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Variation in delayed hypersensitivity in onchocerciasis
372
TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE, VOL. 72, No. 4, 1978
Variation
in delayed
hypersensitivity
in onchocerciasis
A. BARTLETT,Nuffield Laboratories of Comparative Medicine, Zoological Society of London, Regent’s Park, London, NW1 J. TURK, Department of Pathology, Royal College of Surgeons of England, Lincoln’s Inn Fields, London, WC;? J. NGU, Department of Medicine, University Centre for Health Sciences, University of Yaounde, United Republic of Cameroon C. D. i%hxENZIE, Division of Parasitology, National Institute for Medical Research, Mill Hill, London, NW7 H. FUGLSANG* AND J. ANDERSON*,Medical Research Centre, Kumba, SW Province, United Republic of Cameroon Summary
Soluble antigen preparations were made from Onchocerca volr~.& adults recovered from subcutaneous nodules, and from Necator americanus third-stage larvae. Intradermal skin tests were carried out on a total of 100 individuals clinically classified as having either the generalized form of onchocerciasis (86 cases), or the localized sowda form (14 cases). 91 of the people studied produced immediate reactions to the onchocercal antigen, but only those with sowda showed delayed reactions, though one person with generalized disease showed a doubtful delayed reaction. Reactions to N. americanus antigen, both immediate and delayed, were seen in both forms of the disease. The lack of delayed skin reaction in the generalized form of onchocerciasis is discussed, and a comparison is made with other diseases. Preliminary evidence for the existence of a true spectrum of clinical and immunological forms of onchocerciasis is suggested by one casein this study.
skin in the affected area is dark and thickened, intensely itching, with a papular eruption, and there
Introduction
Common skin manifestations of onchocerciasis in Africa are hyperpigmentation, depigmentation, papular eruptions, atrophy and lymphoedema, together with lymphadenopathy, and they have been well illustrated by CONNOR(WHO, 1974). Their prevalence in different geographical areas has been described bv WOODRUFFet al. (1963. 1966a. 1966b), CONNOR‘et ~2. (1970), ANDERSONi& FUGL~ SANG(1973) and by ANDERSONet al. (1974a). The last authors also stated that the majority of people with onchocerciasis in hyperendemic areas had little or no itching and no obvious skin changes. despite the presence of high numbers of microfilariae in diagnostic skin snips. Sowda, Arabic for black or dark, is used as a descriptive term for a common manifestation of onchocerciasis in Yemen (FAWDRY,1957; GASPARINI, 1962; ANDERSONet al., 1973); It is also seen amone the Arabs in the Abu Hamed focus of northern Sudan (MORGAN, 1958; ANDERSONet al., 1974b), and in East Africa (HUTTON, 1954). In contrast to the generalized form described above, sowda in its most typical form is localized in distribution to one lower extremity (Fig. 1). The * Present address: 77 Pennard Road, Shepherd’s Bush, London, W12.
Fig. 1. Male with the characteristic manifestations of localized onchocerciasis, sowda. The skin lesions are localized to one leg and are accompanied by swelling of the regional lymph node.
A. BARTLETT et al. is usually a swelling of the regional lymph nodes. Secondary skin infection is common. Microfilariae of Onchocerca volvulus are usually absent in standard skin snips, but they can be found in full thickness skin biopsies (WHO, 1974). Sowda cases also react with increased itching in response to diethylcarbamazine (DEC). These variations in dermal response, numbers of parasites in the skin, and in the type of pathology may reflect underlying variations in the host immune response to 0. VOZVUZUS.In the present study the immediate and delayed skin reactions to an intradermal injection of a crude antigen prepared from adult 0. volvulus were examined in patients with onchocerciasis in Cameroon. Skin reactions to an antigen solution prepared from Necaror americanus were also examined for comparison. Materials and methods Populations studied Four groups of people from different geographical areas of Cameroon were included, as described below. They were-examined clinically, particularly for changes associated with onchocerciasis, including skin changes, presence of subcutaneous nodules, and occurrence of ocular lesions and microfilariae in the cornea or anterior chamber of the eye. Skin snips were taken with a corneoscleral punch from the buttock, and the number of 0. volvulus microfilariae that emerged in saline was counted. Following the criteria described above, people were then classified into cases of generalized onchocerciasis or localized sowda. Those with either form of the disease developed severe pruritus in response to DEC. Full thickness skin biopsies from two of the people classified as sowda were examined histologically and shown to have microfilariae lying deep in the dermis. Antigens The 0. volvulus antigen used in testing the people in Grouo I was oreoared from fragments of adult 