Abstract

The induction of pathological changes in Onchocerca volvulus infections is directly related to the presence of the microfilarial stage of this filarial nematode. Patients with either of the 2 major forms of the clinical disease (i.e., asymptomatic/mild [n=12] and severe [n=16] dermatopathology) were studied. The cellular immune responses (cell proliferation) of those with severe disease were stronger (stimulation index [SI], 12.3±1.9) than those with mild dermatopathological effects (SI, 2.9±0.6) or control patients (SI, 4.5±0.4). Cytoadherence antibody responses were greatest (grade 4) in the clinically severe group and only weak (grades ⩽1) in the mild group or the control patients. Ivermectin treatment was followed by an increase in immune responsiveness in those with initially poor responses. Thus, the degree of dermatopathological effect is related to the host’s immune response against microfilariae, and ivermectin augments such responses

Onchocerciasis is a filarial infection characterized by a wide variety of ocular and dermal pathological effects, the latter ranging from minor irritation to a severe reactive dermatitis [1]. Microfilariae (Mf) of Onchocerca volvulus are central to the initial acute papular dermal changes, as well as to the punctate keratitis seen in patients with ocular infection [1, 2]. Although various mechanisms have been suggested as possible contributors in pathogenesis of this disease, autoimmunity and secondary bacterial infections (i.e., Wolbachia species), the most likely central pathogenic event involves repeated cycles of inflammation associated with the demise of microfilariae [1]

The clinical variations observed are believed to parallel a variability in the immune response against the Mf [1]. However, it is also generally assumed that a worsening clinical presentation of onchocercal dermatitis reflects an increasing load of Mf. These 2 somewhat contradictory concepts need to be resolved, and the role of immune responses, either in inducing pathological changes or in controlling the number of parasite loads, needs to be investigated further

The present study addresses the immune responses directed against O. volvulus Mf in patients presenting with the 2 major forms of onchodermatitis: the asymptomatic/mild and the severe dermal manifestations. We also assess the effect of the widely used antifilarial treatment, ivermectin (Mectizan), on these immune responses

Patients, materials, and methods The 28 O. volvulus skin-snip positive patients examined in this study became infected in the major onchocerciasis endemic area of southern Sudan [3]. Classification of their onchocercal dermatitis was based on previously developed schemes [3, 4], with 2 categories defined: group A, asymptomatic (no obvious dermal changes) or mild (minor acute changes) onchodermatitis (n=12); and group B, severe (active chronic skin changes) onchodermatitis (n=16)

Nine patients (7 from group A and 2 from group B) were also studied 1–3 months after treatment with therapeutic doses of ivermectin (150 μg/kg). Control serum and cell samples for use in the cytoadherence assays (n=2) and stimulation index (SI) (n=5) were obtained from individuals who were resident outside the endemic areas and never diagnosed as being infected with O. volvulus

Adult O. volvulus worms were isolated from nodules by collagenase digestion [5] and were homogenized. Insoluble material was removed by ultracentrifugation, and the remainder was filtered to make soluble O. volvulus antigen (OvAg) at 1 mg protein/mL [6], a preparation containing Mf antigens derived from uterine Mf. Living Mf for adherence cultures were isolated from onchocercal nodules using an agarose technique [7] and suspended at 1000 Mf/mL in RPMI 1640 medium containing 10% (vol/vol) heat-inactivated fetal calf serum

Eosinophil-rich cell suspensions were prepared from heparinized blood taken from onchocerciasis patients (>43% eosinophilia). Antibody-mediated cell adherence assay to detect antibodies against microfilariae were carried out, in duplicate, in flat bottom culture wells containing 50 Mf and 50 μL of undiluted serum sample from each patient or control subject. After incubation at 37°C for 1 h to allow the antibody to bind to Mf, 4×10 5 cells were added to each well, and the extent of adherence to the surface of at least 30 Mf in each culture was assessed after 3 h. Levels were classified in the following categories: 0, none or only a few cells adherent; 1, up to 25% surface covered with adherent cells; 2, 25%–49% surface covered; 3, 50%–74 % surface covered; and 4, 75%–100% surface covered. Cultures were checked at 24 h to ensure that no false negative assessments had been made

