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Background: Molecular xenomonitoring (MX), the detection of parasite nucleic acid in the vector population, is recommended for onchocerciasis surveillance in elimination settings. However, the sensitivity of MX for detecting onchocerciasis-positive communities has not previously been evaluated. MX may have additional applications for control programmes but its utility is restricted by a limited understanding of the relationship between MX results and human prevalence., Methods: We conducted a systematic review of studies reporting the prevalence of Onchocerca volvulus DNA in wild-caught Simulium spp. flies (MX rate) and corresponding prevalence of microfilaria (mf) in humans. We evaluated the sensitivity of MX for detecting onchocerciasis-positive communities and describe the characteristics of studies with reduced sensitivity. We conducted a linear regression to evaluate the relationship between mf prevalence and MX rate., Results: We identified 15 relevant studies, with 13 studies comprising 34 study communities included in the quantitative analyses. Most communities were at advanced stages towards elimination and had no or extremely low human prevalence. MX detected positive flies in every study area with >1% mf prevalence, with the exception of one study conducted in the Venezuelan Amazonian focus. We identified a significant relationship between the two measurements, with mf prevalence accounting for half of the variation in MX rate (R2 0.50, p<0.001)., Conclusion: MX is sensitive to communities with ongoing onchocerciasis transmission. It has potential to predict human mf prevalence, but further data is required to understand this relationship, particularly from MX surveys conducted earlier in control programmes before transmission has been interrupted., Competing Interests: The authors have declared that no competing interests exist.

In the United States and Europe, human onchocerciasis is a rare disease caused by zoonotic or anthropophilic parasites in the genus Onchocerca . The zoonotic species identified in focal areas of Europe and United States is Onchocerca lupi , and Onchocerca volvulus , the anthroponotic species, may be found among people who had lived in endemic areas of Africa, the Arabian Peninsula, or Latin America. Onchocerciasis due to O. lupi is an emergent parasitic disease, with limited diagnostic methods, in addition to the lack of information on its biology, transmission, and epidemiology. Cutaneous nodules are the disease's most prevalent manifestation but lack diagnostic specificity. To address the diagnosis of onchocerciasis at reference laboratories, we developed a duplex TaqMan real-time PCR (qPCR) method, targeting the cytochrome oxidase subunit I locus which has species-specific probes to identify and differentiate O. lupi from O . volvulus . We determined the performance of the duplex with a panel of 45 samples: 11 positives for O. lupi , six for O. volvulus , five samples with negative results for Onchocerca spp., and 23 non- Onchocerca nematodes. The duplex qPCR correctly detected 10 of 11 O . lupi - and six of six O . volvulus -positive specimens. The new duplex assay allowed the simultaneous detection and discrimination of O . lupi and O . volvulus in clinical specimens, expediting and facilitating the clinical diagnosis of O . lupi in non-endemic settings where the disease is an infrequent finding.

Onchocerciasis is a blinding disease caused by the filarial parasite Onchocerca volvulus , with a worldwide distribution. Onchocerciasis has been targeted for regional elimination based on annual and semiannual mass drug administration (MDA) with ivermectin in endemic communities over several years. This strategy in Ecuador led to the interruption of transmission and suspension of ivermectin MDA in 2009 with certification of elimination in 2014. In the present study, we analyzed sera collected in 2018 from 123 children aged 5-9 years from formerly hyperendemic communities in the Esmeraldas focus, Ecuador, for the presence of antibodies to Ov16 antigen. All samples were negative, indicating no evidence of transmission since MDA was stopped. Ov16-based serology offers an economic and practical alternative for measuring vector infectivity for post-certification surveillance in formerly endemic countries where expertise and capacity to reliably measure fly infectivity rates are costly to maintain.

