Assessment of multiplex Onchocerca volvulus peptide ELISA in non-endemic tropical regions.

BACKGROUND: Currently, serodiagnosis of infection with the helminth parasite Onchocerca volvulus is limited to the Ov-16 IgG4 test, a test that has limited sensitivity and suboptimal specificity. In previous studies, we identified several linear epitopes that have the potential to supplement the diagnostic toolbox for onchocerciasis. METHODS: In this study three peptides, bearing in total six linear epitopes were transferred to a multiplex ELISA platform. This multiplex ELISA was used to assess the clinical utility of the peptide serology markers by analyzing sample sets from both O. volvulus endemic and non-endemic regions. RESULTS: The multiplex platform was shown to be reproducible and data obtained on the multiplex platform were comparable to the singleplex ELISA data. The clinical utility assessment showed that in a population of school-aged children from western Kenya, a virtually O. volvulus-free area, significant cross-reactivity with an as-yet to be determined immunogen was detected. CONCLUSIONS: The observations made in this study invalidate the usefulness of the peptide serology markers for onchocerciasis detection. We discuss what could be the origin of this unexpected serological response, but also highlight the need for better characterized biobanks for biomarker discovery activities.

On-going transmission of human onchocerciasis in the Massangam health district in the West Region of Cameroon: Better understanding transmission dynamics to inform changes in programmatic interventions.

BACKGROUND: Massangam health district (HD), in the West Region of Cameroon, has received ivermectin mass drug administration (MDA) for 20 years, however there is evidence of continued high transmission of Onchocerca volvulus. In order to better understand the transmission dynamics in the HD and inform intervention strategies there is a need to delineate the boundaries of the suspected area of high transmission within the wider transmission zone. METHODOLOGY/PRINCIPAL FINDINGS: Parasitological and entomological surveys were conducted to map out the breeding sites of Simulium damnosum and evaluate the prevalence of onchocerciasis in neighbouring communities, including Makouopsap sentinel community. Potential rapids were prospected for identification of S. damnosum larvae and black flies collected to determine infectivity rates. Adults were assessed for the presence of O. volvulus microfilariae through a skin snip biopsy and examined for the presence of nodules. Anti Ov-16 antibodies were tested for in children. Four perennial breeding sites were identified on the Rivers Mbam and Nja. Large number of flies were collected along the River Mbam, especially in the rainy season, with up to 955 flies per day, suggesting this river is a perennial source of black flies. A total of 0.8% of parous flies were infective across the study area. Parasitological studies provided evidence of high rates of infection in the sentinel community and three neighbouring communities, with 37.1% of adults microfilariae positive in Makouopsap. High Ov-16 seropositivity in children also provided evidence of recent on-going transmission. In comparison, communities sampled further away from the sentinel community and neighbouring breeding sites were much closer to reaching onchocerciasis elimination targets. CONCLUSIONS/SIGNIFICANCE: This study provides evidence of a particular geographic area of high transmission in an approximate 12 km range around the sentinel community of Makouopsap and the neighbouring breeding sites on the River Nja. To eliminate onchocerciasis by 2025, there is a need to explore alternative intervention strategies in this area of high transmission.

Evaluation of the Diagnostic Performance of Onchocerca volvulus Linear Epitopes in a Peptide Enzyme-Linked Immunosorbent Assay.

Diagnostic tools for the detection of infection with Onchocerca volvulus are presently limited to microfilaria detection in skin biopsies and serological assessment using the Ov16 immunoglobulin G4 (IgG4) rapid test, both of which have limited sensitivity. We have investigated the diagnostic performance of a peptide enzyme-linked immunosorbent assay (ELISA) based on immunodominant linear epitopes previously discovered. Peptides that were used in these assays were designated O. volvulus motif peptides (OvMP): OvMP-1 (VSV-EPVTTQET-VSV), OvMP-2 (VSV-KDGEDK-VSV), OvMP-3 (VSV-QTSNLD-VSV), and the combination of the latter two, OvMP-23 (VSV-KDGEDK-VSV-QTSNLD-VSV). Sensitivity (O. volvulus infection), specificity (non-helminth infections), and cross-reactivity (helminth infections) were determined using several panels of clinical plasma isolates. OvMP-1 was found to be very sensitive (100%) and specific (98.7%), but showed substantial cross-reactivity with other helminths. Of the other peptides, OvMP-23 was the most promising peptide with a sensitivity of 92.7%, a specificity of 100%, and a cross-reactivity of 6%. It was also demonstrated that these peptides were immunoreactive to IgG but not IgG4, and there is no correlation with the Ov16 IgG4 status, making them promising candidates to complement this already available test. Combination of the Ov16 IgG4 rapid test and OvMP-23 peptide ELISA led to a sensitivity of 97.3% for the detection of O. volvulus infection, without compromising specificity and with minimal impact on cross-reactivity. The available results open the opportunity for a "clinical utility use case" discussion for improved O. volvulus epidemiological mapping.