0. volv~lus which -were dissected from nodules removed on the same day from patients of Njore village. The fragments of worms were ground in a mortar with about three times their volume of phosphate buffered saline (PBS) at pH 7.3. Cell debris was removed by centrifugation, and the protein content of the extract was estimated spectrophotometrically (taking E 280 of 1.38 = 1 mg protein per ml.) The extract was diluted with PBS to about 1 mg protein/ml and stored frozen until required. The 0. volvulus antigen used for Groups II, III and IV was prepared in London from fragments of adult 0. volvulus dissected from a nodule which had been removed from a patient in Baduma village, and stored at -70°C until dissection. The fragments were ground in a Griffiths tissue grinder in an ice bath. They were frozen and thawed once, and then sonicated at 4°C for 20 seconds at maximum amplitude using a MSE ultrasonicator. The preparation was centrifuged for 30 minutes at lO,OOOg, and the protein concentration of the supernatant fluid was estimated spectrophotoIt was then passed through a 0 *22~ metrically. Millipore filter and freeze-dried in aliquots. The
373
hookworm antigen was prepared in the same way from third-stage larvae of N. americanus. Skin testing The reactivity of clinically characterized patients was assessed by the intradermal injection of 0.1 ml of the antigen solutions into the forearm. The ensuing reactions were examined, as far as possible, after 15 minutes, 4, 24, 48, 72 and 96 hours. The injection sites were observed and any areas of reaction carefully measured using a template (‘Wellcotest’). A reaction with a diameter greater than 1 *O cm was taken as positive at the 1%minute and four-hour assessments, and a reaction greater than 0.5 cm diameter was taken as positive at the later observations. Skin biopsies were taken from four people who showed delayed reactions to the onchocercal antigen preparation, three of them at 24 hours, and one at 72 hours after injection. Tissues for histological examination were fixed in 10% form01 saline. Sections were prepared for routine histology, and subsequently stained with haematoxylin and eosin (H & E). Results Group I This group consisted of 36 people from Njore village in the Mbandjock area of Cameroon, a rainforest focus about 100 km north-east of Yaounde. There were 22 males and 14 females whose ages ranged from 12 to 72 years. All showed microfilariae in buttock snips, the numbers ranging from eight to 364, all but one had palpable subcutaneous nodules and most showed varying degrees of the common onchocercal skin changes, though none exhibited changes suggestive of sowda. All but three of the patients in this group had microfilariae in the cornea and/or anterior chamber of the eye, and 16 had onchocercal eye lesions of varying degree, either of the anterior or posterior segments, or in both locations. Previous examinations showed that the majority had infestations with Ascaris, Trichuris and hookworm, and infections with Dipetalonema perstans and Loa loa were diagnosed in 15 and three of them respectively (RIEDEL et al., 1976; RIPERT et al., 1976). This group was tested with 0.05 microgrammes of the 0. volvulus preparation, and 32 patients showed an immediate reaction (15 minutes). At four hours, five still showed swellings of more than 1 .O cm diameter. No delayed reactions were seen at 24 hours or subsequently (Table I). Group II There were 48 people, 31 males and 17 females aged between eight and 53, from the rain-forest village of Baduma near Kumba, south-west Cameroon, in this group. 45 were similar to the people in Group I in that they manifested generalized onchocerciasis, while the remaining three were clinically defmed as sowda. One of these three, a female aged 20, had lesions that were most marked on the left leg and both buttocks, spilling over to the right thigh and the lower part of the body without sharp limits. When examined five months later she had developed a proven onchocercal nodule, though
TOUBORO, (Mbororo
TOUBORO, savanna (Mboum tribe)
III
IV ONC. NEC. ONC NEC
Generalized Localized l/l l/l
515 515
9110 8110
313 212
41145 29145
32136 ND
a
from
E::
ii:
ii:
213 112
17/40 15140
5136 ND
4
Time
111 O/l
615 l/5
5/10 o/10
313 112
l/45 36145
O/36 ND
at?
Dermal injection
NNE
NNE
zig
313 112
o/45 43145
O/36 ND
$ours)
response**
%
ii::
213 112
o/45 43145
O/36 ND
72
ND ND
iii:
ND ND
O/3 l/2
o/45 41/45
O/36 ND
96
Onchocerca volvulus and Necator americanus in the generalized
NEC. = Necator americanus * ONC. = Onchocerca volvulus ** Results given as: No. of individuals with positive reactions Total No. of individuals tested or seen Positive reactions = + hour and 4 hours : 1 *O cm diameter. Other times: 0.5 cm diameter ND = Not done
savanna tribe) ONC. NEC
ONC. NEC.
Localized Localized
ONC. NEC.
Generalized
BADUMA,
II
rain-forest
ONC. NEC.
Generalized
NJORE, rain-forest
prepared
Antigen*
I
Clinical form
of antigens
Area
response to intradermal infection (sowda) forms of onchocerciasis
Group
Table I-Skin and localized
s, G 8 E
8 2 c 5 2
1
2u
E
;
