Diagnosis and extirpation of nodules in human onchocerciasis.

The clinical search for onchocercal nodules, and their preferred sites on the body in different geographic areas are described and illustrated. The differential diagnosis of onchocercomata is discussed and figures on the frequency of false diagnoses are reported. The prevalence of adult Onchocerca volvulus in relation to the endemicity of infection, the age and sex of the patients, the localisation of nodules on the body, and the measures used to control onchocerciasis are discussed. Recommendations are made on the selection of patients for different medical studies. The technique of nodulectomy and the equipment needed are described.

Histological examination of adult Onchocerca volvulus and comparison with the collagenase technique.

The methods used for the assessment of adult Onchocerca volvulus by histology are described. Based on the results of several studies, mainly in Liberia and Burkina Faso, the morphology of the adult filariae in histological sections is represented as far as it is relevant for the evaluation. Especially are described the morphological alterations due to old age of the worms, to chronic hyperreactivity of the human host (sowda) and effects of the macrofilaricidal suramin and of microfilaricidal drugs. Quantitative results are reported on untreated adult O. volvulus from various countries, the changes of the worm population during 12 years of vector control in Burkina Faso and the effects of suramin, diethylcarbamazine, metrifonate, and ivermectin. The data from the histological examinations are compared with those gained from the examination of worms isolated by the collagenase technique in the same studies.

Effects of high doses of diethylcarbamazine on adult Onchocerca volvulus examined by the collagenase technique and by histology.

Thirty adult male nodule carriers from a hyperendemic onchocerciasis area in the Liberian rain forest were treated with high doses of diethylcarbamazine (30 mg/kg/d) over one week. Another ten patients received placebo tablets and served as a control. All detectable nodules were removed from half of the patients at two months and from the remaining patients at ten months after chemotherapy. The adult worms in the nodules were examined for pathological alterations by the collagenase technique including embryogram, and by histology. No macrofilaricidal effect was observed at either time, and no significant reduction of microfilariae in the uteri of the female worms or in the tissue of the nodules was seen. Two months after the therapy there was a significant increase of degenerated stretched intrauterine microfilariae but this effect was no longer observed after ten months. No pathological effect was seen on the intrauterine coiled microfilariae. On the contrary, their number had significantly increased after ten months which could mean a stimulation of the embryogenesis. No effect on spermatogenesis was observed. Both techniques, the collagenase digestion and the histological examination of the nodules, provided similar results to demonstrate that there was no marked long lasting effect on O. volvulus after a treatment with high doses of diethylcarbamazine.

Histological examination of onchocercomata after therapy with ivermectin.

During chemotherapy trials in hyperendemic onchocerciasis areas in West Africa 15 adult nodule carriers in Liberia and 24 patients in Mali received single doses of ivermectin (150 or 200 micrograms/kg). Nodules were extirpated two, six and ten months after therapy and examined histologically. No macrofilaricidal effect of ivermectin was observed. Two months after therapy, in 93% of all female worms with intrauterine stretched microfilariae nearly all microfilariae were degenerated. The percentage was lower after ten months but still significantly higher than in untreated control groups. Ivermectin did not cause degeneration of the intrauterine coiled microfilariae. But the percentage of the female worms with coiled microfilariae was significantly lower two and ten months after therapy than that in the placebo or untreated control groups. Correspondingly, the percentage of nodules with intact microfilariae in the nodule tissue was also significantly lower throughout the examination period than that of the untreated control groups. There was not observed any effect on the spermatogenesis and spermatozoa were found frequently in the uteri of female worms. Using the method of histology, the long lasting inhibitory effect of a single dose of ivermectin on the intrauterine production of microfilariae could clearly be demonstrated. This proves the value of histology for the assessment of drug effects on adult O. volvulus.

Chemotherapy of onchocerciasis with high doses of diethylcarbamazine or a single dose of ivermectin: microfilaria levels and side effects.

Fifty adult male subjects with moderate to heavy onchocerciasis from the Liberian rain forest were selected for a double-blind placebo-controlled chemotherapy study. The effects of high doses of diethylcarbamazine (DEC) - 30 mg/kg/d - over one week preceded by a one week initial treatment with normal oral doses of DEC or DEC lotion were compared with a single dose of ivermectin (150 micrograms/kg) and placebo. During the initial treatment DEC tablets or lotion caused distinctly more frequent and severe reactions than did invermectin. The reactions to ivermectin did not differ from those of the placebo patients. High doses of DEC caused, in about half of the patients, headache, dizziness, nausea or vomiting. DEC markedly increased the number of corneal microfilariae and of corneal opacities compared to ivermectin. All changes resolved with a return to pretreatment findings two months after treatment. The three treatment groups showed no differences at the ten months follow-up. In all treated patients skin microfilaria counts fell almost to zero by the end of the two week therapy. In the ivermectin group microfilaria counts remained significantly lower than in the DEC patients at the two and ten months examinations. In summary, ivermectin was much better tolerated than DEC and had a longer lasting effect on the microfilariae in the skin. Since high doses of DEC were less effective and caused more frequent and severe side effects, this approach cannot be recommended for treatment of onchocerciasis.

