I I I t' 0[ I

I I'

I Symptomatology, pathology, diagnosis

Edited by A.A. Buck

I

l !

@ World Health Organization Geneva The ll/orld Heolth Organization ( ,yHO ) is one of the specialized agencies in realtion- ship with the United Nations. Through this organization, which came into beine in 7948, the public health and medical professions of more than 130 countries exchange their know- ledge and experience and collaborate in an effort to achieve the highest possible level of health throughout the world. ll/HO is concerned primarily with problems that individual countries or territories cannot solve with their own resources-for example, the eradication or control of malaria, schistosomiasis, smallpox, and other communicable diseases, as well as some cardiovascular diseases and cancer. Progress towards better health throughout the world also demands international cooperation in many other activities: for example, setting up international standards for biological substances, for pesticides andfor pesticide spraying equipment ; compiling aninternationalpharmacopoeia ; drawing up and administer- ing the International Heolth Regulations; revising the international lists of diseases and causes of death ; assembling and disseminating epidemiological information ; recommending nonproprietary names for drugs; and promoting the exchange of scientific knowledge. In many parts ofthe world there is need for improvement in maternal and child health, nutrition, nursing, mental health, dental health, social and occupational health, environmental health, public health administrotion, professional education and training, and health education of the public. Thus a large share oJ'the Organization's resources is devotedtogivingassistance and advice in these fields and to making available-often through publications-the latest information on these subjects. Since 1958 an extensive international programme of colla- borative research and research coordination has added substanlially to knowledge in many fields of medicine and public health. This programme is constantly developing and its many facets are reflected in I|/HO publications. rsBN 92 4 156041 X

@ World Health Organization 1974

Publications of the World Health Organization enjoy copyright protection in accordance with the provisions of Protocol 2 of the Universal Copyright Convention. For rights of reproduction or translation of WHO publications in toto, application should be made to the Office of Publicatrons and Tranlction, World health Organization, Geneva, Switzerland. The World Health Organization welcomes such applications. The 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 or territory or of its authorities, or concerning the delimitation of its frontiers. Authors alone are responsible for the views expressed in this publication. The mention of specific companies or of certain manufacturers' products does not imply that they are endorsed or recommended by the World Health Organization in preference to others of a similar nature that are not mentioned. Errors and omissions excepted, the names of proprietary products are distinguished by initial caprtal letters.

PRINTED IN S'WITZERLAND ONCHOCERCIASIS

Symptomatology, pathology, diagnosis

Edited by

A. A. BUCK Division of Malaria and Other Parasitic Disease.", World Health Organization, Geneva, Switzerland

WORLD HEALTH ORGANIZATION GENEVA 1974 ONCHOCERCTASIS CONTENTS

Page

Preface . 7

Introduction 8 Clinical and pathological aspects, excluding ocular oncho- cerciasis 9 Onchocercomata 9

Onchocercal dermatitis . l0 Lymphatic involvement t4 Systemic onchocerciasis 14 Loss of weight. r5 Diagnosis. l7 Microfilariae in the skin t7 Onchocercomata 20 The Mazzotti test 20

Microfilaruria . 2t

Ocular onchocerciasis and its diagnosis . 23

General comments . 23

Microfilariae in the eye . 24 Keratitis 25

Iridocyclitis . 27 Cataract 28

Choroidoretinal lesions . 28 Postneuritic optic atrophy. 29 Histopathology 29

Annex l. Onchocerciasis survey forms 3r

Annex 2. The measurement of visual acuity 35 Annex 3. Recommended diagnostic facilities for use in field studies . .37 Acknowledgements .38 References .39 Figures l-85 43-80

5 CONTRIBUTORS

J. ANoEnsoN, Ophthalmologist, British Medical Research Council, Helminthiasis Research Unit, Kumba, Cameroon A. A. Bucx, Division of Malaria and Other Parasitic Diseases, World Health Organization, Geneva, Switzerland F. H. BuoopN, Ophthalmologist, South Warwickshire Hospital Group, Birmingham Regional Hospital Board, Stratford-upon-Avon, War- wickshire, England D. H. CoNNon, Chief, Ceographic Pathology Division, Armed Forces Institute of Pathology, Washington, D.C., USA H. FuclsnNc, Epidemiologist, British Medical Research Council, Helminthiasis Research Unit, Kumba, Cameroon J. J. Prcq, M6decin-chef, Section Parasitologie, Centre Muraz, Organisa- tion de Coordination et de Coop6ration pour la Lutte contre les Grandes End6mies (OCCGE), Bobo-Dioulasso, Upper Volta A. RoruNo, Ministry ol Health, Ouagadougou, Upper Y olla; formerly: Directeur-adjoint, Institut d'Ophtalmologie tropicale de l'Afrique occidentale, Bamako, Mali B. B. W.roov, WHO Consultant, Division of Malaria and Other Parasitic Diseases, WHO, Geneva, Switzerland

6 PREFACE

Great interest is at present being shown in onchocerciasis, a serious parositic disease tltat is now' recognized a.e one of tlrc most important public health problems in Af ica and a local problem in some other parts of tlrc w'orld. A major drive to control the disease in seven West Af ican countries is just beginning. WHO is the execuling ogetrcy for an internationalll' assistecl programme to eliminate oncltocerciasis ft'ont the Volta River basin and to reslore the econonic potential of this vast region covering approximatell' 700000 km2. In other countrie.s preparations are being made to carr)'oul surve.t's to a,tse.\.t tlrc onclrccet'ciasis sittetion or to collect infonnation for planning control operatiorts. To prot'ide aJirm basisfor all tlte clinical and epidemiological inrestigalion.r tlrut v,ill be ntade in the course of llte control programme in the Volta Rit'er area and elsev'here, a nteeting v'as convened in Geneva fi"om l8 to 29 June 1973 to discus.s tlte s1'mptomatology, cliagnosis, and pathologl' of tlrc disease, u'ith special reference to the stanclardization of diagnostic methods and tlrc classiJication oftl'pical and atypical ntanifestations ofonchocerciasis. A relised yer.sion of the draft report v'qs subsequently prepared and editedfor publication. This hondbook i.s therefot'e the outcome of much preparator.t' u,ork by an international group of experl.s. Since the last report on onchocerciasis by a WHO Expert Committee was published o't'er 8 reors ago, the opporlunit)'has been taken to include in the handbook o great deal that is new about the patltology of the disease. Althouglt emphasi.s has been placed on tlrc endentic Af ican form of the disease, it should be of considerable interesl to heqlth personnel v'orking in other onchocerciasis infected areas ancl to medical parasitologists generally. The preparation of tltis handbook v,as nrude possible tltough lhe contri- bution of the United Nations Development Programme towords tlrc finan- cing qf the epideniology and chemotherap.r'components of the Onchocerciasis Control Progronrme in the Volta Riyer basin area.

7 INTRODUCTION

Recommendations for classifying the clinical manifestations of oncho- cerciasis and determining the prevalence and intensity of infection with Onchocerca voltulus in clinical and epidemiological studies were made in 1965 by a WHO Expert Committee on Onchocerciasis (l). The present state of knowledge about this parasitic disease is reviewed here and some of the problems that have arisen in recent studies are summarized. In the classical form of the disease the adult filarial worms are located in the subcutaneous tissue, where they are either free or encapsulated in nodules, the onchocercomata. Typically, the filarial larvae (microfilariae) are confined to the subcutaneous tissue and the eye but a number of studies have shown the presence of microfilariae in many other body tissues. In its most severe clinical form onchocerciasis combines various types of skin lesion with ocular manifestations that may involve both the anterior and posterior segments of the eye and may lead to blindness. However, there are also completely asymptomatic infections as well as intermediate clinical manifestations ranging from benign to severe forms of the disease. Only the microfilariae, and not the adult worms, appear to be responsible for the cutaneous and ocular lesions of onchocerciasis. It must also be realized that the clinical features of onchocerciasis may vary according to the different bioclimatic zones and even between different foci in the area, and that within any one focus the severity of individual cases of the disease is Iargely dependent on the duration and intensity of the infection. This intensity can be assessed by quantitative techniques.

8 CLINICAL AND PATHOLOGTCAL ASPECTS, EXCLUDING OCULAR ONCHOCERCIASIS

Onchocercomata

Adult worms are generally found in subcutaneous nodules, but they can also lie free in the subcutis (2, 3), and nodules may be so deeply situated as to be impalpable (4). A nongravid female has even been seen in the aortic wall of an African patient (Fig. I ). Because of the paucity of autopsy studies it is impossible to say what proportion of adult worms lies hidden. Among Africans over 20 years of age who are found to have O. t'olvulu.s microfilariae in a skin snip, 60 \ or more have palpable nodules. In the 10-19 years age group 3V60% of individuals have nodules, while in the 5-10 years age group nodules are found in less than 30 [. Nodules can be found in children as young as 2 years of age. With experience, a typical onchocercoma is easy to recognize. lt is a firm, round or elongated, nontender, well defined subcutaneous tumour. The size may vary from a nodule 0.5 cm in diameter to a mass measuring l0 cm or more across. Nodules are usually freely mobile but may be fixed to the periosteum, deep fascia, or skin. Although isolated nodules are found, nodules are often closely applied to each other in big masses and small satellite nodules, which contribute to the lobulated feel of the tumour, may also be present. The removal of these lobulated masses usually reveals many more nodules than had been palpated, and there is also little doubt that many small isolated nodules, particularly in the scalp, remain undetected. Usually, onchocercomata can easily be distinguished from lipomata, sebaceous cysts, and ganglia. However, lymph nodes in children, as well as juxta-articular nodes in late yaws, can cause difficulties in differential diagnosis. Certainty in diagnosis can only be achieved when the worm within the tumour is identified. In hyperendemic villages the average number of palpable nodules in males over 40 years of age is about 10, but more than 100 have been reported in some individuals (5). In Africa the nodules are commonly distributed around the pelvis, particularly over the iliac crest, femoral trochanter, coccyx, and sacrum (Fig. 2). Another common site is the lateral chest wall. A small proportion of nodules occur around the knee or on the lower leg, ankle, or foot. Less than 2l of onchocerciasis patients, mainly children, have nodules on the head and neck, and nodules are also rare on the arm. In Central America, by contrast, over 50 /, of the nodules are found on the head, about 5 /, on the arm, I 5 /, on the chest wall, and oriy 25 /, around or below the pelvic girdle (6). In patients studied in the Yemen focus nodules were present on the lower part of the body and were relatively uncommon (7).

9 Nodules on the lateral chest wall may appear as soft tissue swellings in X-ray pictures and this may furnish a lead to unrecognized foci of oncho- cerciasis (8). Adult worms are known to remain viable in the human body for 16 years (9, l0). Microscopically, the nodule has an outer shell of hyalinized scar tissue enclosing the adult worms. In some nodules, scar tissue permeates the entire nodule, confining the worms to narrow tunnels and thus immobilizing them (Fig. 3). ln other nodules portions of the worms lie free in a "soft" centre (Fig. 4) composed of variable components including granulation tissue, fibrin, immune complexes, polymorphonuclear neutrophils, lipo- phages, eosinophils, giant cells, lymphocytes, plasma cells, and Russell bodies (Fig. 5). Any combination of these components may be present. Each nodule may contain one or more female worms, nongravid or gravid, intact or degenerating. Male worms may or may not be present. A thin layer of eosinophilic material with the histochemical characteristics of an rmmune complex may form around degenerating adult worms (Fig. 6). Microfilariae, sometimes in great numbers, are present in the centres and walls of the nodules. These microfilariae may degenerate and provoke focal granulomata (Fig. 7). It has been reported from Guatemala (ll) that the adult worms may discharge microfilariae through channels in the nodule wall, but in serially cut African nodules the anterior ends and vulvae of the worms were located within the nodule centre (12) (Fig. 8).

Onchocercal dermatitis

General commenls The following outline, in which clinical and histopathological features are correlated, is presented as a guide to the further study of the variety of clinical and pathological changes in onchocercal dermatitis although many patients develop only a few ofthe changes described and those changes do not necessarily follow the sequence listed. Of the pathological changes, fibrosis is probably the most serious. It begins early, persists throughout the course of infection, and ultimately replaces the specialized dermal structures (the areolar connective tissue supporting the epidermis, the inter- lacing strands of collagen and elastica, and the dermal appendages). The microscopic features that are virtually constant in onchocercal dermatitis include (a) fibrosis, as recognized by an increase of fibroblasts between the collagen fibres, of reticulum fibres throughout the dermis and around the dermal vessels, and the early appearance of scar tissue in the dermal papillae; (b) acid mucosaccharide, as determined by diffuse and focal deposits of alcian-blue-staining material between collagen strands and in proliferating stellate mesenchymal cells; and (c) melanin within phagocytic cells in the upper dermis. Itching and scratching can be the most important early manifestations of onchocercal dermatitis. Any part of the body may be involved, and l0 these may be the only manifestations of the disease in lightly infected persons. The itching may be mild and intermittent or intense and virtually continuous, and excoriations may be prominent. On the other hand, itching is often entirely absent.

