Interleukin-5 and the posttreatment eosinophilia in patients with onchocerciasis.

To understand the role of the eosinophilopoietic cytokine IL-5 in humans, the posttreatment eosinophilic response in a group of microfilaria (mf)-positive patients with onchocerciasis (n = 10) was examined before and after treatment with diethylcarbamazine (6 mg/kg for 7 d). Sequential blood samples were assessed at 24 and 1 h before treatment (baseline values), then at frequent intervals over the next 14 d. Symptom scores, skin microfilariae (mf), and peripheral blood eosinophil counts were recorded as a function of time after treatment, and serum levels of IL-5 were quantitated by a highly sensitive (sensitivity greater than or equal to 20 pg/ml) monoclonal-based ELISA. Pretreatment eosinophil counts ranged from 240 to 1,186 eosinophils/microliter (geometric mean, 675), and the mf counts from 10 to 218 per mg skin (geometric mean, 79). After an initial decline in the peripheral eosinophil count to 28 +/- 8% of pretreatment levels at 8 h after beginning treatment, the eosinophil counts steadily increased over the next 2 wk, reaching a maximum at 14 d (257 +/- 38% of pretreatment levels). Serum levels of IL-5 rose sharply from pretreatment levels to a peak of 70.5 +/- 11 pg/ml by 24 h after treatment. Serum IL-5 remained elevated over the next 2-3 d and declined toward baseline by approximately 6 d after treatment, at which time the eosinophil levels were steadily increasing. IL-3 and granulocyte macrophage colony-stimulating factor, two other cytokines implicated in eosinophilopoeisis, were not detectable in the serum at any time before or after treatment. The rise in serum IL-5 before the posttreatment eosinophilia seen in this group of patients with onchocerciasis demonstrates a temporal relationship between IL-5 and the subsequent development of eosinophilia and implicates IL-5 as an important mediator of eosinophilia in humans.

Ivermectin selection on beta-tubulin: evidence in Onchocerca volvulus and Haemonchus contortus.

Ivermectin resistance is common in trichostrongylid nematodes of livestock, such as Haemonchus contortus. This anthelmintic is the only drug approved for mass administration to control onchocerciasis caused by the nematode parasite, Onchocerca volvulus. In parts of West Africa up to 18 rounds of ivermectin treatment have been administered to communities and there are reports of poor parasitological responses to treatment. Understanding ivermectin resistance and ivermectin selection is an important step to reduce selection pressure for resistance, and to develop molecular markers which can be used to monitor the development of resistance and its spread. Here we report evidence that ivermectin selection changes the frequency of beta-tubulin alleles in both the sheep parasite, H. contortus, and the human parasite, O. volvulus. In O. volvulus we have been able to look at the frequency of beta-tubulin alleles in O. volvulus obtained before any ivermectin was used in humans in Africa, and following its widespread use. In H. contortus, we have been able to look at the frequency of beta-tubulin alleles in a strain which has not seen any anthelmintic selection and in an ivermectin selected strain derived from the unselected strain. We have found ivermectin selects on beta-tubulin in both of these nematode species. In the case of O. volvulus, we had previously reported that ivermectin selects for specific single nucleotide polymorphisms in the O. volvulus beta-tubulin gene. This polymorphism results in three amino acid changes in the H3 helix of beta-tubulin, as well as deletions in an associated intron. We report a simple PCR assay to detect the amplicon length polymorphism, resulting from these intronic deletions, which can be used to monitor the frequency of the beta-tubulin allele selected for by ivermectin in O. volvulus.

Diagnosis of O. volvulus infection via skin exposure to diethylcarbamazine: clinical evaluation of a transdermal delivery technology-based patch.

