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Background: This study examined onchocerciasis transmission in Kwanware and Ottou in the Wenchi Health District of Ghana, where persistent onchocercal microfilariae (mf) levels have been reported since 2012., Methods: This study was conducted from 2019 to 2021 and involved the following: (i) reviewing past records of ivermectin mass drug administration (MDA); (ii) conducting a treatment coverage evaluation survey (CES); (iii) conducting key informant interviews; (iv) prospecting blackfly breeding sites; (v) collecting and dissecting blackflies; and (vi) conducting parasitological and serological surveys., Results: (i) The review indicated ongoing MDA treatment for the past 27 years, with a reported coverage of over 65% in the last 17 yearly rounds; (ii) estimated treatment coverage by the CES in 2019 was 71.3%, with most of those not taking medicine stating that they were not offered; (iii) however, the key informant interviews revealed insufficiencies in reaching a significant number of people for treatment due to remote settlement, mobility, transport logistical issues, failure to register some people for treatment, leading to a false impression of good coverage, and a short distribution time; (iv) the most productive breeding was found within 5 km of Kwanware-Ottou; and (v) blackfly daily biting rates were highest in Kwanware and Ottou, with 199 and 160 bites per day, respectively. Infection in blackflies was found only in Kwanware and Ottou, with infectivity rates of 5.9‰ (per 1000) and 6.7‰, respectively. (vi) The mf prevalence in Ottou and Kwanware, respectively, was 40.0% and 30.0% among adults aged ≥ 20 years, and the anti-(Onchocerca volvulus) Ov16 IgG4 antibodies seroprevalence rates were 8.3% and 13.3% among children aged 5-9 years. These values were reduced to undetectable levels at a radius of 10 km from Ottou., Conclusions: This study confirms that active onchocerciasis transmission centres on Kwanware/Ottou and is confined to a 10 km radius despite 27 yearly treatment rounds. The main contributing factors are suboptimal coverage and high biting rates. Identifying and targeting such a focus with a combination of interventions will be cost-effective in accelerating onchocerciasis elimination in Ghana., (© 2024. The Author(s).)

Skin snip evaluation for onchocerciasis has insufficient sensitivity when skin microfilarial (mf) densities are low, such as following ivermectin treatment. Mf density is suitable for assessing microfilaricidal efficacy but only serves as an indirect indicator of macrofilaricidal activity. We assessed circulating nucleic acids from Onchocerca volvulus as an alternative to skin snips. We screened a plasma sample set of infected individuals followed up at four, 12 and 21 months after microfilaricidal (ivermectin, n = four), macrofilaricidal (doxycycline, n = nine), or combination treatment (n = five). Two parasite-derived miRNAs, cel-miR-71-5p and bma-lin-4, and O-150 repeat DNA were assessed. Highly abundant DNA repeat families identified in the O. volvulus genome were also evaluated. miRNAs were detected in two of 72 plasma samples (2.8%) and two of 47 samples (4.3%) with microfilaridermia using RT-qPCR. O-150 DNA was detected in eight (44.4%) baseline samples by qPCR and the number of positives declined post-treatment. One doxycycline-treated individual remained O-150 positive. However, only 11 (23.4%) samples with microfilaridermia were qPCR-positive. Analysis by qPCR showed novel DNA repeat families were comparatively less abundant than the O-150 repeat. Circulating parasite-derived nucleic acids are therefore insufficient as diagnostic tools or as biomarkers of treatment efficacy for O. volvulus.

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.

