onchocerciasis in sudan: the abu hamed focus

464

TRANSACTIONS OF THE ROYAI. SOCIETY OF TROPICAL MEDICINE AND HYGIENE (1985) 79, 464-468

Onchocerciasis in Sudan: the Abu Hamed focus

J. F. WILLIAMS’, A. H. ABU YOUSIF’, M. BALLARD ‘, R. AWAD’, M. EL TAYEB~ AND M. RASHEED~ ‘Dept. of Microbiology and Public Health, Michigan State University, East Lansing, MI 48824, USA; ‘Khartoum Eye Hospital, Khartoum, Sudan; 3Laboratories of Parasitology, Ministry of Health, Khartoum,

Sudan

Abstract The current status of onchocerciasis in Abu Hamed, Northern Province, Sudan, was studied. Of

208 persons attending out-patient clinics in villages in this region, 71 were microfilariae-positive on skin snips or had palpable nodules. Microfilariae and worms in nodules were identified as Onchocerca volvulus. No microfilariae were seen in peripheral blood. Most nodules and microfilariae were found in the pelvic region, but the intensity of infection was uniformly low (av. <3mf/mg). Despite this, signs of onchocercal dermatitis were common and severe, especially over the buttocks. Papular eruptions and scarring often appeared to lead to black-grey hyperpigmentation, but no cases were seen of the unilateral, hyper-reactive ‘sowda’ described in Arabs in Yemen. No microfilariae were detected in the eyes of any of the patients who had positive outer canthus snips. Serum retinol concentrations were normal but mildly elevated concentrations of serum IgG, IgM and IgA were detected in many patients. Immunoglobulin E values in a sample of 20 microfilariae-positive patients were markedly higher than normal, with most in the 4,000 to 15,000 U/ml range. Eosinophil levels in differential counts of peripheral blood from the 208 villagers were markedly elevated. In skin snip surveys of over 400 villagers and school pupils, sample prevalence rates of 2 to 17.5% were recorded. Simulium biting was seasonal (November to May) and peaked in March. Over-all, the results indicate that 0. volvulus infection persists in the Abu Hamed region as a serious cause of skin disease in the absence of other compli&ting filariases.

Introduction Onchocerciasis is widely distributed in the Demo-

cratic Republic of Sudan (ABDALLA, 1974). It is most prevalent in the southern and eastern provinces which border on Zaire, Uganda and Ethiopia (SATTI & KIRK, 1957; ENARSON, 1977). An additional focus in Northern Province was first described 25 years ago by MORGAN (1958), but since then no formal accounts have been published of the status of the disease in this region. Continuing observations by the Ministry of Health, supplemented by experimental and clinical data recorded by BEIRAM (1974) in his doctoral thesis, form the background to this study of the current characteristics of onchocerciasis at the most northerly focus in the world. Our results indicate that Onchocerca volvulus persists as a serious cause of debilitating and disfiguring skin disease, despite the very low intensity of microfilarial infection demons- trable in most affected patients. Evidence ormarkedly seasonal transmission, uncomplicated by superim- posed infection with other filarial parasites, suggests that this focus offers important opportunities for study of the immunobiology and pathogenesis of onchocercal disease.

Stuay site Materials and Methods

Abu Hamed is situated on the Nile at 19”30’N between the 4th and 5th cataracts in Northern Province (Fig. 1). At this point in its course the river turns sharply westwards and there are many islands, the largest of which is Mograt. Villages on the islands and along the northern and southern banks consist of widely-scattered houses with no clear demarcation of village borders. The river level reaches its peak in late summer (August to September), and ideal conditions for Sin&urn breeding occur when the water

recedes and rocks and vegetation emerge, creating much surface turbulence (November to Mav). Mean maximum and minimum tempiratures in late sur&er are 42.5”C and 28*O”C, and in mid-winter 27*8”C and 12.8”C. There is no significant rainfall in this region, which is flanked on all sides bq the arid Nubian desert.

Residents are Afro-Arab in ethnic type. They tend irrigated crop fields for subsistence and for the production of date palms, for which Abu Hamed and nearby villages are renowned. Medical services are provided by the Civil Hospital in Abu Hamed, and there is an intermediate level boys’ school which draws pupils from villages 100 km up and downstream. The Sudan Railwav line connecting Khartoum and Wadi Halfa runs along the eastern bank 01 the Nile as far as Abu Hamed before turning northwards across the Nubian desert.