BACKGROUND: Elimination of onchocerciasis in Africa is now regarded as an achievable goal in many areas. This makes monitoring changes in infection prevalence a key component of control programmes. Monitoring is currently based on determining the presence of O. volvulus microfilariae in skin snips, an invasive, labour-intensive method. The Onchocerciasis Control Programme (OCP) had established procedures to detect O. volvulus infections via the localized skin reaction induced by killing of microfilariae upon skin exposure to diethylcarbamazine via a patch (OCP-patch). Large scale OCP - patch use is difficult due to labour-intensive patch preparation. At the request of TDR, a manufacturer specialized in transdermal-delivery systems developed a ready-to-use diethylcarbamazine (DEC) containing patch (LTS-2 patch). To qualify this patch for large scale studies of its sensitivity and specificity, this study evaluated its ease of application, ability to detect infection and DEC exposure related adverse reactions compared to the OCP-patch in 30 infected individuals. METHODS: Each participant with 0.2-36.8 O. volvulus microfilariae/mg skin received the OCP-patch and 4 days later the LTS-2 patch at the left and right iliac crest, respectively, for 24 h. Presence and characteristics of local skin reactions were assessed at patch removal and 6 h later. Skin reaction and Mazzotti reaction rates were compared with Fisher's exact and a paired t-test, respectively. RESULTS: The LTS-2 patch could be applied within 10 s. Mild itching occurred at 63.3 % of OCP-patch (duration 8.9 ± 11.8 h) and 26.7 % of LTS-2 patch sites (duration 1.0 ± 2.5 h) and was the most frequent Mazzotti reaction. At patch removal after 24 h, a diagnostic local skin reaction was present under 90 % of OCP-patches and 83 % of LTS-2 patches; 6 h later, it was present at 93 % of OCP-patch and 100 % of LTS-2 patch sites. CONCLUSIONS: The data suggest that safety, tolerability and ability to detect infections of the LTS-2 patch are comparable to those of the OCP-patch. They qualify the LTS-2 patch for field studies to determine LTS-2 patch sensitivity, specificity and utility during large scale use and thus to inform use of the LTS-2 patch by onchocerciasis control programmes to determine prevalence of infection. TRIAL REGISTRATION: Current controlled Trials ISRCTN76875372 .

Diethylcarbamazine disposition in patients with onchocerciasis.

Diethylcarbamazine (DEC), 0.5 mg/kg, was taken orally by six patients being treated for onchocerciasis. Blood samples were taken at timed intervals for 48 hr and urine and feces collected for 4 days. Plasma and urinary concentrations of DEC and DEC N-oxide were measured by gas-liquid chromatography. DEC appeared to be rapidly absorbed, with a peak plasma concentration of 150 to 250 ng/ml reached in 2 to 3 hr. There was a secondary rise in plasma DEC concentration at 5 to 6 hr in all patients. In contrast to the way the drug is eliminated in rats, in man it was by both renal and extrarenal routes, with small amounts (+/- 10%) being excreted as an N-oxide metabolite. DEC kinetics were also investigated in five normal subjects and the result were much the same. Clinical implications are discussed.

The Onchocerca volvulus homologue of the multifunctional polypeptide protein disulfide isomerase.

Protein disulfide isomerase (PDI) functions to catalyze the formation of correct disulfide bonds in nascent proteins, and also acts as one of the subunits of prolyl-4 hydroxylase, the enzyme responsible for the oxidative maturation of procollagen. Since the cuticle of parasitic nematodes consists primarily of a network of collagen molecules which are connected through intermolecular disulfide bonds, PDI might be expected to be involved in the process of cuticle biosynthesis. The isolation and characterization of a cDNA encoding the PDI homologue of Onchocerca volvulus is described. This cDNA contains a single, long open reading frame that encodes sequence motifs identical to the two known active sites of PDI for isomerase activity. The O. volvulus PDI appears to be encoded by a single copy gene. Both in situ hybridization and immunolocalization data suggest that PDI is both spatially and temporally regulated in O. volvulus. The pattern of spatial and temporal regulation is consistent with the involvement of PDI in the biosynthesis of the parasite cuticle. The parasite protein appears to be an antigen recognized by a minority of individuals exposed to O. volvulus.

The Mazzotti reaction following treatment of onchocerciasis with diethylcarbamazine: clinical severity as a function of infection intensity.