Background: Mali has become increasingly interested in the evaluation of transmission of both Wuchereria bancrofti and Onchocerca volvulus as prevalences of both infections move toward their respective elimination targets. The SD Bioline Onchocerciasis/LF IgG4 Rapid Test was used in 2 evaluation units (EU) to assess its performance as an integrated surveillance tool for elimination of lymphatic filariasis (LF) and onchocerciasis., Methodology/principal Findings: A cross sectional survey with SD Bioline Onchocerciasis/LF IgG4 Rapid Test was piggy-backed onto a transmission assessment survey (TAS) (using the immunochromatographic card test (ICT) Binax Filariasis Now test for filarial adult circulating antigen (CFA) detection) for LF in Mali among 6-7 year old children in 2016 as part of the TAS in two EUs namely Kadiolo-Kolondieba in the region of Sikasso and Bafoulabe -Kita-Oussoubidiagna-Yelimane in the region of Kayes. In the EU of Kadiolo- Kolondieba, of the 1,625 children tested, the overall prevalence of W. bancrofti CFA was 0.62% (10/1,625) [CI = 0.31-1.09]; while that of IgG4 to Wb123 was 0.19% (3/1,600) [CI = 0.04-0.50]. The number of positives tested with the two tests were statistically comparable (p = 0.09). In the EU of Bafoulabe-Kita-Oussoubidiagna-Yelimane, an overall prevalence of W. bancrofti CFA was 0% (0/1,700) and that of Wb123 IgG4 antibody was 0.06% (1/1,700), with no statistically significant difference between the two rates (p = 0.99). In the EU of Kadiolo- Kolondieba, the prevalence of Ov16-specific IgG4 was 0.19% (3/1,600) [CI = 0.04-0.50]. All 3 positives were in the previously O. volvulus-hyperendemic district of Kolondieba. In the EU of Bafoulabe-Kita-Oussoubidiagna-Yelimane, an overall prevalence of Ov16-specific IgG4 was 0.18% (3/1,700) [CI = 0.04-0.47]. These 3 Ov16 IgG4 positives were from previously O.volvulus-mesoendemic district of Kita., Conclusions/significance: The SD Bioline Onchocerciasis/LF IgG4 Rapid test appears to be a good tool for integrated exposure measures of LF and onchocerciasis in co-endemic areas., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: Thomas B. Nutman is among the patent holders for Wb123 and Ov16 (no longer under patent) and, through the National Institutes of Health, has received licensing/royalty fees for Wb123.

Background: The study of Onchocerca volvulus has been limited by its host range, with only humans and non-human primates shown to be susceptible to the full life cycle infection. Small animal models that support the development of adult parasites have not been identified., Methodology/principal Findings: We hypothesized that highly immunodeficient NSG mice would support the survival and maturation of O. volvulus and alteration of the host microenvironment through the addition of various human cells and tissues would further enhance the level of parasite maturation. NSG mice were humanized with: (1) umbilical cord derived CD34+ stem cells, (2) fetal derived liver, thymus and CD34+ stem cells or (3) primary human skeletal muscle cells. NSG and humanized NSG mice were infected with 100 O. volvulus infective larvae (L3) for 4 to 12 weeks. When necropsies of infected animals were performed, it was observed that parasites survived and developed throughout the infection time course. In each of the different humanized mouse models, worms matured from L3 to advanced fourth stage larvae, with both male and female organ development. In addition, worms increased in length by up to 4-fold. Serum and urine, collected from humanized mice for identification of potential biomarkers of infection, allowed for the identification of 10 O. volvulus-derived proteins found specifically in either the urine or the serum of the humanized O. volvulus-infected NSG mice., Conclusions/significance: The newly identified mouse models for onchocerciasis will enable the development of O. volvulus specific biomarkers, screening for new therapeutic approaches and potentially studying the human immune response to infection with O. volvulus., Competing Interests: The authors have declared that no competing interests exist.

Onchocerciasis is a neglected parasitic disease targeted for elimination. Current World Health Organization guidelines for elimination include monitoring antibody responses to the recombinant Onchocerca volvulus antigen OV-16 in children to demonstrate the absence of transmission. We report the performance characteristics of a modified OV-16 enzyme-linked immunosorbent assay (ELISA) and describe anti-OV-16 responses in serum samples from laboratory-inoculated nonhuman primates (NHPs) in relation to microfilariae (mf) in skin snip biopsies. This OV-16 IgG4 ELISA had sensitivity and specificity of 88.2% and 99.7%, respectively, as determined by receiver operator characteristic analysis using a serum panel of 110 positive and 287 negative samples from people infected with other filariae or other parasitic infections. Anti-OV-16 responses in inoculated NHP ( N = 9) were evaluated at quarterly intervals for IgM and the four IgG subclasses. Enzyme-linked immunosorbent assay results showed a well-defined IgG4 reactivity pattern and moderate IgG1 antibody responses. Meanwhile, the reactivity by IgG2, IgG3, or IgM did not show a clear pattern. Temporal evolution of IgG4 reactivity was evaluated through monthly testing, showing that NHPs developed anti-OV-16 IgG4 on average at 15 months postinoculation (range: 10-18 months). The average time to detectable mf was also 15 months (range: 11-25). The OV-16 ELISA used in this study was robust and allowed the detection of IgG4 responses, which were observed only among animals with detectable mf ( N = 5), four of which showed declines in antibody responses once mf cleared. These findings also confirmed that the most informative antibody subclass responses to OV-16 are IgG4.