The study population Detailed physical and parasitological examinations were

made on 208 persons, aged 10 to 90, attending out-patient clinics at the Abu Hamed Civil Hospital, and in the villages of El Giraif, El Singerab, El Gezira Portocol and El Gezira Filicol (Fig. 1). Some residents of other small local communities also attended these village-level clinics.

A parasitological survey by skin-snip only was undertaken in 200 persons in Kelesaikal and Kiggi, and in 214 boys at the Abu Hamed School. Method of examination

Each person attending out-patient clinics in September and October, 1981, was given a complete physical examination. Special attention was given to the palpation of nodules and to the assessment of skin lesions or changes compatible with onchocercal dermatitis. Visual acuity was tested using an E chart, and any specific refractive errors were recorded and corrective lenses prescribed. One of us (R.A.) examined all eyes with an ophthalmoscope, and 12 parasitologically positive individuals were also brought in to the Onchocerciasis Research Center in Abu Hamed for further examination with a Haag-Streit 900 slit-lamp.

J. F. WILLIAMS 63 ffl. 465

Blood samples were collected from all patients by vene- puncture and examined for circulating microfilariae by membrane filtration (Nucleporea, 5 mu pore size). One ml samples were lysed in 10 ml 1% formalin in distilled water and the filters were stained with methyl violet before microscopic screening. Thin blood films were stained with Giemsa for differential leucocyte counts and malaria diagno- sis. Serum samples were preserved at -20°C.

Skin snips were taken, using a Walser cornea-scleral biopsy punch, from the outer canthus, shoulder, buttock and calf on the right side only. Snips were incubated in O-2 ml distilled water in 96 well microtitre plates for four hours at ambient temperature (-4(R), before fixation by the addition of 0.05 ml 10% formalin. Preserved snips which were positive were later weighed on a torsion balance, and the numbers of microfiiariae per mg wet weight skin sample computed for each person. Width and length dimensions were recorded for 100 microiilariae. Six positive and six negative formalin-fixed skin snips were paraffin embedded and processed for histological examination.

Serum samples were examined for Vitamin A content by the method of STOWE (1982). Levels of immunoglobulins G, M and A were determined by radio-immunodiffusion using isotype-specific antisera and standards supplied by Kallestad Labs, Inc., Austin, Texas, USA. I~unoglobulin E was measured in selected sera with the Phadebas IgE PristR radio-i~unoassay system (Pharmacia Diagnostics, Upp- sala, Sweden).

Bilateral skin snips from the buttocks only were collected from villagers in Kelesaikal and Kiggi, and from school pupils in Abu Named. Ministry of Health staff participated in the collection of 45 faecal and urine samples from adult villagers in Abu Hamed and El Giraif inorder to assess backgrotmd levels of intestinal parasitism and urinary schistosomiasis. These were examined microscopically after Aotation of 1.0 g faecal samples in 33% &SO,, or centrifug- al sedimentation in the case of urine samples.

Fly collection Fly collection Entomology staff of the Ministry of Health made collec- Entomology staff of the Ministry of Health made collec-

tions and dissection of Silnutiunr three times weekly during tions and dissection of Silnutiunr three times weekly during the breeding season. Only data recorded on biting rates by the breeding season. Only data recorded on biting rates by month are presented below. month are presented below.

Results 60 of 208 outpatients had skin-snips positive for

unsheathed microfihuiae. 15 of these had palpable nodules. An additional 11 persons had at least one nodule, but were negative on skin-snip examination. The youngest person with positive skin snips was 13, and the oldest claimed to be about 90 years. The dimensions of the unsheathed microfilariae were in the ranges of 218 to 303 ym long and 3.7 to 4.9 urn wide. Details on the anatomical distribution of positive snips and nodules are shown in Fig. 2.

Patients with nodules were generally aware of their presence and wished to have them extracted. A nodule removed from the iliac crest of a sin-snip-~sitive patient contained cross sections of adult nematodes, up to 400 urn wide, which were compatible with female worms of 0. volvulus.