To determine definitively whether or not the severity of the Mazzotti reaction was correlated with infection intensity, as determined by skin snip quantification, 21 infected Ghanian patients were evaluated during 7 days of treatment with 200 mg/day of diethylcarbamazine. Serial blood, urine and skin biopsy samples were collected during the progression of the Mazzotti reaction. Hypotension, fever, adenitis and pruritus were all correlated with infection intensity in these patients while arthralgia and tachycardia were not. Peripheral blood eosinopenia and neutrophilia also correlated with intensity of infection and appeared to reflect the accumulation of degranulating eosinophils around "mobilized" microfilariae that migrated from the dermis to the epidermis after diethylcarbamazine (DEC). Other mobilized microfilariae apparently were cleared by the liver and resulted in abnormal liver enzyme levels in the serum which, again, were directly correlated with the patients' microfilarial density. Though the severity of the Mazzotti reaction clearly correlated with intensity of infection, the different times of onset of symptoms, and cellular and serum chemistry changes indicate that there are probably multiple infection intensity-dependent mechanisms responsible for mediating this complex reaction.

Evaluation of ultrasonography for the detection of drug-induced changes in onchocercal nodules.

Clinical trials of macrofilaricidal drugs against Onchocerca volvulus are impeded due to the lack of means for assessing in vivo drug-induced changes in the onchocercomas. The application of ultrasonography in the sequential monitoring of morphologic alterations of onchocercal nodules after six weeks of suramin therapy was evaluated in 20 male patients from Ghana with a total of 64 nodule sites. After each follow-up session, a number of onchocercal nodules were extirpated so that by the end of one year, all nodules had been removed for histologic examination. The sonomorphologic changes observed and their time of appearance correlated well with the histologic findings of the onchocercomas. Eighty-three percent of the onchocercal nodules became hyperechogenic and 22% developed echo-free areas at the end of the follow-up period. Absence of the lateral acoustic shadow increased by more than 30% and the lack of differentiation of the worm center from the capsule and the nodule from its surrounding tissue increased by the end of one-year posttreatment to 100% and 91%, respectively. A mean reduction of nodule size of 27% was also documented. The histologic studies revealed that the proportion of the dead female worms increased from 17% at the end of the suramin therapy to 48% six months later and reached 61% at one year. It is concluded that ultrasonographic monitoring of onchocercomas can provide essential information on drug effects and facilitate clinical trials of macrofilaricidal drugs, limiting histologic evaluation to a few objectively selected onchocercomas.

Comparison of the sensitivity of different geographical isolates of Onchocerca volvulus microfilariae to ivermectin: effects of exposure to drug on development in the blackfly Simulium ornatum.

Four geographical isolates (Ghana forest, Ghana savannah, Cameroon forest, Guatemala) of Onchocerca volvulus microfilariae (mf) and O. lienalis mf (UK) were examined for their sensitivity to ivermectin by incubation in vitro in drug followed by assessing their ability to develop in the blackfly Simulium ornatum after intrathoracic injection. Parasites were incubated for 30 min in ivermectin (10(-6) to 10(-9) M), which resulted in a concentration dependent decrease in the numbers of parasites surviving and developing in the insect; there were significant reductions in parasite recoveries from all isolates in the 10(-6) M to 5 x 10(-8) M ivermectin groups, but no significant effect was seen following incubation in concentrations of 10(-8) M and below. Experiments consistently demonstrated that the 4 isolates of O. volvulus were similarly sensitive to ivermectin (in the 10(-7) M ivermectin groups there was a reduction of 76.3% to 85.1% in numbers of infective larvae, and 60.9% to 85.5% in numbers of all larval stages, compared to controls); O. lienalis mf were significantly more sensitive (100% reduction in infective larvae, 98.7% reduction in all larval stages). This baseline information on drug sensitivity and techniques should prove useful for examining populations of O. volvulus for possible development of drug resistance in the future.

The effects of high-dose ivermectin regimens on Onchocerca volvulus in onchocerciasis patients.