Introduction: onchocerciasis is one of the major infectious diseases caused by Onchocerca volvulus. This parasite is responsible for chronic cutaneous and ocular diseases affecting more than 37 million people of whom 99% are in Africa. The study was conducted in the health district of Ntui from June to September 2016 to determine the prevalence of O. volvulus infection after seven years of massive administration of ivermectin., Methods: two cutaneous snips were made at the iliac crests level in volunteers. These tissues were incubated in physiological saline water and were examined for parasitological investigations in the laboratory., Results: a total of 310 participants were randomly selected, of whom 170 (54.8%) were women and 140 (45.1%) were men aged 6 to 83 years, thus giving a sex ratio of 1.2 in favour of women. After parasitological analysis, 26 participants had microfilaraemia, of whom 15 (10.7%) were men and 11 (6.4%) were women. The most infected age group was 16 to 26 years (12.5%). The highest infection rates were found among farmers (11%) and participants living in the village of Essougly (26.6%). No significant differences in prevalence values between the different groups were noted, whatever the parameter considered., Conclusion: the prevalence of onchocerciasis in the health district of Ntui has declined from a hyperendemic to a hypoendemic state after seven years of massive administration of ivermectin. However, careful monitoring of onchocerciasis should be continued to prevent the area from returning to its original hyperendemicity., Competing Interests: The authors declare no competing interests., (Copyright: Estelle Makou Tsapi et al.)

Background: Onchocerciasis transmission across international borders is not uncommon, yet a coordinated cross border stops mass drug administration (MDA) decision has not been documented., Methods/principle Findings: The Galabat-Metema focus involves neighboring districts on the border between Sudan and Ethiopia. Mass drug administration (MDA) was provided once and subsequently twice per year in this focus, with twice-per-year beginning in Ethiopia's Metema subfocus in 2016 and in the Sudan's Galabat subfocus in 2008. Ov16 ELISA-based serosurveys were conducted in 6072 children under 10 years of age in the Metema subfocus in 2014, and 3931 in the Galabat in 2015. Between 2014 and 2016, a total of 27,583 vector Simulium damnosum flies from Metema and 9,148 flies from Galabat were tested by pool screen PCR for Onchocerca volvulus O-150 DNA. Only 8 children were Ov16 seropositive (all in the Metema subfocus); all were negative by skin snip PCR. The upper limit of the 95% confidence interval (UCL) for Ov16 seropositive was <0.1% for the overall focus and 0.14 positive fly heads per 2000 (UCL = 0.39/2000). However, an entomological 'hotspot' was detected on the Wudi Gemzu river in Metema district. The hotspot was confirmed when 4 more positive fly pools were found on repeat testing in 2017 (1.04 L3/2000 flies (UCL = 2.26/2000). Information exchange between the two countries led to stopping MDA in a coordinated fashion in 2018, with the exception of the hotspot at Wudi Gemzu, where MDA with ivermectin was increased to every three months to hasten interruption of transmission., Conclusion: Coordinated stop MDA decisions were made by Sudan and Ethiopia based on data satisfying the World Health Organization's criteria for interruption of onchocerciasis transmission. Definitions of entomological 'hotspots' and buffer zones around the focus are proposed., Competing Interests: The authors have declared that no competing interests exist.

Objectives: Epidemiological evidence links onchocerciasis with the development of epilepsy. The aim of this study was to detect Onchocerca volvulus microfilariae or its bacterial endosymbiont, Wolbachia, in the cerebrospinal fluid (CSF) of persons with onchocerciasis-associated epilepsy (OAE)., Methods: Thirteen persons with OAE and O. volvulus skin snip densities of >80 microfilariae were recruited in Maridi County (South Sudan) and their CSF obtained. Cytospin centrifuged preparations of CSF were examined by light microscopy for the presence of O. volvulus microfilariae. DNA was extracted from CSF to detect O. volvulus (O-150 repeat) by quantitative real-time PCR, and Wolbachia (FtsZ gene) by standard PCR. To further investigate whether CSF from onchocerciasis-infected participants could induce seizures, 3- and 7-day old zebrafish larvae were injected with the CSF intracardially and intraperitoneally, respectively. For other zebrafish larvae, CSF was added directly to the larval medium., Results: No microfilariae, parasite DNA, or Wolbachia DNA were detected in any of the CSF samples by light microscopy or PCR. All zebrafish survived the procedures and none developed seizures., Conclusions: The absence of O. volvulus in the CSF suggests that OAE is likely not caused by direct parasite invasion into the central nervous system, but by another phenomenon triggered by O. volvulus infection., (Copyright © 2019 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Competing Interests: The authors have declared that no competing interests exist.