8
A. BARTLETT et al.
skin-dwelling microfilariae were still absent from standard diagnostic skin snips. In another of the sowda cases, a 26-year-old male, the lesion was limited to the left shoulder and upper arm, with a very sharp demarcation between normal and affected skin. The third was a 2%year-old female who had been successfully treated two years previously for a similar lesion on her right leg with a course of DEC and suramin, and who was almost symptom-free, although she stated that the previously affected leg had begun to itch again. Skin testing in this group was carried out with both 0. VOZVUZUS (0.1 pg) and N. americanus (0 *OSpg) antigen preparations. At 15 minutes the 0. voZvuZus antigen produced positive reaction in the three sowda cases, as well as in 41 of the 45 generalized cases (Table I). At the same time 29/45 showed positive reactions to the N. americanus antigen, as did both of the two sowda cases (the third was not tested with this antigen). No Arthus-like reactions were seen with either antigen in any individual. At 24 hours, and subsequently, delayed reactions to the hookworm antigen were present in most cases, whether generalized or sowda. Delayed reactions to the 0. voIvulus antigen preparation were absent in all but the three cases of sowda, who showed indurated areas of 0.6, 0.7 and 0.6 cm diameter at their maximum. However one person with the generalized form, an l&year-old male, showed a doubtful delayed reaction at 24 hours, which subsided quickly. The 20-year-old female with sowda was retested after five months and found to have lost the ability to produce a delayed skin reaction; as previously mentioned she had at this time developed a subcutaneous nodule. The male sowda patient with the skin lesion confined to the shoulder was also retested and again produced a positive delayed skin He had reaction to the onchocercal preparation. been treated with DEC and suramin in the meantime. Group III This group comprised 10 males of the nomadic Mbororo tribe in the savanna area of Cameroon. All showed varvine degrees of the localized form of the disease, but sharp-demarcation of the affected area was not always present. Five of them showed from one to three microfilariae in a skin snip, and one of these had a palpable nodule. In the others there were neither nodules nor microfilariae on routine examination of the skin and the eye. Intradermal testing was carried out with the same antigen preparations and amounts as were used in Group-II. -However, due to the nomadic way of life of this tribe. the skin tests could onlv be read at 15 minutes and 24 hours. At 15 minutes the onchocercal antigen preparations gave positive reactions in all but one person, and at 24 hours positive reactions were seen in five of the ten individuals, while the other five had small indurated areas with diameter of less than 0.5 cm. Biopsies were taken at this stage from three people who showed marked indurations. The characteristic feature of the dermal tissues in the delayed reactions was the infiltration of mononuclear cells (Fig. 2), often clustered in islands around the dermal vessels. No
375
Fig. 2. Section through the site of injection of 0. volvulus antigen preparation after 24 hours in an individual defined as suffering from the sowda form of onchocerciasis. Accumulations of cells can be seen in the dermis. (Magnification x 79- 50, H & E stain).
Fig. 3. High power photomicrograph of the accumulation of cells in the delayed reaction. Mononuclear cells predominate in the clusters around the blood vessels. (Magnification x 424, H & E stain). neutrophil leucocytes were present, and only one or two eosinophil leucocytes were seen in the clusters amongst the many mononuclear cells (Fig. 3). The blood vessels were commonly dilated. The N. americanus antigen preparation gave rise to wheals at 15 minutes in eight of the ten patients, but none of the individuals in this group showed any reaction to this preparation at 24 hours. Group IV The last group of people studied belonged to the Mboum tribe from the Cameroon savanna, a tribe in which sowda manifestations of the skin are extremely rare. Three males and three females were examined, and five of them had nodules and microfilariae in diagnostic skin snips. The sixth, a male of 16, had been treated successfully with suramin
376
VARIATION IN DELAYED HYPERSENSITIVITY IN ONCHOCERCIASIS
three years earlier for sow& changes of the buttocks and lower limbs, but the symptoms and signs had recurred. No microfilariae- or-nodules were found in him. All six individuals were tested with the same antigen preparations as were used in Groups II and III, and they all produced an immediate reaction to both preparations. However, only the sowda case developed a delayed reaction to the onchocercal antigen. One of the non-sowda cases had a large soft swelling at the site of injection at 24 hours, which had started about six hours after the injection, and could have been an Arthus type reaction. The N. americanus antigen preparation gave rise to delayed reactions in only one individual in this group, a non-sowda case. Discussion
The results of this study show that people with onchocerciasis produce skin reactions in response to the intradermal injection of a preparation of 0. volvulus adults. Most of the people examined showed an immediate reaction to both the onchocerca and hookworm antigens. Such immediate reactions are known not to be necessarily specific, and they probably reflect the fact that the person has been exposed to nematode antigens (BALL et al., 1971). As can be seen from Table I, all but one of the people with positive delayed reactions (24 hours or more) were those with skin lesions suggestive of sowda. The demonstration of delayed hypersensitivity to onchocerca antigen indicates that cell mediated immunity develops in these individuals. It also suggests that this skin test may be a useful aid to diagnosis of the sowda form of onchocerciasis. An explanation for the lack of response in the generalized disease is not obvious. It is clearly not due to an over-all failure of cell mediated immunity since delayed reactions to the N. americanus antigen preparation were shown by most people in the rainforest areas, where this parasite is endemic. Positive delayed reactions to N. americanus were also seen in a few generalized disease cases in the savanna where the parasite is much less common. A similar failure of cell mediated immunity occurs in lepromatous leprosy, diffuse cutaneous leishmaniasis and kala-azar (TURK & BRYCESON, 1971). In these three conditions, as in generalized onchocerciasis, organisms can be found lying throughout the tissues in the absence of a lymphocyte infiltrate. In addition, delayed hypersensitivity tests to specific antigens are negative. Recently OTTESEN et al. (1977) have shown by lymphocyte transformation, that chronic infection-with lkhkreria bancrofti is characterized by a state of specific cellular immune unresponsiveness to filarial antigens. This contrasted with a vigorous cellular response to these antigens in individuals with the same degree of exposure, living in the same area, but showing no signs of being infected. The differences in response between infected and noninfected was very striking among children, much less so among adults, and the authors suggest that in early exposure to filarial antigens, normal cellular immune responses are stimulated, but with continued and repeated exposure to the antigens many
individuals become specifically tolerant. Subsequent chronic infection leads to the pathological changes associated with disease. Various reasons for soecific failure of cell mediated immune responses have been put forward, including the presence of suppressor B cells (KATZ et al., 1974). Substantial levels of antibody against onchocerca antigens have been demonstrated in people with onchocerciasis (BARTLETT et al., 1975; MACKENZIE et al., in preparation). Further studies on antibody levels in individuals with the different clinical forms of the disease are indicated. Suppressor T cells have been demonstrated in other parasitic infections (JAYAWARDENA
&
WAKSMAN,
1977).
