Comparative characterization of two galectins excreted-secreted from intestine-dwelling parasitic versus free-living females of the soil-transmitted nematode Strongyloides.

Helminths are complex pathogens that ensure their long-term survival by influencing the immune responses of their host. Excretory/secretory products (ESP) can exert immunoregulatory effects which foster parasite survival. Galectins represent a widespread group of ß-galactoside-binding proteins which are involved in a multitude of biological processes operative in parasite-host interaction. We had earlier identified seven galectins in Strongyloides ratti, four of them detected in the ESP of distinct developmental stages of the parasite. In the present report, we focused on the characterization of two of them, Sr-galectin-1 (Sr-Gal-1) and Sr-galectin-3 (Sr-Gal-3). While Sr-Gal-3 expression was strongest in parasitic females, Sr-Gal-1 was predominantly expressed in free-living females. Both proteins were cloned and recombinantly expressed in an E. coli expression system. Their glycan-binding activity was verified by haemagglutination and glycan array analysis. Furthermore, primary immunological activities of the Sr-galectins were initially investigated by the application of an in vitro mucosal 3D-culture model, comprising of mucosa-associated epithelial and dendritic cells. The Sr-galectins stimulated preferentially the release of the type 2 cytokines thymic stromal lymphopoietin and IL-22, a first indication for immunoregulatory activity. In addition, the Sr-galectins dose-dependently fostered cell migration. Our results confirm the importance of these carbohydrate-binding proteins in host-parasite-interaction by indicating possible interaction with the host mucosa-associated cells.

Development and application of three-dimensional skin equivalents for the investigation of percutaneous worm invasion.

Investigation of percutaneous helminth infection is generally based on animal models or excised skin. As desirable replacement of animal experiments, tissue-engineered skin equivalents have recently been applied in microbial and viral in vitro infection models. In the present study, the applicability of tissue-engineered skin equivalents for the investigation of percutaneous helminth invasion was evaluated. Epidermal and a full-thickness skin equivalents that suit the requirements for helminth invasion studies were developed. Quantitative invasion assays were performed with the skin-invading larvae of the helminths Strongyloides ratti and Schistosoma mansoni. Both skin equivalents provided a physical barrier to larval invasion of the nematode S. ratti, while these larvae could invade and permeate a cell-free collagen scaffold and ex vivo epidermis. In contrast, the epidermal and full-thickness skin equivalents exhibited a human host-specific susceptibility to larvae of trematode S. mansoni, which could well penetrate. Invasion of S. mansoni in cell-free collagen scaffold was lowest for all experimental conditions. Thus, reconstructed epidermis and full-thickness skin equivalents confirmed a high degree of accordance to native tissue. Additionally, not only tailless schistosomula but also cercariae could permeate the skin equivalents, and thus, delayed tail loss hypothesis was supported. The present study indicates that the limitations in predictive infection test systems for human-pathogenic invading helminths can be overcome by tissue-engineered in vitro skin equivalents allowing a substitution of the human skin for analysis of the interaction between parasites and their hosts' tissues. This novel tissue-engineered technology accomplishes the endeavor to save animal lives.

Immune recognition of Onchocerca volvulus proteins in the human host and animal models of onchocerciasis.

Onchocerca volvulus is a tissue-dwelling, vector-borne nematode parasite of humans and is the causative agent of onchocerciasis or river blindness. Natural infections of BALB/c mice with Litomosoides sigmodontis and of cattle with Onchocerca ochengi were used as models to study the immune responses to O. volvulus-derived recombinant proteins (OvALT-2, OvNLT-1, Ov103 and Ov7). The humoral immune response of O. volvulus-infected humans against OvALT-2, OvNLT-1 and Ov7 revealed pronounced immunoglobulin G (IgG) titres which were, however, significantly lower than against the lysate of O. volvulus adult female worms. Sera derived from patients displaying the hyperreactive form of onchocerciasis showed a uniform trend of higher IgG reactivity both to the single proteins and the O. volvulus lysate. Sera derived from L. sigmodontis-infected mice and from calves exposed to O. ochengi transmission in a hyperendemic area also contained IgM and IgG1 specific for O. volvulus-derived recombinant proteins. These results strongly suggest that L. sigmodontis-specific and O. ochengi-specific immunoglobulins elicited during natural infection of mice and cattle cross-reacted with O. volvulus-derived recombinant antigens. Monitoring O. ochengi-infected calves over a 26-month period, provided a comprehensive kinetic of the humoral response to infection that was strictly correlated with parasite load and occurrence of microfilariae.