Objectives: To investigate the reasons for the high prevalence of epilepsy (>6%) discovered in 2015 in the Aketi health zone in the north of the Democratic Republic of the Congo., Methods: Persons with epilepsy (PWE) diagnosed in a door-to-door survey in 2015 were traced and re-examined in 2017 by a neurologist. Confirmed PWE were paired with matched controls. For onchocerciasis assessment, children 7-10 years old were tested for IgG4 Onchocerca volvulus (OV16) antibodies, a rapid epidemiological mapping of onchocerciasis (REMO) study was performed, and ivermectin coverage was investigated., Results: Forty-three (61.4%) previously diagnosed PWE were traced; the neurologist confirmed the epilepsy diagnosis in all of them. The overall OV16 positivity rate was 64.5%. Poor ivermectin coverage (55.9%) and a high prevalence of onchocercal nodules (>70%) were observed. The prevalence of epilepsy was 5.7% in Aketi rural town, with nine PWE (13.8%) experiencing head nodding seizures. A case-control study showed that PWE had lower body weight and higher ivermectin coverage in 2017 than healthy controls., Conclusions: The high prevalence of epilepsy in the Aketi health zone, despite 14 years of community-directed treatment with ivermectin (CDTI), was found to be associated with high onchocerciasis transmission and low ivermectin use. An awareness programme to increase ivermectin coverage and the introduction of a bi-annual CDTI programme should be considered., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Onchocerciasis, or river blindness, is a dermal filariasis caused by infection with the nematode parasite Onchocerca volvulus, transmitted to humans through the bites of blackflies of the genus Simulium. Despite the decade-long West African Regional Programme for the Elimination of Onchocerciasis, involving the mass administration of ivermectin to populations in endemic areas, recrudescence has occurred. An example is in the Cascades Region of south-west Burkina Faso where the resumption of transmission had resulted in infection prevalences of up to 70% in some villages. In 2011, a strategy for community-directed distribution of ivermectin (CDTI) was set up to respond to this worrying re-emergence. Here, we report on a study of Onchocerca spp. transmission in the affected area carried out from January to December 2012. Every month, host-seeking adult females of the S. damnosum complex were collected at sites on the River Comoé near the four villages (Bodadiougou, Bolibana, Badara Karaboro and Badara Dogossè) that had recorded the highest prevalences in 2010. Collected blackflies were dissected and infective larvae were identified using the O-150 PCR method., Results: A total of 9114 S. damnosum (s.l.) adult females were collected, of which 5142 were parous (56.4%) and 78 (1.51%) were infective carrying a total of 137 infective larvae. The annual transmission potential (ATP) was calculated as 0, 30, 255 and 771 infective larvae/man/year in Badara Dogossè, Bolibana, Badara Karaboro and Bodadiougou, respectively. Transmission levels in the latter two are of particular concern as they were higher than 100 infective larvae/person/year, the designated minimum threshold required for elimination of severe pathology, including damage to vision., Conclusions: These results confirm that recrudescence of onchocerciasis has occurred, and that transmission of O. volvulus was active at sites on the Comoé River in the Cascades region in 2012. In accordance with WHO recommendations, CDTI should be continued and the situation in the Cascades region should be closely monitored if further spread of this outbreak is to be avoided.

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.

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.

