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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).)

Sudan is endemic for multiple neglected tropical diseases, including trachoma, onchocerciasis (OV), lymphatic filariasis (LF), and schistosomiasis (SCH). In 2019, dried blood spot samples were collected for a baseline trachoma serosurvey in three localities (El Seraif, Kotom, and Saraf Omrah) in North Darfur State. None were classified previously as OV- or LF-endemic, although low levels of SCH had been identified in all three. Approximately 30 households from 25 communities in each locality were selected by multistage cluster random sampling. Collections of DBSs were analyzed by multiplex bead assay for antibodies to multiple pathogens. This paper presents data on OV (Ov16), LF (Wb123, Bm14, Bm33), and SCH (soluble egg antigen [SEA], Sm25) antibodies among 8,322 individuals from 2,119 households. The survey-adjusted seroprevalence estimates for Ov16 were <0.3% in all localities. Lymphatic filariasis-antigen seroprevalences were discordant. Seroprevalence estimates ranged from 4.6-6.0% (Wb123), 0.99-1.4% (Bm14), and 29.2-33.3% (Bm33). Schistosomiasis seroprevalence estimates among school-aged children ranged from 2.7-8.0% (SEA) and 10.9-15.6% (Sm25). Ov16 seropositivity was low and supported the localities' classification as nonendemic. The results suggested LF exposure, but discordance between antigens, challenges defining seropositivity thresholds, and the absence of programmatic guidance based on antibody serology alone for Wuchereria bancrofti indicate a need for remapping surveys to confirm transmission. Schistosomiasis antibody levels were high enough to warrant further mapping to guide treatment decisions. The lack of gold standards limited interpretation of results, particularly for LF, but in resource-challenged areas, integrated serological surveillance offers the possibility of efficient monitoring of exposure to multiple diseases.

The neglected tropical disease onchocerciasis, or river blindness, is caused by infection with the filarial nematode Onchocerca volvulus. Current estimates indicate that 17 million people are infected worldwide, the majority of them living in Africa. Today there are no non-invasive tests available that can detect ongoing infection, and that can be used for effective monitoring of elimination programs. In addition, to enable pharmacodynamic studies with novel macrofilaricide drug candidates, surrogate endpoints and efficacy biomarkers are needed but are non-existent. We describe the use of a multimodal untargeted mass spectrometry-based approach (metabolomics and lipidomics) to identify onchocerciasis-associated metabolites in urine and plasma, and of specific lipid features in plasma of infected individuals (O. volvulus infected cases: 68 individuals with palpable nodules; lymphatic filariasis cases: 8 individuals; non-endemic controls: 20 individuals). This work resulted in the identification of elevated concentrations of the plasma metabolites inosine and hypoxanthine as biomarkers for filarial infection, and of the urine metabolite cis-cinnamoylglycine (CCG) as biomarker for O. volvulus. During the targeted validation study, metabolite-specific cutoffs were determined (inosine: 34.2 ng/ml; hypoxanthine: 1380 ng/ml; CCG: 29.7 ng/ml) and sensitivity and specificity profiles were established. Subsequent evaluation of these biomarkers in a non-endemic population from a different geographical region invalidated the urine metabolite CCG as biomarker for O. volvulus. The plasma metabolites inosine and hypoxanthine were confirmed as biomarkers for filarial infection. With the availability of targeted LC-MS procedures, the full potential of these 2 biomarkers in macrofilaricide clinical trials, MDA efficacy surveys, and epidemiological transmission studies can be investigated., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: Ole Lagatie, Emmanuel Njumbe Ediage, Dirk Van Roosbroeck, Stijn Van Asten, Ann Verheyen, Lieve Dillen, Tom Verhaeghe, Rob Vreeken, Filip Cuyckens and Lieven J. Stuyver are current employees of Janssen Pharmaceutica NV, a Johnson & Johnson company, and may own stock or stock option in that company.

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.