Immunomodulatory effect of diethylcarbamazine in mice infected with Nocardia brasiliensis.

We tested whether diethylcarbamazine (DEC) or ivermectin (IVM), both antiparasitic drugs with reported immunomodulatory properties, were able to affect the immune system to potentiate host defense mechanisms and protect against actinomycetoma in a mouse model. Male BALB/c mice of 10-12 weeks of age were injected with either Nocardia brasiliensis or saline solution. Recorded were the effects of a treatment by DEC (6 mg/kg per os daily for one week) or IVM (200 µg/kg subcutaneously on days 1 and 3) on (i) the development of mycetoma lesion, (ii) the expression of reactive oxygen intermediates (ROI) by phagocytes, (iii) the proliferation index of lymphocytes and (iv) antibody production of IgG and IgM. After an initial lesion in all mice, DEC inhibited a full development and progression of actinomycetoma resulting in a reduced lesion size (p < 0.001). IVM had no inhibitory effect on the development of mycetoma. Furthermore, DEC treatment was associated with a significant enhancement of ROI expression (p < 0.05) by polymorphonuclear neutrophils at day 3 after infection. Lymphocyte proliferation in response to N. brasiliensis antigens and concanavalin A in DEC-treated group was higher than in non-treated group at day 21 and 28 postinfection (p < 0.01). Significant changes in antibody response were not observed. By all parameters tested, DEC was superior to IVM regarding immunostimulatory potency. In conclusion, DEC expressed an in vivo influence on the immune status during the infection by N. brasiliensis leading to retrogression of the mycetoma and increasing cellular immune responses. Our findings may indicate a potential use of DEC as a putative adjuvant in infectious disease or vaccination.

Immunohistological studies on neoplasms of female and male Onchocerca volvulus: filarial origin and absence of Wolbachia from tumor cells.

Up to 5% of untreated female Onchocerca volvulus filariae develop potentially fatal pleomorphic neoplasms, whose incidence is increased following ivermectin treatment. We studied the occurrence of 8 filarial proteins and of Wolbachia endobacteria in the tumor cells. Onchocercomas from patients, untreated and treated with antibiotics and anthelminthics, were examined by immunohistology. Neoplasms were diagnosed in 112 of 3587 female and in 2 of 1570 male O. volvulus. The following proteins and other compounds of O. volvulus were expressed in the cells of the neoplasms: glutathione S-transferase 1, lysosomal aspartic protease, cAMP-dependent protein kinase, alpha-enolase, aspartate aminotransferase, ankyrin E1, tropomyosin, heat shock protein 60, transforming growth factor-beta, and prostaglandin E(2). These findings prove the filarial origin of the neoplasms and confirm the pleomorphism of the tumor cells. Signs indicating malignancy of the neoplasms are described. Wolbachia were observed in the hypodermis, oocytes, and embryos of tumor-harbouring filariae using antibodies against Wolbachia surface protein, Wolbachia HtrA-type serine protease, and Wolbachia aspartate aminotransferase. In contrast, Wolbachia were not found in the cells of the neoplasms. Further, neoplasm-containing worms were not observed after more than 10 months after the start of sufficient treatment with doxycycline or doxycycline plus ivermectin.

Molecular characterisation of kappa-5, a major antigenic glycoprotein from Schistosoma mansoni eggs.

The major immunopathological consequences of infection with Schistosoma mansoni, a T helper type 2 response and granuloma formation leading to fibrotic tissue damage, are caused by the egg stage of the parasite. Three antigens of S. mansoni eggs, termed IPSE/alpha-1, omega-1 and kappa-5, have been found to be the primary targets of the egg-directed antibody response of the host. Here, we report on the isolation, cloning and characterisation of kappa-5. Apart from an uncharacterised mRNA sequence in S. japonicum, no significant similarities of kappa-5 to known sequences from other species were found. In contrast to IPSE/alpha-1 and omega-1, which have been found only in eggs, kappa-5 was present in miracidia as well as in eggs at the mRNA and protein levels. In eggs, isoforms of kappa-5 were observed with both three and four fully occupied N-glycosylation sites, while in miracidia only one isoform with four N-glycans could be detected. Interestingly, in Western blots sera from S. mansoni-infected Africans were reactive against kappa-5 with IgE and IgG isotype antibodies, but against IPSE/alpha-1 and omega-1 only with IgG antibodies. The further characterisation of kappa-5 as one of the three major egg antigens should help to better understand the immunology and immunopathology of schistosomiasis.