Another
explanation may be that the parasite itself, or parasite-derived products, induce specific depression of the host cell mediated immunity. Large amounts of soluble antigen released from the parasite may produce a phenomenon similar to immunological tolerance. The antigenic load of an individual may be increased by simultaneous infection with many other species of nematodes, most of which have antigens in common with 0. volvulus. In Cameroon, sowda casesoccur most commonly in the nomadic tribes of the northern areas, who frequently move in and out of the endemic zones. These people are not subjected to so constant and heavy a rate of infection with 0. volvulus as those who live in areas of hyperendemic onchocerciasis, neither do they appear to suffer the same intensity of infection with other nematode parasites (ANDERSON et al., 1974c). A lower level of parasite-derived substances in these people may be important in the aetiology of sowda. It should also be noted that the effect of concurrent parasitic infections on the host’s response is not yet understood, but under experimental conditions one helminth parasite can modify the response to subsequent infection by another (JENKINS & BEHNKE, 1977).
Genetic variations, both in the parasite and in the infected people, must be considered in the aetiology of sowda. BRYCESON et al. (1976) demonstrated differences in antigenic profile between 0. volvulus strains from the savanna and rain-forest regions. It is likely that the ability of an individual to manifest strong cell-mediated immunity is controlled by an Ir gene related to the major histocompatibility complex (PAUL & BENACERAFF, 1977). Therefore genetic studies, such as HLA typing, may contribute to the understanding of the clinical forms of onchocerciasis. Most of the individuals in this study had either generalized or localized forms of the disease, but, an indication of the existence of a spectrum of clinical forms, such as has been suggested for leprosy (TURK & BRYCESON, 1971), was seen in one of the females in Group II. She was originally diagnosed as a case of sowda and was shown to have a positive delayed skin reaction. On re-examination five months later she had a disease pattern more typical of the generalized form and had lost the ability to produce a delayed skin reaction. However, a definite characterization of a true clinical and immunological spectrum in onchocerciasis remains to be carried out. The determination of host immune responses in various forms of 0. volvulus by the application of specific in vivo and in vitro tests, and their correla-
A. BARTLETT
tion with clinical parameters, are essential for a better understanding of this disease. Acknowledgements
We wish to thank Mr. S. Ayonghe and Mr. C. Fisiy who assisted with the field work in this study. References
Anderson, J. & Fuglsang, H. (1973). Clinical aspects of onchocerciasis in Uganda and Yemen Arab Republic compared with a rain-forest and savanna focus in Cameroon. WHO/ONCHO 73.102 (mimeographed document). Anderson, J., Fuglsang, H. & Zubaidy, A. (1973). Onchocerciasis in Yemen with special reference to sowda. Transactions of the Royal Society of Tropical Medicine and Hygiene, 67, 30-31. Anderson, J., Beiram, M. M., Fuglsang, H. & Gassouma, M. S. S. (1974b). Some aspects of onchocerciasis in northern and southern Sudan. WHO/ONCHO 74.108 (mimeographed document) . Anderson, J., Fuglsang, H., Hamilton, P. J. S. & Marshall, T. F. de C. (1974a). Studies on onchocerciasis in the United Cameroon Republic. I. Comparison of populations with and without Onchocerca volvulus. Transactions of the Royal Society of Tropical Medicine and Hygiene, 68,
190-208. Anderson, J., Fuglsang, H., Hamilton, P. J. S. & Marshall, T. F. de C. (1974c). Studies on onchocerciasis in the United Cameroon Republic. II. Comparison of onchocerciasis in rain-forest and Sudan-savanna. Transactions of the Royal Society of Tropical Medicine and Hygiene, 68, 209-222.
Ball, P. A., Voller, A. & Taffs, L. F. (1971). Hypersensitivity to some nematode antigens. British Medical Journal, i, 210-211. Bartlett, A., Bidwell, D. E. & Voller, A. (1975). Preliminary studies on the application of enzyme immunoassay in the detection of antibodies in onchocerciasis. Tropenmedizin und Parasitologic, 26, 370-374.
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Bryceson, A. D. M., van Veen, K. S., Oduloju, A. 1. & Duke, B. 0. L. (1976). Antigenic diversity among Onchocerca V&X& in -Nigeria, and immunological differences between onchocerciasis in the savanna and forest of Cameroon. Clinical Experimental Immunology, 24, 168-176. Connor, D. H., Morrison, M. E., Kerdel-Vegas, F., Berkoff. H. A.. Tohnson. F.. Turmicliffe. R., Failing, F. C.,’ Hale, L.* N. ’ & Lindquist, K: (1970). Onchocerciasis. Onchocercal dermatitis, lymphadenitis, and elephantiasis, in the Ubangi Territorv. Human Pathology, 1, 553-579. Fawdry, A. L. (1957). Onchocerciasis in South Arabia. Transactions of the Roval Societv of Tropical Medicine and Hygiene, 51; 253-256.-
d
Gasparini, G. (1964). Surgical treatment of ‘sowda’. Dermatologia
et d.
