASIS strains from asymptomatic human subjects are not, and rats respond consistently to standard amoebicide. Monkeys are generally considered unsuitable because of the considerable variation in reaction to experimental infections within different simian species, their high rates of natural infec- tion, and their cost : In general, the origin and breed of the animals used for studying patho- genicity have not been specified, but some observations are available on different strains of rats. OF the 3 strains that have been used, 2 (Wistar and Chester Beatty), have proved fully susceptible, but the third (Sprague Dawley) was less susceptible. In future studies on pathogenicity, therefore, the question of the strain of the experimental host should be considered. Method of infection. Experimental methods that have successfully been ‘employed to induce intestinal infection include : (a) oral administration of amoebic cysts obtained from faeces or cultures, (6) rectal injection of amoebae obiained from freshly passed dysenteric stools, from the pus of amoebic liver abscess, or from cultures, (¢) direct injection of faeces con- taining cysts into the lumen of the large intestine, (d) intra-oesophageal inoculation of cultivated cysts, and (¢) direct injection of trophozoites into the terminal ileum or caecum. ‘The last of these methods has proved the most successful. When a single amoebic lesion is required, the injection of amoebae directly into the caecal wall is a useful technique. (2) Inducing hepatic Infections in animals Selection of host. Young hamsters are the most susceptible of the various hosts that have been examined, and are the only host used at present for producing amoebic liver abscesses. A severe progressive disease is produced in these animals, and death occurs about. 7-14 days after inoculation. Method of infection. Various inoculation routes have successfully been used to produce liver abscesses. They include the injection of trophozoites of E, histolytica (a) direct into the liver, (6) into the portal vein, and (c) into the peritoneum. Placing the inoculum on the surface of the liver lobe with a gelatin sponge has also proved valuable. ‘The method. selected depends on the aim of the experiment. For chemotherapeutic studies where large numbers of animals are used, the gelatin sponge method is preferred because it is the least time-consuming. Since this technique avoids damage by the injection needle, it is also more suitable for histological studies. (3) Methods used to alter pathogenicity Effect of diet. With some animals, such as the guinea-pig and dog, it is essential to use diets that promote ulceration. In the dog, a salmonREPORT OF A WHO EXPERT COMMITTEE a diet acts as a local itritant, causing a non-specific colitis. Guinea-pigs are fed a food designed for rabbits, and show marked atrophy of the caecal ‘mucosa. It is thought that this lowers their resistance to amoebic invasion. It is not certain whether increased susceptibility in the dog and guinea-pig is linked with growth factor deficiencies. Influence of cholesterol. If amoebae are cultured in vitro in the presence of cholesterol, or if the hosts are given high doses of this steroid by mouth, it is possible to convert non-invasive strains isolated from asymptomati hhuman subjects into invasive strains indistinguishable from those isolated from patients with amoebic dysentery. In intestinal infections, the chole- sterol effect may be due to irritation of the mucosa. Animal passage. Strains may become avirulent as a result of prolonged cultivation in vitro. Their virulence can be revived by a technique involving the production of liver abscess in hamsters, and about five transfers from hhamster to hamster. Besides increasing the pathogenicity of the culture to the liver, this method may also lead to greater intestinal ulceration. Direct rat-to-rat passage of material from intestinal uleers has increased the virulence to the caccum. (4) Other in vivo techniques Use of germ-free guinea-pigs. Typical ulcerations are not observed in germ-free guinea-pigs inoculated with £. histolytica and Trypanosoma cruzi cultures, but they do oveur in guinea-pigs infected with certain species of bacteria, This does not mean that the amoebae alone are incapable of invasion, for some tissue invasion has been seen when the mucosa is injured ‘mechanically. It is not known whether, in the absence of bacteria, the caccal environment of axenic guinea-pigs is conducive to the survival of amoebae. Lifect of temperature. The adaptation of 2 strains of E. histolytica to growth at 29°C resulted in increased virulence. The test of virulence used was the production of liver abscesses in hamsters. However, it was, concluded that an increase in bacterial virulence might have also occurred at lower temperatures. (5) In vitro techniques Tissue culture. ‘This approach has not been used with E. histolytica, but has been adopted with the pathogenic reptilian amoeba, E. invadens In experiments using explants of embryonic chick intestine, the tissue remained alive for 10 days at 30°C and the amocbac fed on mucus and dead cells. They ponetraied tissue without any apparent histolytic effect. Ingestion and lysis of cells. Techniques for studying the lysis and phago- eytosis of red cells and leucocytes have been reported.2 AMEBIASIS 3.4 Entamoeba histolytica and E. histolytica-like amoebae 3.4.1, The Laredo-type strains In 1956 a strain resembling Entamoeba histolytica but with the unusual ability to ive and multiply at room temperature as well as at body tempera- ture was isolated, and was named the “ Laredo” strain, Between 1964 and 1966 the isolation of 5 more such strains from human facces was reported. A number of differences between these strains and classical E. histolytica have now been established. 3.4.1.1 Temperature tolerance and growth in hypotonic media. It has now been firmly established that the optimum temperature for growth of trophozoites of the Laredo-type strains is 25-30°C (survival range 0-41°C),, whereas for E, histolytica it is about 37°C (Survival range 20-43°C). The differences in temperature tolerance provide stable, valid, and fairly simple criteria for distinguishing classical E. histolytica from Laredo-type amoebae. Caltured Laredo-type trophozoites are capable of complete cycles of division, encystment and excystment in extremely hypotonic solutions, such as a 1: 64 dilution of culture medium in distilled water. These con- clusions are based on data for 14 strains of E. histolytica and 5 strains of the Laredo-type amoeba. 3.4.1.2 Antigenic make-up. The antigenic relationships between classical E. histolytica and the Laredo-type amoeba have been analysed by the techniques of fluorescent antibody, precipitation in agar gel, and haemagglutination. (Q) Fluorescent antibody reactions. In experiments involving cross- absorption and cross-staining, immunofluorescence studies indicated that the Huff (Laredo-type) strain lacked some of the antigens that are present in a classical £, histolytica strain. When 5 Laredo-type strains were compared with 8 strains of classical F. histolytica, the latter were found to have a distinctly greater ability to react with a human antiserum derived from a patient with amoebic liver abscess, and with a rabbit antiserum against classical E. histolytica. Anti-istolytica serum labelled with fluores- cent stain caused somewhat brighter fluorescence in the corresponding E, histolytica strain than in the Laredo strain. 2) Precipitation in agar gel. Huff and Laredo strains grown at 37°C have shown a different number of precipitin bands from classical E. histo- Iptica. At least 6 distinct bands were found in extracts of axenic cultures of 4 strains of E, histolytica, but only 2 in an axenic culture of the Laredo strain, Characteristically, a prominent slow-moving component that is present in all E. histolytica antigens was absent in the Laredo antigen. In double diffusion experiments, anti-E. histolytica serum reacted much ‘more strongly with £. histolytica antigen than with Laredo antigen.REPORT OF A WHO EXPERT COMMITTEE 23 (3) Haemaggiutination. In tests on a small group of human sera, lower hhaemagelutination titres were obtained with the Laredo antigens than with E. histolytica antigens. 3.4.1.3 Pathogenicity. A limited number of reports indicate that the Laredo-type organisms have little or no pathogenicity to man, and have low or restricted pathogenicity to the common laboratory animals that are susceptible to classical £. histolytica. Nevertheless, since classical E. ‘histolytica strains are sometimes equally non-virulent in animal and human hosts, the pathogenicity factor is of limited value for distinguishing between the 2 groups of amoebae. 3.4.1.4 Sensitivity to. drugs. Laredo-type strains grown at 37°C or 24°C have shown more resistance to drugs than other strains. For example there was a 10-fold resistance to emetine at 37°C and a 30-S0-fold resistance at 24°C, 3.4.1.5 Biochemistry, The biochemical evaluation of Laredo-type strains has not yet proceeded very far, but this group does seem to possess at least 2 biochemical attributes clearly distinct from those characterizing E. histolytica; the differences lie in the character of the enzyme glucokinase ‘and in quantitative amino-acid composition, 3.4.1.6 Nomenclature. There would seem to be justification for con- sidering the Laredo-type amoebae as members of a species separate from E. histolytica. Aside from the morphology of the mature cyst, in fact, the differences between £. histolytica and the Laredo-type strains may be ‘more numerous than those between E. histolytica and E. coli. It is too ‘early to classify the Laredo-type amocbae as a new species, however, for too few strains have as yet been isolated and too few laboratories have carried out comparative studies. 