Feasibility of utilizing the SD BIOLINE Onchocerciasis IgG4 rapid test in onchocerciasis surveillance in Senegal.

As effective onchocerciasis control efforts in Africa transition to elimination efforts, different diagnostic tools are required to support country programs. Senegal, with its long standing, successful control program, is transitioning to using the SD BIOLINE Onchocerciasis IgG4 (Ov16) rapid test over traditional skin snip microscopy. The aim of this study is to demonstrate the feasibility of integrating the Ov16 rapid test into onchocerciasis surveillance activities in Senegal, based on the following attributes of acceptability, usability, and cost. A cross-sectional study was conducted in 13 villages in southeastern Senegal in May 2016. Individuals 5 years and older were invited to participate in a demographic questionnaire, an Ov16 rapid test, a skin snip biopsy, and an acceptability interview. Rapid test technicians were interviewed and a costing analysis was conducted. Of 1,173 participants, 1,169 (99.7%) agreed to the rapid test while 383 (32.7%) agreed to skin snip microscopy. The sero-positivity rate of the rapid test among those tested was 2.6% with zero positives 10 years and younger. None of the 383 skin snips were positive for Ov microfilaria. Community members appreciated that the rapid test was performed quickly, was not painful, and provided reliable results. The total costs for this surveillance activity was $22,272.83, with a cost per test conducted at $3.14 for rapid test, $7.58 for skin snip microscopy, and $13.43 for shared costs. If no participants had refused skin snip microscopy, the total cost per method with shared costs would have been around $16 per person tested. In this area with low onchocerciasis sero-positivity, there was high acceptability and perceived value of the rapid test by community members and technicians. This study provides evidence of the feasibility of implementing the Ov16 rapid test in Senegal and may be informative to other country programs transitioning to Ov16 serologic tools.

Relationship between pro-and anti-inflammatory cytokines profiles and some haematological parameters in some Cameroonians infected with Onchocerca volvulus.

OBJECTIVE: To investigate the relationship between white blood cells, lymphocytes, monocytes, and Interleukin(IL)-1 α, IL-6, IL-10 and IL-13 production in Cameroonians with Onchocerca volvulus (O. volvulus) infection. METHODS: A total of 357 individuals from five sites at Upper Sanga, Lekkie, Nyong, Kelle and Sanaga Maritime divisions and located along Sanaga valley of Sanaga River in South Cameroon were screened for the presence of O. volvulus using the skin snip. The levels of the interleukins (IL-) namely IL-1 α, IL-6, IL-10 and IL-13 were evaluated using enzyme linked immunoabsorbent assay techniques. Haematological parameters were evaluated using standard laboratory automated analyser. RESULTS: O. volvulus microfilariae were found in skin tissues of 85 (23.81%) volunteers. The mean interleukin (IL-) levels in the O. volvulus control and infected individuals were IL-1 α in (1.65 ± 0.79 and 2.31 ± 0.5) pg/mL; IL-6 in (278.36 ± 55.34 and 201.74 ± 34.56) pg/mL; IL-10 in (436.03 ± 208.64 and 418.49 ± 147.88) pg/mL and IL-13 in (8.98 ± 7.28 and 38.06 ± 11.92) pg/mL. There was a negative correlation between monocyte counts and IL-10 concentration in positive individuals. A negative correlation of IL-6 with white blood cell and lymphocyte counts was observed (P<0.05). The level of IL-13 was positively associated with microfilarial load (P<0.05). CONCLUSIONS: We observed depressed IL-6 and raised IL-13 concentrations in the sera of individuals with onchocerciasis which implicate these interleukins in the immunological responses of the disease. Therefore, these IL-6 and IL-13 are associated with O. volvulus infection among Cameroonians.