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Royal Society of Tropical Medicine and Hygiene, 48. 522-525.
Jayawardena, A. N. & Waksman, B. H. (1977). Suppressor cells in experimental trypanosomiasis. Nature, 265, 539-541. Tenkins, S. N. & Behnke, T. M. (1977). Imuairment of primary expulsion bt Trichuris mu& in mice concurrentlv infected with Nematosbiroides d&us. 1 Parasitology, 75, 71-78. Katz, S. I., Parker, D. & Turk, J. L. (1974). B cell suppression of delayed hypersensitivity reactions. Nature, 251, 550-551.
Mackenzie, C. D., Bartlett, A., Butterworth, A. E. & Dinga, J. S. (1970). Studies of antibodies in serum of patients with onchocerciasis. (In preparation.) Morgan, H. V. (1958). Onchocerciasis in the Northern Sudan. Journal of Tropical Medicine and Hygiene, 61, 145-147. Ottesen, E. A., Weller, P. F., Heck, L. (1977). Specific cellular immune unresponsiveness in human filariasis. Immunology, 35, 413. Paul, W. E. & Benaceraff, B. (1977). Functional specificity of thymus-dependent lymphocytes. A relationship between the specificity of T lymphocytes and their functions is proposed, Science, 195, 1293-1300. Riedel, D., Riedel, D. & Ripert, C. (1976). Epidemiologie des filarioses humaines dans la VallCe de la Sanaga. 11” Conference technique de l’OCEAC (mimeographed document). Ripert, C., Riedel, D. & Riedel, D. (1976). Etude Cpidtmiologique des verminoses humaines dam la VillCe de la Sanaga (Cameroon). 11’ Conference technique de 1’OCEAC (mimeographed document). Turk, J. & Bryceson, A. D. (1971). Immunological phenomena in leprosy and related diseases. Advances in Immunology, 13, 209.
Woodruff, A. W., Barnley, G. R., Holland, J. T., Jones, D. E., McCrae, A. W. R. & McLaren, D. S. (1963). Onchocerciasis and the eye in Western Ueanda. Transactions of the Roval Society of ?rapical 50-63.
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57,
Accepted for publication
27th January,
1978.
TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE, VOL. 72, No. 4, 1978
Variation
in delayed
hypersensitivity
in onchocerciasis
A. BARTLETT,Nuffield Laboratories of Comparative Medicine, Zoological Society of London, Regent’s Park, London, NW1 J. TURK, Department of Pathology, Royal College of Surgeons of England, Lincoln’s Inn Fields, London, WC;? J. NGU, Department of Medicine, University Centre for Health Sciences, University of Yaounde, United Republic of Cameroon C. D. i%hxENZIE, Division of Parasitology, National Institute for Medical Research, Mill Hill, London, NW7 H. FUGLSANG* AND J. ANDERSON*,Medical Research Centre, Kumba, SW Province, United Republic of Cameroon Summary
Soluble antigen preparations were made from Onchocerca volr~.& adults recovered from subcutaneous nodules, and from Necator americanus third-stage larvae. Intradermal skin tests were carried out on a total of 100 individuals clinically classified as having either the generalized form of onchocerciasis (86 cases), or the localized sowda form (14 cases). 91 of the people studied produced immediate reactions to the onchocercal antigen, but only those with sowda showed delayed reactions, though one person with generalized disease showed a doubtful delayed reaction. Reactions to N. americanus antigen, both immediate and delayed, were seen in both forms of the disease. The lack of delayed skin reaction in the generalized form of onchocerciasis is discussed, and a comparison is made with other diseases. Preliminary evidence for the existence of a true spectrum of clinical and immunological forms of onchocerciasis is suggested by one casein this study.
skin in the affected area is dark and thickened, intensely itching, with a papular eruption, and there
Introduction
Common skin manifestations of onchocerciasis in Africa are hyperpigmentation, depigmentation, papular eruptions, atrophy and lymphoedema, together with lymphadenopathy, and they have been well illustrated by CONNOR(WHO, 1974). Their prevalence in different geographical areas has been described bv WOODRUFFet al. (1963. 1966a. 1966b), CONNOR‘et ~2. (1970), ANDERSONi& FUGL~ SANG(1973) and by ANDERSONet al. (1974a). The last authors also stated that the majority of people with onchocerciasis in hyperendemic areas had little or no itching and no obvious skin changes. despite the presence of high numbers of microfilariae in diagnostic skin snips. Sowda, Arabic for black or dark, is used as a descriptive term for a common manifestation of onchocerciasis in Yemen (FAWDRY,1957; GASPARINI, 1962; ANDERSONet al., 1973); It is also seen amone the Arabs in the Abu Hamed focus of northern Sudan (MORGAN, 1958; ANDERSONet al., 1974b), and in East Africa (HUTTON, 1954). In contrast to the generalized form described above, sowda in its most typical form is localized in distribution to one lower extremity (Fig. 1). The * Present address: 77 Pennard Road, Shepherd’s Bush, London, W12.