3.4.2. Entamoeba moshkovskii In various parts of the world Entamoeba moshkovskit has been isolated from material in sewage treatment plants and in streams receiving the ‘effluent of sewage plants. Despite its similarity in appearance to E. histo~ Iptica, it possesses 2 distinctive characteristics : it multiplies at tempera- tures from 10°C to 37°C, and it survives exposure to hypotonic solutions, forming contractile vacuoles. The species is therefore clearly distinct from any known strains of classical E. histolytica, but similar to the Laredo-type strains Attempts to infect laboratory animals with E, moshkovskii have proved ‘unsuccessful. ‘The similarities and dissimilarities found in antigen studies are consistent with the distribution of reactions among different strains of a single group. On the basis of known characteristics, therefore, there is24 AMEBIASIS less reason for separating £. moshkovskii from the Laredo group than for separating the latter from classical E. histolytica Finally, itis not certain whether E. moshkovskii has been isolated from the free-living state or indirectly from some animal host. ‘The amoeba ‘unquestionably belongs to the parasitic group of species, as is shown by the absence of nuclear DNA histone, which is present in free-living protozoa, but £. moshkovskii may be a parasitic species that is incidentally encountered in free-living environments, where it is able to live because of its wide temperature and tonicity tolerance. ‘The main reason for not at present designating E moshkovskii as a “ Laredo-type E. histolytica” is that too few strains have been studied in sulffcient detail. Moreover, the moshkovskit group may prove to be a conglomerate of parasitic forms of E. histolytica-like morphology, whose hosts may include species other than man, For the time being, therefore, moshkovskit-type E. histolytica” seems a suitable designation. 4. EPIDEMIOLOGY Study of the epidemiology of amoebiasis can be useful in determining (@ the relative public health importance of the disease, and (5) suitable methods of surveillance and,control. In both cases, attention must first be directed to the transmission of £. histolytica, for amoebiasis is defined (p. 7) as the condition of harbouring this parasite. 4.1 Transmission The parasite enters a new host by the oral route at the eyst stage. The trophozoite need not be considered in connexion with transmission, since it is not infectious and is too fragile to withstand external environmental conditions. For this reason patients with acute amoebiasis, who pass only trophozoites, do not play any part in transmission 4.1.1 The parasite Although the cyst is more resistant than the trophozoite to environ mental conditions, its resistance has limitations that influence the trans- mission and control of the parasite. The cyst of E. histolytica is killed by desiccation, and airborne dust from contaminated areas is not therefore considered to be infectious. The cyst is also killed readily by temperatures above 55°C, so that water can be made safe to drink by boiling it ‘Although chlorine can destroy the cysts, the amount normally used jin water purification is ineffective. The cyst seems more susceptible to iodine than to chlorine, but the amounts of iodine necessary to purify water may be distasteful to some users.REPORT OF A WHO EXPERT COMMITTEE 25 Although man is infected only through contaminated food, drink, and other objects placed in the mouth, the factors that bring about the con- tamination are often difficult to identify. Usually several factors are involved, but their relative importance may vary under different environ- mental and social conditions, so that the epidemiology of amoebiasis varies from place to place and it is not possible to make reliable generaliza- tions for entire countries or large areas. In most cases, multiple factors (eg, contaminated water, food handling, flies, direct faecal contact) are so interrelated that it is difficult or impossible to single out a primary factor. When single sources of infection have been identified, this has generally been in temperate areas where fewer factors are concerned in transmission, 4.1.2 Climate Although F. histolytica is generally more prevalent in the tropics and subtropies, the higher temperature and humidity are not thought to be directly responsible. Indeed, the higher temperatures are likely to have an adverse effect on cysts in the environment. ‘The increased prevalence in tropical countries js generally considered to reflect poor sanitation and health education rather than climatic conditions. Prevalence of E. histo- Iptica is associated with unsanitary conditions in cool and even in arctic regions. This should dispel any notion that amoebiasis is limited to the tropics. 4.1.3 Flies and evekroaches The common housefly and cockroach can retain cysts of £. histolytica in a viable condition within their bodies for several hours or days, and ‘may contaminate food by passing the cysts in their vomitus and excreta. Although arthropods could assist transmission in places where they are abundant and sanitation is poor, it has not been possible to demonstrate their role satisfactorily 4.1.4 Reservoir hosts E. histolytica infection can be established in a number of different host species and occurs naturally in dogs and rats, but primates are the most likely to be important reservoir hosts. Apes, gibbons, Old World and New World monkeys harbour amoebae indistinguishable from £, histo- Iytiea. As cysts occur regularly in the stools of infected primates, such animals in close association with man are a potential source of infection. 4.1.5 Night-soil and sewage irrigation In areas of the world where night-soil is used as fertilizer, vegetables and berries that are eaten raw have been strongly suspected of assisting26 AMOEBIASIS| in the transmission of E. histolytica. Unless night-soil is applied immediately before harvesting, however, it is probably not a significant factor in the spread of intestinal protozoa, although contamination may occur if it is the practice of the producer to “freshen” the vegetables in polluted streams. Vegetables from fields irrigated with polluted water may also be concerned in the epidemiology of amoebiasis, and this source of infection ‘may become more important with the increased pollution of streams and rivers throughout the world and the growing use of irrigation. 4.1.6 Drinking water In all reported outbreaks of amoebiasis, sewage-contaminated water supplies have been the major identified source of infection. When safe chlorinated water supplies become contaminated because defects in sanita- n and plumbing result in leakage or in sewage being siphoned back into the water lines, bacteria will probably be killed by the chlorine, but amoebic cysts will remain infective ‘A piped water supply that is properly installed and operated, therefore, will not transmit infection, but the provision of a safe water supply will not in itself stop transmission of amoebiasis. Water can become contami- nated in numerous ways after it has been drawn, and many people with access to a safe supply continue to use water from unsafe secondary sources. 4.1.7 Direct faecal contact Gross faecal contamination of premises promotes direct contact with faeces containing E. histolytica cysts. Mental institutions in particular, where personal hygiene and sanitation may be poor, frequently have a high rate of infection ; in all probability the infection is transmitted from person to person via contaminated surroundings. Furthermore, some religious practices may increase the frequency of faecal contamination of hands and water. 4.1.8 Food handling Food handlers are often suspected of being concerned in the transmis- sion of amoebiasis, but their role is variable or still uncertain. Cysts of E. histolytica remain viable for 5 minutes on the surfaces of the hands and for up to 45 minutes under the fingernails, so an infected food handler with poor personal hygiene might well convey viable cysts to sandwiches and other foods consumed without further processing. In relatively dry foods they may live for only a short time, but in liquid foods (¢.g., yoghourt, milk) they may survive for as long as 15 days at 4°C. Frozen packaged foods are unlikely to be sources of infection, for the eysts die after 24 hours at —10°C to —15°C.REPORT OF A WHO EXPERT COMMITTEE By 4.1.9 Family contact and crowding In many parts of the world, crowded conditions associated with poor sanitation and personal hygiene have been found to foster transmission ‘of E, histolytica and other intestinal protozoa. Studies have shown that certain families have an unusually high frequency of infection, and the impression has been given that once the infection is introduced into a family it is readily transmitted from person to person. However, most such reports come ftom surveys of rural arcas and the concentration of infec- tions in certein families is probably due to gross faecal contamination of the premises rather than to any person-to-person contact 4.1.10 Miscellaneous factors In addition to the matters already discussed, there are undoubtedly other factors that play a part in transmission. The role of drinking and feeding habits in transmission is essentially related to contaminated hands, food, and drink. It is reported from Nigeria that a high prevalence of amoebic dysentery in infants is associated with hand-feeding by their mothers. Age, sex, and race have frequently been considered in epidemiological studies, but most differences observed have been attributable 10 degrees of exposure and have not indicated a causal relationship with any of these factors. 4.2 Prevalence of infection For many areas of the world there are no recent data on the prevalence of E, histolytica infection, and the data for almost all areas are relatively unreliable, because of either inadequate sampling or faulty methodology Nevertheless, the published data show clearly that prevalence rates vary widely from place to place, and the comparatively low or high infection rates reported for certain areas are probably accurate. ‘The rates for the USA, Canada, and Europe are generally rmuch lower than those for other areas, particularly the Middle East, Mexico, Central and South America, and several African countries. There are wide and ‘unexplained differences among the countries of Asia and the Pacific region. In a few instances exceptionally high rates have been reported for a general population, as for example 57% and 83% for Egypt, 52% for Liberia, and 56% for Ecuador, but it is somewhat unusual for rates to exceed 30% even in communities with the poorest sanitation. Poor sanita- tion obviously favours transmission, but it cannot be taken for granted that communities with equally poor sanitation will have the same rates of infection. Conversely, in two communities that have equally good sanitation, with adequate water supplies, adequate refuse and sewage disposal, and effective control of the marketing of food products and the methods of preparing and serving foods. F. histolytica infection will not28 AMOEBIASIS. necessarily occur at the same rate. It is broadly true, however, that a high prevalence of infection with intestinal protozoa is an indication of frequent ‘transmission, and that frequent transmission is an indication of poor sanitation and education. 