Relevance of ex vivo blood lymphocyte assay for in vivo lymphocyte function.

Determinations of in vitro proliferative and secretory activities of peripheral blood cells are used widely for research in clinical immunology but, to our knowledge, have not been evaluated as to their power to reflect in vivo activities quantitatively. Here, we addressed this question by quantitatively correlating the in vitro secretion of interleukin (IL)-5 by peripheral blood cells to the in vivo activity of IL-5 as reflected by peripheral-blood eosinophil counts. Studying 458 humans exposed to transmission of the nematode Onchocerca volvulus, IL-5 was measured in the supernatants of 0.02-ml whole-blood cells cultured in the presence of O. volvulus extract or mitogen. O. volvulus-reactive IL-5 secretion was correlated significantly to blood eosinophilia in a quantitative manner explaining 15.1% (95% CI 8.3-19.9%) of the variability of eosinophil counts. Interestingly, correlations were obtained only if parasite counts were included in the calculation using multiple regression analysis. The results show that in vitro assays of minute amounts of blood lymphocytes may quantitatively reflect activities of the entire lymphocyte population in vivo.

Circulating concentrations of cardiac proteins in complicated and uncomplicated Plasmodium falciparum malaria.

In an unmatched case-control study of 63 non-immune European patients with uncomplicated (n = 52) and complicated (n = 11) falciparum malaria, serum levels of N-terminal pro-brain natriuretic peptide (NT-proBNP), heart-type fatty acid-binding protein (H-FABP), myoglobin, troponin T and creatin kinase-muscle brain were compared. Elevated levels of NT-proBNP and H-FABP indicated myocardial impairment in complicated but not in uncomplicated falciparum malaria. The clinical impact of these findings remains to be evaluated. The pathophysiology of cardiac impairment in complicated falciparum malaria warrants further investigation.

Promoter haplotypes of the interleukin-10 gene influence proliferation of peripheral blood cells in response to helminth antigen.

Since interleukin (IL)-10 is a key mediator of immunosuppression, and immunosuppression is considered an important element of helminth infection, we studied variants of the putative IL-10 gene promoter in 337 individuals from 130 families heavily exposed to infection by the tissue nematode Onchocerca volvulus. As shown by transmission disequilibrium tests, variants of the IL-10 promoter at positions -1082(G/A), -819(C/T), and -592(C/A) in the haplotype of ATA were significantly associated with high peripheral blood cell (PBC) proliferative responses to O. volvulus antigen (OvAg). No associations were observed using phytohemagglutinin-induced PBC proliferation or with qualitative or quantitative phenotypes of onchocerciasis or onchocerciasis-related skin disease. The findings are compatible with the hypothesis that the ATA haplotype causes a decrease in IL-10 production by OvAg-reactive type-1 regulatory T-lymphocytes, thereby alleviating the suppression of other T cells. To our knowledge, this is the first time that an influence of IL-10 promoter variants is shown on the adaptive immune response.

Pathogenesis and host responses in human onchocerciasis: impact of Onchocerca filariae and Wolbachia endobacteria.

Onchocerca volvulus is a tissue-invasive parasitic nematode causing skin and eye pathology in human onchocerciasis. The filariae habour abundant intracellular Wolbachia bacteria, now recognised as obligatory symbionts, and therefore emerging as a novel target for chemotherapy. Recent research demonstrates that both the filariae and endobacteria contribute to the pathogenesis of onchocerciasis, and molecules have been identified that promote inflammatory or counter-inflammatory immune mechanisms, divert the host's immune response or procure evasion of the parasite.

Wolbachia in filarial nematodes: evolutionary aspects and implications for the pathogenesis and treatment of filarial diseases.