The effect of oral anthelmintics on the survivorship and re-feeding frequency of anthropophilic mosquito disease vectors.

In the Tropics, there is substantial temporal and spatial overlap of diseases propagated by anthropophilic mosquito vectors (such as malaria and dengue) and human helminth diseases (such as onchocerciasis and lymphatic filariasis) that are treated though mass drug administrations (MDA). This overlap will result in mosquito vectors imbibing significant quantities of these drugs when they blood feed on humans. Since many anthelmintic drugs have broad anti-invertebrate effects, the possibility of combined helminth control and mosquito-borne disease control through MDA is apparent. It has been previously shown that ivermectin can reduce mosquito survivorship when administered in a blood meal, but more detailed examinations are needed if MDA is to ever be developed into a tool for malaria or dengue control. We examined concentrations of drugs that follow human pharmacokinetics after MDA and that matched with mosquito feeding times, for effects against the anthropophilic mosquito vectors Anopheles gambiae s.s. and Aedes aegypti. Ivermectin was the only human-approved MDA drug we tested that affected mosquito survivorship, and only An. gambiae s.s. were affected at concentrations respecting human pharmacokinetics at indicated doses. Ivermectin also delayed An. gambiae s.s. re-feeding frequency and defecation rates, and two successive ivermectin-spiked blood meals following human pharmacokinetic concentrations compounded mortality effects compared to controls. These findings suggest that ivermectin MDA in Africa may be used to decrease malaria transmission if MDAs were administered more frequently. Such a strategy would broaden the current scope of polyparasitism control already afforded by MDAs, and which is needed in many African villages simultaneously burdened by many parasitic diseases.

Preparation and characterization of specific monoclonal antibodies for the detection of adult worm infections in onchocerciasis.

Suitable molecular tests for monitoring the viability of adult worms of Onchocerca in vivo are required to accelerate the development of new macrofilaricides in river blindness (onchocerciasis). Hence, three monoclonal antibodies (MAbs) were prepared and evaluated in a sandwich enzyme-linked immunosorbent assay (ELISA) for their abilities to detect circulating adult worm antigens in onchocercal bovine and human sera. The MAbs did not cross-react with a number of control antigens, which included extracts of Ascaris suum, Loa loa, and O. ochengi microfilariae. They were all IgG1 molecules. Their targets in O. ochengi total extract were a set of the same 15 polypeptides with apparent molecular weights of 21-220 Kda. Immunohistochemical studies confirmed their adult worm specificity and showed their binding to the hypodermis of the adult worm. The ELISA could detect as little as 100 pg/mL of the affinity-purified target antigens. It also detected the antigens with 94.1% specificity in 50 out of 56 infected bovine sera (90% sensitivity) and in 21 out of 43 infected human sera (48.8% sensitivity, which could go up to 72.1% on elimination of two skewed control cases). We conclude that the MAbs could be field tested and used in responder populations as described herein or employed as components of more sensitive assays for the evaluation of novel Onchocerca macrofilaricides.

Rapid assessment method for prevalence and intensity of Loa loa infection.

OBJECTIVE: To assess the validity of observations on eye worm and Calabar swellings for the rapid assessment of the prevalence and intensity of loiasis at the community level. METHOD: A total of 12895 individuals over the age of 15 years living in 102 communities in Cameroon and Nigeria took part in the study. A standardized questionnaire was administered to participants from whom finger-prick blood samples were collected and examined for Loa loa microfilariae. Rapid assessments of the prevalence and intensity of loiasis were made on the basis of a history of eye worm or Calabar swellings. FINDINGS: There was a strong correlation between the indices of the rapid assessment procedures and the parasitological indices of L. loa endemicity. The rapid assessment indices were effective in diagnosing high-risk communities (sensitivity 94-100%; specificity 66-92%). The highest sensitivity (100%) and specificity (92%) were obtained with a rapid assessment procedure based on a history of eye worm lasting 1-7 days together with confirmation by the guided recognition of a photograph of adult L. loa in the eye. CONCLUSION: Rapid assessment of the prevalence and intensity of loiasis at the community level can be achieved using a procedure based on the history of eye worm lasting 1-7 days together with confirmation by the guided recognition of a photograph of an adult L. loa in the eye.