4.3 Morbidity and mortality In order to assess the importance of amoebiasis in any area, public health officials will need information on morbidity and mortality. Unfor- ‘tunately, although many sources of data are available, they are at present inadequate and of variable reliability. In many parts of the world amoe- biasis is not a notifiable disease. In some countries “ dysentery” must be reported, but no differentiation as to etiology is required. On the whole, the most useful information will probably come from the records of indus- trial and military hospitals and clinies, and from teaching hospitals. 4.3.1 Variations in prevalence Symptomatic amoebiasis is widely regarded as a “tropical disease”, and in developed countries is thought to be much less common than asymptomatic amoebiasis. However, there is evidence that the apparent ow incidence of symptomatic amoebiasis in certain areas may in part be due to the mildness of symptoms and the lack of laboratory expertise. “Amoebie infection is known to be highly prevalent in some areas where liver abscess is rare, and it is often pointed out that liver abscess may occur in persons who never experience amoebic colitis. In areas where amoebic colitis occurs frequently and in a severe form, amoebic liver abscess is generally also more frequent than usual. According to reported incidence data, however, the ratio of amoebic abscess to amoebic dysentery varies from place to place. ‘Undoubtedly the severity of amoebic colitis varies somewhat between different parts of the world and even between neighbouring districts, but many differences are probably more apparent than real. ‘The evidence is incomplete, for most of it is derived from hospital experience, and many factors determine the type of patient seen. In many areas, for example, only people who are seriously ill will visit a hospital. Such evidence as is available suggests that the general level of hygiene is more important in determining severity than factors such as diet. It seems likely that the frequency of reinfections and the size of the infecting dose are the deciding factors, 4.3.2 Factors influencing morbidity ‘The incidence of symptomatic amoebiasis varies widely from one part of the world to another. Some of the factors that determine whether infecREPORT OF A WHO EXPERT COMMITTEE 29 tion will produce disease in experimental animals have been identified, and are presumed to apply also to natural infections in man, but it has not yet been possible to explain satisfactorily why amoebic colitis or amoebic liver abscess is common in some areas and rare or unknown in others. Some strains of the parasite seem to be more pathogenic than others, Probably, too, there are physiological differences between strains Itis not unreasonable to suggest that differences in pathogenicity may be associated with certain biological features and with geographical factors. Similarly, it can be assumed that the occurrence and severity of the disease will be influenced by host factors, such as age, diet, nutritional state, immune state, previous or existing disease conditions that predispose the host to amoebic disease, and the presence or absence of other organisms ‘The importance of some of these factors is discussed below. (1) Virulence of the parasite. In some tropical and semitropical regions, infection with E, histolytica is more often associated with manifest disease than in the temperate zone. This has led to the belief that there are avirulent and virulent types of E. histolytica, a view supported by experiments with rats. inoculated with the amoeba-bacteria complex cultivated in. vitro. Usually, but not always, strains obtained in the temperate zone are avirulent, whereas those isolated in tropical areas are virulent. Close examination shows that the experimental virulence reflects the clinical status of the individual from whom the strain was isolated When virulent strains are maintained in vitro for many months, their Virulence becomes progressively lower, but can be restored by passage in the liver of hamsters or the caecum of rats. The failure of troops and other travellers to spread the disease when they move from tropical to ‘temperate zones provides circumstantial evidence that a change in virulence may also occur within the human host. Since the invasive properties of the amoeba-bacteria complex can change, the factors influencing these changes must be considered. The influence of bacteria has been extensively studied, but unequivocal evidence is stil lacking. Experiments with germ-free animals have not helped to clarify this influence. The presence or absence of virulence does not appear to be related to completion of the life cycle, for attempts to alter the pathogenicity of virulent and avirulent strains by inducing encystation have been incon- clusive. Reports indicate that pathogenicity in terms of intestinal infection can be altered experimentally in animals by changes in diet, by changes in the temperature at which the amocba-bacteria complex is maintained, and by administration of cholesterol to the host or to the culture medium on which the parasites are grown. Most of these findings have not been confirmed by clinical observations30 AMOEDIASIS| (2) Age of the host. In areas of high endemicity children who become infected show a high rate of symptomatic disease, particularly in the 1-4 years age group, when children are ambulant, difficult to control, and orally inquisitive. The high rate of symptomatic disease may also be related to the size of the infecting dose or to lack of immunity. Although there is serological evidence that antibodies are produced in response to tissue invasion by E. histolytica, it is not known whether these antibodies are protective, (3) Sex of the host. Hepatic amoebiasis shows no sex difference in children, but in adults it is mach more common in males than in females. Hormonal activity and the consumption of alcohol have been offered as possible explanations for this difference. 4.4 Onset of hepatic amoebiasis A distinctive clinical feature of amoebiasis is the long latent period that sometimes seems to occur between the date of original infection and the appearance of clinical symptoms of hepatic involvement. There are 2 possible explanations for this. One is that there is a true latent period, when amoebae lurk somewhere in the tissues without producing any clinical manifestations ; eventually some stimulus causes them to multiply in an area of the liver and produce an abscess. The other explanation is that an original infection induces a state of hypersensitivity in the patient, and a later reinfection produces hypersensitivity reactions in the liver that Tead to an abscess. 5, LABORATORY DIAGNOSIS The diagnosis of amoebiasis in a patient must be based upon both laboratory and clinical evidence. The laboratory can report on the char- acter of the stools and the presence of £. histolytica, and the physician Who has to determine whether the patient has clinical amoebiasis is partly dependent upon the laboratory findings for his diagnosis. Careful atten- tion must therefore be given to the effectiveness of the laboratory procedures and to the competence of the microscopist. Furthermore, close collabora- tion must be maintained between the parasitologist and the clinician. The laboratory procedures that can be carried out to assist with the diagnosis of amoebiasis are the examination of faeces and other materials for E. histolytica and the testing of serum for the presence of specific anti- bodies to E. histolytica. These procedures can be applied for individual patients and in epidemiological investigations.REPORT OF A WHO EXPERT COMMITTEE 31 5.1 Microscopic methods 5.1.1 Collection and preservation of specimens Preferably, faecal specimens should be examined immediately after passage. When this is not practicable, as in population surveys, the speci- ‘mens should be preserved to ensure the recovery of trophozoites and cysts. Formol (510%) can be used to preserve the cysts. Polyvinyl aleohol (PVA) fixative is recommended for the preservation of trophozoites intended for examination in the laboratory using permanently stained preparations. The physician will have to assume responsibility for ensuring that satisfactory specimens are available for examination in the laboratory. He should make certain that the patient does not receive barium, mineral oil, bismuth, kaolin, drugs, and other substances that might interfere with the examination or temporarily reduce the number of organisms in the specimen. In poptlation surveys, 1 stool specimen per person will have to suffice, since it is not practicable to collect more. In the examination of patients suspected of having contracted amoebiasis, however, many specimens are needed before this possibility can be ruled out. "Although specimens obtained by purgation will frequently deliver more organisms, they probably have little advantage’ over a series of normally passed specimens. Further ‘more, trophozoites in purged specimens are frequently difficult to identify. Material collected during sigmoidoscopy should be examined imme- diately for mobile trophozoites in temporary wet mounts, and smears should be prepared for permanent staining. Liver aspirates are difficult to examine, and when necessary should receive special treatment to free the amoebae from the coagulum. Amoebae are most likely to be found in the last of the aspirate, but it is often worth while to add the enzyme streptodornase-streptokinase to portions of the entire specimen in a dilution of 10 units per ml of pus. The mixture should be incubated for 30 minutes, being shaken repeatedly. 