Many parasitic infections have different presenting features in endemic individuals (ENDs) and immunologically naive temporary residents (TRs). Temporary residents with loiasis often display acute symptoms and hypereosinophilia, in contrast to a parasite-induced subclinical state in chronically infected ENDs. Few studies have examined differences in ENDs and TRs infected with the related filarial parasite Onchocerca volvulus . We identified 40 TRs and 36 ENDs with imported onchocerciasis at the National Institutes of Health between 1976 and 2016. All study subjects received an extensive pretreatment medical history, physical examination, and laboratory investigations. We performed additional parasite-specific serologic testing on stored patient sera. Asymptomatic infection occurred in 12.5% of TRs and no ENDs ( P = 0.06). Papular dermatitis was more common in TRs (47.5% versus 2.7%, P < 0.001), whereas more pigmentation changes occurred in ENDs (41.7% versus 15%, P = 0.01). Only endemic patients reported visual disturbance (13% versus 0%, P = 0.03). One TR (3.3%) had onchocercal eye disease, compared with 22.6% of ENDs ( P = 0.053). Absolute eosinophil counts (AECs) were similar in ENDs and TRs ( P = 0.5), and one-third of subjects had a normal AEC. Endemic individuals had higher filarial-specific IgG4 and were more likely to be positive for IgG4 antibodies to Ov -16. Temporary residents and ENDs with imported O. volvulus infection presented with different dermatologic manifestations; ocular involvement occurred almost exclusively in ENDs. Unlike Loa loa , clinical differences appear not to be eosinophil-mediated and may reflect chronicity, intensity of infection, or the presence of Wolbachia in O. volvulus .

There is an urgent global need for a safe macrofilaricide drug to accelerate elimination of the neglected tropical diseases onchocerciasis and lymphatic filariasis. From an anti-infective compound library, the macrolide veterinary antibiotic, tylosin A, was identified as a hit against Wolbachia This bacterial endosymbiont is required for filarial worm viability and fertility and is a validated target for macrofilaricidal drugs. Medicinal chemistry was undertaken to develop tylosin A analogs with improved oral bioavailability. Two analogs, A-1535469 and A-1574083, were selected. Their efficacy was tested against the gold-standard second-generation tetracycline antibiotics, doxycycline and minocycline, in mouse and gerbil infection models of lymphatic filariasis ( Brugia malayi and Litomosoides sigmodontis ) and onchocerciasis ( Onchocerca ochengi ). A 1- or 2-week course of oral A-1535469 or A-1574083 provided >90% Wolbachia depletion from nematodes in infected animals, resulting in a block in embryogenesis and depletion of microfilarial worm loads. The two analogs delivered comparative or superior efficacy compared to a 3- to 4-week course of doxycycline or minocycline. A-1574083 (now called ABBV-4083) was selected for further preclinical testing. Cardiovascular studies in dogs and toxicology studies in rats and dogs revealed no adverse effects at doses (50 mg/kg) that achieved plasma concentrations >10-fold above the efficacious concentration. A-1574083 (ABBV-4083) shows potential as an anti- Wolbachia macrolide with an efficacy, pharmacology, and safety profile that is compatible with a short-term oral drug course for treating lymphatic filariasis and onchocerciasis., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).)

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.

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., 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.