Distribution pattern of peripheral lymphocyte subsets in localized and generalized form of onchocerciasis.

The relationship of peripheral lymphocyte subpopulations to the generalized and localized form of onchocerciasis has been studied in 43 Liberian (West African) patients. Typing revealed a significantly increased proportion of lymphocytes expressing interleukin 2 or transferrin receptors in the localized form of onchocerciasis (African sowda) when compared to subjects with a generalized form and those without signs of onchocerciasis. Patients with African sowda showed a higher number of helper/inducer T cells, a greater ratio of helper/inducer: suppressor/cytotoxic T cells, and an increased number of DR antigen-positive cells and lymphocytes with a natural killer cell phenotype. Furthermore, the serum concentrations of immunoglobulins, particularly IgE an IgG, and antifilarial antibodies were significantly elevated in sowda patients in comparison to the generalized form and the controls. These results indicate that both forms of onchocerciasis represent different immunological types of host responsiveness and that African sowda might be associated with an increased compartment of activated T lymphocytes.

A DNA sequence specific for forest form Onchocerca volvulus.

Onchocerciasis, or river blindness, is caused by infection with Onchocerca volvulus, a filarial parasite which infects about 40 million people in Africa and Latin America. Epidemiological, clinical, entomological and serological studies of African onchocerciasis led to the hypothesis that Onchocerca volvulus exists in different forms in the forest and savannah. It is uncertain if these differences are due to genetic differences within O. volvulus itself, or to epigenetic factors, such as differences in the host populations. To date no basic biochemical differences between the forest and savannah populations of O. volvulus has been found, although isoenzyme studies have shown that differences in allele frequency between forest and savannah populations exist. Here we describe the isolation of a DNA sequence that seems to be specific for the forest form of O. volvulus, the first indication of a basic genetic difference between the savannah and forest forms.

Ivermectin effect on microfilariae of Onchocerca volvulus after a single oral dose in humans.

Ivermectin is a broad spectrum anti-helminthic agent which is currently being tested for use in human onchocerciasis. Its activity is believed to result from its effect on GABA-mediated neurotransmission. We examined the effects of ivermectin on motility of microfilariae of O. volvulus following administration to humans. When ivermectin was given in dosages of 100, 150 and 200 mcg/kg on day 1 there was a clear reduction in motility of microfilariae obtained on day 3 when compared to microfilariae from the placebo group. The mean motility scores in microfilariae from ivermectin recipients were 3.1, 2.3, and 2.2 at 0, 12, and 24 hours of incubation compared to 3.3, 2.9, and 2.5, respectively, in microfilariae from placebo recipients (p less than 0.003, p less than 0.005, and p less than 0.012, respectively). Examination of the effect according to dose suggested a dose-response relationship. Microfilariae in the anterior chamber of the eye 2 days after a single oral dose of ivermectin showed abnormal and reduced winding and coiling. Microfilariae in 50% of ivermectin recipients showed abnormal motility compared to no such effects in subjects examined concurrently who received oral DEC, DEC lotion or placebo. These observations indicate that ivermectin has an effect on motility of microfilariae of O. volvulus following administration to humans.

Phosphoenolpyruvate carboxykinase from Onchocerca volvulus and O. gibsoni.

Phosphoenolpyruvate carboxykinase, a rate-limiting enzyme at the branchpoint of phosphoenolpyruvate (PEP), was demonstrated in Onchocerca volvulus and O. gibsoni. The activity of PEP-carboxykinase from both filarial worms depends absolutely on the presence of divalent cations; in addition to Mg2+ the enzyme activity was strongly activated by Mn2+. The Michaelis constants for PEP, GDP and KHCO3 of the PEP-carboxykinase from O. volvulus were determined to be 0.16 mM, 0.15 mM and 20 mM, respectively; those of the enzyme from O. gibsoni were 0.16 mM, 0.13 mM and 12 mM. Quinolinate was found to be a potent inhibitor of the enzyme from both filarial worms. The inhibition constants were determined to be 11 microM and 15 microM for the enzyme from O. volvulus and O. gibsoni. It is suggested that the activity of PEP-carboxykinase, the initial enzyme of the alternate route from PEP to succinate, may be regulated by ATP-levels. The inhibition constants for ATP were determined to be 0.26 mM and 0.13 mM for the enzyme from O. volvulus and O. gibsoni.

The Testryp CATT (Card Agglutination Test for Trypanosomiasis): a field study on gambiense sleeping sickness in Liberia.