Early changes Altered pigmentation is usually the first objective change. As with pruritus, any part of the body may be involved, but in Africa it is most common on the lower trunk, buttocks, thighs, and shins. In very dark skins, areas of hyperpigmentation may be barely perceptible, even in sun- light, but sharply focused photographs may reveal such changes if intense lighting is used and the film is somewhat overexposed (Fig. 9). The hyper- pigmented areas may be macular-several millimetres to several centimetres in diameter-or irregular in outline, presenting as varied "geographic" patterns. Their margins are not raised or infiltrated and they are not associated with any loss o[ sensation. Rarely, onchocercal dermatitis may present as one or more hypopigmented macules resembling the macules of other dermatoses including streptocerciasis, granuloma multiforme, tuber- culoid leprosy, the treponematoses, and some dermatomycoses. Biopsy specimens taken in the early stages of onchocercal dermatitis reveal normal skin or only minimal changes, including a light infiltrate of inflammatory cells around the dermal vessels and appendages (Fig. l0). Lymphocytes. histiocytes, plasma cells, and eosinophils are usually present in varying numbers but polymorphonuclear neutrophils are absent. Changes in the dermal collagen include slight oedema and increased numbers of fibroblasts between the collagen fibres. Microfilariae may be difficult to find. If onchocerciasis is suspected many sections should be cut, stained by the Giemsa method, and searched carefully for microfilariae (Fig. ll and I2). By the time, a pigmentation change is established there is an infiltrate of inflammatory cells around the vessels and appendages (Fig. l3). In addition, there is usually acanthosis, slight hyperkeratosis, focal para- keratosis, and the presence of a few melanophores in the upper dermis. Fibroblasts are more numerous and they may be large, with a deep baso- philic cytoplasm and conspicuous pointed processes (Fig. 14). A narrow band of scar tissue may be present in the dermal papillae just beneath the basal layer (Fig. l5). The dermal papillae may be slightly broadened and contain acid mucopolysaccharides. Microfilariae may be few or numerous and they tend to concentrate in the upper dermis (Fig. l6).

"Craw crqv:' or "gale filarienne" Papules may develop at any time and on any part of the skin. The papules may be small and closely spaced or large and more widely separated (Fig. 17 and 18). Clear fluid or pus may be extruded. Pruritus is usually severe and leads to scratching, small bleeding points, and sometimes ulcera- rl tions with secondary infections-the appearance known in English as "ctaw craw" and in French as "gale filarienne" (Fig. 19 and 20). Microscopically, these papules are intra-epithelial abscesses composed of varying amounts of fluid, polymorphonuclear neutrophils, and fibrin (Fig. 2l). Serially cut sections usually reveal I or more microfilariae within the abscess (Fig. 22). A thickened parakeratotic scale or crust forms over the pustule.

Sowda Sov'da, the Arabic word for black, is the local name in Yemen for the most important clinical manifestation of onchocerciasis (13). The affected area of skin is intensely pruritic, dark, and thickened. There is a papular eruption (Fig. 23 and 24) and the regional lymph nodes are soft and greatly enlarged, but not tender. The condition is usually localized and typically involves one lower extremity only. A more generalized form involving both legs, or any part of the body, can also be seen. Similar cases have been observed in Africa, but they are rare. Microfilariae are usually absent in skin snips, but they can be found in full thickness skin biopsies (Fig. 25). Hyperkeratosis, parakeratosis, follicular plugging, and acanthosis with some papillary elongation of the rete ridges, are all prominent features. Scattered foci of intraepidermal oedema can be seen. The most striking feature, however, is the extensive inflammatory cell infiltrate composed of many plasma cells, fewer lymphocytes and eosinophils, and a smaller number of histiocytes. This infiltrate forms broad cuffs around dermal appendages and vessels, greatly thickens the dermis, and extends into the subcutaneous fat. Oedema and fibrosis of the dermis are also prominent features, and the dermal papillae are sclerotic. ln the upper dermis there is incontinence of the pigment and the elastic fibres are reduced in number. Specimens taken through papules with formed craters reveal intraepidermal abscesses with an overlying thickened keratin scale. The microscopic appearance of the lymph nodes is dealt with in the section on lymphatic involvement (p. l ).

Oedema In heavily infected patients the skin may be focally swollen ("peau d'orange") or may present a more diffuse lymphoedema (Fig.26). Micro- scopically, the dermis is oedematous and contains many microfilariae, some of which form clusters within dilated lymphatics (Fie.2l). Many fibroblasts are present in the ground substance. These changes may be associated with epidermal atrophy (see Atrophy, p. l3).

Ltcltenoid change

In some patients areas of the skin may be dry and scaling, often with a mosaic pattern. This appearance has been called "lizard skin" (Fig. 28). As with the early changes, the distribution is variable and there may be

l2 intense itching leading to scratching. Uneven pigmentation may also be present. Microscopically, this change is characterized by a separation of the layers of keratin (Fig. 29) and of the keratin from the epidermis. Atrophy of the epidermis is also present and characterized by thinning, especially of the suprapapillary portion, and a reduction in the number and depth of the rete ridges. In the upper dermis, there is also scarring and a reduction in the numbers of elastic fibres (Fig. 30).

Late depigmentatiott or "leopard skin"

A striking feature commonly occurring in long-standing onchocercal dermatitis is localized spotty depigmentation (Fig. 3l ). This is most common on the lower limbs, especially the shins, and is also over the penis and scrotum; more rarely, it occurs in the groin or axilla. The depigmented areas are white and interrupted by foci of persistent pigmentation around the pores and hair follicules. The areas may be slightly depressed or atrophic (Fie. 32). Microscopically, there is an absence of melanin in the epidermis (basal layer, prickle cell layer, and keratin). The underlying dermis may show a variety of mild to severe inflammatory changes, as already described. In the small number of biopsies studied microfilariae were very few beneath the depigmented areas and more numerous in the adjacent pigmented areas.

Atrophl

This is a late stage of onchocercal dermatitis. The surface of the skin has a shiny fragile appearance that has been compared to crushed tissue- paper. The skin is dry and may be unevenly pigmented. It is inelastic, recovering slowly from stretching or pinching, and may be wrinkled and hang in loose redundant folds (Fig. 33). Microscopically, the keratin is thin and, as with the lichenoid skin, separates easily from the epidermis (Fig. 29). The epidermis is also very thin, only 2-3 cells thick in some places, and the rete ridges have vanished, giving it a smooth or "ironed-out" undersurface. The dermis may be oedematous and contain acid mucosaccharide, but the most striking change is replacement of normal dermal structures by scar tissue, which tends to have a circular arrangement around blood vessels. At this stage, silver stains reveal abundant reticulum fibres throughout the dermis and also oriented around blood vessels. These fibres tend to follow the pattern of dermal collagen fibres (Fig. 34). Hair follicles and sweat glands are reduced or absent. Microfilariae vary in number from few to many. Elastic fibres have disappeared from the upper dermis but those in the lower dermis may be thickened and formed into disorganized knots or entanglements (Fig. 35).

t3 Lymphatic involvement

Any of the superficial groups of lymph nodes, including cervical, axillary, epitrochlear, inguinal, femoral, and popliteal, may be involved. The nodes particularly affected are usually those draining areas of onchocercal derma- titis, and they tend to be discrete and not painful or tender. Microscopically, these nodes show fibrosis of the capsule, trabeculae, and medulla. Germinal centres are few and reduced in size, or they may be absent (Fig. 36). Subcapsular sinusoids are dilated, as are lymphatics in the medulla. There are collections of histiocytes in the sinusoids and large numbers of inflammatory cells, including plasma cells, eosinophils, and mast cells throughout the substance of the node (Fig. 37). These nodes contain varying numbers of microfilariae, most frequently in the capsule and the subcapsular sinusoids. Some microfilariae are intact and appear viable, while others are degenerating and surrounded by a granulonatous reaction composed of epithelioid cells, giant cells, and, sometimes, eosino- phils and fibrous tissue (Fig. 38). In endemic areas of Africa this lymphadenitis has features of a competent cell-mediated immune response. A quite different picture was seen in lymph nodes removed from 3 patients with sowda in Yemen. The nodes were much larger than those seen in Africans, measuring up to about 6 cm. Clinically tl.rey were soft nontender tumour-like masses. Macroscopically, the nodes were pale yellow homogeneous and without gross evidence of scarring or other focal lesions (Fig. 39). Microscopically, their capsules were slightly thickened but there was no scarring of the trabeculae or medullae. In contrast to the African nodes with atrophic follicles, there was marked follicular hyperplasia with many histiocytes and large numbers of plasma cells (Fig. 40). Microfilariae were not found in these nodes. The pronounced histopathological differences between the lymph nodes from African and Yemeni patients suggest different host response mecha- nisms to the microfilariae of O. volvulus. Other clinical manifestations of onchocercal lymphadenitis in Africa include hanging groins (Fig. 4l and 42) (14) and other regional lympha- denopathies. An association between onchocerciasis and elephantiasis of the genitalia has been noted in Zaire (15) and Chad (16) (Fig. 43) but further studies are required to determine both the pathogenesis and the apparent geographical differences of this complication (Fig. 4a).

Systemic onchocerciasis

Textbooks of communicable diseases and parasitology refer to oncho- cerciasis as a disease in which the distribution of the infective agent and of the lesion are confined to the skin and the eye. The possible role of O. t'ol'r'ulus as a cause of systemic infection and disease has been questioned' The rapidly increasing number of studies reporting microfilariae of O. vol- tulus in organs other than the skin and the eye requires a critical review,

14 not only of the clinical picture and the diagnosis of onchocerciasis, but also of its pathogenesis. A list of the sites other than the skin and the eye from which the parasites have been recovered is given in Table l, and the geographical distribution of microfilaruria is shown in Table 2. Systematic examinations of blood and lymph have not yet been carried out but large numbers of O. ,toh'ulus microfilariae have been seen in the superficial dermal lymphatics (Fig. 5l) and the regional lymph nodes. The microfilariae have also been seen occasionally in blood vessels (Fig. 52). These observations, together with deep organ involvement, suggest that the pathway of microfilarial dissemination is via the haemolymphatic system. To determine the frequency and clinical significance of disseminated oncho- cerciasis, detailed comprehensive clinical and pathological studies are needed.

Loss of weight

More recently, systemic effects of chronic onchocerciasis have come under closer scrutiny. Rolland & Balayr found that marked differences in the body weight of adult males and females were associated with the intensity of infection. Comparing hyperendemic with mesoendemic and hypoendemic villages, they observed an average difference in body weight of 4 kg in males and of 3 kg in females. Buck et al. (16) followed-up oncho- cerciasis patients with different infection loads, as determined by standardized skin snips, for 3 years and found significant weight losses in heavily infected

Table 1 . Evidence of systemic onchocerciasis

Organ or O. volvulus Frequency and type Geographrcal area srte adults mlcrofrlarrae of observatron and reference

Blood + Occasronal rn hrstopathologrcal spectmens Zarre ('l 5) + 5 hosprtal patrents Mexrco (39) Blood vessel + 1 autopsy Zarc (40) Aorta (Frg. 1) + + 1 autopsy Zarre (1 5) Lrver (Frg. 45 + autopsy Zare (15) and 46) + aulopsy Zane (4Ol Krdney (Frg. 47 + autopsy Zare (15) and 48) + autopsy Zaie (40) Lungs (Frg. 49) + autopsy Zaie (15) Spleen + autopsy Zave (4O) Lymphatrc system + Routrne frndrng tn htstologtcal (Frs. 50) examrnations of lymph nodes Troprcal rn endemrc areas Afnca Cubrtal nerve + 1 autopsy Zare (4O) Cerebrosptnal flurd + 5 cases Mexrco (39) a number of cases Zaire (5) Urine + See Table 2 Vaginal smear + 2 9$ ot Papancolaou smears rn Cameroon (41 ) a hyperendemrc focus (savanna) Sputum + 3 cases followrng frrst dose of Cameroon (42) drethylcarbamazrne (treatment (savanna) tflal ) -. ' 9!ta1A,4. & Balay, G. (1969) L'oilchocercose dans lefoyer Bra. Unpublished document No. lll/oNCHo. of the centre Muraz of-the organisatlon de Coordinitron et de Coop6- ratlon po--ur la Lutte contre les Grandes End6mies and the Minrstry of Public Health and Socral Affarrs of Upper Volta.

t5 Table 2. Geographical occurrence of microfilaruria in onchocerciasis from tropical Africa' and Mexico