Fig. 1. Male with the characteristic manifestations of localized onchocerciasis, sowda. The skin lesions are localized to one leg and are accompanied by swelling of the regional lymph node.
A. BARTLETT et al. is usually a swelling of the regional lymph nodes. Secondary skin infection is common. Microfilariae of Onchocerca volvulus are usually absent in standard skin snips, but they can be found in full thickness skin biopsies (WHO, 1974). Sowda cases also react with increased itching in response to diethylcarbamazine (DEC). These variations in dermal response, numbers of parasites in the skin, and in the type of pathology may reflect underlying variations in the host immune response to 0. VOZVUZUS.In the present study the immediate and delayed skin reactions to an intradermal injection of a crude antigen prepared from adult 0. volvulus were examined in patients with onchocerciasis in Cameroon. Skin reactions to an antigen solution prepared from Necaror americanus were also examined for comparison. Materials and methods Populations studied Four groups of people from different geographical areas of Cameroon were included, as described below. They were-examined clinically, particularly for changes associated with onchocerciasis, including skin changes, presence of subcutaneous nodules, and occurrence of ocular lesions and microfilariae in the cornea or anterior chamber of the eye. Skin snips were taken with a corneoscleral punch from the buttock, and the number of 0. volvulus microfilariae that emerged in saline was counted. Following the criteria described above, people were then classified into cases of generalized onchocerciasis or localized sowda. Those with either form of the disease developed severe pruritus in response to DEC. Full thickness skin biopsies from two of the people classified as sowda were examined histologically and shown to have microfilariae lying deep in the dermis. Antigens The 0. volvulus antigen used in testing the people in Grouo I was oreoared from fragments of adult 0. volv~lus which -were dissected from nodules removed on the same day from patients of Njore village. The fragments of worms were ground in a mortar with about three times their volume of phosphate buffered saline (PBS) at pH 7.3. Cell debris was removed by centrifugation, and the protein content of the extract was estimated spectrophotometrically (taking E 280 of 1.38 = 1 mg protein per ml.) The extract was diluted with PBS to about 1 mg protein/ml and stored frozen until required. The 0. volvulus antigen used for Groups II, III and IV was prepared in London from fragments of adult 0. volvulus dissected from a nodule which had been removed from a patient in Baduma village, and stored at -70°C until dissection. The fragments were ground in a Griffiths tissue grinder in an ice bath. They were frozen and thawed once, and then sonicated at 4°C for 20 seconds at maximum amplitude using a MSE ultrasonicator. The preparation was centrifuged for 30 minutes at lO,OOOg, and the protein concentration of the supernatant fluid was estimated spectrophotoIt was then passed through a 0 *22~ metrically. Millipore filter and freeze-dried in aliquots. The
373
hookworm antigen was prepared in the same way from third-stage larvae of N. americanus. Skin testing The reactivity of clinically characterized patients was assessed by the intradermal injection of 0.1 ml of the antigen solutions into the forearm. The ensuing reactions were examined, as far as possible, after 15 minutes, 4, 24, 48, 72 and 96 hours. The injection sites were observed and any areas of reaction carefully measured using a template (‘Wellcotest’). A reaction with a diameter greater than 1 *O cm was taken as positive at the 1%minute and four-hour assessments, and a reaction greater than 0.5 cm diameter was taken as positive at the later observations. Skin biopsies were taken from four people who showed delayed reactions to the onchocercal antigen preparation, three of them at 24 hours, and one at 72 hours after injection. Tissues for histological examination were fixed in 10% form01 saline. Sections were prepared for routine histology, and subsequently stained with haematoxylin and eosin (H & E). Results Group I This group consisted of 36 people from Njore village in the Mbandjock area of Cameroon, a rainforest focus about 100 km north-east of Yaounde. There were 22 males and 14 females whose ages ranged from 12 to 72 years. All showed microfilariae in buttock snips, the numbers ranging from eight to 364, all but one had palpable subcutaneous nodules and most showed varying degrees of the common onchocercal skin changes, though none exhibited changes suggestive of sowda. All but three of the patients in this group had microfilariae in the cornea and/or anterior chamber of the eye, and 16 had onchocercal eye lesions of varying degree, either of the anterior or posterior segments, or in both locations. Previous examinations showed that the majority had infestations with Ascaris, Trichuris and hookworm, and infections with Dipetalonema perstans and Loa loa were diagnosed in 15 and three of them respectively (RIEDEL et al., 1976; RIPERT et al., 1976). This group was tested with 0.05 microgrammes of the 0. volvulus preparation, and 32 patients showed an immediate reaction (15 minutes). At four hours, five still showed swellings of more than 1 .O cm diameter. No delayed reactions were seen at 24 hours or subsequently (Table I). Group II There were 48 people, 31 males and 17 females aged between eight and 53, from the rain-forest village of Baduma near Kumba, south-west Cameroon, in this group. 45 were similar to the people in Group I in that they manifested generalized onchocerciasis, while the remaining three were clinically defmed as sowda. One of these three, a female aged 20, had lesions that were most marked on the left leg and both buttocks, spilling over to the right thigh and the lower part of the body without sharp limits. When examined five months later she had developed a proven onchocercal nodule, though
TOUBORO, (Mbororo
TOUBORO, savanna (Mboum tribe)
III
IV ONC. NEC. ONC NEC
Generalized Localized l/l l/l
515 515
9110 8110
313 212
41145 29145
32136 ND
a
from
E::
ii:
ii:
213 112
17/40 15140
5136 ND
4
Time
111 O/l
615 l/5
5/10 o/10
313 112
l/45 36145
O/36 ND
at?