Peripheral blood mononuclear cells (PBMC) were separated from the whole blood of each subject by centrifugation over Ficoll-Hypaque (Histopaque 1077; Sigma). After washing, cells were suspended at the required cell concentration in RPMI 1640 medium, supplemented with 10% heat-inactivated fetal calf serum (Hyclone), 2 mMl-glutamine, 1 mM HEPES, 0.04 mM 2-mercaptomethanol, and 80 μg gentamycin

The blastogenic responses was measured using a standard 3H-thymidine incorporated technique. One-hundred thousand cells in 100 μL RPMI 1640 medium were stimulated by 10 μL of either 5 μg/mL phytohemagglutinin (PHA) mitogen (Sigma) or 20 μg/mL OvAg. Cultures were incubated, in triplicate, for 5 days at 37°C in a humidified atmosphere of 5% CO2 in air. Eighteen hours before harvesting, 1 Ci of 3H-thymidine 5 Ci/mmol (Amersham International) was added. Cells were harvested on fiber filters (Titertek; Flow Laboratories), the radioactive uptake were measured, and the results were expressed as an SI. The latter was calculated, as follows: the average counts per minute of 3 similar wells containing antigen-average counts per minute of 3 nonstimulated wells×10 −3

The Student’s t test, under unequal variance assumption, was used to compare the SI of the different groups, the Mann-Whitney U nonparametric test for the comparison of the groups in terms of cytoadherence, the paired Student’s t test to compare the pre- and posttreatment results, and, finally, the Pearson correlation test was used to assess the relationship between the assays and severity of disease

Results Twelve patients (group A) presented with essentially normal skin or with minor acute papular reactions (i.e., the mild onchodermatitis group). Sixteen individuals (group B) had skin changes typified by chronic indurated papules, extensive pigment variation, thickening of the epidermis, edema of the dermis, and severe persistent pruritus (i.e., the severe onchodermatitis group). O. volvulus Mf loads were highest in the asymptomatic/mild skin disease group (mean of those measured, 48.7±16.3 cells), whereas markedly lower Mf loads were evident in the severe skin disease group (mean of those measured, 2.1±1.6). The Mf loads in group A were significantly higher than those in group B (P<.005), as determined by Student’s t test. Thus, there was an inverse relationship between the Mf count and the severity of clinical manifestation (table 1)

Table 1

Assessment of immune responses in 2 clinical forms of onchodermatitis

Table 1

Assessment of immune responses in 2 clinical forms of onchodermatitis

The 2 groups differ significantly in the ability of serum to mediate cell adherence to the surface of Mf (z=-2.615; P=.008, Mann-Whitney U test). Patients from the mild dermatitis group showed only a minimal response (mean group, 1) in the cell adherence assay (table 1), whereas serum samples of patients with severe dermatitis mounted strong responses in the cell adherence assay (mean group, 3)

Cell proliferation responses to OvAg (table 1) were higher (12.3±1.9) in patients with more-severe skin manifestations. The mean proliferative index (SI) of the mild dermatitis group was 2.9±0.6, which was statistically different from the severe patient group (P<.005). The responses to OvAg in control individuals (4.5±0.4) were similar to those in patients with mild skin disease (group A). PHA responses are within the range expected and indicate that the culture system was technically adequate

A positive relationship between the presence of the ability to mount an active cell-adherence response and an active cell-mediated response was seen (Pearson’s correlation, r=0.689; P=.003). These responses were found to be inversely correlated to the Mf loads: the lower the Mf load, the greater the cytoadherence activity and cell proliferation responses

The cell-mediated responses of 9 patients (7 from group A and 2 from group B) were assayed after treatment with ivermectin, and responses increased in all cases, most significantly in those with mild disease (group A), whose average increase was >2.5. The increase in 2 severe dermatitis patients was minimal (figure 1). The clinical condition of all patients improved after treatment

Figure 1

Effect of ivermectin treatment on the lymphocyte responses to Onchocerca volvulus antigen. P1–P7, patients with asymptomatic/mild dermatitis; P8 and P9, patients with severe dermatology; white bars before treatment; black bars after treatment. All tests were performed 1–3 months after standard treatment with ivermectin

Figure 1

Effect of ivermectin treatment on the lymphocyte responses to Onchocerca volvulus antigen. P1–P7, patients with asymptomatic/mild dermatitis; P8 and P9, patients with severe dermatology; white bars before treatment; black bars after treatment. All tests were performed 1–3 months after standard treatment with ivermectin