5.1.2 Methods of examination ‘The procedures available for the demonstration of E. histolytica in clinical materials are : (1) Temporary wet mounts. These can be made directly from stool specimens, sigmoidoscopie scrapings, and liver aspirates. Mounts prepared with physiological saline are examined for trophozoites and cysts. Those prepared with Dobell’s or Lugo!’s solutions are examined for stained cysts, and those prepared with buffered methylene blue solution are examined for stained trophozoites. Unless indisputable characteristics of F. histo- Iytica ace observed, however, identification by this method should be considered only as tentative.32 AMOEBIASIS (2) Concentration procedures. These are useful in revealing light infections if cysts are present, but as yet there is no satisfactory method. for concentrating trophozoites. Both the formol-ther (FE) sedimentation technique and the zinc sulfate flotation procedure are effective when per- formed accurately. The FE technique is less subject to technical errors, ‘and can be used with specimens preserved in formol. (3) Permanently stained faecal slides. Amoebae can be accurately identified with the aid of slides prepared from fresh faecal specimens or from specimens fixed in PVA. Besides helping to confirm the identity of organisms in doubtful cases, this method will reveal organisms not found by other techniques. Heidenhain’s iron haematoxylin staining procedure, using fresh spec- imens, gives the best results for critical work, but there are many shorter procedures that are satisfactory for diagnostic purposes (¢.g., a modification of Gomori’s trichrome method, the method of Mukherjea, Ray, and Chakraborty: and the chlorazol black method). The trichrome method hhas been extensively used and is satisfactory for specimens fixed in PVA as well as for unpreserved specimens. (4) Staining of amoebae in biopsy or autopsy material. Amocbae are not easily identified in routine sections stained by haematoxylin and eosin. A useful screening method is to fix sections in formol and stain them with PAS (periodic acid Schiff) reagent. The amoebae stand out as bright red bodies. ‘The exact morphology is rarely clear, but the presence of such bodies is sufficient to warrant careful scarch of sections treated with stains that are better suited to the demonstration of morphology (¢.g.. iron haematoxylin and trichrome). (8) Cultivation. If appropriate media and due care arc used, amoebae can be cultured from fresh stool specimens, but rarely from specimens that are several hours old. Specimens that cannot be cultured immediately should be refrigerated. At present there is no procedure that can guarantee the recovery of amoebae from stool specimens, and no medium that will support the growth of E. histolytica alone, so when growth occurs it is still necessary to make the identification on the basis of morphology. Permanently stained slides prepared from culture sediments fixed in PVA. will assist with examination, Of the several media available, the modified Boeck & Drbohlav medium and Balamuth’s medium are frequently used. With most media itis advisable to add small quantities of penicillin, strepto- mycin, or acriflavine prior to inoculation in order to suppress bacterial multiplication in the primary culture. * Mukherjea, A. K., Ray, H. N, & Chakraborty, B. M. (1987) Bull. Calewta Sch. trop. Meds, 5, 84REPORT OF A WHO EXPERT COMMITTEE 3 5.1.3 Standard stool examination for E. histolytica In order to obtain more reliable information on the prevalence of E. histolytica in various parts of the world, it is important to standardize the laboratory procedures that are used in hospitals and clinics and, more particularly, for population surveys. Recognizing that the facilities and time available will vary from one laboratory to another, the Committee suggests the following procedures, Hospitals and clinies () Collect a series of normally passed specimens. Examine for con- sistency, colour, abnormal elements (e.g., blood, mucus), and the presence of E. histolytica (wophozoite and cysts). If no organisms are found, sigmoidoscopy or even catharsis may be indicated in order to demonstrate the etiological agent. 2) If prompt delivery of stool specimens to the laboratory cannot be assured, mix a portion of each specimen in PVA to preserve any tropho- zoites. It may also be desirable to preserve a portion in formol to stain the chromotoidal bodies in the cyst. In either case, the untreated portion should be delivered to the laboratory so that its character can be noted. (3) Examine several temporary wet mounts from each specimen. The saline mount is very useful, and in appropriate cases the temporary stains are helpful for observing the internal structures, (4) Concentrate the specimens that are likely to contain cysts (formed ‘and soft. specimens), (5) Prepare permanently stained slides from the unpreserved or PVA- fixed portions of (a) all £. histolyrica-positive specimens, for a permanent record, (6) soft or diarrhoeic specimens, which are likely to contain tropho- zoites, and (c) any specimens that reveal unidentifiable organisms by the above techniques. Surveys (2) Collect 1 specimen from individuals selected at random, recording age, sex, and information from which each person can subsequently be identified. 2) Examine each specimen grossly, and record its consistency, colour, and the presence of abnormal elements (e.g., blood and mucus) @) Preserve portions of each specimen in formol and PVA fixative. (@) Perform the FE concentration technique on portions in formol and ‘examine them in unstained and in iodine-stained wet mounts, (3) Prepare permanently stained slides from PVA-fixed portions of (@) specimens positive for protozoa on concentration, (4) specimens con-34 AMOEBIASIS taining blood andjor mucus, and (c) soft and loose specimens, which are likely to contain trophozoites. 5.1.4 Criteria for the identification of E. histolytica Microscopists who assist in the diagnosis of amoebiasis must be able to find and identity all species of intestinal protozoa that man may harbour. Furthermore, they must be able to differentiate between amoebae and various artefacts that may be present. Since size is an important factor in identification, the microscope should be equipped with a calibrated ocular micrometer. It should also have a low-power objective for searching wet mounts, a highpower, dry objective for more careful examination of organisms encountered, and an oii sion objective for examining permanently stained films and, occasionally, wet mounts (sealed with petroleum jelly). It is rarely possible to identify E. histolytica from the observation of a single organism or a single charac- teristic. In most instances the identification can be made if one of the following characteristies is observed in several organisms. Wet mount preparations :* () Trophozoites with directional locomotion, containing red blood cells; 2) Trophozoites exceeding 12 yin diameter, with directional locomo- tion and relatively clear eytoplasm ; (3) Trophozoites exceeding 12 in diameter, with relatively clear cytoplasm. Buffered methylene blue stain reveals nuclei that have evenly distributed peripheral chromatin granules and small centrally placed. karyosomes 5 (4) Cysts exceeding 10 y1 in diameter, with 1-4 Entamoeba-type nuclei (iodine stain) and distinct rod-shaped chromatoidal bodies ; (9) Cysts exceeding 10 jx in diameter, most of them containing 4 Entamoebactype nuclei (iodine stain). Permanently stained sides : (1) Trophozoites containing red blood cells and with Entamoeba-type nuclei ; (2) Trophozoites over 10 jin diameter, with relatively clear cytoplasm and with nuclei that have evenly distributed peripheral chromatin granules and small centrally placed karyosomes ; 2 the findings are positive, permanently stised sides should be prepared whenever practicable to provide confirmation and a permanent record.REPORT OF A WHO EXPERT COMMITTEE 35 G) Cysts over 10 pin diameter, with 1-4 Enamoeba-type nuclei and istinct rod-shaped chromatoidal bodies ; (4)" Cysts over 10 yc in diameter, most of them containing 4 Entamoeba~ type nuclei 5.1.5 Reporting of laboratory results ‘To assist him in reaching his diagnosis, and to supplement the informa- ‘tion obtained from the patient's case history and from physical examination, ‘the physician requires a detailed laboratory report. Statements such as “amoebae found”, “cysts found”, or ‘amoebic trophozoites found ™ are of little value and may lead to etroneous interpretations. The labora tory report should contain the following information : (0) Character of the specimen—consistency (¢.g., formed, soft, loose, watery), colour, and abnormal elements (¢.g., blood and mucus) ) Genus and species of parasite found (c.g., E. histolytica, E. cali, ete.) (3) Stages of the amoeba (e.g., trophozoites, cysts) (4) Distinctive cytoplasmic inclusions (¢.g., red blood cells in the trophozoites, rod-shaped chromatoidal bodies in the cysts) (3) Character of the cellular exudate (e.g., pus cells, pyknotic bodies, ring nuclei, etc.) Before the examination of a specimen is undertaken, notes should be ‘made on the specimen for the laboratory records and for use in the report. The information in point (1) is required so that the significance of the findings can be better assessed. It is important that close collaboration bbe maintained between the clinician and parasitologist. 