Dermal injection
NNE
NNE
zig
313 112
o/45 43145
O/36 ND
$ours)
response**
%
ii::
213 112
o/45 43145
O/36 ND
72
ND ND
iii:
ND ND
O/3 l/2
o/45 41/45
O/36 ND
96
Onchocerca volvulus and Necator americanus in the generalized
NEC. = Necator americanus * ONC. = Onchocerca volvulus ** Results given as: No. of individuals with positive reactions Total No. of individuals tested or seen Positive reactions = + hour and 4 hours : 1 *O cm diameter. Other times: 0.5 cm diameter ND = Not done
savanna tribe) ONC. NEC
ONC. NEC.
Localized Localized
ONC. NEC.
Generalized
BADUMA,
II
rain-forest
ONC. NEC.
Generalized
NJORE, rain-forest
prepared
Antigen*
I
Clinical form
of antigens
Area
response to intradermal infection (sowda) forms of onchocerciasis
Group
Table I-Skin and localized
s, G 8 E
8 2 c 5 2
1
2u
E
;
8
A. BARTLETT et al.
skin-dwelling microfilariae were still absent from standard diagnostic skin snips. In another of the sowda cases, a 26-year-old male, the lesion was limited to the left shoulder and upper arm, with a very sharp demarcation between normal and affected skin. The third was a 2%year-old female who had been successfully treated two years previously for a similar lesion on her right leg with a course of DEC and suramin, and who was almost symptom-free, although she stated that the previously affected leg had begun to itch again. Skin testing in this group was carried out with both 0. VOZVUZUS (0.1 pg) and N. americanus (0 *OSpg) antigen preparations. At 15 minutes the 0. voZvuZus antigen produced positive reaction in the three sowda cases, as well as in 41 of the 45 generalized cases (Table I). At the same time 29/45 showed positive reactions to the N. americanus antigen, as did both of the two sowda cases (the third was not tested with this antigen). No Arthus-like reactions were seen with either antigen in any individual. At 24 hours, and subsequently, delayed reactions to the hookworm antigen were present in most cases, whether generalized or sowda. Delayed reactions to the 0. voIvulus antigen preparation were absent in all but the three cases of sowda, who showed indurated areas of 0.6, 0.7 and 0.6 cm diameter at their maximum. However one person with the generalized form, an l&year-old male, showed a doubtful delayed reaction at 24 hours, which subsided quickly. The 20-year-old female with sowda was retested after five months and found to have lost the ability to produce a delayed skin reaction; as previously mentioned she had at this time developed a subcutaneous nodule. The male sowda patient with the skin lesion confined to the shoulder was also retested and again produced a positive delayed skin He had reaction to the onchocercal preparation. been treated with DEC and suramin in the meantime. Group III This group comprised 10 males of the nomadic Mbororo tribe in the savanna area of Cameroon. All showed varvine degrees of the localized form of the disease, but sharp-demarcation of the affected area was not always present. Five of them showed from one to three microfilariae in a skin snip, and one of these had a palpable nodule. In the others there were neither nodules nor microfilariae on routine examination of the skin and the eye. Intradermal testing was carried out with the same antigen preparations and amounts as were used in Group-II. -However, due to the nomadic way of life of this tribe. the skin tests could onlv be read at 15 minutes and 24 hours. At 15 minutes the onchocercal antigen preparations gave positive reactions in all but one person, and at 24 hours positive reactions were seen in five of the ten individuals, while the other five had small indurated areas with diameter of less than 0.5 cm. Biopsies were taken at this stage from three people who showed marked indurations. The characteristic feature of the dermal tissues in the delayed reactions was the infiltration of mononuclear cells (Fig. 2), often clustered in islands around the dermal vessels. No
375
Fig. 2. Section through the site of injection of 0. volvulus antigen preparation after 24 hours in an individual defined as suffering from the sowda form of onchocerciasis. Accumulations of cells can be seen in the dermis. (Magnification x 79- 50, H & E stain).