Ivermectin, at the standard dose of 150 micrograms/kg bodyweight, does not kill the adult worms of Onchocerca volvulus and does not disrupt embryogenesis or spermatogenesis. Repeated standard doses, if maintained, arrest microfilarial production but result in only a mild-to-modest macrofilaricidal effect. We investigated whether high doses would effectively kill the adult worms, and whether cessation of microfilarial production could be reproduced by an equivalent, single, high dose. One hundred men participated in a double-blind placebo-controlled trial and received increasing doses of ivermectin from 150 micrograms/kg to 1600 micrograms/kg bodyweight. Nodules were excised at day 180 and examined by histopathology. Total doses of ivermectin up to 1600 micrograms/kg were not significantly more effective than 150 micrograms/kg. Moreover, they did not reproduce the marked inhibitory effects of the repeat standard-dose regimens on embryogenesis, nor the modest effect on adult worm viability, at comparable total doses. These effects may be functions of multiplicities of dosages rather than of the total dose. Our findings also suggest that repeated high-dose regimens are unlikely to be more effective than a similar number of 150 micrograms/kg doses. This deficiency of ivermectin requires that the search for macrofilaricides remains a top priority.

Comparison of the sensitivity of different geographical races of Onchocerca volvulus microfilariae to ivermectin: studies in vitro.

This study was designed to provide baseline information on the sensitivity of 4 geographical isolates of Onchocerca volvulus microfilariae (mf) (Ghana forest, Ghana savanna, Cameroon forest and Guatemala) to ivermectin, and to develop an in vitro system with which to examine parasites for the possible development of drug resistance. Drug effects were best visualized in the presence of monkey kidney (LLCMK2) feeder cells in the culture system (MEM medium+20% serum), since mf maintained in the absence of cells declined in condition rapidly. Incubation of Ghana forest mf (+cells) in ivermectin (10(-5)-10(-10) M) caused a decrease in motility index (MI) scores in a concentration-dependent fashion; drug effects could be observed as early as 6 h, but cultures maintained for up to 8 d showed greater differences between control and drug groups with increasing time. All 4 O. volvulus isolates and O. lienalis (bovine) were compared for their response to ivermectin (10(-7) M): O. lienalis mf were significantly more sensitive (78%) reduction in MI scores on day 8) than the O. volvulus isolates (33.4-47.7% reduction). O. volvulus microfilariae ex utero generally displayed lower levels of motility and were slightly less inhibited by ivermectin than were skin mf. The in vitro system described can distinguish between the populations of mf studied on the basis of differing MI responses to ivermectin and, when combined with assays to test the infectivity of mf to blackflies following exposure to drug, will provide methods with which to examine parasites for the possible development of resistance.

Ocular findings in a double-blind study of ivermectin versus diethylcarbamazine versus placebo in the treatment of onchocerciasis.

The effect of ivermectin, a new microfilaricide, was assessed in a double blind trial against diethylcarbamazine citrate (DEC) and placebo. Fifty-nine adult males with moderate to heavy infection with Onchocerca volvulus and with eye involvement were recruited from an area under Onchocerciasis Control Programme (OCP) vector control in Northern Ghana. They were randomly assigned to an eight-day treatment with ivermectin as a single dose of 12 mg on day 1 followed by placebo for the remaining seven days, or DEC, total dose 1.3 g, or placebo, and ophthalmological review was undertaken over a period of one year. DEC acted quickly to eliminate microfilariae from the eye and was associated with reactive ocular changes and in a few cases functional deficit. Ivermectin eliminated microfilariae slowly from the anterior chamber of the eye over a period of six months. The ocular inflammatory reaction was minimal and no functional deficit occurred. It is postulated that the observed slow action of ivermectin on the eye may be attributed in part to its instability to cross the blood-aqueous humour barrier because of its molecular size as a macrocyclic lactone causing microfilariae to leave the eye gradually along a newly created gradient. Ivermectin is an effective microfilaricide with minimal ocular adverse effect and could therefore be suitable for widespread application without strict supervision.

Useful field of view as a reliable screening measure of driving performance in people with Parkinson's disease: results of a pilot study.