The intensity of infection was uniformly low. The mean number of microfilariae per mg in positive snips was 2.9, with a range from one to 28. Microfilariae were not detected in histological sections of the positive or negative skin snips made after the incubation was complete. Nine patients had microfilariae in snips taken at the outer canthus. These plus three other parasitologically positive patients were examined with the slit-lamp but no microfilariae were detected in the anterior chamber or cornea.

Of the 71 patients who had microfihtriae and/or nodules, 375% had changes in the anterior segment of the eye. Most common were comeal opacities, trichiasis, conjunctival follicles and scars and Her- bert’s pits; most of the lesions of the anterior segment were therefore considered to be attributable to trachoma. The corresponding figure for anterior segment changes jn negative patients was 25%. Abnormahties in pigment deposition in the posterior

A. -

LIRYA , =“.*I I \ \

___--___ -.__

i---

.-

Fig. 1. Map illustrating the location of Abu Hamed (A), and the regional villages from which the patient samples were drawn for onchocerciasis surveys (B).

466 ONCHOCERCIASIS IN SUDAN

segment were found in four patients, all of whom were parasitologically negative. Visual acuity scores of 6/18 or worse were assigned to 28% of eyes in negative patients and 42.3% of those with microfilariae or nodules. Cataractous lesions were present in 55 patients and were probably responsible for a significant proportion of visual deficits. No cases of classical onchocercal ocular lesions (e.g., sclerosing keratitis, choroidoretinitis) were seen in any patients.

Of those attending out-patient clinics, 84% com- plained of chronic pruritus. Many bore self-inflicted marks of recent scratching. The following lesions were recorded and considered likely to be due to onchocercal dermatitis and its consequences: chronic papular eruptions and associated scars (27.5%); prem- ature atrophic changes, especially over the trunk and legs (21.7%); pigmentary abnormalities with intense darkening, and patches of depigmentation (57.5%); hyperkeratotic thickening (29.4%). Lesions were most severe in almost all cases over the buttocks (Fig. 3), but the thighs, lower legs and arms were also affected. This set of skin lesions was no more frequently seen in parasitologically positive than in negative patients. There were no cases seen of unilateral limb involvement comparable to the syndrome of ‘sowda’ seen in Arab peoples of the Arabian peninsula (CONNOR et al., 1983).

No microfilariae were detected on any of the membrane iilters used for examination of peripheral blood. In Giemsa-stained smears no malaria parasites were seen. Although we were unable to collect faecal and urine specimens from all villagers at the time, Ministry of Health staff later assisted in the procure- ment of these from 45 individuals. There were no cases of schistosomiasis detected on examination of these samples, and only three cases of intestinal helminthiasis were found (two Ascaris hmbricoides; one Trichuris rrichiura and Hymenolepis nana).

Differential leucocyte counts revealed remarkably high proportions of eosinophils; mean values for the 208 individuals examined were: EOS 14.7% 2 13.9 (SD), NEUT 42.5% + 13.5, LYM 369% + 12*4%, MONO 4.7% + 3.4 and BASO 0.2% +_ 1.0. The range of eosinophil values was from 0 to 75%. Mean serum IgG, IgM and IgA levels were 1887 + 529 SD mg/dl, 223 + 128 mg/dl, and 239 + 96 mg/dl, respectively, for the 208 patients. Mean normal values in westerners are 1047, 126 and 177 for these isotypes. Serum IgE levels were measured in 20 skin-snip-positive patients, and in 10 asymptomatic individuals who had neither palpable nodules nor microfilariae. In five of the parasitized patients IgE levels ranged from 47 to 315 U/ml; up to approximately 500 U would be considered normal in westerners. In the remaining 15 patients values were markedly elevated, extending from 3,700 to 27,000 U/ml, with most in the 4,000 to 15,000 U/ml range. In the 10 non-parasitized sub- jects, six samples had levels well below 500 U/ml, but four were slightly higher (600 to 630).

Serum levels of retinol palmitate and retinol were determined on a randomly selected sample of 18 parasitologically positive individuals and 18 negatives. No values fell outside the accepted normal range; the mean vitamin A level was computed to be 1.14 i.u./ml for the whole group.