The presence of intracellular bacteria in the body of various species of filarial nematodes, including important parasites such as Brugia malayi, Dirofilaria immitis, and Onchocerca volvulus, was observed as early as the mid-1970s. These bacteria were shown to be transovarially transmitted (from the female worm to the offspring) and to be present in significant amounts in the body of the nematode. As highlighted by their discoverers, the potential importance of these bacteria is fairly obvious: (1) bacteria-derived molecules should be considered as having an immunological and pathological role in filarial diseases; (2) the interaction between the bacteria and the filarial host deserves investigation, in view of the possibility that the bacteria are needed by the host nematode and could thus represent a target for therapy. Other authors, independently from the discovery of these intracellular bacteria, showed that the antibiotic tetracycline (which is well known for its efficacy on intracellular bacteria) had detrimental effects on two species of filarial nematodes (Brugia pahangi and Litomosoides sigmodontis). It is therefore surprising that for more than 20 years, no further investigations focused on the bacteria of filarial nematodes, nor on the anti-filarial properties of tetracycline. Recently, the bacteria of filarial nematodes have been independently "rediscovered" by research groups from the schools of Hamburg, Liverpool and Milan. These bacteria are now classified as Wolbachia, and the basic aspects of their phylogenetic history and relationship with the Wolbachia of arthropods have been reconstructed. In addition, their implications for the pathogenesis and treatment of filarial diseases have started to be uncovered. This paper, which is authored by representatives of the three European schools who reopened this research area, reviews our present knowledge of these fascinating microorganisms, highlighting the complexity of a symbiotic system which involves, in addition to the nematode and its bacterium, the vertebrate host.

Neutrophil accumulation around Onchocerca worms and chemotaxis of neutrophils are dependent on Wolbachia endobacteria.

Unlike in many other helminth infections, neutrophilic granulocytes are major cellular components in the hosts immune response against filarial worms. The pathways that drive the immune response involving neutrophils are unclear. This study shows that Wolbachia endobacteria (detectable by polyclonal antibodies against endobacterial heat shock protein 60 and catalase and by polymerase chain reaction being sensitive to doxycycline treatment) are direct and indirect sources of signals accounting for neutrophil accumulation around adult Onchocerca volvulus filariae. Worm nodules from untreated onchocerciasis patients displayed a strong neutrophil infiltrate adjacent to the live adult worms. In contrast, in patients treated with doxycycline to eliminate the endobacteria from O. volvulus and to render the worms sterile, the neutrophil accumulation around live adult filariae was drastically reduced. Neutrophils were absent in worm nodules from the deer filaria Onchocerca flexuosa, a species which does not contain endobacteria. Extracts of O. volvulus extirpated from untreated patients showed neutrophil chemotactic activity and in addition, induced strong TNF-alpha and IL-8 production in human monocytes, in contrast to filarial extracts obtained after doxycycline treatment. Thus, neutrophil chemotaxis and activation are induced directly by endobacterial products and also indirectly via chemokine induction by monocytes. These results show that the neutrophil response is a characteristic of endobacteria-containing filariae.

Humoral responses to a secretory Onchocerca volvulus protein: differences in the pattern of antibody isotypes to recombinant Ov20/OvS1 in generalized and hyperreactive onchocerciasis.

The Onchocerca volvulus secretory protein Ov20/OvS1 represents a dominant antigen expressed in the infective larvae, microfilariae and adult stages of the parasite. The humoral responses to this protein have not yet been analysed in the polar clinical and immunological forms of onchocerciasis. Analysis by ELISA of class and subclass antibodies to Ov20/OvS1 in persons with the generalized or the hyperreactive form of onchocerciasis revealed similar strong responses of IgG1, IgG4 and IgM antibody levels in both forms of onchocerciasis and significant differences were observed in the IgE and IgA antibody classes. Computation of the ratios of antibodies showed that persons with the generalized form exhibited significantly higher ratios of IgG4 to IgG1, IgG4 to IgE, and IgM to IgE than patients with the hyperreactive form. To investigate the isotype recognition of antigenic sites on Ov20/OvS1 protein, three recombinantly expressed fragments (F1-3) of Ov20/OvS1 were probed using sera which strongly reacted with intact recombinant Ov20/OvS1. Epitope(s) on F1 comprising amino acid residues 1-63 were significantly recognized by IgG1 and IgE, while IgM recognized epitopes on all three fragments. The strongest reaction of IgM occurred with epitope(s) formed by residues 108-171 (F3). In contrast, IgG4 type antibodies were not reactive with either of the three OvS1 fragments, but they reacted with intact Ov20/OvS1 protein. Generalized onchocerciasis, unable to eliminate microfilariae, and hyperreactive onchocerciasis, with a high potency to eliminate or to reduce parasite loads, can be distinguished by a distinct pattern of isotype responses to Ov20/OvS1.