Background: Onchocerciasis, also known as river blindness, is a parasitic disease. More than 99 percent of all cases occur in Africa. Bioko Island (Equatorial Guinea) is the only island endemic for onchocerciasis in the world. Since 2005, when vector Simulium yahense was eliminated, there have not been any reported cases of infection. This study aimed to demonstrate that updated WHO criteria for stopping mass drug administration (MDA) have been met., Methodology/principal Findings: A cross-sectional study was conducted from September 2016 to January 2017. Participants were 5- to 9-year-old school children. Onchocerciasis/lymphatic Filariasis (LF, only in endemic districts) rapid diagnostic tests (RDTs) were performed. Blood spots were collected from RDT positive children and 10 percent of the RDT negatives to determine Ov16 and Wb123 IgG4 antibodies through enzyme-linked immunosorbent assay (ELISA). Skin snips were collected from RDT positives. Filarial detection was performed by PCR in positives and indeterminate sera. Black fly collection was carried out in traditional breeding sites. A total of 7,052 children, ranging from 5 to 9 years of age, were included in the study. Four children (0.06%) were Ov16 IgG4 RDT positives, but negative by ELISA Ov16, while 6 RDT negative children tested positive by ELISA. A total of 1,230 children from the Riaba and Baney districts were tested for LF. One child was Wb123 RDT positive (0.08%), but ELISA negative, while 3 RDT negative children were positive by Wb123 ELISA. All positive samples were negative by PCR for onchocerciasis and LF (in blood spot and skin snip). All fly collections and larval prospections in the traditional catching and prospection sites were negative., Conclusions/significance: WHO criteria have been met, therefore MDA in Bioko Island can be stopped. Three years of post-treatment surveillance should be implemented to identify any new occurrences of exposure or infection.

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.

In Africa, onchocerciasis and lymphatic filariasis (LF) are co-endemic in many areas. Current efforts to eliminate both diseases are through ivermectin-based mass drug administration (MDA). Years of ivermectin distribution for onchocerciasis may have interrupted LF transmission in certain areas. The Kédougou region, Senegal, is co-endemic for LF and onchocerciasis. Though MDA for onchocerciasis started in 1988, in 2014 albendazole had not yet been added for LF. The objective of this study was to assess in an integrated manner the LF and onchocerciasis status in the three districts of the Kédougou region after ≥10 years of ivermectin-based MDA. The study employed an African Programme for Onchocerciasis Control (APOC) onchocerciasis-related methodology. In the three districts, 14 villages close to three rivers that have Simulium damnosum breeding sites were surveyed. Convenience sampling of residents ≥5 years old was performed. Assessment for LF antigenemia by immunochromatographic testing (ICT) was added to skin snip microscopy for onchocerciasis. Participants were also tested for antibodies against Wb123 (LF) and Ov16 (onchocerciasis) antigens. In two districts, no participants were ICT or skin snip positive. In the third district, 3.5% were ICT positive and 0.7% were skin snip positive. In all the three districts, Wb123 prevalence was 0.6%. Overall, Ov16 prevalence was 6.9%. Ov16 prevalence among children 5-9 years old in the study was 2.5%. LF antigenemia prevalence was still above treatment threshold in one district despite ≥10 years of ivermectin-based MDA. The presence of Ov16 positive children suggested recent transmission of Onchocerca volvulus. This study showed the feasibility of integrated evaluation of onchocerciasis and LF but development of integrated robust methods for assessing transmission of both LF and onchocerciasis are needed to determine where MDA can be stopped safely in co-endemic areas., Competing Interests: The authors have declared that no competing interests exist.