Country where Type ot Frequency of Type ol Method of were seen poPulatton mtcrofrlarufla study urine cases examrnalton routrne Nrgena (44) hospltal Patlents 1 case clrnrcal study Mexrco (39) hospital patrents 5 cases clrnical study routrne routrne Uganda nonrandom sample of 71300:23% survey wrth routlne (45) Zare ) general populatron uflne examinattons Ethiopra (46) hosprtal rnpatrents 1 case rncrdental findrng routrne in hosprtal lab. roulrne Lrberra (47) hospltal Patrents 1 case rncidental frnding rn hosprtal lab. lflple Chad (8) total vrllage 441386 = 11.4% eprdemtologtcal populatron study concentlatton tilple Chad(1 6) selected vrllage 461135 = 34.1 % clrnrcal study oI populatron mrcrofrlaruria concenliatton : routrne Cameroon Patlents selected 3140 7.5o/o routtne unne (forest)b lor treatment examrnatton tnple outpatrents 1 3/1 00 = 1 3.0 % chnrcal studY UpperVolta' concentratton trrple lvory Coasl" nonrandom sample of 5/100:50% survey populatron concentrallon not specrfied routrne N rgen ad hosprtal pattents occasronal observatrons representatlve sample 1a847 = 52% eprdemrologrcal sludY tflple Cameroon concentratton (savanna) (41 )of general populatlon wrth tflple Ghana (48) hosprtal Patrents 81176 = 4.5 Yo c|nrcal studY routrne urlne concentratton examrnatton 22/85 = 259ok chntcal studY tfl ple Malr' outpattents concentration : routtne Cameroon nonrandom sample of 35194 37 % survey (savanna) vrllage populatton tn (42]. hyperendemrc area

ln Buck (43) ^ from Dr B O, L Duke, 1972' 'i;Loonit'"oi".unrcatroni. i-lin,tna,l studics on tlrc prest'nce ofntttrcrtlanae oJ Otchocerca volvulus F;;:i"i ['il;rii, iiszii 96 A.lrmrted number tn th? uttne ol onchocerctosts plr*rirl ,"p,jOri.'n"a wnO'a""u'ent ONCHO/72 ' orrLirrrv or Jrofessronaliv rnterested rn the subiect of copres of rhrs document ,," 1211 Geneva2T' from rhe orvrsron of rvr"ru,,a unj.bli'-"t"iliiiuiii.-p"i.ont Fa*.itiCb,.eas"s, wbrld tiJatth orgairrzauon, --';p"ri6"rtSwrtzerland. communrcalron from Dr O. O Kale, 1972- --- . i;;;;;;i io..rn,"ut,on from Dr. A Rousemont. 1973'

the 1967 and 1970 Table 3. Changes- of the weight: height ratio-(W:H) between siuiies in Chad in relation io microfilarral counts in skin snips

for No. rn Mean W:H Drflerence tn I Skrn snip drfference count group for 1 970 w.H 1967-70

0.5 -: 25 1 .74 +0 01 50 1.1 14 1 .67 -0.05 50-1 00 3.5 - 100 4 1 .27 -o.27

persons (Table 3). These preliminary studies by Rolland & Balay have utro p.orid.d the information that severe onchocerciasis may lead to excessive mortality in adults.

l6 DIAGNOSIS

Microfilariae in the skin

Most authorities agree that the best method for the parasitological diagnosis of onchocerciasis is the skin snip. The method can be standardized and used for both qualitative and quantitative assessment. The number of microfilariae emerging from a skin snip is a reflection of the intensity of infection. Skin snips can be taken with a needle and razor blade or with forceps and curved scissors. These techniques produce snips of varying size and shape and it is therefore necessary to weigh the snips to calculate the number of microfilariae per mg (17). Scheffell introduced the corneo-scleral punch for taking skin snips. With this instrument it is possible to take snips of uniform size and depth and an average weight of 0.8 mg (range, 0.4-l .2 mg) (18). The method is fast and painless, and leaves a small lesion that heals rapidly. It is therefore well accepted. The available instruments are shown in Fig. 53, and the recommended sizes are indicated. Instruments with ring handles like those of scissors are easier to manipulate than those with spatulate handles because they allow sufficient pressure to be applied to produce the ideal thickness of skin snip. Whatever method is used, the specimen should be of such a thickness that the outer parts of the dermal papillae are sampled, this being the level at which microfilariae are most commonly found. Blood staining should develop in the wound after a few seconds (Fig. 5a).

Qual itat ive assessme n t In prevalence surveys it is sufficient to taken one snip from each person at the site most likely to be heavily infected. The site of choice may have to be determined by taking multiple snips from different sites in a random sample of the population. In Africa the best site is just above the iliac crest, in Central America over the scapula or iliac crest, and in Yemen over the lower calf. The snip is transferred immediately to a drop of normal saline or distilled water on a microscope slide. To prevent the evaporation of the examination medium the slide can be kept in a covered dish on a sheet of wet foam rubber. The microscopic examination is made with the low power (,< l0 objective; ,,<5-x l0 eyepiece). If normal saline is used a search can be made for microfilariae after l0 min., but with distilled water it is necessary to wait 30 min. Since microfilariae emerge slowly in distilled water, snips

' Unpublrshed data. t7 are teased and re-examined after a further 30 min. if no microfilariae are seen at first. II the presence of microfilariae of Dipelalonema streptocerca is suspected, the snip is allowed to dry, and then fixed and stained for the identification of the infective species (see below, differential diagnosis). After taking a skin snip, the instrument is disinfected.

Quant i tat ive ossess men t The same technique is used for quantitative assessments, but in order to obtain comparability between surveys the following procedure is strongly recommended as standard. The snip is placed io distilled v'ater and all emerging microfilariae of O. volvulu.r are counted with a tally counter after 30 min. Distilled water is used instead of physiological saline for the following reasons (18): (a) the emergence of microfilariae in relation to time seems to be less variable; (b) with reasonable consistency, 50f of the microfilariae will have emerged at the end of 30 min. (Fig. 55); (c) the counting of microfilariae is easier because they move more slowly and do not form clumps; (d) there is no risk of variations in the salt concentration resulting from evaporation. The recommended sites for obtaining snips are, again, over the scapula, both iliac crests, and the lower calf (Fig. 56). The total number of micro- filariae found at each site should be reported separately. If many snips are taken from the same site it is necessary to leave a distance of I cm between cach snip (Fig. 57). This method allows the measurement of variations in the microfilarial density, not only between individuals but also at different sites in the same individual, under varying conditions and at different times. It is also sufficiently sensitive to be used for the evaluation of drug trials and control measures. Periodicity in the appearance of O. volvulus microfilariae in the superficial layers of the dermis does occur, but the amplitude is small in comparison with that of Wuchereria bancrofti, for example. Also, variations in the physiological rhythm of individuals, and between different parts of the body in the same individual, reduce the effect of periodicity-as observed in a prevalence survey-below the level of practical significance. Picq et al.1 showed that in a patient infected with W. bancrofti, Dipetalonema perstqns, and O. tol'tulus, the maximum to minimum ratios for microfilarial patency during 24 hours were, respectively, gg:1,2.1: l, and 1.7:1. The influence of the ambient temperature, the skin temperature, and the season on the microfilarial density in the superficial layers of the skin may be important and require to be studied.

'picq. J. J. & Jardel, J. P. (1973) LIne nithode d'ivaluation des densitts nicrofilariennes d'Onchiterca volvulus Leuckart l'893, chez des onchocerquiens. Part 2(Frenchonly), unpublished WHOdocumentONCHO/71.10-1. Altmttednumberofcopiesofthisdocumentare-avatlableto persons officially or professionally interested rn the subject from the Division of Malaria and bther Parasrtic b,scisct, World Health Organtzatton' l21l Ceneva 27, Swrtzerland' l8 Thc dffirential diagnosis of microJilariae scert in skin snips

Besides the microfilariae of O. t,olvulus, those of D. .streptocerco may also inhabit the human dermis. lf blood is taken with the skin snip, the possibility of finding other microfilariae, particularly D. perstans and Loa loa, must be remembered. The distribution of D. streptocerca, as at present known, is confined to Africa and the affected area includes Cameroon, Ghana, Nigeria, Zaire, and probably Angola. The species seems to be more prevalent in forest than in savanna country. The adult worms live in the dermal tissue (Fig. 58) (19). The microfilariae are usually much less numerous than in oncho- cerciasis and they are found almost exclusively above the waist; Kershaw et al. (20) stated that they are commonest at the shoulder, next on the torso, and few in number or absent at the extremities. The distribution of O. volvulus microfilariae in the body varies according to the geographical location of endemic foci. ln Africa, the highest con- centrations of these microfilariae are found over the iliac crests. High concentrations are also found over the calf and ankle, where D. streptocerca microfilariae seldom, if ever, penetrate. The movements of the two species of microfilariae are different, with a tendency to more active writhing by O. t,olvulus and a slower, more concertinaJike action in D. streplocerca. There should be no difficulty in distinguishing stained specimens (see Table 4). Apart from the size difference, the different lengths of cephalic space, the clear space at the tail, and the different shape and grouping of the nuclei are characteristic. The sharply pointed tail of O. volvulus is very different from the rounded tip of D. streptocerca (Fig. 59 and 60).

Table 4. Diagnostic characters of microfilariae oI D. strcptocerca and O. volvulus (Fig. 61)

D. strcptocerca O. volvulus

Geographical distnbution West and Central Afflca, forest Troprcal Afflca, Central and South country; lare rn dry savanna Ameilca, Yemen, forest and savanna Anatomical drstnbutron Shoulder, then trunk, seldom ln any part of the body: common or on extremrtres at least present at calf or ankle Numbers of microfrlarrae in snrp Few Few - many Movemenls Slow, concertrna-lrke Vrolent wrthrng (less so rn drstrlled water than rn salrne) Tip of tail rn stained specrmens Bluntly rounded Frnely pornted

Characteflstrcs of starned specrmens (1 9) Length (pm) 230-250 300-350 Maxrmum wtdth (Em) 2 5-5 5-9 Cephahc space (length) (pm) 3-5 7-1 1 Antelior end to nerve rng ( pm) 60-70 60-70 Anterior nuclei (shape and Oval, staggered but not Elongate, frrst 4 overlapprng, even arrangement) overlappi ng 3 abreast

Features of tatl Nucler rn row at end 9-1 2, round or quadrate 4-8 elongate Shape of termrnal nucleus Oval or round elongate Length of clear space ( um) 1 9-l 5 Shape at trp Bluntly rounded Frnely pointed

19 Onchocercomata

The correlation between onchocercomata and the microfilarial density in the skin and eye varies between individuals and areas. However, valuable information on the prevalence and intensity of infection can be obtained by finding and counting nodules. The examination is carried out both by inspection and palpation, with the subject as nearly naked as possible. The number and distribution of nodules are recorded and in conglomerate onchocercomata the number of palpable lobules is counted.

The Mazzotti test

In 1948 Mazzotti (21) described allergic reactions following the admin- istration of a single dose of diethylcarbamazine. This procedure, using a dose of 50 mg, has since been referred to as the "Mazzotti test" and has been extensively used in some areas as a diagnostic aid. The reaction usually starts within 15-30 min, but, rarely, may be delayed up to 24 hours or more. The first and most prominent feature of the reaction is pruritus, and this tends to be most intense where the microfilariae are most numerous. Pruritus is soon followed by erythema, and this in turn by the outbreak of a papular eruption. The papules are often discrete but may coalesce to give a more generalized brawny induration. In heavy infections the skin may become oedematous and hot after some hours. These changes are particularly prominent in the skin of the groin and axilla, where the lymph nodes often become swollen, tender, and painful, some- times severely enough to render the patient almost immobile (Fig.62 and 63). Oedema can also involve the skin of the head and neck, and may be pro- minent in the eyelids, where it is accompanied by epiphora and conjunctival hyperaemia. At this stage the patients are extremely miserable and may also suffer from general effects including fever, headache, and joint and muscle pain. The reactions usually reach a maximum within 24 hours and then subside gradually over the following 48 hours. The affected skin often desquamates (Fig. 64). In other cases the reactions are far less marked and the skin changes are more Iike an urticaria. A small proportion of infected persons show no reaction. However, the administration of 50 mg of diethylcarbamazine to very heavily infected persons has caused cough, tachypnoea, tachycardia, and, more dramatically, shock with pulmonary oedema, collapse, and loss of consciousness. Clinical changes in the skin during the Mazzotti reaction can be cor- related with the microscopic changes. Biopsy specimens taken t hour after the administration of diethylcarbamazine reveal hyperaemia, increased oedema, and an accumulation of eosinophils around degenerating micro- filariae (Fig. 65). This phenomenon is seen even in subjects who have few or no eosinophils in the dermis before taking diethylcarbamazine. Between I and 3 days after the diethylcarbamazine the microfilariae show advanced

20 degenerative changes. Sometimes only fragments of the microfilarial nuclei remain and these are surrounded by eosinophils, many of which have ruptured and released their granules into the tissues (Fig. 66). [n other patients, eosinophils are inconspicuous and epithelioid cells and giant cells surround and phagocytose the degenerating microfilariae (Fig. 67). There is also a third fate for microfilariae following the administration of diethyl- carbamazine. Some migrate, cephalic end first, into and through the epidermis (Fig. 68), reaching the keratin layer after several days (Fig. 69). Here, they remain coiled and are subsequently lost by desquamation. Some of these microfilariae provoke little reaction during their transepidermal migration but others provoke intra-epidermal oedema (Fig. 70) and an aggregation of polymorphonuclear neutrophils, resulting in the formation of intra-epidermal abscesses (Fig. 7l). It has also been shown that diethylcarbamazine causes the migration of O. volvulus microfilariae into the cornea (22). Furthermore, there is a considerable increase in the number of microfilariae excreted in the urine, and they have been seen in the sputum of patients who reacted with respir- atory distress and productive cough. Microfilariae have also been found in the blood and cerebrospinal fluid following the administration of diethyl- carbamazine. Although Mazzottl's test is specific and sensitive, it yields a small pro- portion of both false positives and false negatives. In addition, its inter- pretation is to some extent subjective. It rs not, therefore, recommended as a routine test in epidemiological studies. However, because of its "mobilization" of microfilariae into the epidermis, cornea, urine, sputum, lymphatic system, blood, and cerebrospinal fluid, the procedure is of considerable interest in research. In view of the severe reactions in some patients the dose of diethyl- carbamazine used for this test, as well as for the start of treatment, must be carefully considered. In some areas 25 mg is sufficient, and even 12.5 mg is adequate for small heavily infected patients. The drug should not be used indiscriminately, and particularly not in severely debilitated old persons, pregnant women, and those suffering from acute conditions. Symptomatic relief from the unpleasant effects of a Mazzotti reaction may be obtained with a combination of acetyl salicylic acid and codeine. Corticosteroids and antihistamines should be available to treat patients with severe reactions.