5.1.6 Interpretation of laboratory findings The following presumptive interpretations can be made from data in the laboratory report : Findings Interpretation Dysenterie stool (gross blood and mucus); Probably acute amoebic dysentery hhaematophagous £- histolytica; very lite cellar enudate Active tissue pathology (acute amoebic “dysentery if history or other observations contirm passage of dysenteric stools) Stools mre of less diarshovie, containing quantity of mucus, with or ‘without geass blood36 AMOEBIASIS Findings Incerpretation (@) Haematophagous E. Hirolyica pre- Amoebiasis, probably with haemorrhagic sent uleration () Non-hacmatophagous E, histolytica Amoebiasis, possibly without significant present invasion of mucoss (© B. histolytica cysts present, but no Colony of trophozoites in upper part of trophozoites ‘colon, possibly no significant tissue in- volvement Formed or semi-formed stools with E.his- Colony of trophozoites in upper part of ‘olbtica cysts, But not necessarily with colon, possibly no sigifcant. tisue ‘trophozoites involvement It is not satisfactory to regard the presence of E. histolytica cysts as clinically unimportant. If there are cysts in the stool, there are trophozoites somewhere in the intestine. These may produce deep local lesions extension to the liver, without giving rise to noticeable intestinal symptoms. Similarly, if E. histolytica trophozoites are found in a dysenteric patient, they should not be accepted without question as the etiological agent, since a concomitant Shigella or Salmonella infection. may be responsible for the dysenteric symptoms. Attention must therefore be given, particularly in areas with a high prevalence of asymptomatic amoebiasis, to the character of the associated cellular exudate. If a typical “ bacillary exudate” is present, the physician should be cautious about concluding that there is a causative relationship between the disease and the amoebae 5.1.7 Competence of the diagnostic laboratory Any laboratory procedure employed for the demonstration of amoebae is only as reliable as the microscopist who uses it. No one should attempt to identify intestinal amoebae independently before undergoing intensive training and working for some time under supervision. Programmed instruction in the laboratory diagnosis of amoebiasis has been developed as an aid in the training of students who wish to specialize in this area of laboratory work.! In itself, no academic degree or certificate should be taken as proof of competence. Heads of laboratories. should establish adequate quality control to ensure that the procedures are properly carried. out, provide their technicians with opportunities for refresher training, and institute_proficiency testing programmes so that they have an objective check on the competence of their laboratories 5.2 Immunodiagnostic methods It is becoming increasingly evident that antibodies are produced in invasive amoebiasis. This realization, coinciding with the advent of better 1 US Department of Health, Education, and Welfare (1964) Amoebiasis : laboratory diagnosis, Washington D.C., US Government Printing Ofice (Public Health Service Publication No. 1187)REPORT OF A WHO EXPERT COMMITTEE 37 antigens and advances in serological methods, has led to widespread interest in the use of immunodiagnostic techniques in amoebiasis. These methods appear to be highly specific for amoebic antibodies, but vary in sensitivity. Since antibodies may persist for an ill-defined period after termination of infection, a positive serological result is not in itself an adequate basis for the diagnosis of active amocbic infection. However, failure to detect antibodies by the more sensitive methods helps the physician to rule out appreciable tissue invasion. Serological procedures are reliable aids to the diagnosis of amoebic disease. In recent years their potential value in the assessment of tissue- invasive amoebiasis in population groups has been demonstrated. Because the tests seem to permit objective interpretation of the relative importance of amoebiasis in population groups, their use in epidemiological studies ig recommended. 5.2.1 Gebsiffusion precipitin test A. micro-adaptation of the Ouchterlony double-diffusion technique using agar gel is likely to prove a good test for routine use. It has the advantages of being simple, reproducible, economical in material, and suitable for use with haemolysed or sometimes even with contaminated sera. The method has been fully evaluated in areas where amoebiasis is endemic ; experienced workers have found it adequately sensitive for amoebic liver abscess and amoebic dysentery. Positive results usually appear within 8 hours, but occasionally take longer, so a final reading is made 48 hours after setting up the test. In a positive reaction, the number of bands formed depends upon the purity of the antigen used. 5.2.2 Indirect haemagglutination test This method is highly sensitive in the hands of skilled workers, but technical difficulties are frequently encountered. Owing to modifications in the preparation of the reagents used by different workers, the titres observed vary, and there is no general agreement regarding the precise titre that indicates « positive reaction 5.2.3 Complement-fixation rest This is one of the oldest procedures for the serological diagnosis of amoebiasis, but is being superseded by more modern methods, 5.2.4 Fluorescent antibody tests ‘These procedures appear to be highly sensitive, but they require further evaluation in endemic areas, They have the advantage that the nevessary38 AMOEBIASIS| antigen can be prepared easily from ordinary cultures of amoeba, but they require special equipment, and considerable experience is needed for inter- pretation, A. soluble-antigen fluorescent antibody (SAFA) test has been used recently in amoebiasis, with promising results. This test is read with a fluorimeter and interpretation is less subjective than’ with other ffuorescent antibody procedures. 5.2.5 Other methods Procedures based upon the immobilization of living amoebae or the phagocytosis of erythrocytes by amoebae are not widely used. Immuno-electrophoretic techniques are useful in research, but less suitable for routine diagnostic use. 5.2.6 Preparation of standard antigen Much work has been done during the past few years on the preparation of antigens from E. histolytica grown in monoxenic or other simplified cultures. These efforts have shown that it is feasible to develop useful immunodiagnostic procedures for amoebiasis, but the complexity of the antigen preparations has made standardization difficult. The recent development of methods for the axenic cultivation of E. histolytica has provided the basis for the preparation of standard antigens. Substantial amounts of such antigens have been prepared from 2 strains of E. histolytica, and have satisfactory stability in lyophilized form. Both antigens have proved their usefulness in a variety of serological tests (indirect haemagglutination, agar-gel, complement-fixation, and SAFA) in several geographical areas. These standard antigens represent the entire lysate recovered from washed amoebae. ‘The results of exploratory efforts to produce antigens with a wider range of application by means of fractionation procedures hhave not been particularly promising, although they have suggested that certain fractions may be more suitable for use in indirect haemagglutina- tion tests. 6. THERAPY Although there have been reports of unusual and resistant forms of amoebiasis, there is no evidence of natural or acquired resistance to amoe- bicides in E. histolytica. Most therapeutic failures are the result of incorrect diagnosis, unsuitable choice of drug, or failure to observe certain principles of treatment. The following sections are concerned principally with specific aspects of drug therapy. However supporting measures, such as attentionREPORT OF A WHO EXPERT COMMITTEE 9 to environmental factors, diet, correction of malnutrition, and health edu- cation, should not be neglected in clinical management 6.1 Present drug situation ‘The aim of chemotherapy is to rid the patient of infection with E. histolytica, which may be present in the bowel lumen, in the bowel wall, or systemically (chiefly inthe liver). All amocbicides used until recently varied in efficacy at the 3 sites where the parasites commonly exist; consequently, it was Frequently neces- sary to use combinations of drugs for adequate therapy. The development of certain new drugs that are highly active at all sites is simplifying treatment and changing the belief that complete cure of most forms of amoebiasis requires several drugs and possibly repeated courses of treatment. The commonly used drugs may be classified as follows. 6.1.1 Direct-acting amoebicides that act principally in the bowel lumen There are many such preparations, c-2(@) quinoline derivatives (diiodo- hydroxyquinoline, iodochlorhydroxyquin, chlorhydroxyquin, and chinio- fon) ; (6) arsenical derivatives (acetarsol, carbarsone, thioarsenites, glyco- biarsol, and diphetarsone) ; and (c) miscellaneous drugs (diloxanide furoate, chlorbetamide, chlorophenoxamide, 5,6-quinone-4,7-phenanthroline, glau- carubin, dichloracetylethylaminoethylbenzene, and paromomycin). 6.1.2 Indirect-acting amoebicides that act in the bowel lumen and wall but not in the liver This group comprises antibiotics, of which there are many that have some ameobicidal activity. By far the most effective are tetracycline, chlortetracyeline, and oxytetracycline of which the usual dosage is 250 mg every 6 hours for 10 days. 6.1.3 Tissue amoebicides that act principally in the bowel wall and liver The principal members of this group are emetine hydrochloride, emetine and bismuth iodide, and dehydroemetine. (1) Emetine hydrochloride. Emetine hydrochloride is administered at a dosage of 1 mg per kg of body weight daily (not exceeding 60 mg per day) for 10 days. Owing to its effect on the myocardium, it is contraindicated when heart disease is present, and it should be administered with care to the elderly and those who are underweight Q) Emetine and bismuth iodide. Emetine and bismuth iodide is admin- istered at a dosage of 195 mg daily (given at night) for 10 days. Concurrent administration of phenobarbital, to allay nausea and vomiting, is advisable Emetine and bismuth iodide frequently causes diarrhoea.40. AMOEBIASIS (3) Dekydroemetine. Dehydroemetine, which was synthesized in 1959, is excreted more rapidly than is emetine, and has been shown experimentally to be concentrated more in the liver and less in the heart than is emetine However, the activity of the two drugs against £, histolytica is similar in vitro, Although dehydroemetine is less toxic than emetine, the same pre cautions and contraindications should be observed in its use. The usual dosage is 1.5 mg per kg of body weight daily for 10 days. When administered parenterally, emetine and dehydroemetine are excel- lent tissue amoebicides. However, if they are used alone to treat amoebic dysentery, there is a high relapse rate, owing to their failure to eradicate amoebae in the bowel lumen. 