Background: Of increasing importance to the medical and veterinary communities is the zoonotic filarioid nematode Onchocerca lupi. Onchocercosis, thus far found in wolves, dogs, cats and humans, is diagnosed via skin snips to detect microfilariae and surgical removal of adults from the eye of the host. These methods are time-consuming, laborious and invasive, highlighting the need for new tools for the diagnosis of O. lupi in susceptible hosts. Symptoms related to the presence of the adults in the eye can range from none apparent to severe, including blindness. No reliable chemotherapeutic protocols are available, as yet, to eliminate the infection. Paramyosin, an invertebrate-specific protein, has been well-studied as an allergen, diagnostic marker and vaccine candidate. The aim of this study, therefore, was to isolate and characterise paramyosin from O. lupi to assess its suitability for the development of a serological diagnostic assay., Methods: The adult and microfilarial stages of O. lupi were isolated from the eyes and skin of a 3-year-old male dog. Total RNA was extracted and reverse transcribed into single stranded cDNA. Reverse-transcription PCR was used to isolate a full-length paramyosin cDNA from adult worms and to investigate the temporal expression patterns of this gene. All amplicons were sequenced using dideoxy chain termination sequencing. Bioinformatics was used to predict the amino acid sequence of the gene, to compare the DNA and protein sequences with those available in public databases and to investigate the phylogenetic relationship of all molecules. Antibody binding sites were predicted using bioinformatics and mapped along with published antigenic epitopes against the O. lupi paramyosin protein. The native protein, and three smaller recombinantly expressed peptides, were subjected to western blot using serum from dogs both positive and negative for O. lupi., Results: Paramyosin of O. lupi was herein molecularly characterized, encoded by a transcript of 2,643 bp and producing a protein of 881 amino acids (101.24 kDa). The paramyosin transcript was detected, by reverse transcription PCR, in adults and microfilariae, but not in eggs. Phylogenetic analysis indicates that this molecule clusters with paramyosins from other filarioids to the exclusion of those from other taxa. A total of 621 unique antibody binding epitopes were predicted for this protein and another 28 were conserved in other organisms. This information was used to design three peptides, for recombinant expression, to identify the antibody binding epitope(s) and reduce potential cross-reactivity with serum from dogs infected with other filarioid nematodes. Native paramyosin, purified from microfilariae and adults, was detected by antibodies present in serum from dogs with known O. lupi infections., Conclusions: Data provided herein may assist in the development of a serological diagnostic test, based on antibodies to O. lupi paramyosin, for the diagnosis of this infection, in order to gain more information on the real distribution of this little known filarioid of zoonotic concern.

Background: Diagnostics provide a means to measure progress toward disease elimination. Many countries in Africa are approaching elimination of onchocerciasis after successful implementation of mass drug administration programs as well as vector control. An understanding of how markers for infection such as skin snip microfilaria and Onchocerca volvulus-specific seroconversion perform in near-elimination settings informs how to best use these markers., Methods: All-age participants from 35 villages in Togo were surveyed in 2013 and 2014 for skin snip Onchocerca volvulus microfilaria and IgG4 antibody response by enzyme-linked immunosorbent assay (ELISA) to the Onchocerca volvulus-specific antigen Ov16. A Gaussian mixture model applying the expectation-maximization (EM) algorithm was used to determine seropositivity from Ov16 ELISA data. For a subset of participants (n = 434), polymerase chain reaction (PCR) was performed on the skin snips taken during surveillance., Results: Within the 2,005 participants for which there was Ov16 ELISA data, O. volvulus microfilaremia prevalence and Ov16 seroprevalence were, 2.5 and 19.7 %, respectively, in the total population, and 1.6 and 3.6 % in children under 11. In the subset of 434 specimens for which ELISA, PCR, and microscopy data were generated, it was found that in children under 11 years of age, the anti-Ov16 IgG4 antibody response demonstrate a sensitivity and specificity of 80 and 97 %, respectively, against active infections as determined by combined PCR and microscopy on skin snips. Further analysis was performed in 34 of the 35 villages surveyed. These villages were stratified by all-age seroprevalence into three clusters: < 15 %; 15-20 %; and > 20 %. Age-dependence of seroprevalence for each cluster was best reflected by a two-phase force-of-infection (FOI) catalytic model. In all clusters, the lower of the two phases of FOI was associated with a younger age group, as reflected by the seroconversion rates for each phase. The age at which transition from lower to higher seroconversion, between the two phases of FOI, was found to be highest (older) for the cluster of villages with < 15 % seroprevalence and lowest (younger) for the cluster with the highest all-age seroprevalence., Conclusions: The anti-Ov16 IgG4 antibody response is an accurate marker for active infection in children under 11 years of age in this population. Applying Ov16 surveillance to a broader age range provides additional valuable information for understanding progression toward elimination and can inform where targeted augmented interventions may be needed. Clustering of villages by all-age sero-surveillance allowed application of a biphasic FOI model to differentiate seroconversion rates for different age groups within the village cluster categories.