Differential cytokine and antibody responses to adult and larval stages of Onchocerca volvulus consistent with the development of concomitant immunity.

The possibility of concomitant immunity and its potential mechanisms in Onchocerca volvulus infection were examined by analyzing cytokine and antibody responses to infective larval (third-stage larvae [L3] and molting L3 [mL3]), adult female worm (F-OvAg), and skin microfilaria (Smf) antigens in infected individuals in a region of hyperendemicity in Cameroon as a function of age. Peripheral blood mononuclear cell interleukin 5 (IL-5) responses to F-OvAg and Smf declined significantly with age (equivalent to years of exposure to O. volvulus). In contrast, IL-5 secretion in response to L3 and mL3 remained elevated with increasing age. Gamma interferon responses to L3, mL3, and F-OvAg were low or suppressed and unrelated to age, except for responses to Smf in older subjects. IL-10 levels were uniformly elevated, regardless of age, in response to L3, mL3, and F-OvAg but not to Smf, for which levels declined with age. A total of 49 to 60% of subjects had granulocyte-macrophage colony-stimulating factor responses to all O. volvulus antigens unrelated to age. Analysis of levels of stage-specific immunoglobulin G3 (IgG3) and IgE revealed a striking, age-dependent dissociation between antibody responses to larval antigens (L3 and a recombinant L3-specific protein, O. volvulus ALT-1) which were significantly increased or maintained with age and antibody responses to F-OvAg, which decreased. Levels of IgG1 to L3 and F-OvAg were elevated regardless of age, and levels of IgG4 increased significantly with age, although not to O. volvulus ALT-1, which may have unique L3-specific epitopes. Immunofluorescence staining of whole larvae showed that total anti-L3 immunoglobulin levels also increased with the age of the serum donor. The separate and distinct cytokine and antibody responses to adult and infective larval stages of O. volvulus which are age related are consistent with the acquisition of concomitant immunity in infected individuals.

Clinical pharmacokinetics of suramin in patients with onchocerciasis.

OBJECTIVE: Ten male patients with onchocerciasis received six weekly infusions of suramin according to the WHO-recommended regimen. RESULTS: In no case did the plasma concentration of suramin exceed 300 mg x l(-1), and serious toxicity was not observed. The apparent volume of distribution (median 20.6 l) was comparable to that reported for patients with prostatic carcinoma. Elimination from patients with onchocerciasis was relatively slow (median plasma clearance 6.2 ml x h(-1), median terminal elimination half-life 91.8 days). CONCLUSION: Microfilariae were eliminated in eight out of ten patients. Spontaneous nodule regression was noted in four patients.

Eosinophils are the major effector cells of immunity to microfilariae in a mouse model of onchocerciasis.

Mice inoculated with microfilariae of the filarial nematode Onchocerca lienalis clear their parasites over a period of 3-4 months and are highly resistant to re-infection. We have investigated the comparative roles of the eosinophil, macrophage and neutrophil in effecting this parasite clearance, employing agents specifically to perturb cell function in vivo. Using the anti-IL-5 monoclonal antibody TRFK-5, we show that eosinophils are of primary importance in effecting resistance to re-infection. Ablation of macrophages (with carbon) and neutrophils (with the monoclonal antibody NIMP-R14) had no effect on parasite clearance following re-infection. Neutralization of these 3 cell types during a primary infection showed that while the removal of both eosinophils and macrophages caused a small but significant delay in parasite clearance, the depletion of neutrophils had no effect. This report describes the first direct evidence for eosinophil-mediated killing of microfilariae in vivo, and is consistent with Th-2 cell responses previously described in this model.

Identification of recombinant filarial proteins capable of inducing polyclonal and antigen-specific IgE and IgG4 antibodies.