Lipopolysaccharide-like molecules derived from Wolbachia endobacteria of the filaria Onchocerca volvulus are candidate mediators in the sequence of inflammatory and antiinflammatory responses of human monocytes.

The majority of Onchocerca volvulus-infected persons show signs of cellular anergy, and long-time survival of adult and larval parasites in subcutaneous tissue is observed. The mechanisms leading to immunological hyporesponsiveness are poorly understood. Monocytes/macrophages represent a link between the innate and acquired immune system and are candidate cells to promote inflammatory and antiinflammatory processes. In the present study we have shown that products of microfilarial (O. volvulus) and adult (O. volvulus and O. ochengi) parasites affect monocytes in vitro. An early production of TNF-alpha by exposed monocytes was followed by the production of IL-10 and a reduced expression of HLA-DR and the costimulatory molecules B7-1 and B7-2, while other adhesion receptors remained unaffected. Downregulation of the functional membrane receptors failed to occur after treatment of the cells with anti-IL-10 antibodies. The engagement of CD14, a dominant membrane receptor on monocytes and major binding protein for lipopolysaccharides, was indicated by partial blocking of monocyte modulation by neutralizing antibodies to CD14 and by the antagonistic lipid A analog compound 406. Lipopolysaccharide-like molecules were detected in sterile products of O. volvulus stages which could originate from Wolbachia bacteria related to Gram-negative Rickettsiales, known to be abundant in the hypodermis and the female reproductive organs of O. volvulus. The present results indicate that the monocyte/macrophage may be a major target cell for immunomodulatory parasite-derived and intraparasitic, bacteria-derived molecules, thereby contributing to the host's cellular hyporesponsiveness.

The secretory Onchocerca volvulus protein OvS1/Ov20 exhibits the capacity to compete with serum albumin for the host's long-chain fatty acids.

The mechanism by which filarial parasites derive fatty acids bound to the host's carrier protein is poorly understood. The capacity of a secretory protein of Onchocerca volvulus (OvS1/Ov20) to compete with serum albumin for arachidonic and other fatty acids was investigated in this study. Binding affinities of the two proteins for the long-chain fatty acids were determined using displacement assays. The fluorescent probes used included 11-((5-dimethylaminonaphthalene-1-sulfonyl)amino) undecanoic acid (DAUDA) and cis-parinaric acid. OvS1 protein bound arachidonic acid with an affinity five-fold greater than the affinity exhibited by serum albumin. Oleic acid was bound by the parasite protein with an affinity two-fold greater than the affinity shown by serum albumin. Furthermore, the affinities exhibited by OvS1 protein in binding arachidonic and linoleic acid were about two times higher than the affinity for oleic acid. The results suggest that the OvS1 protein has the capacity to compete with the main host's fatty acid carrier protein for the long-chain fatty acids, in particular arachidonic acid, the precursor for eicosanoids.

Use of the recombinant Onchocerca volvulus protein Ov20/OvS1 for the immunodiagnostic differentiation between onchocerciasis and mansonelliasis and for the characterization of hyperreactive onchocerciasis (sowda).

The protein Ov20/OvS1 was used as antigen in ELISA and Western blot in order to differentiate onchocerciasis from African mansonelliasis and to characterize the hyperreactive form of Onchocerca volvulus infection (sowda). The specificity of the IgG4 Western blot was 98% for the differentiation between persons with onchocerciasis and Mansonella microfilariae (mf) carriers (125 persons with M. perstans and 92 with M. streptocerca), whereas the IgG4 ELISA showed a specificity of 81% in 137 M. perstans mf carriers and 85% in 94 M. streptocerca mf carriers. The sensitivity of Ov20/OvS1 in identifying onchocerciasis using the IgG4 ELISA was 75% for 103 O. volvulus mf carriers with the generalized and 89% for 44 patients with the sowda form of onchocerciasis. IgE antibodies against OvS1 were found in 95% of 39 patients with hyperreactive onchocerciasis but only in 15% of 47 persons with the generalized form. Thus, Ov20/-OvS1 appears a promising candidate antigen for the diagnosis of onchocerciasis and in particular for the detection of the sowda type of disease.