Background: The immunochromatographic test (ICT) for lymphatic filariasis is a serological test designed for unequivocal detection of circulating Wuchereria bancrofti antigen. It was validated and promoted by WHO as the primary diagnostic tool for mapping and impact monitoring for disease elimination following interventions. The initial tests for specificity and sensitivity were based on samples collected in areas free of loiasis and the results suggested a near 100% specificity for W. bancrofti. The possibility of cross-reactivity with non-Wuchereria bancrofti antigens was not investigated until recently, when false positive results were observed in three independent studies carried out in Central Africa. Associations were demonstrated between ICT positivity and Loa loa microfilaraemia, but it was not clearly established if these false positive results were due to L. loa or can be extended to other filarial nematodes. This study brought further evidences of the cross-reactivity of ICT card with L. loa and Onchocerca ochengi (related to O. volvulus parasite) using in vivo and in vitro systems., Methods: Two filarial/host experimental systems (L. loa-baboon and O. ochengi-cattle) and the in vitro maintenance of different stages (microfilariae, infective larvae and adult worm) of the two filariae were used in three experiments per filarial species. First, whole blood and sera samples were prepared from venous blood of patent baboons and cattle, and applied on ICT cards to detect circulating filarial antigens. Secondly, larval stages of L. loa and O. ochengi as well as O. ochengi adult males were maintained in vitro. Culture supernatants were collected and applied on ICT cards after 6, 12 and 24 h of in vitro maintenance. Finally, total worm extracts (TWE) were prepared using L. loa microfilariae (Mf) and O. ochengi microfilariae, infective larvae and adult male worms. TWE were also tested on ICT cards. For each experiment, control assays (whole blood and sera from uninfected babon/cattle, culture medium and extraction buffer) were performed., Results: Positive ICT results were obtained with whole blood and sera of L. loa microfilaremic baboons, culture supernatants of L. loa Mf and infective larvae as well as with L. loa Mf protein extracts. In contrast, negative ICT results were observed with whole blood and sera from the O. ochengi-cattle system. Surprisingly, culture supernatant of O. ochengi adult males and total worm extracts (Mf, infective larvae and adult worm) were positive to the test., Conclusions: This study has provided further evidence of L. loa cross-reactivity for the ICT card. All stages of L. loa seem capable of inducing the cross-reactivity. Onchocerca ochengi. can also induce cross-reactivity in vitro, but this is less likely in vivo due to the location of parasite. The availability of the parasite proteins in the blood stream determines the magnitude of the cross-reactivity. The cross-reactivity of the ICT card to these non-W. bancrofti filariae poses some doubts to the reliability and validity of the current map of LF of Central Africa that was generated using this diagnostic tool.

Background: microRNAs (miRNAs), a class of short, non-coding RNA can be found in a highly stable, cell-free form in mammalian body fluids. Specific miRNAs are secreted by parasitic nematodes in exosomes and have been detected in the serum of murine and dog hosts infected with the filarial nematodes Litomosoides sigmodontis and Dirofilaria immitis, respectively. Here we identify extracellular, parasite-derived small RNAs associated with Onchocerca species infecting cattle and humans., Methods: Small RNA libraries were prepared from total RNA extracted from the nodule fluid of cattle infected with Onchocerca ochengi as well as serum and plasma from humans infected with Onchocerca volvulus in Cameroon and Ghana. Parasite-derived miRNAs were identified based on the criteria that sequences unambiguously map to hairpin structures in Onchocerca genomes, do not align to the human genome and are not present in European control serum., Results: A total of 62 mature miRNAs from 52 distinct pre-miRNA candidates were identified in nodule fluid from cattle infected with O. ochengi of which 59 are identical in the genome of the human parasite O. volvulus. Six of the extracellular miRNAs were also identified in sequencing analyses of serum and plasma from humans infected with O. volvulus. Based on sequencing analysis the abundance levels of the parasite miRNAs in serum or plasma range from 5 to 127 reads/per million total host miRNA reads identified, comparable to our previous analyses of Schistosoma mansoni and L. sigmodontis miRNAs in serum. All six of the O. volvulus miRNAs identified have orthologs in other filarial nematodes and four were identified in the serum of mice infected with L. sigmodontis., Conclusions: We have identified parasite-derived miRNAs associated with onchocerciasis in cattle and humans. Our results confirm the conserved nature of RNA secretion by diverse nematodes. Additional species-specific small RNAs from O. volvulus may be present in serum based on the novel miRNA sequences identified in the nodule fluid. In our analyses comparison to European control serum illuminates the scope for false-positives, warranting caution in criteria that should be applied to identification of biomarkers of infection.