6.1.4 Tissue amoebicides that act prineipally in the liver ‘The most important member of this group is chloroquine, of which the dosage is 600 mg of the base statin, a further 300 mg after 6 hours, and then 150 mg 2-3 times daily for 28 days. Chloroquine may cause nausea ‘and vomiting ; less commonly, skin eruptions, headache, and blurring of vision 6.1.5 Amoebicides that are effective at all sites Two recently developed compounds, niridazole and metronidazole, are ‘unique in that they are effective against all forms of amoebiasis. Niridazole, or 1-(5-nitro-2-thiazolyl)-2-imidazolidinone, has considerable activity against amoebae at all sites when administered at a dosage of 25 mg per kg of body weight daily for 7-10 days. However, its toxic effects include gastrointestinal disorders, psychoses, and epileptiform fits. It may also ‘cause electrocardiographic changes, although these are of doubtful signifi- ‘cance. Metronidazole, or 2-methyl-S-nitroimidazole-1-ethanol, has been widely used with safety since 1959 for the treatment of urogenital trichomoniasis. More recently, it has been used to treat giardiasis and acute ulcerative gingivitis. In the treatment of amoebiasis, itis unique in that it has powerful intestinal and systemic amoebicidal activity, is well tolerated, and does not produce serious toxic effects. For the treatment of intestinal amoebiasis, it is administered at a dosage of 800 mg 3 times daily for 5 days. For the treatment of amoebic liver abscess, it may be administered either in a single dose of 2.4 g, or in doses of 400 mg 3 times daily for 5 days. Few reports of its efficacy in the treatment of asymptomatic intestinal amoebiasis have been published. 6.2. Drugs in practical use In the following sections, methods of therapy for the different clinical forms of amoebiasis are describedREPORT OF A WHO EXPERT COMMITTEE 41 6.2.1 Asymptomatic intestinal amoebiasis When E. histolytica is confined to the bowel lumen, cure may be achieved by the use of “luminal " ot contact amoebicides. However, of the many such drugs that are available, none is completely reliable in eradicating amoebae. Few have been thoroughly evaluated and, as there is probably little to choose among them, they may be selected on the basis of satisfac- tory tolerance, lack of toxicity, and expense. It should be pointed out that if significant tissue invasion has occured, contact amocbicides do not provide adequate treatment, although they may be of value for adjuvant therapy ‘Amocbicides that are active in the bowel lumen in addition to other sites are also effective in the treatment of asymptomatic intestinal amoe- biasis. Such drugs include the tetracyclines, oral emetine preparations, and possibly metronidazole. 6.2.2. Nondysenteric colitis Misleading claims readily arise with respect to nondysenteric colitis, since the borderlines between asymptomatic intestinal amoebiasis and amoe- bic dysentery are obscure. If the amoebae are situated predominantly in the bowel lumen, tissue amoebicides alone are unsatisfactory and mild ‘cases, with minimal or no tissue invasion, respond to luminal amoebicides. However, nondysenteric colitis should as’a general rule, be treated in the same Way as amoebic dysentery. 6.2.3. Amoebie dysentery The treatment of amoebic dysentery should, in addition to eradicating amoebae in the bowel lumen and bowel wall, protect the liver from invasion. Several combinations of drugs yield cure rates exceeding 95%. The fol- owing are recommended : (a) For mild to moderate cases (e.g., ambulant patients), a combination ‘of tetracycline, a luminal amoebicide, and chloroquine provides a safe, nontoxic, well-olerated, and effective treatment. Ifthe etiology is in doubt, the tetracyclines have the useful advantage of efficacy against bacillary dysentery. (B) For severe cases with much tissue invasion, emetine hydrochloride ‘or dehydroemetine together with tetracycline and a luminal amocbicide may be used. In such patients the rapid and potent tissue amoebicidal properties of emetine preparations may be lifesaving. Furthermore, it is sometimes impossible to administer drugs orally, and these drugs remain. the best for parenteral use. When they are used for the treatment of amoebie dysentery, the inclusion of chloroquine is unnecessary (©) The administration of emetine hydrochloride and, subsequently. ‘of emetine and bismuth iodide is a long-established form of treatment for amoebic dysentery. Tt is effective but, unless carefully administered, itis2 AMOEBIASIS| liable to cause nausea and vomiting. Furthermore, it regularly causes diarrhoea or exacerbates dysentery. (@) Metronidazole is safe, well-tolerated, and highly effective. It is the only single, nontoxic drug that is effective at all sites and is the treatment of choice in most instances. 6.2.4 Relapse in intestinal amoebiasis If relapse occurs, the patient may be treated with one, or a combination, of the alternative drugs that are available. If circumstances permit, the source of reinfection should be sought whenever an adequately treated patient suffers a relapse ; it is frequently another member of the patient's household who has an asymptomatic infection. 6.2.5 Hepatic amoebiasis Although relatively few drugs are effective against hepatic amoebiasis, ‘cure rates approximating 100% can be achieved. It must be assumed that intestinal infection is also present in all patients, who should be treated accordingly. Aspiration is not required in all cases, but adequate drainage is essential in the management of large abscesses. Although chloroquine is inferior to others drugs recommended for the treatment of hepatic amoebiasis, its use in combination with emetine pre- parations renders a second course of the latter unnecessary. Initial loading doses of chloroquine should be given to attain an adequate concentration in the liver. Metronidazole is highly effective against hepatic amoebiasis and, if given in sufficient dosage, will also eradicate the parasite from the bowel. For the treatment of hepatic amoebiasis, the Committee recommends the use of either (a) metronidazole or (b) a combination of either emetine hydrochloride or dehydroemetine with chloroquine and a luminal amoe- bicide. If the patient also suffers from dysentery, the treatment should be modified as follows : (a) if metronidazole is used, the daily dosage should bbe increased, and (6) if the alternative regimen is used, tetracycline should be added. 6.3 Clinical trials and assessment of cures 6.3.1 General principles * It is recommended that clinical trials of new drugs be undertaken in as many different areas as possible, since variations in tolerance and possibly response may occur. * Information on the conduct of drug trials will be found in Wid Hlth Org. techn Rep. Ser 1966, Nos. 317, 341 and 1968, No. 403REPORT OF A WHO EXPERT COMMITTEE B ‘The type of amoebiasis studied must be clearly defined. If the studies include trials on two or more forms of the disease, the different groups ‘ust be carefully distinguished. The diagnostic criteria that are used should be fully described and, if possible, should include serological confirm: ‘The dosage and method of administration of the drug should be stated, and any other medication received by the patients that may have affected the outcome should be described. Patients should have uncomplicated amoebiasis without other concom- itant disease. If possible, trials involving out-patients and other ambulant patients should be avoided. The number of patients necessary to provide significant results should be determined by sequential analysis. Criteria of eure and failure must be defined. Adequate post-treatment and follow-up studies are essential, In assessing long-term results, the possibilities of both reinfection and relapse should be considered. 6.3.2 Asymptomatic and non-dysenteric amoebie colitis ‘The diagnosis of this condition is commonly based solely on the identi- fication of eysts of E. histolytica, which must be made by skilled workers, Faeces should be examined by the methods outlined in seetion 5.1.3 Many amoebicides tend only to suppress the passage of cysts, which reappear shortly after the completion of treatment, Furthermore, cysts are often passed intermittently, although they usually reappear within 6 weeks of the cessation of treatment. Ideally, stool specimens should be examined daily for 3 weeks after the completion of treatment, and follow-up specimens should be examined after ! month. 2 months, and 3 months. 6.3.3 Amoebic dysentery 6.3.3.1 Selection of subjects. Only patients in hospital with active dysentery should be studied, Sigmoidoscopy should reveal definite rectal ulceration. Prior to treatment, haematophagous trophozoites should be demonstrable in the stools and ulcer scrapings, and a stool culture should indicate the absence of pathogenic bacteria. The patients should not have received amocbicides, previously 6.3.3.2 Conduct of treatment. All treatment should be administered under the supervision of a clinician by a trained member of the nursing staff, who must ensure that the drugs are taken by the patient. ‘The patient must be visited daily by the clinician who, in addition to observing general progress, should note signs of intolerance or toxicity. Daily stool examinations should be made by direct saline smears and by a concentration technique. When the results are doubtful, staining procedures should be used. Sigmoidoscopy should be performed before treatment and at S-day intervals. Healing of ulcers and mucosal changes should be noted. If“4 AMOEBIASIS active open ulceration is present, scrapings should be taken and examined by direct smear ; staining procedures should also be used if necessary. 