Microfilaruria

The recommended method of making urine examinations for the pre- sence of the microfilariae of O. volvulus is the triple-concentration tech- nique (16). The subject should be instructed to empty his bladder completely into a spillproof polypropylene container with a snap-on lid. Containers calibrated in millilitres are preferable. The volume of urine should be recorded and tincture of thiomersal is added (about I ml per 100 ml of

2t urine). The specimen is then transferred to a 150-ml funnel fitted with a length of plastic tubing and a clamp. The specimen is allowed to settle overnight and then l0 ml are withdrawn from the sediment layer into a centrifuge tube. After centrifugation, 3-4 ml of the sediment resulting are transferred to small storage vials. At con- venience, these specimens are washed from the vials into 15-ml graduated tubes and centrifuged again. All but 0.5 ml of the supernatant fluid is drawn off. The sediment is mixed by shaking, and is then examined micro- scopically for O. volvulas microfilariae. This method permits an estimation to be made of the number of microfilariae in the specimen. In field studies with limited manpower and equipment, the following simple method of urine examination can be employed. Urine specimens are shaken immediately before l0-ml samples are transferred to l5-ml conical tubes and spun in a manually operated centrifuge for 3 min at about I 500 rev/min. The supernatant fluid is decanted and 0.5 ml of the sediment is examined microscopically for microfilariae. The sensitivity of either method can be increased considerably by examining the urine of patients who have received a small dose (12.5-25 mg) of diethylcarbamazine. Studies by Roux & Picql and by Anderson & Fuglsang (22) to compare the frequency of microfilaruria in a population sample of a hyperendemic area before and after diethylcarbamazine treat- ment have shown that the prevalence of microfilaruria can be boosted to more than twice its original level.

I Roux, J. & Prcq, J. J. (1973) Microfiloruria and the administrotion of drethylctrbanruzina m onchocerriasis cascs. Unpublished WHO document ONCHO/73.104. A limrted number of copies of thrs document are available to persons officially or professionally rnterested in the subject from the Drvrsion of Malarra and Other Parasitic Dtseases, World Health Organizatton, l2l1 Ceneva, 27, Switzerland.

22 OCULAR ONCHOCERCIASIS AND ITS DIAGNOSIS

General comments The appearance and development of ocular lesions in onchocerciasis depend on the duration and intensity of the infection. It seems probable that they result from the local death of microfilariae. The age at which ocular lesions appear, their severity, and the frequency of impaired vision and blindness are closely related to the intensity of infection in the com- munity. Males are normally more heavily infected than females of cor- responding age, and ocular lesions therefore tend to appear at an earlier age and to be more serious in males. The same ocular lesions are found in all the major endemic areas of Africa and Latin America. However, in African savanna villages sclerozing keratitis and anterior uveitis are more frequent and severe than they are in rainforest villages with comparable levels of endemicity. This may be explained by the tendency for the anterior segment of the eye to contain greater numbers of microfilariae in the savanna areas and by differences in the parasite strains. In hypoendemicl villages onchocercal punctate keratitis is found in all age groups but permanent ocular lesions are uncommon. They develop only in adults and are rarely gross. In mesoendemicl villages punctate keratitis is common in young persons but is seen less often in the older age groups. Permanent lesions are found in young adults and become progressively more common and serious in older persons. Blindness and impaired vision associated with both anterior and posterior segment lesions is an appreciable, but not overwhelming, problem. In hyperendemicr villages heavy invasion of the eye by microfllariae is common in early childhood; permanent lesions may develop in youth and may result in impaired vision and blindness at this age. By the time adult life is attained lesions of the anterior and posterior segment of the eye are common and usually coexist. These lesions progress, sometimes with acute exacerbations, so that there is an increasing prevalence of impaired vision and blindness in each decade. However, in elderly patients the intensity of microfilarial infection tends to fall off, so that although the end result is often a seriously damaged eye, the disease process remains quiescent and microfilariae can no longer be demonstrated.

r Based on the prevalence rates in representatrve population samples:.- l0i;,, sporadrc cases; lO-39%, hypoendemrc; 40-69%, mesoendemrc; > 70olo hyperendermrc.

23 Microfilariae in the eye

The cornea Onchocerciasis can be diagnosed by the presence of microfilariae in the cornea. With a good slitJamp at a magnification of x 25 it is possible for the experienced observer to see living, often actively moving, microfilariae (23). The microfilariae are frequently coiled up and are thus almost invisible at lower magnifications. They are mainly peripheral and are seen most easily by retroillumination in the reflected beam. Dead microfilariae can be seen easily at a magnification of ,t 16, but sometimes they show up only by retroillumination. The larvae often lie perfectly straight but other postures-S-shaped, C-shaped, or angulated- are also seen. They can be located at any depth in the stroma and, although more common nearer the corneal periphery, they are often seen at the centre. In a heavily infected savanna village in Cameroon, microfilariae were seen in the cornea of 6l I of the total population over the age of 5 years. The corresponding prevalence in a heavily infected rainforest village in Cameroon was 39 ft.

The anterior cltamber Onchocerciasis can also be diagnosed by finding microfilariae in the anterior chamber of the eye. Their characteristic wriggling movements are easy to see in the slit lamp beam. The microfilariae usually follow the thermal currents of the aqueous humour and may be seen anywhere in the anterior chamber. During prolonged examinations with the slit lamp they tend to sink and disappear into the lower angle. Digital massage of the eye or bending the head down for about a minute both increase the numbers of circulating microfilariae. Dead microfilariae have not been observed free in the anterior chamber. The frequency of invasion of the anterior chamber seems to be less subject to geographic variation. In hyperendemic villages in Cameroon, the prevalence was similar in both rainforest and savanna,i.e., 40-501 of the total population over the age of 5 years. No doubt this proportion would have been higher with repeated observations following massage and head posturing. Microfilariae have been observed in the anterior chamber in a 3-year-old child. Living or dead microfilariae are sometimes found adhering by one extremity to the anterior lens capsule. They have not been seen within the lens in the living patient but the presence of a microfilaria in a cataractous lens removed at operation has been described (24).

The retrolental space and vitreous By means of the slit lamp beam, microfilariae are often seen in the retro- lental space and in the vitreous. Owing to the magnification of the lens

24 they appear larger at these sites than do microfilariae in the cornea or anterior chamber.

Con junctiva

Microfilariae may also be found in conjunctival biopsies, but we do not recommend this procedure. A skin snip taken at the outer canthus provides adequate information on the load of microfilariae in the vicinity of the eye. and a slit lamp examination of the cornea and anterior chamber provides more direct and important evidence of ocular involvement.

Keratitis

Punctole keratitis

Following the death of a microfilaria within the cornea, a reaction usually starts to form within days or weeks at some point along the larval body (F19. 72). The resulting opacities vary slightly in appearance according to their depth in the cornea. In the anterior third of the stroma, a typical lesion results. It is circular and has a whitish broken-up surface, a diameter of about 0.5 mm, and an indistinct edge that does not show up in the reflected beam. It has been likened to a snowflake (Fig. 73). This type of opacity is particularly common in younger people and non-African expa- triates, in whom it may be somewhat larger. The epithelium over these opacities is intact, there is no staining with fluorescein, and corneal sensation is unimpaired. Deeper in the stroma, a similar lesion results but the colour is a dull grey and the surface more homogeneous. The diameter is up to 0.5 mm and the edges are indistinct. The lesions of punctate keratitis vary in number from I to 50 or more. They are distributed most commonly within 2-3 mm ol the Imbus, particularly in the nasal or temporal inter- palpebral areas, but they may be seen anywhere in the cornea. The lesions are usually bilateral. These opacities do not interfere appreciably with vision and they disappear without trace. Many descriptive terms have been given to these lesions, including "cracked ice", "linear", "fluffy", "snowflake", "morula", "feathery", and "nummular". It is recommended that wherever possible they should be recorded as dead microfilariae surrounded by reaction, but that when the larval body has been absorbed the term "fluffy"1 should be used. Di.fibrential diagrosrs. Disc-shaped corneal opacities of unknown etiology are common, particularly in the savanna areas. They have a diameter of 1.0-1.5 mm and are characterized by a sharp margin that shows up clearly in the reflected slit Iamp beam. Such opacities are seen with equal frequency in persons with and without onchocerciasis. Certain adenoviruses produce

'The term rn French rs "cotonneuse"

25 corneal lesions very similar to those of onchocerciasis, but these have not been frequently diagnosed in tropical Africa. They tend to be centrally located, there may be a slight epithelial roughening over them, and they are not infrequently unilateral. Other discrete, but much larger, lesions caused by viruses are also seen, but rarely. Small irregular superficial scars resulting from trauma can resemble fluffy opacities.

Sclerozing keratitis

Sclerozing keratitis is an important cause of blindness and results from the presence of large numbers of microfilariae in the cornea. The first detectable change is a haziness of the anterior third of the stroma, showing as a strikingly white segment involving the peripheral millimetre or so. It starts typically in the 2-4 or 8-10 o'clock position (Fie.7q. A superficial migration of pigment on to the cornea follows, and this is accompanied by the ingrowth of thin superficial vessels. The underlying changes are thus often obscured. A white area of stromal keratitis of variable extent is usually seen in front of the pigmented zone ( Fig. 75). Thick, straight stromal vessels may also be seen growing into the area ol active keratitis (Fig. 76). Sclerozing keratitis usually progresses from the sides and below, and a confluent lesion is commonly seen (referred to as "k6ratitesemilunaire" by French writers) (Fig.77). The pupil area is often covered and total opacifi- cation may result, but the central and upper areas frequently remain relatively clear (Fig. 78 and 79). These clearer areas may contain many hundreds of microfilariae, and the cornea is sometimes so saturated with them that any reactions around individual larvae are swallowed up in the overwhelming stromal response. Sclerozing keratitis, particularly when advanced, is fre- quently accompanied by anterior uveitis. Degenerative changes including plaques of calcification may occur in longstanding cases. Dffirential diagnosis. Trachomal pannus, although frequently seen at the lower limbus, is invariably more pronounced above. Pterygium, with its corneal apex and wing-like extension to a conjunctival base, bears little resemblance to sclerozing keratitis. Pronounced pigmentation at the limbus together with a slight irregularity and heaping up of nasal and temporal limbal tissue are common in Africans, both with and without onchocerciasis. Bulbar involvement in vernal conjunctivitis is not particularly localized to the lower cornea, and eversion of the upper lid will often reveal the additional presence of the characteristic palpebral form. Keratoconjunctivitis may leave small vascularized flanged limbal infiltrates in their wake, and these can mimic sclerozing keratitis. The stromal haze of arcus senilis, if mainly localized nasally or temporally, can look very like the earliest stages of sclerozing keratitis. If there is total corneal opacification diagnosis is usually conjectural. Microfilariae are then, of course, not visible. The differential diagnosis at this stage includes trachoma, advanced glaucoma, degenerations, and scarring from smallpox, measles, xerophthalmia, and injuries.

26 Iridocyclitis

Like sclerozing keratitis, iridocyclitis is found particularly in the more heavily infected patients. This lesion may present with varying degrees of severity from mild torpid to severe plastic inflammation. There are few if any features, particularly in the less severe inflammations, that distinguish the anterior uveitis of onchocerciasis from that due to other causes. How- ever, the association of uveitis with the presence of microfilariae in the eye, together with other onchocercal lesions of the anterior and posterior segments, indicates the likely etiology.

Milcl uveitis

Mild uveitis is often seen in a noninjected eye. Although flare and cells are present in the anterior chamber keratic precipitates are fine, and few if any synechiae form. Slight degrees of pupil distortion are common. Some- times, the pupil is somewhat dilated and reacts poorly to direct light.

S e ve re p last ic infl antntat i o n

Ciliary injection occurs in the active phase but it is often partially masked by limbal pigment. Keratic precipitates of a nongranulomatous type and deposits of pigment are found on the corneal endothelium. Exudate and even fibrin strands may be seen in the anterior chamber and are often deposited on the anterior lens capsule or in the base of the anterior chamber. Deposits on the lens may leave a thin film that sometimes produces the appearance of an exfoliation of the capsule. However, in intense infections a thick felt of exudate may occlude the pupil; this is often pigmented and may be vascularized by vessels from the iris. Whitish deposits in the base of the anterior chamber ("pseudohypopyon") tend to be drawn upwards and forwards on to the posterior surface of the cornea at the 6 o'clock position. Moreover, the iris is sometimes drawn down into the mass, producing a piriform distortion of the pupil. Posterior synechiae are often marked and the pupil may be completely secluded. Peripheral anterior synechiae are frequently found at the 6 o'clock position but in some eyes they may be more extensive. The synechiae may only be visible with a gonioscope, but sometimes extend far enough on to the cornea to be seen directly with the slit Iamp beam. In other eyes, even though the peripheral anterior synechiae are extensive they are masked by sclerozing keratitis. In some intense infections a large sucker-like attach- ment of the iris to the posterior surface of the cornea at the 6 o'clock position is seen.

Secondary glaucoma

Severe iridocyclitis may lead to secondary glaucoma. The end result may be hypotension, corneal degeneration, or even phthisis bulbi.