A combination of deep-sequencing and bioinformatics analysis enabled identification of twenty-two microRNA candidates of potential nematode origin in plasma from Loa loa-infected baboons and a further ten from the plasma of an Onchocerca ochengi-infected cow. The obtained data were compared to results from previous work on miRNA candidates from Dirofilaria immitis and O. volvulus found in host circulating blood, to examine the species specificity of the released miRNA. None of the miRNA candidates was found to be present in all four host-parasite scenarios and most of them were specific to only one of them. Eight candidate miRNAs were found to be identical in the full sequence in at least two different infections, while nine candidate miRNAs were found to be similar but not identical in at least four filarial species., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Uganda is the only African country whose onchocerciasis elimination program uses a two-pronged approach of vector control and mass drug distribution. The Ugandan program relies heavily upon the use of serosurveys of children to monitor progress toward elimination. The program has tested over 39,000 individuals from 11 foci for Onchocerca volvulus exposure, using the Ov16 ELISA test. The data show that the Ov16 ELISA is a useful operational tool to monitor onchocerciasis transmission interruption in Africa at the World Health Organization (WHO) recommended threshold of < 0.1% in children. The Ugandan experience has also resulted in a re-examination of the statistical methods used to estimate the boundary of the upper 95% confidence interval for the WHO prevalence threshold when all samples tested are negative. This has resulted in the development of Bayesian and hypergeometric statistical methods that reduce the number of individuals who must be tested to meet the WHO criterion.

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.)

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., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Human onchocerciasis, caused by the filarial nematode Onchocerca volvulus, is controlled almost exclusively by the drug ivermectin, which prevents pathology by targeting the microfilariae. However, this reliance on a single control tool has led to interest in vaccination as a potentially complementary strategy. Here, we describe the results of a trial in West Africa to evaluate a multivalent, subunit vaccine for onchocerciasis in the naturally evolved host-parasite relationship of Onchocerca ochengi in cattle. Naïve calves, reared in fly-proof accommodation, were immunised with eight recombinant antigens of O. ochengi, administered separately with either Freund's adjuvant or alum. The selected antigens were orthologues of O. volvulus recombinant proteins that had previously been shown to confer protection against filarial larvae in rodent models and, in some cases, were recognised by serum antibodies from putatively immune humans. The vaccine was highly immunogenic, eliciting a mixed IgG isotype response. Four weeks after the final immunisation, vaccinated and adjuvant-treated control calves were exposed to natural parasite transmission by the blackfly vectors in an area of Cameroon hyperendemic for O. ochengi. After 22 months, all the control animals had patent infections (i.e., microfilaridermia), compared with only 58% of vaccinated cattle (P = 0.015). This study indicates that vaccination to prevent patent infection may be an achievable goal in onchocerciasis, reducing both the pathology and transmissibility of the infection. The cattle model has also demonstrated its utility for preclinical vaccine discovery, although much research will be required to achieve the requisite target product profile of a clinical candidate.