6.3.3.3 Assessment of results and follow-up. Iis desirable that patients should remain in hospital for 15-25 days after the completion of treatment. ‘At the time of discharge, an initial assessment of results should be made, fon the basis of the following criteria (@) Failure: persistence -or recurrence of cither trophozoites or cysts. of E. histolytica after completion of therapy. (®) Probable failure : persistent rectal ulceration, despite the disappear- ance of E, histolytica (©) Cure : disappearance of symptoms and of E. histolytica, and healing. of ulcers. When a failure occurs, the patient should be given other, effective treat ment. Patients who are apparently cured should be requested to return I month after discharge, and at once should symptoms recur. The follow-up examination after 1 month should include sigmoidoscopic and stool exam- ination, and the results of the trial should be reassessed. If possible, further follow-up examinations should be made 2 and 3 months after discharge. However, in endemic areas it becomes progressively more difficult to dis tinguish between relapse and reinfection as the length of time after discharge increases. Trials at regular intervals with a standard reference drug are advisable, to ensure that neither the disease nor the criteria has changed ; emetine | hydrochloride has been found useful for this purpose. Control groups receiving placebos only should not be used in drug trials involving patients with invasive amoebiasis, 6.3.4 Amoebie liver abscess 6.3.4.1 Selection of subjects. Patients must have uncomplicated amoe- bic liver abscess and should not have recently received amoebicides. No co-existing disease should be present. The diagnosis must be proved by the aspiration of characteristic pus, which should be bacteriologically sterile. Ideally, £. histolytica should be isolated from the pus. 6.3.4.2 Conduct of treatment, Thorough aspiration, which may have to be repeated, should be viewed as part of the treatment. Stool examina- tions must be carried out. Intestinal amoebiasis must be presumed to be present in all cases and, if the drug under trial is believed to be effective only against hepatic amocbiasis, additional treatment should be given with a luminal amoebicide. Procedures of drug administration and manage ment similar to those described under amoebic dysentery are advisable.REPORT OF A WHO EXPERT COMMITTEE 4s 6.3.4.3 Assessment of results and follow-up. The criteria of failure or ‘cure are as follows : (@) Failure: persistence or recurrence of symptoms or signs after the completion of therapy. Complete proof of failure is obtained by the aspira- tion of pus containing E. histolytica. (®) Cure: disappearance of symptoms. It should be noted that eleva- tion of the diaphragm may be observed radiographically for months after cure. Although the liver usually rapidly regresses in size in response to aspiration and therapy, it is not uncommon for a small, non-tender lump to persist for some weeks at the site of aspiration, particularly if the abscess was situated in the epigastrium. For many years the return of the erythro- cyte sedimentation rate to normal Ievels has been used as an indication of healing of a liver abscess. ‘Most patients can be discharged on the 20th day. Follow-up examina- tions should be made 1 month, 3 months, and 6 months after discharge: ‘The vast majority of relapses or recurrences occurs within the first 6 weeks. 6.4. Mass treatment and chemoprophylaxis Since man himselt is the principal reservoir of E. histolytica, co-ordinated mass treatment repeated at intervals of a few months should be wseful in reducing the endemicity of amoebiasis in a given population group. If the problem of drug administration can be solved satisfactorily should be possible to carry out such mass treatment of intestinal amoebi by the use of one of the available chemotherapeutic agents of low toxi ‘The drug used should be one of the direct-acting intestinal amoebicides or possibly metronidazole. Cases of active amoebic disease should, of course, be treated individually. It has been shown that single doses of paromo- mycin and metronidazole have considerable efficacy ; if further evaluation demonstrates the effectiveness of intermittent single-dose treatment, the otherwise substantial problem of drug administration may be greatly sim- plified. Studies have shown the prevalence of amoebiasis to be greatly reduced by the administration of drugs such as diiodohydroxyquinoline, iodohy- droxyquinoline, and glycobiarsol, suggesting that the chemoprophylaxis of amoebiasis is feasible. Relatively low daily doses of glycobiarsol were sufficient to afford protection against experimental exposure. Good results Ihave recently been achieved by the twice-weekly administration of clefamide to a rural population in Mexico. Such results indicate that the amount of 4 drug required for prophylaxis against natural exposure may be consider- ably less than that recommended for the treatment of established infections. ‘The use of drugs consisting of a mixture of an amoebicide and an46 AMOEBIASIS| antibiotic for the prevention of both amoebiasis and bacterial infections is deprecated, since it is likely to result in the emergence of resistant strains of bacteria, 7. CONTROL, Of the many factors that influence the morbidity and mortality rates for amoebic colitis, amoebic liver abscess and other forms of amoebiasis, the most important are the frequency and intensity of transmission. In fact, transmission is the only factor that is sufficiently well understood in all its aspects to be considered in a control effort at either individual or com- munity level. No other measure, such as immunization or the provision of an adequate and well-balanced diet, is known to be effective, Since there is no fundamental difference in transmission between amoe- biasis and other filth-borne enteric infections, control measures that are effective for enteric bacterial infections are also effective for amoebiasis, 1, Individual measures There ate few preventive measures that can be used effectively by the individual. Primarily, the prevention of enteric infection by an individual oor a group of persons residing briefly in an endemic area is a matter of selecting the foods and fluids that are least likely to be contaminated. To a large extent the same procedure should be followed by the family residing in the area for several months to a year, or even longer. When food is being prepared for such groups, itis possible to institute hygienic kitchen practices that will minimize the risk of exposure. Measures to prevent contamination of cooked foods, the disinfection of uncooked fruits and vegetables, and the provision of a safe and adequate supply of water (¢.g., by boiling or chemical disinfection), can be carried out to whatever extent seems feasible or necessary. Laboratory studies indicate that, as a prophylactic measure for the individual family or closed population (e.g., club, mess-hall), uncooked fruits and vegetables can be disinfected with’ aqueous solutions of iodine (around 200 ppm) or acetic acid (5-10% or full-strength vinegar). No results of practical trials to investigate the disinfection of food with these chemicals have been reported. In most instances, thorough washing with detergents will remove amoebic cysts from fruits and vegetables Except in special circumstances, routine examination of food handlers is not a practical method of control ; only a small proportion of those infected will be identified and a high rate of reinfection is likely. Intermittent treatment of all food handlers may be justified in highly endemic areas. Anti-amoebic drugs given periodically in curative dosages, or daily in doses smaller than those recommended for routine treatment (approximately one-REPORT OF A WHO EXPERT COMMITTEE a half of less), have been reported to be effective, This method does not appear to have been widely used, and is not recommended except for special conditions of very high risk 7.2 Community measures In communities with poor sanitation, amoebiasis and other enteric ‘microbial infections may constitute the major cause of morbidity. In some communities amoebic liver abscess is a major cause of death among children and adult males, From the economic point of view the medical management of the disease represents a high expense that can be only roughly estimated ‘when the cost of hospitalization, examination and treatment is taken into account, together with the loss of productivity. Patients with hepatic amoebiasis may be required to remain in hospital for long periods. Con- sequently, although the importance of the disease varies greatly from place to place, amoebiasis is likely to be among the major health problems in a developing community in the tropics. ‘The means of control available to a community fall roughly under the following headings : public services and utilities, sanitary regulations, and health education. Usually control is made the responsibility of a local government public health department and administered at a higher level. In a community without an effective local health organization, a control programme is unlikely to be effective. 7.2.1 Publie services and utilities The minimum services for the health of an urban community are a safe and adequate water supply and some form of sanitary disposal of sewage wastes and refuse, A water system that provides standard chlorina- tion and piped delivery will not ordinarily be safe as regards amocbiasis without effective filtration and professionally planned plumbing, since the water ean carry amoebic cysts when no viable enteric bacteria are present. Hyperchlorination is required to kill amoebic eysts, and this is unacceptable fon 2 counts : high cost and objectionable flavour. Filtration is therefore essential whenever chlorination is required. Sewage disposal systems can be satisfactory and yet range from the crude to the highly refined and complicated, but no system will serve its purpose fully unless the population is properly prepared by health education ‘The requirements for the prevention of amocbic infection are the same as for other types of enteric microbial infection. Utilization of night-soil may provide a means of transmission in special circumstances, but not to the same extent as for the soil-transmitted helminths. One of the services that can be provided by the community to prevent amoebiasis is fly control, although this is to a large extent accomplished48 AMOEBIASIS through regulations. The organized removal of organic refuse from the community will assist in the control of fly breeding. Flies are important mechanical vectors of infecting organisms in faeces. 