Yemen is a country that has been treating severe cases of oncho-dermatitis since 1992 and is now moving to a program aimed at the elimination of the transmission of Onchocerca volvulus . It is important to ensure that the currently acceptable tools used in epidemiological assessment of onchocerciasis in Africa and Latin America also apply to Yemen. Five hundred and ten blood samples from three known O. volvulus -endemic areas, locations that have never been under a mass treatment program, were tested for the presence of antibodies against a panel of O. volvulus -specific antigens using enzyme-linked immunosorbent assay (Ov16) and luciferase immunoprecipitation system (Ov-FAR-1 and Ov-MSA-1) assays. Overall, 31.4% of the samples tested were positive, with positivity increasing with age. Positivity was seen in 76.5% of those presenting with clinical onchocerciasis but importantly also in more than 28.5% of those defined as free of oncho-dermatitis; these latter individuals are likely to be serving as a source for persistent reinfection. This study supports the use of the current O. volvulus -specific serologic methodology in Yemen.

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.

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.

Background: Onchocerciasis is targeted for elimination in Africa through annual or biannual ivermectin mass drug administration (MDA). An immunodiagnostic test, based on the detection of human IgG4 antibodies in the blood to the Onchocerca volvulus-specific antigen Ov16, is one of the recommended tools for determining whether transmission is interrupted and mass treatment can stop. For different transmission settings, the relationship between post-MDA Ov16 antibody prevalence in children (measured 1 year after the last round of MDA) and the duration and coverage of MDA, the mf prevalence in the population, and the probability that onchocerciasis is eventually eliminated is explored through mathematical modelling., Methodology: The ONCHOSIM model was extended with new output on the Ov16 antibody serostatus of individuals. Seroconversion was assumed to be triggered by the first worm establishing in the host, with seroconversion occurring either before maturation, after maturation or only after the start of mf production. We are mainly interested in seroconversion rates in children, and for now ignore the possibility of seroreversion to simplify the model., Principal Findings: Yearly repeated MDA leads to a strong reduction in the parasite acquisition rate in humans. This reduces the seroconversion rate in newborns and young children, while those who seroconverted before the start of control remain antibody positive. Both the microfiladermia prevalence in the population aged 5 years and above and the Ov16 antibody prevalence in children under 10 declined with increasing duration of MDA. The association between either of these indicators and the model-predicted probability of elimination was not influenced much by the assumed treatment coverage levels, but was found to depend on baseline endemicity levels, assumptions regarding the trigger of seroconversion, and diagnostic test characteristics (sensitivity and specificity)., Conclusions: Better understanding of the dynamics of Ov16 antibody responses is required for accurate interpretation of seroprevalence data and more precise estimation of endpoint for MDA. Our study demonstrates that this endpoint will be dependent on baseline endemicity levels, which should be taken into account in guidelines for defining when to stop MDA., Competing Interests: The authors have declared that no competing interests exist.

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., (Copyright © 2012 Hainan Medical College. Published by Elsevier B.V. All rights reserved.)

Onchocerciasis, also known as river blindness, is the second leading infectious cause of blindness worldwide. In order to successfully control this disease, the development of efficient diagnostic tools as well as effective treatments is imperative. A number of proteins have been proposed as vaccine and diagnostic candidates, yet none have been successfully advanced to the point of general clinical use. We have prepared major sperm protein 2 (MSP2) from Onchocerca volvulus as a possible diagnostic antigen for onchocerciasis. Importantly, recombinant MSP2 is dimeric in solution, identical to alpha-MSP from the roundworm, Ascaris suum. A panel of sera obtained from Cameroonian individuals afflicted with onchocerciasis positively responded to the recombinant MSP2. Our data suggest that MSP2, like the previously described antigen Ov16, can be utilized as a diagnostic onchocerciasis antigen for monitoring the interruption of transmission.