The study was conducted to investigate a variation in the distribution of endemic elephantiasis previously determined to be of geochemical origin in three neighbouring and essentially homogenous villages, Bambili, Bambui and Finge of the Bambui Health District of NW Cameroon. A total of 301 subjects were examined for onchocerciasis and lymphatic filariasis in the area using standard procedures. The onchocercal microfilarial prevalence varied from 6.5% in Bambili through 20.4% in Bambui to 60.4% in Finge. The onchocercal serological prevalence based on IgG4 detection followed a similar trend. By contrast, blood microfilariae were absent in the area as verified by use of sensitive techniques. The community prevalence of elephantiasis varied from 1.1% in Bambili to 4.4% in Bambui and 10.4% in Finge. The correlation between the parasitological prevalence of onchocerciasis and the prevalence of lymphedema in the three villages was strong (r=0.99, p<0.05). We confirm that the elephantiasis in the area is of geochemical origin and the results suggest that it is being exacerbated by onchocercal lymphadenitis.

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.

Background: A previously developed, specific, rapid-format immunochromatographic card test that detects immunoglobulin G4 to the recombinant Onchocerca volvulus antigen Ov-16 was modified to detect antibodies in whole blood., Methods: Ov-16 card test results were assessed in 1511 subjects > or =2 years of age in 7 West African villages with varying histories of onchocerciasis control measures., Results: In villages in which control measures had been implemented, anti-Ov-16 antibody prevalence rates ranged from 5.2% to 65.1%. Antibody prevalence rates were close to zero among subjects born after effective control measures had been implemented. In 2 villages without a history of control measures where onchocerciasis was endemic, microfilariae (MF) prevalence rates were 82.8% and 65.1%, and antibody prevalence rates were 73.1% and 62.1%. In these 2 villages, the sensitivity of the Ov-16 card test was 81.1% and 76.5%, the specificity was 100%, and the positive predictive value was 91.8% and 80.5%. MF and antibody prevalence rates were correlated (Spearman's r=0.815; P<.038)., Conclusions: The Ov-16 card test is field applicable, exhibits high sensitivity and specificity for O. volvulus infection, and has great potential as a tool for surveillance and for evaluating the success of onchocerciasis control measures.

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.

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.

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.

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.

Objective: To determine the incidence of serious adverse events (SAEs) after mass treatment with ivermectin in areas co-endemic for loiasis and onchocerciasis, and to identify potential risk factors associated with the development of these SAEs, in particular encephalopathic SAEs., Methods: We retrospectively analysed SAEs reported to have occurred between 1 December 1998 and 30 November 1999 in central-southern Cameroon by chart review, interview and examination of a subset of patients., Results: The overall incidence of SAEs for the three provinces studied was 6 per 100,000. However, for Central Province alone the incidence of SAEs was 2.7 per 10,000 overall, and 1.9 per 10,000 for encephalopathic SAEs associated with Loa loa microfilaremia (PLERM). The corresponding rates for the most severely affected district within Central Province (Okola) were 10.5 per 10,000 and 9.2 per 10,000 respectively. Symptoms began within the first 24-48 h of ivermectin administration but there was a delay of approximately 48-84 h in seeking help after the onset of symptoms. First-time exposure to ivermectin was associated with development of PLERM., Conclusion: In Cameroon, the incidence of SAEs following ivermectin administration in general, and PLERM cases in particular, varies substantially by district within the areas co-endemic for loiasis and onchocerciasis. More intense surveillance and monitoring in the first 2 days after mass distribution in ivermectin-naïve populations would assist in early recognition, referral and management of these cases. The increased reporting of SAEs from Okola is unexpected and warrants further investigation. Research is urgently needed to find a reliable screening tool to exclude individuals (rather than communities) at risk of PLERM from the mass treatment program.

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.

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.

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.