27 Iris atrophy

lris pigment atrophy is often seen in onchocercal patients, but is also common as a senile change in noninfected persons. Some workers believe that complete loss of the iris pigment frill associated with a smoothing over of the iris crypts to produce an appearance suggestive of golden yellow blotting paper is suggestive of onchocerciasis. A fine fibrin deposit may form on a pupil margin that has been denuded of its pigment frill.

Cataract

Uncomplicated senile cataract is seen with equal flrequency in persons with and without onchocerciasis. However, the overall prevalence of cataract in all age groups is higher in populations in endemic areas. In infected persons it is commonly associated with, and secondary to, anterior uveitis.

Choroidoretinal lesions

Choroidoretinal lesions are found in all areas where onchocerclasts ls endemic, though they have yet to be reported from Yemen. They are usually associated with anterior segment lesions, particularly of the anterior uvea (9). Like the latter, they are often found in both eyes but usually the lesions have progressed to different degrees and are not bilaterally symmetrical. The lesions usually appear at the outer side ol the macula (Fig. 80) and they are also commonly found encircling the optic disc. They may coalesce to produce a large lesion at the posterior pole of the eye that surrounds both the optic disc and macula (25). The macula appears to be capable of resisting the disease process rather longer than the surrounding retina and, except in severe cases, it olten retains a relatively normal appearance asso- ciated with good visual acuity. The following clinical signs should be looked for: (l) True retinal oedema may be difficult to diagnose. The normal red reflex is lost and the retina may appear to be raised and mottled. Some- times, the nerve fibres radiating from the optic disc become abnormally prominent and this appearance is often exaggerated in fundus photographs (Fie.8l). (2) The retinal pigment epithelium is disrupted and becomes irregular. In more advanced lesions the retinal pigment has migrated, exposing the choroidal vascular pattern, usually over areas with a clearly defined and often scalloped edge. Occasionally, the retinal pigment may disappear almost completely, but usually clumps of retinal pigment remain, often at the edge of the denuded area or alongside retinal blood vessels. Two types of retinal pigment are found: (a) dense black pigment that tends to form clumps (Fig. 82), and (6) red-brown pigment spots (Fig. 83). Consecutive optic atrophy is lrequently associated with extensive choroidoretinal lesions,

28 and the optic disc presents the typical waxy appearance. However, there is often coincident inflammation of the optic nerve head, leading to typical postneuritic optic atrophy. (3) Choroidal pigment disturbance produces a granular appearance between the choroidal vessels (Fig. 8a) and the more pronounced disturbance shown in Fig. 80. Changes in the choroidal vessels lead to a progressive alteration in their colour from red to orange, yellow, and eventually white. (4) Fine yellowish dots are commonly seen lying deep in the retina in patients with and without onchocerciasis. A particular type of punctate lesion has been described in onchocercal patients and it is reported that it increases or appears for the first time following diethylcarbamazine or suramin therapy (26). Differential diagnosis. When these lesions are early and isolated none of them is pathognomonic of onchocerciasis. However, as the full picture of a Hissette-Ridley type lesion develops, particularly when associated with microfilariae in the eye and with evidence of inflammation in the cornea, iris, or optic nerve, the diagnosis becomes increasingly evident. Some diseases that should be considered in the differential diagnosis are toxoplasmosis, histoplasmosis, diffuse choroiditis, degenerative lesions, and abiotrophies such as primary retinal pigmentary degeneration (retinitis pigmentosa) and Sorsby-type lesions.

Postneuritic optic atrophy

Postneuritic optic atrophy is common in heavy infections and normally associated with lesions elsewhere in the eye. Scarring and pigment disturb- ance at the disc margin are often striking. Dense sheathing may extend along retinal vessels for several disc diameters beyond the optic disc and the retinal arterioles are usually markedly narrowed. Fine sheathing of the retinal vessels is common in normal eyes and is not of diagnostic significance. tt is likely that these lesions are preceded by episodes of papillitis or neuroretinitis; however, this stage is rarely seen (Fig. 85). Congestion of the nerve head without swelling (Fig. 8l) is more frequently encountered and may be a sign of pathological significance.

Histopathology

Reports on histological material have been published by a number of authors.r Microfilariae have now been reported in every tissue of the eye in association with ocular lesions. The microfilariae have frequently been found in perivascular spaces associated with round-cell inflammatory reac- tions. Neumann & Gunders (37) have reported a case in which microfilarial

'See, lor example, relerences 5,24,26,28,29,30,31, 32, 33,34, 35, j6,37

29 debris and round-cell infiltration lvere found around the ciliary arteries penetrating the sclera. Live microfilariae were present in the choroid and retina. They concluded that the typical posterior pole lesions are caused by microfilariae or their toxins reaching the choroid and retina by penetrating the sclera alongside the posterior ciliary arteries and nerves. Unfortunately, most eyes examined have been blind eyes, and in these late cases scarring of the tissues has occurred. There is a need to obtain eyes for histological examination at an early stage of the disease. This need might be met by an ophthalmologist attached to a general hospital situated in an area where onchocerciasis is endemic. It would be necessary to arrange a close liaison between the ophthalmologist and both the local medical staff and an ocular pathologist. It might then be possible to make an ocular examination of patients prior to death from other causes, enucleate eyes at autopsy, and later correlate the clinical and histological findings.

30 Annex I

ONCHOCERCIASIS SURVEY FORMSI

ONCHOCERCIASIS SURVEY FORM (Part 1) Identilication Country Canton, District, etc.: ...... Village ...... Geographical coordinates latitude north or south (degrees and minutes) longitude east or west (degrees and minutes) Family number...... Individual identification number Name...... Age .... (for children under I year use OO for 0 - less than Sex 6 months OX lor 6 - 12 months) Tribe ...... Place of birth

Exaninatiott Body height ...... cm Body weight kg Cross deformity and obvious incapacitation: 0 normal I blindness 2 loss of limbs 3 dwarfism 4 skeletonism 5 others: I ...... 2...... 3...... 4 ...... 5 ...... Nodules: Present (estimated total number ) Absent (give distribution diagram if needed) Other severe manifestations : 0 none I hanging groin 2 hydrocele 3 elephantiasis: site ..... other oedema: specify 5 anasarca 6 ascites 7 hernia (specrfy

1 ... 2 ... 3 ... 4... 5 ... Skin condition (list only maior changes) 1 ...... 2 ...... 3 ......

I Thcsc forms are only provisional and may be rcviscd latcr in thc light of ficld cxpcriene.

3l Laboralor y e.raninat io ns Skin snips: done not done number of microfilariae l-T-fl (examples: l6lTltl, rcft I0l,lt Iololt Urine: specimen available )es n no [--l volume l-T-|--lml haematuria present absent chyluria present absent albumen present absent blood present absent sediment microfilariae of O. volvulur present absent other microfilariae present absent (Specify ...) Schislosona hactnalobiunt eggs present E absent l---l ONCHOCERCIASIS SURVEY FORM (Part 2) ( Eye exanination hy an ophthalmologist ) Micro/ilariae in llte anterior chantberl Risht Left 0 microfilariae absent 0 I 14 microfilariae seen I 2 5-19 microfilanae seen 2 3 2049 microfilariae seen 3 4 50+ microfilariae seen 4 8 microfilariae not visible because of corneal opacities 8 9 not examined 9 Living nicrofilariae in the cornear Right Lcft 0 microfilariae absent 0 I l-4 microfilariae seen I 2 5-19 microfilariae seen 2 3 2049 microfilariae seen 3 4 50-99 microfilariae seen 4 5 100+ microfilariae seen 5 8 microfilariae not visible because ol opacificatron 8 9 not examined 9 An1, "7"r0" microfilariae in the cornea with or rtilhout reqclio,t, including nticrofilarial I bod ies w i t hin opaci t ies Right LeIt 0 microfilariae absent 0 I l4 microfilariae seen I 2 5-19 microfilariae seen 2 3 204.9 microfilariae seen 3 4 50-99 microfilariae seen 4 5 l00T microfilariae seen 5 8 microfilariae not visible because of opacification 8 9 not examined 9 Fluffy opacities (coltonwool, snowfloke) in the corneal Risht Left 0 absent 0 1 14 opacities I 2 5-19 opacities 2 3 20+ opacities 3 8 opacities not visible because of corneal opacification 8 9 not examined 9 r In ficld rnvcstigations whcre counting of mrcrofilarrae or opacitics is rmpractrcablc, recordings may be srmphfied as follows O = absent; I = few; 3 = numerous.

32 Olher torneal opacities (specily)

Rieht Left 0 absent 0 I present I 8 opacities not visible because ofcorneal opacification 8 9 not examined 9 Sclerozing keratitis Risht Left 0 absent 0 I nasal or temporal encroachment I 2 confluent semilunar, pupil free 2 3 confluent, pupil covered 3 8 cornea unrecognizable from other causes 8 9 not examined 9 Trachotna Right Lcft 0 corneal signs of trachoma absent 0 I Herbert's pits present and/or pannus encroaching less than 2 mm I 2 pannus encroaching more than 2 mm 2 -t entropion tnchiasis present, any degree 3 8 cornea unrecognizable from other causes 8 9 not examined 9 Pupil Risht Lett 0 normal 0 I pupil drlated and poorly reacting I 2 posterior synechiae present, any extent up to seclusio puprllae 2 3 seclusio pupillae 3 8 not visible 8 9 not examined 9 h'itis Right Left 0 absent 0 I acute intis, any degree I 2 torpid iritis, any degree 2 8 unrecordable because ol corneal opacification 8 9 not examined 9

Lens Rieht Left 0 clear lens 0 I early cataract, fundus detarls mostly visible I 2 advanced cataract, fundus details largely obscured 2 3 mature cataract J 4 subluxation or dislocation 4 8 not visible because ofcorneal opacification 8 9 not examined 9

Vitreous Risht Lelt 0 normal 0 I microfilariae on posterior lens surface I 2 microfi lariae in vitreous 2 3 both I and 2 3 8 not visible 8 9 not examined 9

JJ Optic disc Right Left 0 normal 0 I pahsh disc, (?) pathological condition I 2 primary optic atrophy 2 3 postneuritic optic atrophy 3 4 paprllitis 4 5 congestion of nerve head without swelling, (?) pathological 5 condition 6 glaucomatous cupping 6 8 not visible 8 9 not examined 9 Retinal vessels Rieht Left 0 normal 0 I sheathing of arteries I 2 sheathing of verns 2 3 combination of 1 + 2 J 4 narrowing of arteries 4 5 combination of 1 * 4 5 6 combination of 2 -l 4 6 7 combination ol 3 * 4 7 8 not visible 8 9 not examined 9 Choroidoretinitis ( Ridley type) Right Left 0 absent 0 I early disruption of retinal pigment I 2 as I but with exposure of choroid and clumping of retinal 2 pigment, any degree 8 not visible because of opaque media 8 9 not examined 9

Choro idore t init is ( d i s t r ibut ion ) Rieht Left 0 absent 0 I temporal to macula I 2 peripapillary 2 3 bothl*2 J 4 other locations 4 5 combrnation of I f 4 5 6 combination of 2 * 4 6 7 combrnation ol 3 * 4 7 8 not visible because of opaque media 8 9 not examined 9 Focal choroiditis ol toxoplasmic type Risht Lcft 0 absent 0 I macular I 2 elsewhere 2 3 combination of I + 2 3 Probable anolomical location of main cause of blindness or impaired vision Risht Left 1 cornea I 2 lens 2 3 anterior uvea J 4 choroidoretinal 4 5 optic nerve 5 6 phthisis bulbi 6 7 other (specify) 7

34 Annex 2

THE MEASUREMENT OF VISUAL ACUITY

Assessment of vision under field conditions is difficult. To be compre- hensive it should include visual acuity, refraction, visual fields, colour vision, and dark adaptation. When examining unsophisticated patients some of these investigations are impossible, even when the best equipment is avail- able. However, many cases of serious visual impairment caused by oncho- cerciasis will be missed if only patients with impaired visual acuity are examined ophthalmologically. The measurement of visual acuity in the open air under field conditions is not easy, but if attention is paid to the following factors it should be possible to obtain useful information. (l) The selection of testers who have the required temperament and skill. (2) The effective occlusion of the opposite eye. (3) The use of illiterate "E" or "C" tests, preferably with each size of letter on a separate square card. ( ) The satisfactory and, as far as possible, constant illumination of the test card. (5) The shading of patients from glare. Visual acuity could be measured more accurately, and other more elaborate examinations would be possible, if a specially equipped van were available to eye units. Such a van should include facilities for blackout, air conditioning, the standard illumination of tests, refraction tests, etc. Wherever the local terrain permits, it is strongly recommended that such vans should be provided.

Ocular examination lbr visual acuityt Right IrIt 0 616 0 I 6le I 2 6lt8 2 J 6160 3 4 counting fingers at 3 m, 3/60 4 5 counting fingers at I m, l/60 5 6 perception of light 6 7 no perception of light '7 8 not able to understand test 8 9 not examined 9

I Thc WHO Study Oroup on thc Prcvcntion of Blndncss (38) defincd visual impairment rn tems of vrsual acuity with both cyes. Thc corrclation betwecn thc two rcales rs as follows:

35 Yrsron wtth the bell"r Coruesponding WHO seeing eye, using Slud! Group category the above scale oJ vtsual impairtnent 3 I 4 2 5 3 6 4 7 5

36 Annex 3

RECOMMENDED DIAGNOSTIC FACILITIES FOR USE IN FIELD STUDIES

Slit lamp (in special travelling case) with accessories applanation tonometer triple mirror contact lens single mirror contact lens (gonioscope) Direct ophthalmoscope Binocular indirect ophthalmoscope with condensing lens Loupe E-test cards Blackout curtains. It is implicit in the appointment that an ophthalmologist recruited for onchocerciasis surveys should have received preliminary training in the field. If ophthalmological work is to be performed by general practitioners, it is assumed that they will have had adequate training in both general ophthal- mology and the problems of onchocerciasis. Some work can be delegated to specially trained paramedical personnel. but only under medical super- vision.