Fig. 3. High power photomicrograph of the accumulation of cells in the delayed reaction. Mononuclear cells predominate in the clusters around the blood vessels. (Magnification x 424, H & E stain). neutrophil leucocytes were present, and only one or two eosinophil leucocytes were seen in the clusters amongst the many mononuclear cells (Fig. 3). The blood vessels were commonly dilated. The N. americanus antigen preparation gave rise to wheals at 15 minutes in eight of the ten patients, but none of the individuals in this group showed any reaction to this preparation at 24 hours. Group IV The last group of people studied belonged to the Mboum tribe from the Cameroon savanna, a tribe in which sowda manifestations of the skin are extremely rare. Three males and three females were examined, and five of them had nodules and microfilariae in diagnostic skin snips. The sixth, a male of 16, had been treated successfully with suramin
376
VARIATION IN DELAYED HYPERSENSITIVITY IN ONCHOCERCIASIS
three years earlier for sow& changes of the buttocks and lower limbs, but the symptoms and signs had recurred. No microfilariae- or-nodules were found in him. All six individuals were tested with the same antigen preparations as were used in Groups II and III, and they all produced an immediate reaction to both preparations. However, only the sowda case developed a delayed reaction to the onchocercal antigen. One of the non-sowda cases had a large soft swelling at the site of injection at 24 hours, which had started about six hours after the injection, and could have been an Arthus type reaction. The N. americanus antigen preparation gave rise to delayed reactions in only one individual in this group, a non-sowda case. Discussion
The results of this study show that people with onchocerciasis produce skin reactions in response to the intradermal injection of a preparation of 0. volvulus adults. Most of the people examined showed an immediate reaction to both the onchocerca and hookworm antigens. Such immediate reactions are known not to be necessarily specific, and they probably reflect the fact that the person has been exposed to nematode antigens (BALL et al., 1971). As can be seen from Table I, all but one of the people with positive delayed reactions (24 hours or more) were those with skin lesions suggestive of sowda. The demonstration of delayed hypersensitivity to onchocerca antigen indicates that cell mediated immunity develops in these individuals. It also suggests that this skin test may be a useful aid to diagnosis of the sowda form of onchocerciasis. An explanation for the lack of response in the generalized disease is not obvious. It is clearly not due to an over-all failure of cell mediated immunity since delayed reactions to the N. americanus antigen preparation were shown by most people in the rainforest areas, where this parasite is endemic. Positive delayed reactions to N. americanus were also seen in a few generalized disease cases in the savanna where the parasite is much less common. A similar failure of cell mediated immunity occurs in lepromatous leprosy, diffuse cutaneous leishmaniasis and kala-azar (TURK & BRYCESON, 1971). In these three conditions, as in generalized onchocerciasis, organisms can be found lying throughout the tissues in the absence of a lymphocyte infiltrate. In addition, delayed hypersensitivity tests to specific antigens are negative. Recently OTTESEN et al. (1977) have shown by lymphocyte transformation, that chronic infection-with lkhkreria bancrofti is characterized by a state of specific cellular immune unresponsiveness to filarial antigens. This contrasted with a vigorous cellular response to these antigens in individuals with the same degree of exposure, living in the same area, but showing no signs of being infected. The differences in response between infected and noninfected was very striking among children, much less so among adults, and the authors suggest that in early exposure to filarial antigens, normal cellular immune responses are stimulated, but with continued and repeated exposure to the antigens many
individuals become specifically tolerant. Subsequent chronic infection leads to the pathological changes associated with disease. Various reasons for soecific failure of cell mediated immune responses have been put forward, including the presence of suppressor B cells (KATZ et al., 1974). Substantial levels of antibody against onchocerca antigens have been demonstrated in people with onchocerciasis (BARTLETT et al., 1975; MACKENZIE et al., in preparation). Further studies on antibody levels in individuals with the different clinical forms of the disease are indicated. Suppressor T cells have been demonstrated in other parasitic infections (JAYAWARDENA
&
WAKSMAN,
1977).
Another
explanation may be that the parasite itself, or parasite-derived products, induce specific depression of the host cell mediated immunity. Large amounts of soluble antigen released from the parasite may produce a phenomenon similar to immunological tolerance. The antigenic load of an individual may be increased by simultaneous infection with many other species of nematodes, most of which have antigens in common with 0. volvulus. In Cameroon, sowda casesoccur most commonly in the nomadic tribes of the northern areas, who frequently move in and out of the endemic zones. These people are not subjected to so constant and heavy a rate of infection with 0. volvulus as those who live in areas of hyperendemic onchocerciasis, neither do they appear to suffer the same intensity of infection with other nematode parasites (ANDERSON et al., 1974c). A lower level of parasite-derived substances in these people may be important in the aetiology of sowda. It should also be noted that the effect of concurrent parasitic infections on the host’s response is not yet understood, but under experimental conditions one helminth parasite can modify the response to subsequent infection by another (JENKINS & BEHNKE, 1977).
Genetic variations, both in the parasite and in the infected people, must be considered in the aetiology of sowda. BRYCESON et al. (1976) demonstrated differences in antigenic profile between 0. volvulus strains from the savanna and rain-forest regions. It is likely that the ability of an individual to manifest strong cell-mediated immunity is controlled by an Ir gene related to the major histocompatibility complex (PAUL & BENACERAFF, 1977). Therefore genetic studies, such as HLA typing, may contribute to the understanding of the clinical forms of onchocerciasis. Most of the individuals in this study had either generalized or localized forms of the disease, but, an indication of the existence of a spectrum of clinical forms, such as has been suggested for leprosy (TURK & BRYCESON, 1971), was seen in one of the females in Group II. She was originally diagnosed as a case of sowda and was shown to have a positive delayed skin reaction. On re-examination five months later she had a disease pattern more typical of the generalized form and had lost the ability to produce a delayed skin reaction. However, a definite characterization of a true clinical and immunological spectrum in onchocerciasis remains to be carried out. The determination of host immune responses in various forms of 0. volvulus by the application of specific in vivo and in vitro tests, and their correla-
A. BARTLETT
tion with clinical parameters, are essential for a better understanding of this disease. Acknowledgements
We wish to thank Mr. S. Ayonghe and Mr. C. Fisiy who assisted with the field work in this study. References
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Accepted for publication
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