Severe adverse events (SAEs) following ivermectin treatment may occur in people harbouring high Loa loa microfilarial (mf) densities. In the context of mass ivermectin distribution for onchocerciasis control in Africa, it is crucial to define precisely the geographical distribution of L. loa in relation to that of Onchocerca volvulus and predict the prevalence of heavy infections. To this end, we analysed the distribution of mf loads in 4183 individuals living in 36 villages of central Cameroon. Mf loads were assessed quantitatively by calibrated blood smears, collected prior to ivermectin distribution. We explored the pattern of L. loa mf aggregation by fitting the (zero-truncated) negative binomial distribution and estimating its overdispersion parameter k by maximum likelihood. The value of k varied around 0.3 independently of mf intensity, host age, village and endemicity level. Based on these results, we developed a semi-empirical model to predict the prevalence of heavy L. loa mf loads in a community given its overall mf prevalence. If validated at the continental scale and linked to predictive spatial models of loiasis distribution, this approach would be particularly useful for optimizing the identification of areas at risk of SAEs and providing estimates of populations at risk in localities where L. loa and O. volvulus are co-endemic.

Onchocercosis or riverblindness, caused by the filaria ochnocerca volvulus, is endemic in many countries of central and Western Africa. Symptoms of the disease can occur years after the infection, chronic itching dermatitis is the first sign, without treatment blindness may develop after years. Onchodermatitis is a hyperreactive course of onchocercosis with massive eosinophilia and elevated IgE, which suppresses a microfilarial spread through the body. Here, we report about the case of an 9-year-old girl who immigrated from the republic of Congo at the age of seven and has been living in Germany for more than two years. Presumably she suffered from onchodermatitis. She presented papular, indurated and itching skin lesions with pigmentary changes, predominantly located at the limbs. Remarkable results of blood tests were 11,000/microl (60 %) eosinophils and IgE 28 000 KU/l, ECP > 200 mg/l, without a history of atopic diseases. HIV, Strongylosis and Loa Loa were excluded. Anti filaria antibodies were detected in a concentration of 51 AKE, microscopy of skin samples failed to detect the parasites. After a single dose of Ivermectin the dermatitis improved, after two weeks the itching was absent, results of repeated blood tests tend to normalize in the following months. Due to the long lifespan of filaria in humans, the disease occurs years after infection in endemic areas. The differential diagnosis for itching skin lesions with high eosinophils in children from developing countries should include onchocercosis.

Studies to determine the prevalence of antibodies to Onchocerca volvulus, prior to and after actions carried out to interrupt transmission, are scarce in Mexico. Here we report the prevalence of immunoglobulin G (IgG) and IgG4 antibodies in an enzyme-linked immunosorbent assay (ELISA) against a crude extract of O. volvulus adult worm in serum samples from persons under noninterrupted biannual treatment with ivermectin in areas of onchocercosis endemicity in Mexico. To perform the prevalence studies, the ELISA procedures were first evaluated. Serological studies were performed with serum samples from skin microfilaria carriers from Guatemala and from people microfilariodermic negative living in the same area as the Guatemalan patients. Sensitivity values for IgG or IgG4 detection were 71 and 86%, while specificities were 92 and 100%, respectively. No anti-O. volvulus antibodies were found in samples from nonendemic controls from Mexico, but 3 of 71 samples from residents in the onchocercosis area of Oaxaca, Mexico, and who have been under ivermectin treatment during the last 10 years were only positive to IgG. Notwithstanding that the IgG4 isotype was not detected and a low (4.2%) anti-O. volvulus IgG antibody prevalence was found, a seroepidemiological follow-up must be performed in order to confirm interruption of onchocercosis transmission in the area of Oaxaca, Mexico, in which onchocercosis is endemic.

In the present study, the cytokines interleukin (IL)-12 and IL-18 were evaluated for their capacity to modulate and to re-direct in vitro parasite antigen-specific cellular responsiveness in patients exposed to Onchocerca volvulus and Entamoeba histolytica infection. We found that IL-18 was highly capable of reducing parasite antigen-induced IL-10 production by PBMC. In contrast, addition or neutralization of IL-12, also in combination with IL-18 and the interferon-gamma-inducible chemokine IP-10 did not affect IL-10 production. Interestingly, the highest IL-10 levels were measured when IL-18 and IP-10 were both neutralized. Although having no effect on IL-10, IL-12 strongly promoted spontaneous and parasite antigen-driven IFN-gamma production by PBMC, whereas IL-18 was only moderately affecting IFN-gamma release by PBMC re-stimulated with E. histolytica- or O. volvulus-specific antigens. Both IL-12 and IL-18 diminished the cellular production of IL-13, and a synergistic effect was observed when the cytokines were combined. Likewise, neutralization of IL-12 enhanced Entamoeba and Onchocerca antigen-driven IL-13 production, but no further increase of IL-13 was observed, when anti-IL-12 and anti-IL-18 were used together. This study disclosed that IL-18 will significantly down-regulate parasite-specific IL-10 production, whereas IL-12 induced IFN-gamma and inhibited IL-13 production by PBMC from humans exposed to O. volvulus and E. histolytica. Such selective immune-regulatory capacity of IL-12 and IL-18 may comprise an important tool to re-direct polarized cytokine responses towards a balanced Th1/Th2 cytokine profile, which may prevent pathology and promote immunity against helminth and protozoan parasite infections.