JI ACKNOWLEDGEMENTS

The World Health Organization wishes to express its appreciation to Dr H. Connor lor providing the photographs for Fig. l-52, 58-60, and 62-'71 (distinguished by a Unrted States Armed Forces Institute of Pathology number (AFIP)), and Dr J. Anderson for providing all the colour photographs (Fig. 73 85). Fig. 61 is reproduced from worms in disease; a manual of medical helminthology by Dr R. L. Muller by kind permission of the publishers, Heinemann Medical Books, London. The photograph used for Fig. 23 was kindly made available by Dr H. Fuglsang.

38 REFERENCES

(l) WHO Expert Committee on Onchocerciasis (1966) Wld Hlth Org. techn. Rep. Ser., No. 335 (2) Nnochiri, E. (1964) Observations on onchocercal lesions seen in autopsy specimens in Western Nigeria, Ann. top. Med. Parasit.,58, 89-93 (3) Israel, M. S. (1959) The nodule in onchocerciasis, Trans. ro1,. Soc. trop. Med. Hy,g., 53, 142-147 (4) Duke, B. O. L. (1970) Onchocerciasis : deep worm bundles close to hrp joints, T'arrs. t'q,. Soc. trop. Med. Hyg.,64,791-792 (5) Hissette, J. (1932) M6moire sur l'Onchocerca volvulus "Leuckart" et ses manrfesta- tions oculaires au Congo belge, Ann. Soc. belge Mid. trop., 12,433-529 (6) Lagraulet, J., Monjusiau, A. & Durand, B. ( I 964) Etude des localisatrons des nodules dans l'onchocercose; leurs relations avec le lieu de piq0re des simulies, Mid. trop.. 24, 566-572 (7) Anderson, J., Fuglsang, H. & Zubaidy, A. (1973) Onchocerciasis in yemen with special reference to Sowda, Trans. roy. Soc. trop. Med Hyg.,6j,3C_3l (8) Buck, A. A. et al. (1969) Onchocercrasis: some new epidemiologic and clnical findings, Anter. J. trop. Med. Hyg.,18,217-230 (9) Quer6, M. A. et al. (I963) Etude statistique sur la fr6quence et la sp6cificit6 des compli- catrons oculaires de I'onchocercose, Bull. Soc. mid. Afr. noire Langue frang., B, l-26 (10) Roberts, J. M. et al. (1967) Onchocerciasis in Kenya, 9, Il, and lg years afrer elrmrnation of the vector, Bull. I4/ld Hlth Org., 37, 195-212 (ll) Figueroa, M. H. (1972) Enfermedad de Robles. como saler Ias microfilarras del oncocercoma, Rey. Invest. Salud pilbl. (Mex.),32,9 l9 (12) Neafie, R. C. (1972) Morphology of Ont.hocerta.t'olt'ulu.s, Amer. J. ctin. path., S7, 574 586 (13) Gaspannr, G. (1962) "sowda" nuova malattra o forma inedita di onchocercosi?, Arch. ital. Sci. med. trop.,43,635-646 (14) Nelson, G. s. (1958) Hanging groin and hernia, compLcations of onchocerciasrs, Trans. ro)'. Soc. trop. Med. Hyg.,52,272-275 (15) connor, D. H. et al. (1970) onchocercrasis: onchocercal dermatitrs, lymphadenitis and elephantiasis rn the Ubangi Territory, Hum. path., 1,553-579 (16) Buck, A. A. et al. (1971) Microfilaruria in onchocerciasis, Bull. Wtd Hlth Org.,45, 353-369 (17) Duke, B. o. L. (1962) A standard method olassessing mrcrofilarral densitres on onchocerciasis surveys, Bull. Vr'ld Hlth Org.,27,629-632 (18) Ptcq, J. J., Coz, J. & Jardel, J. P. (1971) Une m6thode d'6valuation des densit6s mrcrofilarres d'o. volvulus Leuckart, 1893 chez des onchocerquiens: technique et temps de lecture des biopsies cutan6es, Bull. llld Hhh Org.,45, 517-52O (19) Meyers, W. M. et al. (1972) Human streptocerciasis, Anter. J. trop. Med.Hyg.,2l, 528-545 (20) Kershaw, W. E., Duke, B. O. L., & Budden, F. H. (1954) The distribution of micro- filariae of O. yolvulus in the human skin, Brir. med. I.,2,724-729 (21) Mazzotti, L. (1948) Possibilidad de utilizar como medio dragnostico en la oncho- cercosis, las reacciones alergicas consecutivas a Ia administracron de "Hetrazan", Rev. Inst. Salubr. Enferm trop. (Me.r.),9,235,231- (22) Anderson, J. & Fuglsang,H. (1973) variation in numbers of microfilariae of oncho- cerca volvulus in the anterior chamber of the human eye, Trans. roy. soc. trop. Med. Hye., 67, 544-548 (23) Anderson, J. & Fuglsang,H. (1973) Living microfilariae of onchocerca volvulus in the cornea, Brit. J. Ophthal.,57,712

39 (24) Cordero-Moreno, R. (1973) Etrologic factors in tropical eye diseases. Anter. J. Ophthal., 75, 349-364 (25) Rldley, H. (1945) Ocular onchot'erciasis, including an investigatiott in the Gold Coasr. London, Pulman (monograph supplement to the Brit. J' Ophthal') (26) Vedy, J., Sirol, J. & coulm, J. (1971) La r6tinopathie ponctu6e albescente oncho- cerquienne, Bull. Soc. Poth. exot.,9,479486 (27) Giaquinto, M. (1934) Pr6sence de microfilaires d'Onchocerca (aecutiens dans Ie nerf optique, Rif. med., 50, 858-861 (28) Biyant, J. (1935) Endemic retino-choroidrtis in the Anglo-Egyptian Sudan and its poisible relationship to Onchocerca t'olvulus, Trans' roy' Sot" trop' Med' Hl's'' 28' 523-532 (29) Semadeni, B. (1943) Histologischer Befund bei einem Fall von zahlreichen Mikro- filarien beider Atgen, Schweiz. med- Lltschr.' 73,75-7'7 (30) Clark, W. B. (1947) Ocular onchocerciasis in Guatemala: an investigation of 215 natives infected with Ont.hocerca volt'ulus, Trans. Amer. J. Ophthal. Soc., 45,461 501 natives infected with onchocerca volvulus, Trans. Amer. J. Ophthal. soc.,45,461-501 (31) Lavier, c. et al. (1956) Etude anatomo-pathologique d'un eil presentant une chorio- retinite onchocerqulenne, Bull. Soc. Path. exot., 49, 434438 (32) Offret, c. et al. (1958) Etude histopathologique d'un ceil onchocerquien, Bull. Sot. Ophtal. Paris,71, ll0 ll3 (33) D'Haussy, R. et al. (1958) Contribution ?r l'6tude des l6sions du fond de l'ceil dans

f 'onchocercose, Mid. trop., 18' 340-367 (34) Lagraulet, J. (1958) A propos de I'onchocercose oculatre en Am6rique et en Afrique, Bull. Soc. frang. Ophtal.,7l,266 276 (35) Rodger, F. C. (1960) The pathogenesis and pathology of ocular onchocercrasis. lv The pathology, Amer. l. Ophrhal.' 49,560-594 (36) paul, E. V. tZimmerman, L. E. (1970) Some observations on the ocular pathology of onchocerctasis, Hunr. Path., l, 581-594 (37) Neumann, E. & cunders, A. E. (1973) Pathogenesis of the posterior segment lesion ofocular onchocerciasrs, Anter. J. Ophthal.,75' 82-89 (38) WHO Study Group on the Prevention of Blindness (1973) Wld Hlth Org. te<'hn' Rep' Ser., No. 518 (39) Mazzottr, L. (1959) Presencia de microfilarias de onchocerca tolvulus en el liquido celalorraquideo de enfermos tratados con hetrazan, Rev. Ittst. Salubr. Enfarn. trop' ( Mex.) , 19, I 5 (40) Rodhain, J. & Cavfllov' W. (1935) Un cas de localisation profonde de"MicroJilaria volvulus", Ann. Soc. belge Mdd. trop.,15,551-560 (41)Thomas,D.B..Anderson,R.l.&MacRae,A.A.(1973)Microfilariaeofonchocerca volvulus tn a vaginal irrigation specimen' Anter. J' Parasit',59,941 942 (42) Fuglsang, H. & Anderson, J. (1973) The effect of diethylcarbamazine and suramin on Oncltocerca yolt,ulus microfilariae in the urine-preliminary results, Lanrct, 2, 321-322 (43) Buck. A. A. (1973) Mrcrofilaruria in onchocerciasis in Africans, Z. Tropennted. Parasir.,24, 336-339 (44) Dyce Sharp, N. A. (1926) Communtcations: contribution to the study of onchoterca til,ulus Leuckart, with some observattons on its prevalence rn Nigeria, Trans' ro.r" Soc. trop. Med. HYg.,19, 373-388 (45) Price,O. L.rtsotlTheoccurrenceof microfilariae of oncho(ert'at'olvulus inurineof rnfected individuals, J. Parasit., 47, 572 Haarlem. (46) Oomen, A. P. (1969) Srudies on onchocerciosis and elephantiasis in Ethiopia, De Erven F. Bohn N. V. (411LberiaResearchInstrtute(1969)In: FourthSenti-qnnualReportoJtheBernhard-Notht Inliruta lbr Maritime and Tropit'al Diseases, Hamburg (48) Anteson, R. K. (1972) Studies in filariasis in Ghana. l. Preliminary survey of human filarral inlections. concurrence of diurnal and non-periodic forms, Ghana nted. J.. 1t.146-148

40 I L LU STRATI ON S Fig. 1. AcoilednongravidadultfemaleO.volvulusinthewall ofanaorta. Theworm lies in the media and has disrupted the smooth muscle an$ the elastic fibres; scartissue has formed around the worm, thickening the overlyin! intima (Movat stain, x11' AFrP 73-61 57)

r3

Fig.2. A patient resident in the Ubangi Territory of Zai{e with onchocercal nodules over the elbows, iliac crests, trochanters, and the left knee. The papules on the arms and thighs and the depigmentation of the penis are manifestations of onchocercal dermatitis (AFl P 68-7638-3)

-43- iit ::i

Fig. 3. Several adult O. volvulus worms coiled within a nodule. The hyalinized scat tissue surrounding the worms incarcerates them, preventing their migration (Movar stain, x7.6; AFIP 69-3639)

,' ,, -.. . r -l .3' E3-t. ik.-. :lM&f i-t- \W)

#ry$f'1., tf

Fig. 4. Mass of entangled and intertwined male and female O. volvulus worms remain- ing after the host tissue has been digested away from a nodule ( x3.6; AFIP 69-9753)

Fig. 5. Fragments of degenerating adult O. volvulus worm within an abscess in the centre of a nodule (Movat stain, x19; AFIP 69-501 8) _44_ s

i s *$ Fig. 6. A layer of acidophilic material, presumably immune complex, forming around a worm (arrowed). Clusters of polymorphonuflear leucocytes are digesting this material (Movat stain, x145; AFIP 69-5022)

ra'" Fig.7. Degenerating microfilariae within a nodule; thes! microfilariae are surrounded by epithelioid and giant cells (Movat stain, x485; AFIP 72 -1729O) rs. qE b a t

r{} t # \ {.", ft& q LJ

*. 1 k rt

Fig.8. Transverse section through the anterior end of an apult gravid female O. volvulus worm, showing a small digestive tube and the vulva (arrovled) (haematoxylin and eosin, x450; AFIP 69-5925) -45- .'.

Fig. 9. Uneven pigmentation of the skin is an early change in onchocercal dermatitis. As this figure shows, uneven pigmentation may be seen more clearly in overexposed photographs (AFIP 68-1 0067-1 )

Fig. 10. ln early onchocercal dermatitis there is usually, as seen in this figure, slight acanthosis with elongation of the rete ridges, uneven pigmentation of the basal layer, inflammatory cell infiltrates mainly about the small dermal vessels, and a few micro- filariae (arrowed) within the dermis (haematoxylin and eosin, x100; AFIP 67-3526)

-46- t * k' i*t,

''|k l rq 'a, /r, l ,rk&1,,*** &

Fig. 1 1. The anteriorend of an O- volvulusmicrofilaria has a cephalic clearspace (seen here) between 7 gm and 13 pm long; the anterior nuclei (arrowed) are situated side by side (haematoxylin and eosin, x1080; AFIP 71-10O72)

r

A # w , r I .', ., w l! -- !- .b q , '*;'',, & h ...&, L ry{ F ; * *.*

#l' -; 1r * t Fig. 12. The posterior end of the O. volvulus microfila is finely tapered and there is a caudal clear space 9-18 pm long. The terminal nu form a single row (arrowed) and are thin and elongated (haematoxylin and eosin, x 080; AFIP 71 -10073)

-47- t ,

Fig. 13. Collection of lymphocytes around the small dermal vessels, the first micro- scopic change usually detected in early mild onchocercal dermatitis (haematoxylin and eosin, x395; AFIP 69-4951 )

Fig. 14. Fibrosis of the dermis with the presence of large numbers of fibroblasts (anowed), some of which are large and have pointed processes, is a prominentfeature of onchocercal dermatitis (haematoxylin and eosin, x600; AFIP 69-4939)

-48_ Fig. 15. A band of scar tissue (arrowed) has replace the norma loose areolar connective tissue of the dermal papillae (Movat stain, x 1 ; AFIP 69-

,' ,ls

T .}

?