Eosinophil granule proteins in serum and urine of patients with helminth infections and atopic dermatitis.

Eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EPX) are cytotoxic molecules involved in helminth infections and allergic reactions. Hitherto most clinical chemical studies have been concerned with the analysis of serum ECP in allergic diseases. The aim of this study was to examine whether serum as well as urine levels of these proteins are useful clinical chemical parameters in helminthiases and allergic diseases such as atopic dermatitis. Comparing these diseases under the same methodological conditions, levels of ECP and EPX were generally higher in helminthiases than in atopic dermatitis and non-helminth, non-allergic diseases. The highest levels of both proteins occurred in tropical worm diseases, in particular hookworm disease and onchocerciasis. When comparing helminthiases with allergic disorder, only hookworm disease (ECP and EPX) and onchocerciasis (EPX) exhibited significantly higher eosinophil cationic protein serum levels than atopic dermatitis. In patients with schistosomiasis mansoni and egg loads of > 1000-10 000 eggs/g stool (epg) EPX serum levels were significantly higher than in patients exhibiting loads < 1000 epg. Urinary analyses revealed only EPX to be present in measurable amounts. Levels of this protein were much higher in urine of patients with hookworm disease and onchocerciasis than in those with atopic dermatitis and in healthy controls. The results suggest that besides serum EPX, urinary EPX may be a useful clinical chemical parameter in eosinophilia of helminth and allergic aetiology.

Hyperreactive onchocerciasis exhibits reduced arachidonate and linoleate levels in serum triglycerides.

The mechanism by which the minority of patients with onchocerciasis exhibiting the hyperreactive (sowda) form of the disease may be able to kill the microfilariae of Onchocerca volvulus is still poorly understood. In this study, the relative amounts of arachidonate and linoleate in serum phospholipids and triglycerides were investigated by gas chromatography both in patients infected with O. volvulus who exhibited either a hyperreactive or a generalized form of onchocerciasis and in persons with no filarial infections. Remarkable differences were observed in the serum triglycerides but not in the phospholipids. In comparison to persons without any filarial infection, significantly lower relative amounts of arachidonate--indicated by elevated triene-tetraene ratios--and of linoleate--indicated by lower diene + tetraene - triene values--were detected in patients with hyperreactive onchocerciasis, and less pronounced differences were found in persons with generalized onchocerciasis. The relationship between reduced amounts of arachidonate and linoleate in serum triglycerides and possible implications on the eicosanoid production in the host-parasite relationship leading to parasite elimination are discussed.

Delayed-type hypersensitivity reactions to Onchocerca volvulus antigens in exposed and non-exposed African individuals.

Although considered of critical importance, the mode of helper T-lymphocyte function in Onchocerca volvulus infection is still unclear including the role of the Th1/Th2 dichotomy. We studied the delayed-type hypersensitivity (DTH) reaction, which is the classical Th1 response, to O. volvulus antigens in Africans exposed and not exposed to the infection. DTH reactions were found in a small percentage of patients with generalized onchocerciasis, but in a high percentage of patients with localized onchocerciasis, in putatively immune subjects, and also in non-exposed individuals, which may be due to cross-reactivity with other nematodes. These findings support the notions of (i) prenatal influence of maternal O. volvulus infection preventing development of Th1 responses and/or (ii) suppression of Th1 responses by the infection itself.

Analysis of renal function in onchocerciasis patients before and after therapy.

The occurrence of renal abnormalities was investigated in patients with onchocerciasis in comparison to individuals without onchocerciasis in Guinea. Serum creatinine levels, excretion of urinary marker proteins, and kidney size by ultrasound were determined. A high prevalence of glomerular as well as tubular dysfunctions was observed; however, no association with onchocerciasis could be detected. We also hypothesized that patients with hyperreactive onchocerciasis might be prone to develop immune-mediated glomerular disorders; however, this could not be verified. Following treatment with ivermectin, a slight but significant increase in the excretion of urinary albumin and alpha1-microglobulin was seen five days after treatment in all treated patients, whereas levels of proteinuria were significantly higher five days after treatment only in patients with high microfilarial densities. Our results indicate that ivermectin can cause glomerular and tubular disturbances in patients with onchocerciasis; however, these are minor and do not seem to be clinically relevant.