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.

To identify potential vaccine candidates for the prevention of infection with the filarial nematode Onchocerca volvulus, we screened an O. volvulus L3 stage cDNA library with sera from putatively immune (PI) subjects, and a prominent immunogenic clone of 1,184 nucleotides was identified. It contained an open reading frame of 363 amino acids encoding the glycolytic enzyme fructose 1,6 bisphosphate aldolase (Ov-fba-1). Immunolocalization experiments demonstrated that the protein was most abundantly expressed in metabolically active tissues, including body wall muscle and the reproductive tract of adult female worms. Immunoelectron microscopy of L3 demonstrated binding in the region where the cuticle separates during molting, in the channels connecting the esophagus to the cuticle, and in the basal lamina surrounding the esophagus and the body cavity. Among subjects from areas where this organism is endemic specific humoral and cellular immune responses to recombinant protein were observed in both PI and infected subjects, whereas responses were not observed among subjects who had not been exposed to O. volvulus. Despite the absence of differential responsiveness in parasite-exposed human populations, when the recombinant was tested for protective efficacy in a mouse chamber model, a reduction in survival of larvae by ca. 50% was seen. This observation provides support for the further study of this parasite enzyme as a vaccine candidate in larger animal models.

Onchocerciasis is caused by the filarial nematode Onchocerca volvulus and is a major public health problem in West and Central Africa. With only partial and long-term treatment currently available, there is a need to develop a suitable vaccine. We analysed the antibody response to infective L3 larvae because this stage is thought to be associated with host protective immunity. In addition, we have related our findings to the age, gender and current infection intensity of our participants: variables that may significantly influence antibody production. Interestingly, whilst 90% of our study group were seropositive for adult specific immunoglobulin (Ig)E, only 23% produced L3 specific IgE. This is in contrast to IgG4 where seropositivity was comparable at 96% and 92%, respectively. Furthermore, IgG levels were significantly affected by age and the intensity of infection but unaffected by host gender. This finding is independent for the IgG subclass (IgG1, IgG2, IgG3 and IgG4) and its specificity (L3 versus adult antigen). In summary, we show that L3 larvae induce little specific IgE and the antibody response shows a different isotype balance than that against adult antigens. Both host and parasite variables can influence antibody production in this disease.

Improved methods are needed for field diagnosis of onchocerciasis, to support efforts aimed at elimination of the disease. A rapid-format card test was evaluated that detects IgG4 antibodies to recombinant Onchocerca volvulus antigen Ov16 with serum samples from patients with onchocerciasis and with various types of control serum samples. The sensitivity of the test with serum samples from 106 microfilariae-positive subjects was 90.6%. The test was equally sensitive with serum samples obtained from patients in Africa and Latin America. Specificity was excellent; positive tests were observed for 2 of 38 serum samples from patients with other filarial infections and for 1 of 23 serum samples from patients with nonfilarial helminth infections. The 3 "false-positive" serum samples were from West Africans who could have been coinfected with onchocerciasis. No positive tests were observed with nonendemic serum samples from normal adults, patients with autoimmune disorders, or patients with the hyper-IgE syndrome. This new test holds great promise as a simple tool for diagnosis of onchocerciasis.