Fig. 16. A coiled microfilaria in the u pper dermis immediately beneath the basal lay3r (haematoxylin and eosin, x350; AFIP 69-8688)

-49- < ) l-.'

,

t \

Fig. 17. A female patient from the Ubangi region of Zaire with papular eruptions of the upper trunk, arms, and neck; this is a less common manifestation of onchocercal dermatitis (AFIP 68-7835-6)

t

c..

Fig. 18. The forearm of a patient from Guatemala with a papular eruption. The papules are more widely separated than in the patient shown in Fig. 17 and some of them are pustular and scaling (AFIP 72-17116)

-50 W

&

Fig. 19. This male patient has severe onchocercal dermatitis with depigmentation of the legs and secondary infection resulting from scratching (AFIP 68-8080-1 )

Fig.20. ThesamepatientasinFig. 19. Treatmentofboththeonchocercal dermatitis and the secondary infection has produced rapid healing (AFIP 68-8080-2)

_ -sl _ Fig.21 . Section of a papule revealing an intra-epidermal abscess containing many polymorphonuclear leucocytes, fibrin, and degenerating epithelial cells (haematoxylin and eosin, x12; AFIP 69-5428)

F..!r

Fig. 22. A small intra-epidermal abscess containing a microfilaria. Two tangential sections of a single microfilaria are visible; one part is entirely within the abscess (upper arrow), the other part (lower arrow) traverses the wall of the abscess. The roof of the abscess is the keratin layer (haematoxylin and eosin, x250; AFIP 72-17172)

<1 w { - sr, $.ii::i

auilr , f;

.J

--*l Fig. 23. A boy from Yemen with the characteristic manifestations of sowda, i.e., involvement of a single extremity, darkened skin, a papular eruption, swelling of the limb, and enlarged femoral nodes

, .:]

Fig.24. Papular eruption of sowda ("1.22; AFIP 72-17181) 53- [,

$

\ \

\

* Fig.25. Microfilariae are only rarely seen in biopsy specimens taken from patients with sowda. This microfilaria is in the mid-dermis and has provoked no focal reaction. Two cross-sections (arrowed) and a long tangential section of the microfilaria are visible (Giemsa, x750; AFIP 72-4538)

,:.:" -: '.t i

Fig. 26. The skin of this patient is oedematous and focally depigmented; his femoral and inguinal nodes are enlarged. One inguinal node when removed revealed the advanced changes of onchocercal lymphadenitis (AFIP 68-7912-1)

-54- :"'{4"

Fig. 27. section of skin from a boy revealing atrophy of the epidermis, diff use oedema of the dermal collagen, many large fibroblasts, and large numbers of microfilariae (haematoxylin and eosin, x145; AFIP 68-9448)

(

Fig. 28. "Lizard skin" characterized by scaling and altered pigmentation. An oncho- cercal nodule is present on the leftside of the ihest (arrowedf (nrrp 69-9769)

-55- (-

Fig. 29. "Lizard skin" is associated microscopically with the separation of the keratin (a"n almost constant feature), atrophy of the epidermis, and chronic inflammation of the dermis (haematoxylin and eosin, x50; AFIP 73-6043)

.-*

Fig. 30. ln "lizard skin" there is also a reduction of the elastic fibres in the upper dermis. Th-e elastic fibres (black) visible in this section are in the mid-dermis in the lower part of the picture (Movat stain, x 115; AFIP 71-12155)

-56- |:

Fig.31. "Leopard skin", characterized by focal depigmentation, is most common on the shin. Small foci of persistent pigment are visible around the pores and hairfollicles (AFIP 72-17223)

Fig. 32. The depigmented areas are depressed, reflecting atrophy and un- derlying fibrosis (AFIP 68-10071-2)

-57- Fig. 33. The skin of the lower thighs and knees hangs in loose redundant folds (AFIP 72-4500-A)

Fig. 34. Reticulum (precollagen) is a prominent feature of onchocercal dermatitis. ln this section the reticulum fibres, stained black, are distributed throughout the dermal collagen and have a circular orientation around the small dermal vessels (reticulum stain, x115; AFIP 67-3532)

-58- Fig.35. The reduced elastica in the upperdermis is associated with the disorganization of the elastica in the mid and lower dermis; knots or entanglements (arrowed) sometimes form (Movat stain, x 130; AFIP 67 -7485)

Fig. 36. Lymph node from a patient with severe onchocercal dermatitis and elephan- tiasis of the genitalia revealing fibrosis of the capsule and the trabeculae (large arrows). The superficial and deep lymphatics are dilated (small anow) and the subcapsular lymphatics contain histiocytes (haematoxylin and eosin, x12; AFlp 69-9521)

-59- .{ *

t.

*' il .x

Fig.37. ln this section of a lymph node cross-sections of a degenerating microfilaria are visible (arrowed) and are surrounded by eosinophilic leucocytes, plasma cells, and a few lymphocytes (haematoxylin and eosin, x485; AFIP 69-8694) -t* ....,a .t t) ,tare 3r,' {b! ;.\ _b

r{ it r; , .-,4 'i&& 'N43 "r#

Fig. 38. A degenerating microfilaria (arrowed) surrounded by epithelioid cells, giant cells, and fibrous tissue (Giemsa, x375; AFIP 69-8464)

-60- *

I

R

lse

Fig. 39. Femoral and inguinal lymph nodes removed from patients with sowda ate large,soft,andwithoutgrossevidenceof scarringorfocal lesions(x!.21; AFlP72-4555)

I

Fig. 40. Section of lymph nodes from patients wilh sowda reveal follicular hypoplasia (in contrast to the nodes of onchocercal lymphadenitis in African patients) and an infiltrate of plasma cells and histiocytes (haematoxylin and eosin, x50; AFIP 73-4620)

-61 - ."*r a

:-'-;fu :. F,

Fig.41 . Hanging groin may be unilateral or bilateral and overlies a cluster of fibrotic femoral or inguinal nodes (AFIP 70-3172)

I

&. l

Fig. 42. A patient with unilateral hanging groin overlying femoral'lymph nodes showing the advanced changes of onchocercal lymphadenitis (AFIP 68-1 0066-1 )

-62 .l:,

Fig. 43. Elephantiasis of the genitalia and severe lymphoedema of the skin in a boy. They are associated with advanced onchocercal lymphadenitis of the inguinal lymph nodes (AFIP 68-7912-2)

I t I t

Fig. 44. Enlarged scrotum in a patient with severe onchocercal dermatitis. The enlargement had continued steadily over a period of several years and at the time of its removal the scrotum weighed 18 kg (AFIP 68-8582)

-63- 4' .* t r ;

I

, t ,L

Fig.45. A coiled microfilaria in a hepatic sinusoid; it has provoked no reaction (haema- toxylin and eosin, x650; AFIP 73-4628)

t

( Fig.46. Section through the capsule of the liver revealing numerous microfilariae (arrowed) that have apparently caused the fibrous thickening of the capsule and an inflammatory cell infiltrate (haematoxylin and eosin, x195; AFIP 72-17568)

t

-64- w

Fig. 47. A glomerulus containing many microfilariae of O. volvulus (arrowed). All lie within the capillary tuft. The glomerulus and the surrounding tissues also contain an inflammatory cell infiltrate (haematoxylin and eosin. x305; AFIP 73-4627)

:d

Fig.48. Section of kidney with a microfilaria in a tubule (haematoxylin and eosin, x675; AFIP 71-12149)

_65_ \ a

L

a .r? .'. .> a fi # t /. t, *$ l ,CL

Fig. 49. Section of lu ng with a microf ilaria ol O. volvulus surrou nded by a few inf lam- matory cells (haematoxylin and eosin, x675; AFIP 71-1215'l)

Fig.50. Section of a lymph node revealing marked interstitial fibrosis with bands of fibrous tissue containing dilated lymphatics. The scar tissue tends to have a circular arrangement around the blood vessels (arrowed); this is seen also in the skin (haemato- xilin and eosin, x60; AFIP 70-9313)

-66- ;*^

. *{; f..\

Fig. 51. Section of skin with dilated lymphatics in the upper dermis containing micro- filariae of O. volvulus (haematoxylin and eosin, x 195; AFIP 70-7349)

Fig. 52. Vessel containing a microfilaria in a lymph node. The anterior and posterior ends of the microfilaria (large and small arrows, respectively) are both visible (haemato- xylin and eosin, x650; AFIP 70-11370)

-67 - !;t.55 ,,

,. :t ,, ai la: !) t( s ,t 0r

Fig. 53. lnstruments for obtaining skin snips: Holth type (left) and Walsertype (centre and right) sclerocorneal punches (diameter of punch approximately 2.3-2.5 mm)

Correct depth for skin snip

wHo 30513 Fig. 54. ldeal depth of skin snip for the assessment of O. volvulus microfilariae

-68 90

80

70 o

60

N o50 I E

.9 40 =

30

20 o

10

tyHo 40030 0 0 10 20 40 50 60 70 80 90 100 110 120 Emergence lime (min)

Fig. 55. Time distribution of O. volvulus Fig. 56. Recommended sites for skin microfilariae emergence from skin snips biopsies in routine examinations lor and corresponding cu mulative freq uencies O. volvulus microfilariae

( skin snips

/ cn wHo 40031 /t Fig. 57. Schema for multiple skin snips from the same general site

-69- Fig. 58. Section of skin through a papule revealing a coiled adult Dipetalonema strcptocerca in the superficial dermis surrounded by an inflammatory cell exudate (haematoxylin and eosin, x110; AFIP 73-1230)

#ry, n s \&M

Fig. 59. The microfilariae ol Dipetalonema strcptocerca have a short cephalic clear space about 5 pm long and the anterior nuclei are staggered (arrowed); they are not side by side or overlapping as the anterior nuclei of O. volvulus microfilariae (see Fig. 1 1 ), Furthermore, the nuclei approaching the anterior end of the D. streptocerca microfilariae form a single column; this is quite different from the arrangement of these nuclei in O. volvulus microfilariae (haematoxylin and eosin, x1080; AFIP 71 -10075) 70_ ffi W;'WN

W W Y ,xp 3r

,& w ,A 7 ',.,,W

& r'! m Y r *d.4&,x r*" . * w Fig.60. ln the posterior end of a D. strcptocerca microfilaria the nuclei form a single column. These nuclei are short and quadrate and go to the tip of the tail, leaving a caudal clear space of only 1 pm (anowed); this is in contrast to the posterior end of O.volvulus microfilariae (see Fig. 12) (AFIP 71-8341)

50!m

Drawr by D'f. L. Mull-"r, Londo. Schoo o{ Hv(rene and Troorcal Medrcrne

Fig. 61 . Microfilariae of O. volvulus (top) and D. stteptocerca (bottom)

- 7t - 4- -

Fig. 62. Section of lymph node removed from a patient after a course of diethylcarba- mazine; the capsule is oedematous and inflamed and there are abscesses in the sub- capsular lymphoid tissue (arrowed) (haematoxylin and eosin, x45; AFIP 73-5695)

I

Fig. 63. Enlargement of the abscesses in Fig. 62 revealing large numbers of degener- ating microfilariae surrounded by inflammatory cells including polymorphonuclear neutrophils, histiocytes, and a few lymphocytes (Giemsa, "440; AFIP 73-5693) -72- Fis. 64. Extensive desquamation of the skin of the thigh after the patient was treated with diethylcarbamazine (AFl P 7 2-17 247 )

C\ \i ? ,i a

q \ sl &

q' aa-., .$.

'n{n*,*. ix-_* &*$* t Fig.65. Biopsy specimen taken t hour after an initial dose of diethylcarbamazine revealing numerous sections of a degenerating microfilaria in the dermis. Eosinophilic leucocytes surround the microfilaria (haematoxylin and eosin, x575; AFIP 69-9527)

-73- .8 '- .-i8$s-i:.i$'" ...*- ; :$&; ''' ,.rt d *':. I ) -! "f,r: -.r (F "c 'rt d! v .*, !& i'' ti . 'lqp - ,;T.+ I ^**d' /,.sr -.d!$::: ' '!....\ &,- -$si|I!}l' f { $ alt , .(d t \ ]u $,a 3 Fig. 66. Section of skin sample taken 3 days after the patient received diethylcarba- mazine revealing fragments of a degenerating microfilaria (arrowed) surrounded by eosinophilic leucocytes that have disintegrated releasing granules into the adjacent tissues (haematoxylin and eosin, x525; AFIP 70-7347)

i rp r !r.