PURPOSE: To determine the correlations of the Useful Field of View (UFOV), compared to other clinical tests of Parkinson's disease (PD); vision; and cognition with measures of on-road driving assessments and to quantify the UFOV's ability to indicate passing/failing an on-road test in people with PD. METHODS: Nineteen randomly selected people with idiopathic PD, mean age = 74.8 (6.1), 14 (73.7%) men, 18 (94.7%) Caucasians, were age-matched to 104 controls without PD. The controls had a mean age of 75.4 (6.4), 59 (56.7%) men, 96 (92.3%) Caucasians. Both groups were referred for a driving evaluation after institutional review board approval. RESULTS: Compared to neuropsychological and clinical tests of vision and cognition, the UFOV showed the strongest correlations (r > .75, p < 0.05) with measures of failing a standardized road test and number of driving errors. Among PD patients, the UFOV Risk Index score of 3 (range 1-5) was established as the optimal cutoff value for passing the on-road test, with sensitivity 87 percent and specificity 82 percent, AUC = 92 percent (SE 0.61, p = .002). Similarly, the UFOV 2 (divided attention) optimum cutoff value is 223 ms (range 16-500 ms), sensitivity 87.5 percent, specificity 81.8 percent, AUC = 91 percent (SE 0.73, p = .003). The UFOV 3 (selected attention) optimal cutoff value is 273 ms (range 16-500 ms), sensitivity 75 percent, specificity 72.7 percent, AUC = 87 percent (SE 0.81, p = .007). CONCLUSION: In this pilot study among PD patients, the UFOV may be a superior screening measure (compared to other measures of disease, cognition, and vision) for predicting on-road driving performance but its rigor must be verified in a larger sample of people with PD.

The chemotherapy of onchocerciasis II. Quantitation of the clinical reaction to microfilaricides.

A method of quantitation of the clinical reaction to microfilaricides has been devised at the Onchocerciasis Chemotherapeutic Research Centre at Tamale, in the savanna area of Northern Ghana. The method employs commonly occurring reactions--pruritus, rash, glandular reactions, musculoskeletal, febrile and cardiovascular--and a scoring system with a built-in weighting factor. Although the method is time-consuming and requires close, continuous observation of patients, it should permit more specific statements to be made about the severity of clinical reactions in individual patients or in groups of patients taking similar or different doses of the same drug or taking different drugs of the same drug or taking different drugs on a comparative basis. It is recommended that quantitative methods of the clinical reaction to microfilaricides form a basic part of the assessment of any drug used in the control of onchocerciasis.

Research notes from the Onchocerciasis Chemotherapy Research Centre, Ghana.

Brief notes are given on drugs which have been tested at the Onchocerciasis Chemotherapy Research Centre at Tamale and Hohoe and found to have activity against Onchocerca volvulus. Ivermectin in single doses as high as 800 micrograms/kg was found to be no more effective than the standard dose of 150 micrograms/kg. The benzimidazole carbamates, mebendazole and albendazole, differ in their effects on O. volvulus. The former has microfilaricidal effects and is toxic to developing embryos surrounded by an egg shell but not the stretched microfilariae. Albendazole has no microfilaricidal activity but is toxic to all intra-uterine stages. The reasons for these differences are unclear. Early studies with amocarzine are described; the maximum tolerable dose is 20 mg/kg and the predominant activity, against the microfilariae, is marked only at doses greater than 12 mg/kg. None of the drugs tested has macrofilaricidal activity.

The conquest of 'river blindness'.

Studies carried out at the WHO/OCP Onchocerciasis Chemotherapeutic Centre in Tamale, Ghana, are reported, culminating in the clinical trials of ivermectin (Mectizan), which has revolutionized our approach to the treatment of the disease. It is concluded that the conquest of 'river blindness' is an eminently achievable goal.

The chemotherapy of onchocerciasis IV. Further trials with metrifonate.