The inhabitants of three Liberian villages in a known gambiense sleeping sickness area with a low prevalence were examined comparatively with the haematocrit centrifugation technique (HCT), the miniature anion exchange centrifugation technique (m-AECT), the indirect fluorescent antibody test (IFAT) and the Testryp CATT (Card Agglutination Test for Trypanosomiasis). Among 418 persons examined was one individual from whom trypanosomes could be isolated with the HCT and m-AECT. This positive case and another 21 individuals had positive titres of 1:40 and more in the IFAT. The same number, but not the same individuals, showed positive reactions in the CATT in the 1:5 serum dilution. The 1:25 dilution of blood and serum was inferior to the corresponding 1:5 dilution and could therefore be omitted. The 'false positive' rate in the CATT was 5.0% in the serum, 10.1% in the blood, and 11.5% in blood or serum. The combination of HCT and m-AECT in the field did not achieve better results than the CATT or IFAT. Thus the CATT can be recommended as the preferable technique for screening of gambiense trypanosomiasis in the field.

Differences in the distribution of HLA antigens in localized and generalized form of onchocerciasis.

Fifty-nine unrelated Liberians have been studied for human leukocyte antigens (HLA). Twenty-three individuals were classified as localized and 24 as generalized onchocerciasis, and the remaining 12 subjects showed no signs of the infection. HLA-typing documented differences in phenotype frequencies for class I (A, B, C) and class II (DR) antigens between the 3 groups of tested subjects. The strongest differences were observed between the localized form and the group without infection with a predominance of HLA-B35 (52% vs. 25%), Cw4 (48% vs. 25%), DR3 (57% vs. 17%), DR5 (65% vs. 8%, DRw52 (96% vs. 33%), and a decrease of DR1 (4% vs. 42%). Differences could also be stated for distinct HLA antigen combinations comprising these relevant antigens. The findings in part hold also true for the comparison of the total group of onchocerciasis (localized plus generalized form) versus the group without infection, however, at a lower level of difference. This pilot study indicates that both forms of onchocerciasis represent different immunological types of host responsiveness.

Onchocerca volvulus: enzyme polymorphism in relation to the differentiation of forest and savannah strains of this parasite.

Isozyme analysis was carried out on Onchocerca volvulus worms collected from Liberia, Ivory Coast, Burkina Faso and Sudan to see whether this technique could detect differences between forest and savannah populations of this parasite. A total of 243 forest and 189 savannah individual female worms were electrophoresed and stained for seven enzymes. Four showed some polymorphism, LDH, MDH, PGM and MPI and the other three, GAPDH, PEP and GPI were invariant. Statistical analysis of the results showed that the relative proportions of genotypes from within the different countries conformed to Hardy-Weinberg expectations. Pairwise comparisons of allele frequencies between countries showed that populations from Liberia and Ivory Coast had a very similar composition; there was some divergence between all the other pairs of populations and the genetic distance was calculated to summarize the degree of divergence. The number of loci examined was small and the genetic distances were within the range expected for separate geographical populations of the same species. The usefulness of this technique in worm identification is discussed.

Prevalence of Paragonimus uterobilateralis infection in children in a Liberian village.

Stool and sputum samples from 127 children in an endemic paragonimiasis area in Liberia, West Africa, were examined for the presence of lung fluke eggs. Samples from nine children (7%) were positive. The eggs were identified as those of Paragonimus uterobilateralis.

Detection of serum antibodies and circulating antigens in a chimpanzee experimentally infected with Onchocerca volvulus.

The course of the humoral immune response was followed in a chimpanzee experimentally infected over 27 weeks with a total of 168 Onchocerca volvulus 3rd-stage larvae obtained from naturally infected wild-caught blackflies. Antibodies against an adult worm extract could be detected by ELISA from week 16 onwards (after the inoculation of 44 larvae). Peak antibody levels were observed between weeks 66 and 74 (about one year after the last larval injection). Thereafter, antibody levels markedly decreased but rose again after week 120. First microfilariae could be detected from week 124 onwards. Microfilarial counts remained low (not more than two microfilariae per skin snip) until the end of the observation period. High levels of IgM antibodies against adult O. volvulus antigens were detectable between weeks 26 and 80 by ELISA. Total IgE levels were found to be only marginally elevated during the course of the infection. Circulating parasite antigens were only detectable for a short time (weeks 34 to 44) of the prepatent period by immuno-radiometric assays (IRMAs) using monoclonal antibodies which were raised against O. gibsoni eggs. Competitive radio-immuno-assays detected host antibodies inhibiting binding of 125I-labelled monoclonal antibodies to parasite antigens from week 28 onwards. Host antibodies clearly interfere later in infection with the detection of circulating antigens.

Humoral and cellular immune responses to Onchocerca volvulus infection in humans.