Defining the optimal diagnostic tools for evaluating onchocerciasis elimination efforts in areas co-endemic for other filarial nematodes is imperative. This study compared three published polymerase chain reaction (PCR) methods: the Onchocerca volvulus -specific qPCR-O150, the pan-filarial qPCR melt curve analysis (MCA), and the O150-PCR enzyme-linked immunosorbent assay (ELISA) currently used for vector surveillance in skin snip biopsies (skin snips) collected from the Democratic Republic of the Congo. The pan-filarial qPCR-MCA was compared with species-specific qPCRs for Loa loa and Mansonella perstans . Among the 471 skin snips, 47.5%, 43.5%, and 27.0% were O. volvulus positive by qPCR-O150, qPCR-MCA, and O150-PCR ELISA, respectively. Using qPCR-O150 as the comparator, the sensitivity and specificity of qPCR-MCA were 89.3% and 98.0%, respectively, whereas for O150-PCR ELISA, they were 56.7% and 100%, respectively. Although qPCR-MCA identified the presence of L. loa and Mansonella spp. in skin snips, species-specific qPCRs had greater sensitivity and were needed to identify M. perstans . Most of the qPCR-MCA misclassifications occurred in mixed infections. The reduced sensitivity of O150-PCR ELISA was associated with lower microfilaria burden and with lower amounts of O. volvulus DNA. Although qPCR-MCA identified most of the O. volvulus -positive skin snips, it is not sufficiently robust to be used for stop-mass drug administration (MDA) evaluations in areas co-endemic for other filariae. Because O150-PCR ELISA missed 43.3% of qPCR-O150-positive skin snips, the qPCR-O150 assay is more appropriate for evaluating skin snips of OV-16 + children in stop-MDA assessments. Although improving the sensitivity of the O150-PCR ELISA as an alternative to qPCR might be possible, qPCR-O150 offers distinct advantages aside from increased sensitivity.

Background: The Esmeraldas focus of onchocerciasis in Ecuador expanded geographically during the 1980s and was associated with severe ocular and skin disease. Mass drug administration (MDA) with ivermectin started in 1991, initially once but later twice a year, in the principle endemic focus followed by all satellite foci. Treatment was stopped in 2009 when entomological assessments determined that transmission of Onchocerca volvulus had been interrupted., Methods: Three years after the cessation of ivermectin treatment in 2012, as defined by the WHO guidelines for onchocerciasis elimination, blackfly collections were done in four sentinel sites in former hyperendemic areas. The presence of infective larvae in local vectors, Simulium exiguum and Simulum quadrivittatum, was assessed by detection of O. volvulus DNA by PCR. Additional flies captured in four extra-sentinel sites located in former hyper- and mesoendemic dispersed isolated areas were also assessed., Results: The results from 68,310 captured blackflies, 40,114 from four sentinel villages in the previously hyperendemic areas (Corriente Grande, El Tigre, San Miguel on Río Cayapas and Naranjal on Río Canandé) and 28,197 from extra-sentinel locations, were all negative for the presence of O. volvulus. These extra-sentinel sites (Hualpí on Río Hoja Blanca, Capulí on Río Onzole, La Ceiba on Río Tululví and Medianía on Río Verde) were included to provide additional evidence of the impact of MDA on the transmission of O. volvulus in isolated endemic areas., Conclusions: Our data indicate that transmission of O. volvulus has been stopped in all endemic areas in Ecuador, including all satellite foci outside the main focus. These findings indicate that a strategy of ivermectin distribution twice a year to over 85% of the treatment-eligible population was effective in eliminating the infection from Ecuador in a focus with a highly competent primary vector, S. exiguum, and where the infection rates were equal to or greater than observed in many onchocerciasis foci in Africa.

Background: Onchocerciasis is a chronic parasitic infection originally endemic in 13 discrete regional foci distributed among six countries of Latin America (Brazil, Colombia, Ecuador, Guatemala, Mexico and Venezuela). In Colombia, this disease was discovered in 1965 in the Pacific Coast of the country. The National Onchocerciasis Elimination Program was established in 1993 with the aim of eliminating disease morbidity and infection transmission. In 2013, the World Health Organization (WHO) verified Colombia as free of onchocerciasis, becoming the first country in the world to reach such a goal. This report provides the empirical evidence of the elimination of Onchocerca volvulus transmission by Simulium exiguum (s.l.) after 12 years of 6-monthly mass drug administration of Mectizan® (ivermectin) to all the eligible residents living in this endemic area., Methods: From 1996 onwards, a biannual community-based mass ivermectin administration programme was implemented, complemented by health education and community participation. In-depth parasitological, serological and entomological surveys were conducted periodically between 1998 and 2007 to evaluate the impact of ivermectin treatment according to the 2001 WHO guidelines. When the interruption of parasite transmission was demonstrated, the drug distribution ceased and a three-year post-treatment surveillance (PTS) period (2008-2010) was initiated., Results: After 23 rounds of treatment, parasitological and ophthalmological assessments showed absence of microfilariae in skin and anterior chamber of the eyes. Serological tests proved lack of antibodies against O. volvulus in children under 10 years-old. A total of 10,500 S. exiguum flies tested by PCR had no L3 infection (infectivity rate = 0.0095%; 95% CI: 0.0029-0.049) during 2004, indicating interruption of parasite transmission. However, biannual ivermectin treatments continued until 2007 followed by a 3-year PTS period at the end of which 13,481 flies were analyzed and no infective flies were found (infectivity rate = 0%; 95% CI: 0.0-0.014)., Conclusions: These results fulfilled the WHO criteria for onchocerciasis elimination. Consequently, in 2013 Colombia was verified as free of onchocerciasis, demonstrating that elimination of this neglected tropical disease is an achievable goal and paving the way for an elimination agenda to be followed by other endemic countries in Latin America and Africa.