Thirty-four healthy adult males, moderately to heavily infected with Onchocerca volvulus, were treated daily with metrifonate at 10 mg/kg body weight for either three or six days. The patients' reactions and the effects on the microfilariae were measured on a 'single-blind' basis. Muscarinic effects of acetylcholine were prominent despite the use of belladonna alkaloids, and nicotinic effects--leading to a proximal muscle weakness--occurred in one patient after the fifth dose. A syndrome of polyarthritis, fever and elevated erythrocyte sedimentation rate occurred in 11 patients, in two of whom microfilariae were demonstrated in the joint fluid. The six-day regime destroyed 83% of the microfilarial load and the three-day regime destroyed 72%, but the difference was not statistically significant (as assessed one week after the completion of treatment). It seems unlikely that metrifonate will replace DEC as the 'reference' microfilaricide and attempts to improve its efficacy by employing higher dosage must employ an intermittent dose regime.

The chemotherapy of onchocerciasis iii. A comparative study of diethylcarbamazide (DEC) and metrifonate.

In a single-blind comparative study of diethylcarbamazine (DEC) and metrifonate against onchocerciasis, 20 patient received a total dose of 6.6 g DEC over a two-week period and 20 patients each received one dose of metrifonate 10 mg/kg body weight at ten-day intervals, three times. Both the efficacy and the severity of reaction to treatment were measured. DEC proved the more effective, destroying 98.9% of the microfilarial load while metrifonate destroyed only 75.4% (assessed one week after completion of treatment). The reaction to treatment was much more severe in patients treated with DEC. The persistence of significant postural cardiovascular effects for two weeks after the completion of DEC needs to be considered in mass therapy. The pattern of re-emergence of skin microfilariae indicated no difference between the two drugs over a six-month period. It is concluded that DEC is more effective than metrifonate and remains the 'reference' microfilaricide although its effects are more severe than those of metrifonate.

Quantification of the ocular reactions to microfilaricides in the chemotherapy of onchocerciasis.

Severe adverse systemic and ocular reactions have complicated the chemotherapy of onchocerciasis. A method for the quantification of ocular reactions to chemotherapy has been devised at the Onchocerciasis Chemotherapy Research Centre, at Hohoe, in Ghana. Symptoms, visual function, anterior segment inflammation and disease of the posterior segment are graded. The information is entered into a computerised database which allows reaction scores to be calculated both for individual patients, and for treatment groups. This system enables comparison of adverse ocular reactions to various new chemotherapeutic regimens.

Immunoregulation in onchocerciasis: persons with ocular inflammatory disease produce a Th2-like response to Onchocerca volvulus antigen.

To examine the role of specific cytokines in mediating the clinical manifestations of human onchocercal disease, microfilariae-positive Ghanaian subjects with inflammatory ocular disease were compared with microfilariae-positive subjects without ocular disease. Onchocerca volvulus antigen (OvAg)-stimulated peripheral blood mononuclear cells (PBMC) from subjects with disease produced significantly more interleukin (IL)-10 (with disease = 447.34 vs. without disease = 292.22 pg/mL; P < .01) and IL-5 (with disease = 33.36 vs. without disease = 27.26 pg/mL; P = .02). OvAg-stimulated IL-4 and interferon (IFN)-gamma levels were essentially undetectable in either group. When cytokine mRNA levels were measured by reverse transcriptase-polymerase chain reaction ELISA, persons with disease produced significantly more OvAg-stimulated IL-4, IL-5, and IL-10 mRNA (P = .03, < .01, .05, respectively). No difference in IFN-gamma mRNA production by either group was seen. Addition of neutralizing alpha IL-10 antibody to OvAg-stimulated PBMC increased TFN-gamma production to detectable levels in 20 of 24 persons.

The chemotherapy of onchocerciasis V. A standard method for the determination of microfilarial density in skin snips.

The microfilarial density in 400 skin snips from 100 patients was determined using the standard technique of the Onchocerciasis Chemotherapeutic Research Centre (OCRC method), the method used by the Onchocerciasis Control Programme (OCP method), and also after collagenase digestion. The OCRC method was fairly consistent and detected 84% of the total microfilariae. The OCP method gave a density which was consistently 20% less than the OCRC method, indicating that the increase in weight in skin snips following incubation in saline was fairly predictable. It is concluded that collagenase digestion is not worthwhile as a routine technique in chemotherapeutic trials, for which the OCRC method is recommended.