Chronic helminth infection induces a type-2 cellular immune response. In contrast to this, mycobacterial infections commonly induce a type-1 immune response which is considered protective. Type-2 responses and diminished type-1 responses to mycobacteria have been previously correlated with active infection states such as pulmonary tuberculosis and lepromatous leprosy. The present study examines the immune responses of children exposed to both the helminth parasite Onchocerca volvulus and the mycobacterial infections, Mycobacterium tuberculosis and M. leprae. Proliferation of peripheral blood mononuclear cells (PBMC) and production of IL-4 in response to both helminth and mycobacterial antigen (PPD) decreased dramatically with increasing microfilarial (MF) density. Although interferon-gamma (IFN-gamma) production strongly correlated with cellular proliferation, it was surprisingly not related to MF density for either antigen. IL-4 production in response to helminth antigen and PPD increased with ascending children's age. IFN-gamma and cellular proliferation to PPD were not related to age, but in response to helminth antigen were significantly higher in children of age 9-12 years than children of either the younger age group (5-8 years) or the older group (13-16 years). Thus, there was a MF density-related down-regulation of cellular responsiveness and age-related skewing toward type 2 which was paralleled in response to both the helminth antigen and PPD. This parasite-induced immunomodulation of the response to mycobacteria correlates with a previous report of doubled incidence of lepromatous leprosy in onchocerciasis hyperendemic regions. Moreover, this demonstration that helminth infection in humans can modulate the immune response to a concurrent infection or immunological challenge is of critical importance to future vaccination strategies.

Afro-Ecuadorian individuals from an area where Onchocerca volvulus is hyperendemic have been monitored for infection over the past 16 years. To determine whether in utero exposure to O. volvulus biases a child's subsequent immune responses, children (9 to 16 years old) for whom the mother's infection status was known were chosen for study. Children of infected mothers (n = 19) had significantly higher levels of skin microfilariae than children of uninfected mothers (n = 13; P = 0.021). While the serum levels of O. volvulus-specific immunoglobulin G (IgG), IgG subclasses, and IgE showed no significant differences between the two groups of children, peripheral blood mononuclear cells of children of infected mothers produced higher levels of Th2-type cytokines to several parasite antigens and lower levels of Th1-type cytokines to nonparasite antigens than those of children of uninfected mothers. Thus, in utero exposure to O. volvulus has a long-term effect on the child's subsequent cellular immune response that may render the child more susceptible to O. volvulus infection postnatally.

The diagnostic value of ELISAs based on recombinant Onchocerca volvulus antigens OC 3.6 and OC 9.3 was evaluated with sera from endemic areas in West Africa, Guatemala and Ecuador. IgG assays were slightly more sensitive than those that detected IgG4, and the antigen combination was significantly more sensitive than either antigen alone (OC 3.6, 93%; OC 9.3, 84%, combined 98%). These assays were also evaluated with sera from 2 villages in the Onchocerciasis Control Programme area of West Africa including one village (Pendie) with recent recrudescence of infection and one (Niarba) where transmission had been interrupted for 15 years by vector control. The OC 3.6 IgG antibody assay was sensitive for new infections and exposure in Pendie; 24/24 (100%) of people with positive skin snips and 15/74 (20%) of sera from MF negative people had IgG antibodies to this antigen. In addition, antibodies to OC 3.6 often preceded the onset of skin snip positivity in Pendie. In contrast, IgG antibodies to OC 3.6 and OC 9.3 were rarely seen in children born during the 15 years since transmission was interrupted by vector control in Niarba. These encouraging results suggest that antibody assays based on OC 3.6 and OC 9.3 may be valuable tools for surveillance of onchocerciasis and also for monitoring the efficacy of control programmes.

Objective: To determine the distribution of ivermectin in plasma and tissues of onchocerciasis patients following a single oral dose of 150 micrograms kg-1., Setting: Medical Department at Soba University Hospital, Khartoum., Patients: Twenty five patients and fourteen healthy volunteers., Methods: Serial blood samples were obtained from both groups. Tissue samples were removed from various patients as full thickness skin punch biopsies or during nodulectomy. Ivermectin concentration was determined by radioimmunoassay., Results: The plasma pharmacokinetic variables for patients were; maximum plasma concentration 52.0 ng ml-1; time to achieve maximum concentration, 5.2 h.; elimination half life, 35.0 h; and the area under the plasma concentration curve versus time, 2852 ng.h.ml-1. In healthy volunteers, the plasma ivermectin distribution was similar to that in patients, and both groups showed a tendency for a second rise in plasma concentration of the drug suggestive of enterohepatic recirculation. Ivermectin was detected in tissues obtained from patients. Fat showed the highest and most persistent levels, whilst values for skin, nodular tissues, and worms were comparable. Subcutaneous fascia contained the lowest concentrations., Conclusions: Infection with O. volvulus does not affect the pharmacokinetics of ivermectin, and filarial infected tissues and parasites themselves do take up the drug. There may be prolonged retention of ivermectin because of depot formation in fat tissue.