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.

Background: Onchocerciasis is a parasitic disease caused by the filarial nematode Onchocerca volvulus. In endemic areas, the diagnosis is commonly confirmed by microscopic examination of skin snip samples, though this technique is considered to have low sensitivity. The available melting-curve based quantitative real-time PCR (qPCR) using degenerated primers targeting the O-150 repeat of O. volvulus was considered insufficient for confirming the individual diagnosis, especially in elimination studies. This study aimed to improve detection of O. volvulus DNA in clinical samples through the development of a highly sensitive qPCR assay., Methods: A novel hydrolysis probe based qPCR assay was designed targeting the specific sequence of the O. volvulus O-5S rRNA gene. A total of 200 clinically suspected onchocerciasis cases were included from Goma district in South-west Ethiopia, from October 2012 through May 2013. Skin snip samples were collected and subjected to microscopy, O-150 qPCR, and the novel O-5S qPCR., Results: Among the 200 individuals, 133 patients tested positive (positivity rate of 66.5%) and 67 negative by O-5S qPCR, 74 tested positive by microscopy (37.0%) and 78 tested positive by O-150 qPCR (39.0%). Among the 133 O-5S qPCR positive individuals, microscopy and O-150 qPCR detected 55.6 and 59.4% patients, respectively, implying a higher sensitivity of O-5S qPCR than microscopy and O-150 qPCR. None of the 67 individuals who tested negative by O-5S qPCR tested positive by microscopy or O-150 qPCR, implying 100% specificity of the newly designed O-5S qPCR assay., Conclusions: The novel O-5S qPCR assay is more sensitive than both microscopic examination and the existing O-150 qPCR for the detection of O. volvulus from skin snip samples. The newly designed assay is an important step towards appropriate individual diagnosis and control of onchocerciasis.

Nodding Syndrome (NS) is a childhood neurological disorder characterized by atonic seizures, cognitive decline, school dropout, muscle weakness, thermal dysfunction, wasting and stunted growth. There are recent published information suggesting associations between Nodding Syndrome (NS) with cerebrospinal fluid (CSF) VGKC antibodies and serum leiomidin-1 antibody cross reacting with Onchocerca Volvulus ( OV ). These findings suggest a neuro-inflammatory cause of NS and they are important findings in the search for the cause of Nodding Syndrome. These observations perhaps provide further, the unique explanation for the association between Nodding Syndrome and Onchocerca Volvulus . Many clinical and epidemiological studies had shown a significant correlation between NS and infestation with a nematode, Onchocerca volvulus which causes a disease, Onchocerciasis , some of which when left untreated can develop visual defect ("River Blindness"). While these studies conducted in Northern Uganda and Southern Sudan indicate a statistically significant association with ( OV infection (using positive skin snips), we observe that ( OV is generally endemic in many parts of Sub Saharan Africa and Latin America and that to date, no NS cases have been recorded in those regions. This letter to the Editor is to provide additional information on the current view about the relationship between Nodding Syndrome and Onchocerca Volvulus as seen in Northern Uganda.