Discussion It is well accepted that the pathological changes in onchocerciasis are related to the microfilarial stage of this nematode. Immune responses to O. volvulus are well documented [8–12 ], but the relationship between such host responses and an individual patient’s clinical status has remained uncertain at best. The results of the present study show that 2 types of immune response mounted against O. volvulus Mf antigens differ between 2 major forms of onchodermatitis. This finding is in keeping with the implications of various published studies [8, 12, 13] and clearly identifies immune responses against the Mf stage as directly related to the dermal presentation

The positive correlation between immune response and clinical severity seen in this present study in all likelihood reflects an increase in inflammation associated with increased Mf destruction; thus, a decrease in detectable Mf load parallels an increase in severity of skin disease. Low dermal living Mf loads are often seen in individuals with severe pathology and clinically obvious skin disease, such as those with the form of onchocerciasis known as sowda (or reactive onchodermatitis) [2, 8]. These patients may have significant burdens of dying or nonmotile parasites that are not detected in the skin-snip assay, which demonstrates only live parasites. These observations contrast with the popular belief that detectable Mf loads simply increase with duration of infection, and that disease severity parallels increases in parasitic loads, as measured by the skin-snip assay. The ability of the host’s immune system to destroy the Mf, together with the duration of infection, are clearly both important factors affecting the eventual clinical outcome

Infected individuals with mild dermatopathology (group A) appeared to be less able to mount antibody and cellular responses specifically directed against Mf. Antibodies against many of the antigens of O. volvulus have been described including those specifically involved in the destruction of O. volvulus Mf through antibody-dependent cell cytotoxicity mechanisms [9–11 ]. Cell mediated immunity (CMI) has also been described and is known to be central to certain clinical forms, such as reactive onchocercal dermatitis (sowda) [8]. It is assumed that in vitro cellular responses to adult worm antigen cocktails made from adult worms also include activities stimulated by Mf stage antigens, as Mf are a significant component of adult female worms

Immunosuppression towards Mf has been suggested elsewhere [12], and the poor cell adherence to O. volvulus Mf in the mild pathology group suggests that the antibody-mediated immune responses in these individuals are ineffective at destroying Mf. This finding may explain, in part, their clinically quiescent state, when compared with the severe pathologically effected group, whose serum mounted stronger responses against the worm’s surface. Adherence has been shown to correlate with clinical manifestations of Mf destruction [1]

Ivermectin appeared to significantly enhance cell-mediated responses in the mild onchodermatitis patients examined 3 months after treatment. This supports the findings of Soboslay et al. [13], who suggested the immune system is affected by ivermectin chemotherapy. Immune mechanisms may be involved not only in the initial induction of Mf killing but also in maintaining an ongoing process of Mf destruction in an individual. This concept is supported by the recent finding that ivermectin is sometimes less effective in immunocompromised individuals [14]. Resurgence of Mf loads in persons previously treated with ivermectin may be influenced by the immune responses resulting from that treatment. Mf loads are, as a rule, maintained at low levels after treatment [15], and any such active immune responses do not appear to induce further pathological effects

The exact sequence of events and consequences associated with the death, breakup, and removal of Mf are still open to conjecture. Immune, nonspecific and drug-induced destruction of Mf can all occur at different times during this infection. When death of Mf is induced by chemotherapeutic agents, severe anaphylactoid (Mazzotti) reactions can occur [1]; fortunately, these occur less with the current microfilariacidal agent, ivermectin. Nevertheless control of the associated pathogenic side reactions is important, especially in those individuals who react severely, such as sowda patients [16]. Understanding the role of the immune response in the induction of these adverse reactions will help achieving this goal

The finding of an inverse relationship between Mf load and severity of skin disease has important implications for control programs. Such programs are often guided by Mf loads and are based on the assumption that the Mf load directly relates to disease status. As Mf loads are often low in patients with the most severe disease, such control programs are failing to address these needy patients. This exclusionary policy should be revisited to take account of those most severely affected with this disease. The findings presented here also underline the importance of including clinical evaluations in epidemiological assessments of disease prevalence instead of relying on parasitological parameters alone

Acknowledgments

We thank Mohammed Satti and Moawia Muhktar for their assistance

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Presented in part: Annual meeting of the American Society of Tropical Medicine and Hygiene, Atlanta, 11–15 November 2001 (abstract 074).
Informed consent was obtained from each volunteer. The study protocol adhered to the guidelines of the US Department of Health and Human Sciences and was approved by appropriate institutional review boards.
Financial support: National Institutes of Health (grant AI-16312)