A major antigen recognized by human sera in Onchocerca volvulus infections is a parasite eggshell protein. The cDNA clone for this antigen was isolated from a lambda gt11 O. volvulus cDNA library using antisera from patients with high microfilarial counts. Sequence analysis of the cDNA clone predicts a polyglutamine repeat near the 5' end of the cDNA, and a motif of four arginines near the 3' end, reminiscent of that found in many regulatory proteins. The cDNA was subcloned into a yeast expression vector and reagent quantities of recombinant antigen produced in Saccharomyces cerevisiae. Antisera produced to the recombinant purified protein localized the antigen to the eggshell of developing microfilariae within the adult female uterus. No other sites of Oveg1 expression were noted in adult worms, but labeling was seen in internal membrane structures of L3 larvae. Sera from infected chimps recognized Oveg1 only after infections became patent. Sera from infected humans showed reactivity to Oveg1 that varied from 39 to 95%, depending upon the geographic location.

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.

In vitro neutrophil-mediated microfilarial killing, humoral responses to crude adult worm antigens (OVAg) and to four recombinant Onchocerca antigens (GST-OV7, GST-OV103, GST-OV3.6, and MBP-OV16), as well as Western blot analysis to stage-specific Onchocerca proteins were studied in individuals from Bassa County, Liberia, infected with onchocerciasis and in endemic normals, defined as individuals without microfilaridermia. Both groups exhibited high levels of specific serum-dependent killing of microfilariae but could not be differentiated on the basis of these results. However, infected individuals had a significantly higher frequency of antibody response to OVAg (P = 0.0001) except GST-OV103. Based on the pattern of response to the different antigens, 17 categories of specific recognition were observed. Nine of these categories were common to both the infected individuals and the endemic normals, 2 were unique to the infected individuals, and 6 were unique to the endemic normals. Among the endemic normals, we identified a subcategory of individuals who had nondetectable levels of total IgG to OVAg by ELISA and had significantly lower IgG4 responses to OVAg. These same individuals demonstrated evidence of past infection, based on serum antibodies detectable by Western blot analysis to male and female adult worms and skin microfilariae, and had a positive response to two or less of the recombinant antigens. We believe that this subcategory may represent individuals with a unique status of immunity.

We report the clinical and parasitological effects of a modified treatment regimen for suramin. Twenty adult males received up to 5 g (72.5 to 84.7 mg/kg) of suramin over 36 days. Detailed clinical and laboratory examinations were done before treatment and then at intervals over 2 years. Nodules were removed at 6, 13, 26 and 52 weeks for histology. Systemic tolerance was good. Anterior segment inflammation was however common and 2 patients required intervention to prevent posterior synechiae. No new posterior segment lesions developed; a rare improvement occurred in one patient with papillitis. Proteinuria, mostly mild, occurred in nearly all patients. Previously unreported renal glycosuria was documented in one patient. Microfilariae in the skin and anterior chamber did not change significantly for 5 or more weeks after which rapid reductions occurred. Ocular parasites were absent at 2 years and skin microfilariae were near zero. Peripheral blood eosinophil counts fell in parallel with those of microfilariae in the skin and anterior chamber and were normal at one and two years. These findings at 2 years may provide indirect evidence of a macrofilaricidal or a permanent chemosterilant effect on the adult worms. Nodule examination revealed an embryotoxic effect from week 6, a lethal effect on the male worms from month 3 and on the female worms from month 6 after treatment started. At one year 34% of the female worms examined were alive. Thus total doses of suramin in the range 72.5 to 84.7 mg/kg have only a modest lethal effect on the female worms. Suramin remains a restricted drug and a suitable replacement is urgently needed.

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.

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.

A double-blind clinical trial was conducted in Monagas State, Venezuela to assess the tolerance and efficacy of albendazole in the therapy of Onchocerca volvulus infection. Forty-nine patients (26 treated and 23 controls) received a 10-day course of albendazole (400 mg/day) or a placebo. Consistent with the excellent tolerance observed, albendazole did not kill microfilariae. However, analysis of changes in microfilarial densities (mf/mg of skin) over one year showed that albendazole was active against O. volvulus, presumably by interfering with embryogenesis. The nature, degree, and duration of this effect remain to be determined.