Eosinophils, eosinophil cationic protein (ECP), eosinophil-derived neurotoxin (EDN/EPX), myeloperoxidase (MPO) and IgE were measured in blood, serum and/or urine in Schistosoma haematobium- and Onchocerca volvulus-infected Guineans and O. volvulus- and S. haematobium-negative Guineans coinfected or infected with intestinal nematodes. The number of eosinophils and levels of eosinophil granule proteins but not of MPO were found to be strongly elevated in all Africans as compared to European controls. The highest serum ECP and serum and urinary EDN/EPX levels were observed in the hyperreactive form of onchocerciasis (sowda). Onchocerciasis patients and O. volvulus-negative Africans coinfected or infected with intestinal nematodes (hookworm and/or Ascaris lumbricoides) revealed higher serum granule protein concentrations and/or absolute eosinophil counts and urinary ECP than those without nematode infections. Statistical differences between both sections were found for the absolute eosinophil counts and for serum EDN/EPX and IgE in generalized onchocerciasis, and for urinary ECP in sowda, indicating stimulation of the eosinophil potential of O. volvulus-positive patients by coexistent hookworm infection. This worm species, in contrast to A. lumbricoides, causes especially high eosinophil counts and EDN/EPX and IgE levels. From these results it is concluded that in nematode diseases, ECP and EDN/EPX levels reflect the degree of antigenic stimulation, eosinophil activation and eosinophil turnover rates. Serum ECP and serum and urinary EDN/EPX may, therefore, serve as parameters to monitor helminth infection. Urinary ECP may be a marker of eosinophiluria secondary to urogenital manifestation of S. haematobium. It is elevated in hyperreactive onchocerciasis activated by intestinal nematodes.

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.

The serum levels of three major granulocyte proteins were measured in patients with onchocerciasis, bancroftian filariasis and intestinal schistosomiasis and compared to controls from patients with malaria, Africans living in areas not endemic for these infections and healthy Germans. The investigation comprised the determination of the eosinophil granule proteins eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN/EPX), and the neutrophil/monocyte granule protein myeloperoxidase (MPO). ECP and EDN/EPX levels were found elevated only in the three helminth infections that are associated with eosinophilia, while MPO was found elevated in all tested disease groups. The levels of eosinophil granule proteins observed in the helminth diseases by far exceeded those described for bronchial asthma and atopic dermatitis. ECP, EDN/EPX and MPO serum levels reflect the ongoing disease and are related to functional activity of the respective leukopoetic system. ECP and EDN/EPX appear to be markers of the eosinophil effector system and MPO a marker of the neutrophil and/or monocyte/macrophage effector system. Significantly higher ECP levels in chronic hyperreactive onchodermatitis (sowda) versus generalized onchocerciasis seem to reflect an augmented degree of antigenic stimulation, eosinophil activation and eosinophil turnover rates, indicating a more active mechanism of parasite clearance in sowda patients.

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.

We have investigated various aspects related to managing wide-scale ivermectin distribution schemes within randomized controlled trials in communities where onchocerciasis is endemic. Multiple logistic regression analysis of determinants of compliance to five doses of ivermectin in 589 people in Sierra Leone showed independent significant associations with leopard skin depigmentation, the severity of side effects of treatment, fulfilling the exclusion criteria for treatment, and long-term residence in the community. These results are useful for tailoring health promotion messages in Sierra Leone, but the associations may differ in other West African societies. In Nigeria 1847 people were interviewed about various subjective responses, including itching. None of these showed clear improvement after three years of ivermectin treatment. Positive comments about treatment were generally non-specific and similar in the placebo and ivermectin groups. Negative comments were usually related to adverse reactions, especially itching and rash, and were more common after ivermectin. The lack of any benefit attributable to ivermectin that is discernible to its recipients may make it difficult to maintain the high compliance rates needed for long periods if mass dosing programmes are to have a lasting impact on onchocerciasis. In addition, no consistent effects of ivermectin were found by measuring visual acuity, height, weight or haematocrit in comparison with placebo. This may indicate that evidence of clinical impact is very slow to develop and is hard to measure using simple objective methods after only three doses of treatment. At present it seems that parasitological, entomological and detailed ophthalmological or dermatological methods are required to demonstrate the impact of ivermectin treatment in the medium-term.

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.