In the small community of Kelesaikal, 24 of 137 volunteers who submitted to bilateral skin snip

TOTAL NO. OF PENSDNS EKIN.SNIP

POSITIVE Al EACN SITE (SO PATIENTS~

To&A~nL~i~P

NDOULES (26 PATIENTS)

Fig. 2. Anatomical distribution of microfilariae and palpable nodules in 0. w&&s-infected patients. Some patients were positive at multiple skin sites, and in a few individuals several nodules were detected.

Fig. 3. Typical chronic onchocercal dermatitis over the buttocks in patient from Abu Hamed. This was the most common lesion seen in the study.

240

J .J -A.S’O N.D’J’F‘M’A’M’

Fig. 4. Adult Simulium damnosum collections at Abu Hamed illustrating the intensity of biting by moth.

J. F. WILLIAMS et Cd 467

sampling were positive. The youngest was 15 years old. In Kieai. an island close to Mograt (see Fig. I) only four % 63 volunteers were pos%ve.‘ Again the youngest was 1.5 years old. Of 214 pupils at the Abu Hamed boy’s school, four were positive. All four were at least 17 years old.

Ministrv of Health data on Si~u~~u~ collections at Abu Ha&ed permit construction of the picture of seasonal occurrence of blackfIies illustrated in Fix. 4. The data are representative of the situation in 1973, 74,75 and 76. There were no important differences in collection data between these years.

Discussion In Morgan’s description of onchocercal disease in

Abu Hamed in 1958 few details were offered of the parasitological characteristics of the patients examined, although some of the clinical signs he described seem very likely to have been due to OS volvulus. He detected microfilariae in skin biopsies from only nine patients, but palpated nodules in 10 of 70 adult residents of the Abu Hamed district, and six of 73 boys at the intermediate school. The morpholo- gical characteristics of microfilariae which we collected from snios were compatible with those of 0. volvu1us, although the parasites were rather narrower than is usual. This may have been an artefact oroduced bv dilute formalin fixation at high ambient iemperatures (generally 40°C). The worms in the extracted nodule appeared to be 0. vu~v~~~s adults. In view of the negative results of the nerinheral blood filter tests, it- seems unlikely that other filarial parasites were present in these patients at levels which would have affected the clinical picture. On the other hand, the medominance of trachoma-related lesions makes it difficult to ascribe ocular disease to the influence of 0. vo~v~~~s infection. MORGAN (1958) diagnosed most cases of onchocerciasis in Abu Hamed on the basis of cornea1 opacities, rather than subject individuals to the painful biopsy procedure he used; his estimates of the frequency of infection may therefore have been too high.

The distribution of nodules around the pelvic region in our study was similar to that seen in southern provinces of Sudan (ENARSON, 1977), although the intensity of ~crohlarial counts was remarkably low. Since microfilariae were not detectable in snip sections examined post-incubation, it did not appear that they were being retained within the biopsied tissue. Despite the low numbers of organ- isms present, the spectrum and severity of the skin lesions suggest that onchocerciasis is none the less an ~~rtant cause of morbidity in these patients. The frequent papular eruptions over the buttock area, together with subsequent pigmentary changes, hyper- keratosis and atrophy, make up a clinical syndrome which is entirely comparable to that seen in heavily infected people of the southern region. The intense blackish-grey hyperpigmentation in the pelvic area was very striking in pale skinned Arab patients, but there was no evidence of the peculiar, unilateral ‘sowda’ process in any of the individuals we examined.

Concentrations of IgG, IgM and IgA were elevated mildly by western standards. Nowever, the values for IgE in a sample of parasitized patients were abnorm-

ally high. H~r~unoglobul~ae~a-E is a charac- teristic of hehninthiasis and has been described in most human filariases (HUSSEIN et al., 1981). The allergen composition of 0. valvulus is very complex (WEISS et al., 1982) and many IgE binding antigens have been detected. The relationship between elevated IgE and pathogenetic processes in onchocerciasis is not clear, although some of the manifestations of disease and the adverse responses to therapy are likely to involve IgE-mediated events. The high number of eosinophils suggests a role for these cells in the immunopathology of onchocerciasis, especially since it appears that eosinophils can partici- Date as effector cells in microfilarial destruction in man (MCKENZIE, 1980; Rpicz et al., 1982).