Onchocerciasis is one of the major filarial diseases affecting humans and a leading cause of blindness. Control of the disease by chemotherapy and by elimination of the vector is not feasible in most areas of endemicity. The host immune response is thought to play a major role in the pathogenesis of complications. However, there is no clear evidence of protective immunity to reinfection in individuals who continue to be exposed to infective larvae. Antigens of Onchocerca volvulus are complex and show extensive cross-reactivity with other filarial parasites of humans and animals. Infection in humans results in the production of precipitating and reaginic antibodies to the parasite and in increases in levels of immunoglobulins that have no apparent specificity for parasitic antigens. Chronic antigenic stimulation in the presence of an antibody response leads to increased levels of circulating immune complexes. Cell-mediated immunity to parasite-derived antigens, as measured by migration inhibition, lymphocyte blastogenesis, and delayed skin-test reactivity, is decreased during infection. In addition, there is a decrease in delayed skin-test reactivity and in lymphocyte blastogenesis in response to unrelated antigens.

Calcification in adult Onchocerca volvulus.

Calcified worm fragments of adult Onchocerca volvulus from patients in West Africa were collected for a chemical analysis. The material contained predominantly calcium carbonate. Some results of investigations on the occurrence of calcified worms in untreated populations are reported on. Relations between the numbers of old parasites and those of calcified worms are discussed. The significance of possible resorption of calcified worm fragments for the interpretation of the effects of drug trials or other control measures are mentioned.

Onchocerca volvulus: transplantation of adult worms into chimpanzees.

Adult Onchocerca volvulus were transplanted into chimpanzees with or without complete or partial digestion of associated nodule tissue. Survival of adults worms was limited, but low level microfilarial densities remained detectable in skin over partially digested nodule transplants for 12 months. Human nodule tissue transplanted with adult worms showed enhanced survival as compared to subcutaneous tissue transplanted without worms. It is postulated that this reflects the action of an immunosuppressive factor utilized by the parasite to permit its own survival.

Effects of a single complete nodulectomy on nodule burden and microfilarial density two years later.

In hyperendemic onchocerciasis areas of the Liberian rain-forest it was possible to re-examine 210 microfilaria carriers two years after a first examination. At the first examination of these patients 143 had presented with palpable nodules, and 110 nodule carriers had been completely nodulectomized. Two-thirds of the operated patients had an average of one newly detected nodule two years later, and their microfilarial densities were slightly increased. In the remaining third of the operated patients no nodules could be palpated two years later, and the average microfilarial densities were slightly decreased. The 33 nodule carriers who had not undergone nodulectomy had, however, significantly higher numbers of nodules and microfilariae in the skin two years after the first examination. The possible benefit of a single, complete nodulectomy is discussed.

Dolichol kinase in Ascaris suum and Onchocerca volvulus.

Dolichol kinase was demonstrated in the microsomal fraction of Ascaris suum and Onchocerca volvulus. The enzyme from nematodes exhibited specificity for CTP as phosphoryl donor and was found to be inhibited by the reaction product CDP. Enzyme activity was optimal at pH 7.4, in the temperature range between 30 degrees and 37 degrees C, and in the presence of 0.5% Triton X-100. In addition, the enzyme was found to depend on divalent cations for activity; Mg2+ being more effective than Mn2+ and Ca2+. The dolichol kinase from both nematodes was shown to be independent of Ca2+-calmodulin for activity. The apparent Km values for dolichol were determined to be 7.5 and 9.0 micrograms ml-1 for the enzyme from A. suum and O. volvulus, respectively. Those for CTP were estimated to be 0.85 and 0.75 mM, respectively.

Development of onchocerca lienalis and O. volvulus from the third to fourth larval stage in vitro.

Third-stage larvae of Onchocerca volvulus and O. lienalis were observed to molt to the fourth stage in various cell-free in vitro systems. The percentage of O. lienalis completing the molt was similar in the three culture media and two gas phases tested ranging from 44.8% (1:1 IMDM:NCTC + 5% CO2: 95% N2) to 56.7% (L-15 + 5% CO2: 95% air). Percent molting in O. volvulus ranged from 0% (F12(K) + 5% CO2: 95% N2) to 33.3% (L-15 + 5% CO2: 95% N2). All media were supplemented with either 20% FCS or 20% horse serum. Molting by O. lienalis occurred on days 2-5 in culture. Molting by O. volvulus was observed as early as day 5 and as late as day 10. Incomplete casting of the third-stage cuticle was frequently observed in O. volvulus. Larvae of both species entered a lethargus 24-48 hours prior to the onset of molting. Maximum survival in culture was 42 days for O. lienalis and 25 days for O. volvulus. Significant growth of larval O. lienalis was noted early in the culture period, but neither species continued development to the fifth stage.