7.2.2. Health regulations When a community is prepared to accept strict regulations regarding food vendors, food handlers, and the facilities for the preparation and dis- pensing of food, it generally will already have brought the transmission of filth-borne infections to a low rate, A ban on the keeping of livestock within the immediate area of a city will further reduce transmission by removing breeding sites for flies. Control of cockroaches and other vermin may also reduce the transmission of amoebiasis, and is desirable in any case. Regulations for slum clearance and the building of new houses should include provisions regarding proper toilet facilities and water supply 1.2.3 Health education People should be taught about the relations between sanitary conditions and amoebiasis. The importance of practising good personal hygiene should be stressed, and all members of the health team should be trained to demonstrate the proper way of washing hands, cleaning and protecting ‘vegetables and fruit, and controlling insects. ‘These educational activities can be illustrated by audiovisual materials The primary school can play an important role in training children in the prevention of diseases Hospitals, out-patient services and health centres should also participate in educational activities. Administrators, together with medical, paramedi- cal and auxiliary personnel, should be trained to provide patients and their families with information on health practice. 8 RESEARCH SUGGESTIONS 8.1 The amocbae of man and animals (@) Cytochemical studies should be carried out in order to explain the function of various subcellular organelles observed by electron microscopy ; in particular, a study should be made of the localization of membrane and cytoplasmic enzymes in relation to invasiveness, (b) Efforts should be continued to develop a defined medium for axenic culture of E. histolytica (©) Biophysical, biochemical and physiological studies should be made in order to elucidate the basis of encystment under both in vitro and in vivo conditions.REPORT OF A WHO EXPERT COMMITTEP 49 (@) Additional biochemical and immunological studies should be per- formed with E. histolytica and E. histotyticasiike strains in order to establish their metabolic and other biological differences. 8.2. Epidemiology ‘The following investigations are particularly needed : (@) Study of the amoebae of primates and other mammals ”closely associated with man, comparing their morphology and pathogenicity with that of E, histolytica, in order to determine whether these animals are reservoir hosts in amoebiasis. @)_A survey of intestinal protozoa in different parts of the world, carried out in such a way as to provide reliable comparative data for areas representative of different types of community with different types of climate. (©) The collection of reliable information from different geographical areas on the frequency of asymptomatic and symptomatic intestinal infee- tions in relation to the frequency of liver abscess. The data should include records of age, sex, and race (d) Surveys for antibodies of F. histolytica, using standard procedures and reagents, to be conducted in different parts of the world in an effort to ‘obtain information on the relative importance of amoebiasis in different localities. Wherever possible, such investigations should be combined with examinations for the etiological agent and with clinical studies (see (4) and (c) above). (©) Study of the effects of change in environment on the prevalence and natural history of the disease by means of a continuous survey of populations migrating from a highly endemic area to an area of low endem- icity. This study could be associated with the testing of amoebicides and anthelminthies. (Cf) Further experimental work in man and animals to study the factors invoived in the production of lesions and the prevention of invasion. In particular, this work should include studies on the effects of sensitization and immdnity on host-parasite relationships. (g) Attempts to isolate additional strains of E. histolsicalike amocbae from as many geographical areas as possible (up to now these strains have been found only in the USA) and 10 test their virulence as soon as prac- ticable (i) A histochemical study of E. histofvica, especially in relation to ‘the tissue of the host.50 AMOEBIASIS, (i). Study of the relationship between the susceptibility of various hosts, including axenic (germ-free) animals, and the production of antibodies. ({) The development of miore effective methods for the recovery and identification of E. histolytica from water supplies. (i) Comparative studies of the various immunodiagnostic techniques for the diagnosis of amocbiasis, using a standard antigen (Studies on the persistence of antibodies. (1m) Use of serological methods to clarify the confused situation regarding the diagnosis of the illdefined conditions of “nondysenteric: amoebic colitis" and “ amoebic hepatitis (1) Further research on the fractionation of antigens (o) Study of the role of hypersensitivity inthe severity of colic lesions and in the extension of the disease. Such investigations should be performed (i in man, and deal with the rclationship between the severity ofthe disease and the importance of delayed hypersensitivity to amoebic antigens ; and (i) in animals, écaling with the influence of preimmunization on the devel- ‘opment of colic of hepatic amoebiasis in animals reinfected with amocbae. (p) Investigation ofthe roe of the variations in the colonie flora induced by bacterisl immunization. Studies should be performed to determine the relationship between the variations in infection with amoebae in experimen tal animals and the variations in the colonic flora induced by immunization with diffrent strains of Escherichia col (q) Study of the variations in the protective effect of antisamocbie anti- bodies according to the animal species. The aim of this research is to determine whether severe disease in some species is related to their devel- oping antibodies against a few antigenic determinants only, and whether the mild forms of disease observed in other species are due to their produe- ing antibodies against a greater number of antigenic determinants. (F)_ Study of anti-colon auto-antibodies. ‘These auto-antibodies could be studied by immunofluorescence methods, in sera from a large population of patients with various forms of amoebiasis. Comparative studies should bbe performed in experimental animals. (5) Studies should be performed on antigenic differences, not only between amocbue from different species, but also between amoebae of the same species but of different origin 8.3 Diagnosis (@) A method for the specific staining of E. histolytica in faeces should be developed, e.g., using a fluorescent antibody technique.REPORT OF A WHO EXPERT COMMITTEE sl () Since amoebae are difficult to identity correctly, and E. histolytica may be at or below the threshold level of ordinary techniques, it has been suggested that the * amoebic index "? and the “ amioebie prevalence rate”? may be useful in assessing epidemiological factors. The validity of these concepts should be tested. 8.4 Therapy (@) The comparative evaluation of amoebicides in endemic and non- endemic areas of amoebiasis is recommended ; the trials should cover all forms of the disease, but particularly non-dysenteric amoebic colitis and asymptomatic intestinal amoebiasis, (@) Further research is needed on the use of suitable direct-acting amoebicides for the chemoprophylaxis of amoebiasis in situations of high endemicity, e.g, in certain institutions 8.5 Control (@) The social and anthropological factors involved in amoebiasis should be assessed in communities with high and low endemicity. (®) Estimation of the cost of amoebiasis in different cities or areas should be encouraged : the estimates, which should at least cover dysentery and amoebic liver abscess, should be based on criteria to be established by WHO, and take into consideration the cost of professional attention, drugs, hospitalization, loss of working time, deaths, ete (©) A study should be undertaken of the effect of rehousing (or stum learance) projects on the incidence and prevalence of intestinal parasites in urban communities of developing countries. 9. RECOMMENDATIONS. is a disease of public health importance that might become more important with increasing tourism, immigration and emigration, and movement of labour, the Committee makes the fol- Jowing recommendations (1) ‘The control and prevention of amoebiasis should be integrated into public health and social services, and should be included in programmes The term “amocbie index" refers to a procedure in which the prevalence of Ento- ‘moeba coli is used as an index tothe prevalence of E. hstlyica in © community * ‘The “amoebic prevalence rate” (APR) isthe percentage of individuals examined in ‘an epidemiological study who are found to be infected with one or more of the following species of amocba : F.histofstica, E- hartmann, £. colic and Eidolimax nana. The APR has been suggested in particular for stags where the preveleace of E.hrvalvtr is aw2 AMOEBIASIS for surveys or control of diarrhoeal diseases, where such programmes exist. (2) WHO should study the possibility of initiating and organizing the surveys and data-collecting activities in different parts of the world that fare suggested in section 8.2 (6), (¢), and (d). ©) Support should be given to basic research on the factors that are involved in conditioning the invasiveness of E. histolytica and in subsequent host-tissue reaction, as described in section 8.2 (e), (f), (h), and (0). (4) Trials of new drugs should continue. (5) Comparative evaluation of immunobiological tests should be under- taken, (©) Continuing professional education on amoebiasis and other para- sitic infections should be promoted by every possible means, including the establishment of diagnostic and training centres. ACKNOWLEDGEMENT ‘The Committee acknowledges the special contribution made to its deliberations by Dr F. Biagi, Parascologist, Parasitic Diseases, WHO.