Filarial infection is characterized by an immune response associated with the production of Ag-specific IgG4 and IgE and IL-4 and IL-5. To identify filarial Ags capable of inducing such responses and to analyze the role Ags themselves play in sustaining it, 24 recombinant filarial parasite proteins were screened for their ability to be recognized by sera from 67 individuals with tissue-invasive filarial infections. Among the recombinant proteins that were recognized by IgG4 or IgE Abs in 25% of the sera or more, two were selected on the basis of their ability to elicit polyclonal and Ag-specific IgE/IgG4 Abs in vitro. Ov27 (analogous to Ov7/cystatin, a cysteine protease inhibitor) and OvD5B (analogous to Ov33, an aspartyl protease inhibitor) induced both a polyclonal and Ag-specific IgE/IgG4 response that was blocked by neutralizing Abs to IL-4 and to IL-13 or by soluble IL-4 receptors. Recombinant human IFN-gamma and IL-12 also led to a decrease in the production of polyclonal and Ag-specific IgE/IgG4 Abs. In addition, these two recombinant proteins preferentially stimulated the secretion of IL-4, IL-5, and IL-10 (in contrast to IFN-gamma). The data suggest that certain epitopes on filarial Ags preferentially elicit a Th2-type response and provide an in vitro model for dissecting the mechanisms underlying this preferential response.

Cell-adhesion molecules expressed by activated eosinophils in Onchocerca volvulus infection.

Cell-adhesion receptors mediate the interaction between host effector cells and cellular or multicellular targets, including opsonized parasites. Our recent finding of a deposition of plasma proteins, including fibronectin, on the surface of infective larvae of the helminthic parasite Onchocerca volvulus led us to investigate the possible expression of cell-adhesion molecules (CAM), particularly fibronectin receptors, on eosinophilic granulocytes from persons infected with O. volvulus. Immunofluorescence analyses showed that freshly isolated eosinophils strongly expressed the beta 2-integrin CR3 and exhibited to a lower degree CR4 and the integrin-associated carbohydrate determinant Le(x), as well as antigen p24 (CD9). Eosinophils exposed to the eosinophil-active cytokines recombinant human interleukin 3 (rhIL-3) and granulocyte/macrophage colony-stimulating factor (rhGM-CSF) in addition to CR3, CR4, and CD9 expressed the beta 1-integrins VLA-4 and to a lesser extent VL-5 (both fibronectin receptors) as well as the intercellular adhesion molecule ICAM-1. Low-level expression of these adhesins was also observed on eosinophils cultured in the presence of these interleukins on confluent fibroblasts. The presence of VLA-4 as well as VLA-5 on activated eosinophils was confirmed by demonstration of the formation of immune rosettes using antibody-coated microspheres and by their attachment to fibronectin-coated surfaces. These results indicate the possibility of an involvement of previously unidentified antibody- and complement-independent mechanisms in cellular interactions with the parasite O. volvulus.

Investigation of cross-reactions against Trichinella spiralis antigens by enzyme-linked immunosorbent assay and enzyme-linked immunoelectrotransfer blot assay in patients with various diseases.

Data regarding cross-reactions against Trichinella spiralis in humans are scarce and controversial. For this reason, we tested serum samples from patients with typhoid fever, brucellosis, toxoplasmosis, amoebiasis, cysticercosis, trichocephaliasis, ascariasis, and onchocerciasis against an antigenic extract of T. spiralis infective larvae in an enzyme-linked immunosorbent assay (ELISA) and an enzyme-linked immunoelectrotransfer blot (EITB) assay. All except one serum sample from the group of patients with onchocerciasis were negative in the ELISA; in the EITB assay, only faint bands were observed with the samples from patients with onchocerciasis and ascariasis and negative results were obtained with the samples from patients with other diseases. In conclusion, cross-reactions were found only in the groups of patients with other nematode infections and were of very low magnitude, most of them virtually negative.

C-reactive protein and interleukin-6 are elevated in onchocerciasis patients after ivermectin treatment.

Ivermectin treatment of onchocerciasis can induce adverse reactions. Mechanisms underlying these reactions are poorly understood but may include activation of neutrophils. This study investigated the acute-phase response in onchocerciasis patients during 2 days after ivermectin treatment. The acute-phase protein C-reactive protein (CRP) and cytokines that mediate the acute-phase response (tumor necrosis factor-alpha [TNF alpha] and interleukin-6 [IL-6]) were measured in 144 skin snip-positive onchocerciasis patients and 12 skin snip-negative controls who received one dose of ivermectin (150 micrograms/kg). No elevated TNF alpha levels were found, but IL-6 and CRP were elevated in 25.7% and 50.7% of the patients, respectively, after ivermectin treatment. Most patients (89.2%) with raised IL-6 also had raised CRP. Such increases were not observed in controls and in patients were correlated with adverse reactions and microfilarial densities. These findings suggest a possible role of the acute-phase response in microfilarial destruction following ivermectin treatment.