Visual acuitv was obviouslv much affected in this patient groupby the combined effects of trachoma and cataracts, and without further detailed examination it is not possible to incriminate 0. volvulus directly in the ocular disease processes. With such low levels of microfilariae in the skin it might prove very difficult to detect any which enter the eye. It is worth noting, however, that some patients did have microfilariae at the outer canthus. Moreover, the occurrence of cases of retinal pigment atrophy and pigment clumping is consistent with changes associated with 0. voZvuZus infection in other parts of Africa. Over-all, patients in Abu Hamed did not have abnormal serum retinol levels, and avitaminosis-A is therefore unlikely to be a causal factor in posterior segment pathology.

The co~u~ty prevalence rates which we measured may be falsely low given the difficulty of detecting small numbers of parasites. Microfilariae did not reach detectable numbers until about the mid-teens, although some very severe skin problems were seen in children in this age group who were parasitologically negative. The low rate of infection in the schoolboys may reflect the fact that these pupils come from areas up and downstream where infection rates may be much lower than in the Abu Hamed region.

Many patients commented that their skin disease was much aggravated by the bites of the local Simulium damnosum, commonly known as ‘kunteb’. The intensity of biting at the peak of the season (Fig. 4) is formidable. Our findings suggest that continued efforts should be made to characterize host-parasite relationships in Abu Hamed. The results should serve to devise strategies for alleviating the local problem and may also clarify pathogenetic mechanisms of significance in the biology of this disease elsewhere.

Acknowledgements This work was supported by NIH grant 5 PO1 AI 16312

and by the resources of the Ministry of Health, Sudan, and the Khartoum Eye Hospital. We are grateful to Dr Howard Stowe, Michigan State University, for performing the retinol analyses.

References Abdalla, R. E. (1974). Filariasis in the Sudan. Transactions of

the Roy& Society of Tropical Medicine and Hygiene, 68, 53-55.

Beiram, M. M. (1974). M, D. Thesis. University of Khartoum, School of Medicine, Khartoum, Sudan.

Connor, D. H., Gibson, D. W., Neaiie, R. C., Merighi, B. & Buck, A. (1983). Sowda-Onchocerciasis in North Yemen: A clinicopathologic study of 18 patients. Amer- ican Journal of Tropical Medicine and Hygiene, 32, 123-137.

468 ONCHOCERCIASIS IN SUDAN

Enarson, D. A. (1977). Observations on onchocerciasis in the Sudan Republic: Endemicity, intensity of infection and clinical features. Annals of Tropical Medicine and Parasiwlogv, 71, 465-468.

Hussein, R., Hamilton, R. G., Kumaraswami, V., Adkin- son, N. F. & Otteseon, E. A. (1981). IgE responses in human 6lariasis. I. Quantitation of filaria-specific IgE. Journal of Immunology, 27, 1623-1629.

Mackenzie, C. D. (1980). Eosinophil leucocytes in filarial infections. Transactions of the Royal Society of Tropical Medicine and Hygiene, 74 (Suppl.), 51-57.

Morgan, H. V. (1958). Onchocerciaais in the Northern S$a;47Twmal of Tropical Medtcane and Hygiene, 61,

Racz, P., Tenner-Racz, K., Buttner, D. W. & Albiez, E. J. (1982). Ultrastructural evidence for eosinophil-parasite adherence (EPA) reaction in human onchocercal lym-

phadenitis in the early period following diethylcarbama- zine treatment. Tropenmedizin and Parasitologic, 33, 213-218.

Satti, M. H. & Kirk, R. (1957). Observations on the chemotherapy of onchocerciasis in Bahr El Ghazal province, Sudan. Bulletin of the World Health Organiza- tion, 16, 531-540.

Stowe, H. D. (1982). Vitamin A profiles in equine serum and milk. Journal of Animal Science, 54, 76-81.

Weiss, N., Gvalzata, M., Wyss, T. & Betschart, B. (1982). Detection of IgE-binding Onchocerca volvulus antigens after electrophoretic transfer and immuno-enzyme reaction. Acta Tropica, 39, 373-377.

Accepted for publicatiotl 28th August, 1984.

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onchocerciasis in sudan: the abu hamed focus

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