; M l \ N n

Fig. 67. Skin specimen taken 24 hours after the patient was treated with diethylcarba- mazine. The degenerating microfilaria is surrounded by histiocytes, epithelioid cells, and giant cells (haematoxylin and eosin, x850; AFIP 69-4938)

-74- ..*

I

Fig. 68. The anterior ends of several microfilariae (arrowed) have penetrated the basal Iayer and are beginning their migration into the epidermis. This specimen was obtained 5 hours and 20 minutes after the patient received an initial dose of diethylcarbamazine (haematoxylin and eosin. x300; AFIP 73-5689)

rJr t,

I :.n'\ ,,.f .'f -j'';

Fig.69. Aspecimen obtained Tdaysaftertheinitialdoseof diethylcarbamazinereveals a clearing of the microfilariae from the dermis but occasional coiled microfilariae (arrowed) are trapped within the keratin (haematoxylin and eosin, x 145; AFlp 68-9446) _75_ t t,

\)

Fig.70. Skin specimen obtained 5 days after the initial dose of diethylcarbamazine revealing a microfilaria migrating into the epidermis (arrowed). This migration has caused inter-epidermal oedema and an intra-epidermal migration of polymorphonuclear neutrophils (haematoxylin and eosin, x450; AFIP 72'3237)

I

Fig.71. Migrating microfilariae in this skin specimen have provoked the formation of intra-epidermal abscesses. The specimen was obtained 48 hours after the initial dose of diethylcarbamazine (haematoxylin and eosin, x 1 1 5; AFIP 73-5682)

!::. lT Ttffffiffi '.:t',2,i::z::; (a) (b) (c) (d) (e) (f) (s) (h) (i) (i) wHo 40033 Fig.72. Schematic representation of reactions in the cornea forming around individual deid microfilariae. (a) Microfilaria without reaction; (b)-(f) varying degrees of reaction; (g) and (h) fully developed fluffy opacity within the body, almost absorbed in (h); (l) and (i) resulting opacities.

-76- 'il

.!i

{ t

.-"!

ily,q1qn*1,*1',

Fig. 73. Punctate keratitis Fig.74. Limbal haze of early sclerozing keratitis

\ \

I ,fn' I ;

I I t*&-, * I j

Fig. 75. White area of stromal keratitis in front Fig. 76. Thick stromal vessels in the cornea of pigmented zone containing over 1 000 microfilariae

-77 - {

Fig. 77. Confluent opacification in sclerozing keratitis

f til

J a

. :G" .-. i '''

I

Fig.78. Advanced sclerozing keratitis

3 i*iltts. ] ttril|!, ,r., t -

Fig. 79. Slit lamp photograph showing corneal opacifications of the lower half with only discrete opacities above

-78- Fig. 80. Extensive fundus lesion temporal to macula

ire

Fig. 81. Congestion of the optic disc and the prominent sweep of nerve fibres

=-

F55 * E: +ts- : i'';.' i*'

Fig. 82. Large clumps of retinal pigment associated with extensive m* ch oroidoretin itis

-79- Fig. 83. Red-brown pigment spots in inflammatory patch temporal to the macula

'/ I /

E

Fig, 84. Exposure of choroidal pigment temporal to the macula

Fig. 85. Papillitis

-80- WHO publications may be obtained, direct or through booksellers, from

ALGERIA Soci6t6 Nationale d'Edition et de Diffusion. 3, bd Zirout Youcef, Arcrrns. ARGENTINA Libreria de las Naciones, Cooperativa Ltda, Alsina 500, BupNos Arnrs Editorial Sudamericana S.A., Humberto 1o 545, BurNos AInrs. - AUSTRALIA Australian Government Publishing Service, Sales and Distribution, P.O. Box 84, CANBERRA, A.C.T. 2600 (mail orders); AGPS Book Centre, ll3-115 London Circuit, CANBERRA Ctrv; 347 Swanston St., MELBoURNT; Commonwealth Centre, l-3 St George's Terrace, Penru; Bank House, 315 George St., SyoNry- Hunter Publi- cations, 58a Gipps Street, Cou-rNcwoop, Vic. 3066. AUSTRIA Gerold & Co., I. Graben 31, VrrNNl l. BELGIUM Office international de Librairie, 30 avenue Marnix, BRUssELs. BRAZIL Biblioteca Regional de Medicina OMS/OPS, Unidad de Venta de Publicaciones, Caixa Postal 20.381, Vila Clementino, 01000 Sio Pruro, S.P. BURMA see lndJa, WHO Regional Office. CANADA Information Canada Bookstore, l7l Slater Street, OrrAwA, Ontario KIA OS9 1683 Barrington Street, HALTFAX, Nova Scotia B3J 129; Edifice Aeterna-Vie, I182; ouest, rue Ste-Catherine, MoNrnrlr, ll0 (Qu6.); 221 Yotge Street, ToRoNro 205, Ontario;657 Granville Street, VANcouvrn 2, B.C.; 393 Portage Avenue, WrNNlerc, Manitoba. CHINA China National Publications Import Corporation, P.O. Box 88, PEKrNc. COLOMBIA Distrilibros Ltd, Pio Alfonso Garcia, Carrera 4a, Nos 36-119, CrnucrNr. COSTA RICA Imprenta y Libreria Trejos S.A., Apartado 1313, SaN Jos6. CYPRUS MAM, P.O. Box 1674, Nrcosrl. DENMARK Ejnar Munksgaard, Ltd, Norregade 6, ConrNHacrN. ECUADOR Libreria Cientifica S.A., P.O. Box 362, Luque 223, Guayaeurr-. EGYPT Nabaa El Fikr Bookshop, 55 Saad Zaghloul Street, ALEXANDRTA. FIJI The WHO Representative, P.O. Box 113, Suve. FINLAND Akateeminen Kirjakauppa, Keskuskatu 2, HrlsrNxr 10. FRANCE Librairie Arnette, 2, rue Casimir-Delavigne, PARrs 6". GERMANY, Govi-Verlag GmbH, Beethovenplatz l-3, FnaNxrunr a. M. 6 W. E. Saarbach, FEDERAL Postlach 1510, Follerstrasse 2, 5 ColocNr I Alex. Horn.- Spiegelgasse 9, 62 REPUBLIC OF WTESBADEN- - GREECE G. C. Eleftheroudakis S.A., Librairie internationale, rue Nikis 4, ATHENS (T. 126\. HAITI Max Bouchereau, Librairie "A la Caravelle", Boite postale 111-8, Ponr-au-PnrNcs. HUNGARY Kultura, P.O.B. 149, BuDApEsr 62 - Akad6miai K6nyvesbolt, Vdci utca 22, BuoaprsrV. ICELAND Snaebjorn Jonsson & Co., P.O. Box l l3l, Hafnarstraeti 9, Revxra.vx. INDIA WHO Regional Office for South-East Asia, World Health House, Indraprastha Estate, Ring Road, Nrw Drlur 1 Oxford Book & Stationery Co., Scindia House, New DelHr; 17 Park Street, Cllcurra- l6 (Sub-agent). INDONESIA see India, WHO Regional Office. IRAN Mesrob Grigorian, Naderi Avenue (Arbab-Guiv Building), TEHERAN. IRELAND The Stationery Office, Dunr-rN. ISRAEL Heiliger & Co., 3, Nathan Strauss Street, JERUsALEM. ITALY Edizioni Minerva Medica, Corso Bramante 83-85, TuRtN; Via Lamarmora 3, Mrur.t. JAPAN Maruzen Co, Ltd, P.O. Box 5050, ToKyo International, 100-31. KENYA The Caxton Press Ltd, Head Office: Gathani House, Huddersfield Road, P.O. Box 1742, Narnosr. KHMER The WHO Representative, P.O. Box ll1, PHNoU-PrNu. REPUBLIC LAOS The WHO Representative, P.O. Box 343, VrrNrrlNr. LEBANON Documenta ScientificaiRedico, P.O. Box 5641, Bsrnur. LUXEMBOURG Librairie du Centre,49, bd Royal, Luxruaounc. MALAYSIA The WHO Representative, P.O. Box 2550, Ku,ll,r. Luupun Jubilee (Book) Store Ltd,97 Jalan Tuanku Abdul Rahman, P.O. Box 629, Kuara- Luupun. Parry's Book Center, K.L. Hilton Hotel, Kuua. Lunpun. MEXICO La Prensa M6dica Mexicana, Ediciones Cientificas, Paseo de las Facultades 26, MExrco Crrv 20, D.F. MONGOLIA see lr,dia, WHO Regional Office. MOROCCO Editions La Porte, 281 avenue Mohammed V, R.ln,lr.

Price: Sw. fr. 72.- Prices are subject to change without notice. WHO publications may be obtained, direct or through booksellers, from

NEPAL sae India, WHO Regional Office. NETHERLANDS N.V. Martinus Nijhoff's Boekhandel en Uitgevers Maatschappij, Lange Voorhout 9, THE H.acrrE. NEW ZEALAND Government Printing Office, Government Bookshops at: Rutland Street, P.O. Box 5344, Aucrl.rrNo; 130 Oxford Terrace, P.O. Box 1721, CHRrsrcHuncr; Alma Street, P.O. Box 857, HnurlroN; Princes Street, P.O. Box 1104, Durr,rorN; Mulgrave Street, Private Bag, WrrrrNcroN R. Hill & Son Ltd, Ideal House, Cnr. Gilles Avenue & Eden St., Newmarket, AucruNn- S.E. l. NIGERIA University Bookshop Nigeria, Ltd, University of Ibadan, Ia,loa.N. NORWAY Johan Grundt Tanum Bokhandel, Karl Johansgt. 43, Osr-o 1. PAKISTAN Mirza Book Agency, 65 Shahrah Quaid-E. Azam, P.O. Box 729, Llnons 3. PARAGUAY Agencia de Librerias Nizza S.A., Estrella No. 721, AsuNcr6N. PERU Distribuidora Inca S.A., Apartado 3l15, Emilio Althaus 470, Lrrve. PHILIPPINES World Health Organization, Regional Office for the Western Pacific, P.O. Box 2932, MlNrr,r The Modern Book Company Inc., P.O. Box 632, 926 Rizal Avenue, MaNra. - POLAND Skladnica Ksiggarska, ul. Mazowiecka 9, W.r,rlstw ( except periodicals) Ruch, ul. Wronia 23, WARsAw (periodicals only). -BKWZ PORTUGAL Livraria Rodrigues, 186 Rua Aurea, LrssoN. REPUBLIC OF The WHO Representative, Central P.O. Box 540, Sroul. KOREA REPUBLIC OF The WHO Representative, P.O. Box 242, SrrcoN. VIET.NAM SINGAPORE The WHO Representative, 144 Moulmein Road, G.P.O. Box 3457, SrNclpoxB l. SOUTH AFRICA Van Schaik's Bookstore (Pty) Ltd, P.O. Box 724, PREroRrA. SPAIN Comercial Atheneum S.A., Consejo de Ciento 130-136, BARcELoNA 15; General Moscard6 29, Meouo 20 - Libreria Diaz de Santos, Lagasca 95, M,lonlo 6. SRI LANKA see lndia, WHO Regional Office. SWEDEN Aktiebolaget C.E. Fritzes Kungl. Hovbokhandel, Fredsgatan 2, SrocrHor.u 16. SWITZERLAND Medizinischer Verlag Hans Huber, Liinggassstrasse 76,3012 BsnNs 9. THAILAND see India, WHO Regional Office. TUNISIA Soci6t6 Tunisienne de Diffusion, 5 avenue de Carthage, TuNrs. TURKEY Librairie Hachette, 469 avenue de I'Ind6pendance, Isr,rNnur-. UGANDA see address under Ksxyn. UNITED H. M. Stationery Office; 49 High Holborn, LoNpoN WCIV 6HB; l3a Castle Street, KINGDOM EotNsuncH EH2 3AR; I09 StMaryStreet,CARDrFFCFl IJW; 80Chichesterstrect, Brrr,c.sr BTI 4JY; Brazennose Street, MANcHEsrrn M60 8AS; 258 Broad Street, BtnMrNcg.ql"l Bt 2HE; 50 Fairfax Street, BRrsroL BSI 3DE. All mail orders should be sent to P.O. Box. 569, London SEI 9NH. UNITED REP. see address under Kr:^qyt. OF TANZANIA UNITED STATES Single and bulk copies of individual publications (No, subscriptiozsl.'Q Corporation, OF AMERICA 49 Sheridan Avenue, Ar-n,c.Ny N.Y. 12210. Subscriptioni : Subscription orders, accompanied by check made out to the Chemical Bank, New York, Account World Health Organization, should be sent to the World Health Organization, P.O. Box 5284, Church Street Station, Nrw Yonx, N.Y. 10249. Correspondence concerning subscrip- tions should be forwarded to the lVorld Health Organization, Distribution and Sales Service, 121 I GrNrv,c, 27, Switzerland. Publications are also available from the United Nations Bookshop, NEw YoRK, N.Y. lOOlT ( retail only) . USSR For readers in the USSR requiring Russian editions .' Komsomolskij prospekt 18, Medicinskaja Kniga, Moscow For readers outside the USSR requiring Russian editions : Kuzneckij most 18, MeZdunarodnaja- Kniga, Moscow G-200. VENEZUELA Editorial Interamericana de Venezuela C.A., Apartado 50785, C,lucls Libreria del Este, Av. Francisco de Miranda 52, Edificio Galip6n, C,{n,c.cA.s. - YUGOSLAVIA Jugoslovenska Knjiga, Terazije 27/II, Brrcuoe.

Orders from countries where sales agents have not yet been appointed may be addressed to: World Health Organization, Distribution and Sales Service, l2ll Gercva27, Switzerland, but must be paid for in pounds sterling, US dollars, or swiss francs.