[The evaluation of endemic onchocerciasis in the Amou prefecture (Togo)]. / Contribution à l'évaluation de l'endémie onchocerquienne dans la préfecture de l'Amou (Togo).

We have evaluated the endemicity of the onchocerciasis in two villages of the district of Amou in Togo, Okpahoué et Onglo. We have studied 294 subjects including 136 males and 158 females representing the quarter of the population of the two villages (clinical investigation and exsanguine cutaneous biopsy): 111 persons are parasited by Onchocerca volvulus that means a prevalence rate of 37.7%. We can therefore say that the villages are in a state of "meso-endemicity". We have been able to find nodules by 20 persons among the 111 that give a percentage of 6.8 of the examined persons. Among children of 3 to 9 years old, the prevalence and the average microfilaremia are respectively 8.86 and 3.54%. Among the persons of 50 or older than 50, we found a prevalence rate of 65.95% and an average microfilaremia of 28.37.

Onchocerca volvulus. Monoclonal anti-idiotype antibody as antigen signal for the microfilaricidal cytotoxicity of diethylcarbamazine-treated platelets.

Over the past 35 yr, diethylcarbamazine (DEC) has been the most widely used agent for the treatment of filarial diseases, particularly in onchocerciasis. The microfilaricidal action of DEC has been recently shown to be mediated by blood platelets with the additional triggering of a filarial excretory Ag (FEA). This FEA could be detected by using mAb in the serum of infected patients. By using one mAb (IA2(23] directed against Onchocerca volvulus and recognizing circulating Ag (Ab1), we purified by affinity chromatography the target molecule of IA2(23) (an O. volvulus glycoprotein recognized by IA2(23) mAb). This compound had a dose-dependent effect on the cytotoxic action of DEC-treated platelets. We subsequently produced an anti-idiotype mAb to Ab1 (Ab2), and considered the possibility of replacing the O. volvulus glycoprotein recognized by IA2(23) mAb by Ab2. Ab2 was selected according to its ability to inhibit the binding of radioiodinated Ab1 to the filarial target Ag. It induced the production of anti-O. volvulus antibodies (Ab3) in rats. At a constant concentration of DEC platelets, the addition of increasing amounts of Ab2 led to a dose-dependent cytotoxic effect against parasite larvae. Experiments performed with Ab2 on detergent solubilized surface proteins of platelets identified four bands of Mr 18, 26, 43.5, and 100 kDa, supporting the idea of the presence of binding sites on the platelets for a FEA required for the microfilaricidal cytotoxicity of DEC-treated platelets.

[Serodiagnosis of onchocerciasis using micro-ELISA. Study of 450 sera and comparison with indirect immunofluorescence]. / Séro-diagnostic de l'onchocercose par micro-Elisa. Etude de 450 sérums et comparaison avec l'immuno-fluorescence indirecte.

The microtest ELISA has been used for human Onchocerciasis serological study. The antigens employed were adult Onchocerca volvulus extracts, collected from dissected nodules, delipidized and cleared from human proteins by affinity Chromatography. Under the circumstances, the positivity limit of the test seems excellent (maximum )D: 0,23) defined with 171 negative sera, 66 of them taken from Africans. Specificity controls were studied with 56 heterologous sera; cross-reactions occurred with hydatidosis and especially wit various nematode infections, in particular loasis. With reagents and technical conditions used, the specificity limit of the test corresponds to an OD of 0,4 (measured with a 3 mm optical course). The diagnosis value of the test was verified by studying sera from 90 individuals wit a positive skin biopsy and with sera from 233 adults living in endemic areas. For all the infected people, the global percentage of positivity with ELISA is not greater than that with indirect immunofluorescent antibody test (85%). On the other hand, the micro-test ELISA seems slightly more sensitive in detection of high serological positivities. We did not find any statistically relationship presence and quantity of microfilarial worms in skin biopsy and positivity with the microtest ELISA. Likewise, in some polyinfested patients (with Onchocerca volvulus and Dipetalonema perstans or Wuchereria bancrofti), we did not observe any correlation between the results given the microtest ELISA and the quantity of microfilariae in the blood stream.