Background: After more than a decade of community-directed treatment with ivermectin (CDTI) in the West Region of Cameroon, epidemiological evaluation conducted in 2011 showed that onchocerciasis endemicity was still high in some communities. The conceptual framework for onchocerciasis elimination recommends in such case, to conduct additional phase 1A surveys at intervals of three to four years. Therefore, to assess the progress made towards the elimination of onchocerciasis in the West CDTI projects, we conducted a cross-sectional survey in May 2015 in 15 unevaluated communities where the highest baseline endemicity level were found in 1996. All volunteers living for at least five years in the community, aged five years or more, underwent clinical and parasitological examinations. Individual adherence to ivermectin treatment was also assessed. Analyses of data were weighted proportionally to age and gender distribution in the population., Results: The mean age was 28.4 ± 22.2 years and there were 55% of women among the 2058 individuals examined. The weighted prevalences were 5.5%, 2.1% and 1.7% for microfilaridermia, nodule and cutaneous signs, respectively. The weighted microfilaridermia prevalences varied from 4.0 in 5-9 years old to 11.6% in 40-49 years old. In the 30 children under 10 years examined in Makouopsap, the weighted prevalences were 49.9% for microfilaridermia and 13.3% for nodule. In surveyed communities, the weighted prevalences varied from 0 to 41.6% for microfilaridermia, with 11 (73.3%) communities having <5%. Except Makouopsap which had 41.6%, all the surveyed communities were below 15% for microfilaridermia prevalence. The community microfilarial load (CMFL) expressed in microfilariae/skin snip (mf/ss), also significantly dropped by 98-100%, from 3.75-33.16 mf/ss in 1996 to 0-0.94 mf/ss in 2015. The weighted therapeutic coverage in 2014 was 69.4% and the 5 years' adherence was only 39.3% among participants., Conclusions: After more than 15 years of CDTI, there is an important progress towards the elimination of onchocerciasis in the communities surveyed. Innovative strategy like semi-annual ivermectin treatment plus vector control or the adjunction of a vector control strategy to the current annual treatment should be implemented in the bordering districts of the Centre and West Regions, as well as in other parts of the country with persistent high prevalences in the sight of onchocerciasis elimination.

The search for new macrofilaricidal drugs against onchocerciasis that can be administered in shorter regimens than required for doxycycline (DOX, 200mg/d given for 4-6 weeks), identified minocycline (MIN) with superior efficacy to DOX. Further reduction in the treatment regimen may be achieved with co-administration with standard anti-filarial drugs. Therefore a randomized, open-label, pilot trial was carried out in an area in Ghana endemic for onchocerciasis, comprising 5 different regimens: the standard regimen DOX 200mg/d for 4 weeks (DOX 4w, N = 33), the experimental regimens MIN 200mg/d for 3 weeks (MIN 3w; N = 30), DOX 200mg/d for 3 weeks plus albendazole (ALB) 800mg/d for 3 days (DOX 3w + ALB 3d, N = 32), DOX 200mg/d for 3 weeks (DOX 3w, N = 31) and ALB 800mg for 3 days (ALB 3d, N = 30). Out of 158 randomized participants, 116 (74.4%) were present for the follow-up at 6 months of whom 99 participants (63.5%) followed the treatment per protocol and underwent surgery. Histological analysis of the adult worms in the extirpated nodules revealed absence of Wolbachia in 98.8% (DOX 4w), 81.4% (DOX 3w + ALB 3d), 72.7% (MIN 3w), 64.1% (DOX 3w) and 35.2% (ALB 3d) of the female worms. All 4 treatment regimens showed superiority to ALB 3d (p < 0.001, p < 0.001, p = 0.002, p = 0.008, respectively), which was confirmed by real-time PCR. Additionally, DOX 4w showed superiority to all other treatment arms. Furthermore DOX 4w and DOX 3w + ALB 3d showed a higher amount of female worms with degenerated embryogenesis compared to ALB 3d (p = 0.028, p = 0.042, respectively). These results confirm earlier studies that DOX 4w is sufficient for Wolbachia depletion and the desired parasitological effects. The data further suggest that there is an additive effect of ALB (3 days) on top of that of DOX alone, and that MIN shows a trend for stronger potency than DOX. These latter two results are preliminary and need confirmation in a fully randomized controlled phase 2 trial., Trial Registration: ClinicalTrials.gov #06010453., Competing Interests: The author has declared that no competing interests exist.

Background: Diagnostic procedures for the diagnosis of infection with the nematode parasite Onchocerca volvulus are currently based on the microscopic detection of microfilariae in skin biopsies. Alternative approaches based on amplification of parasitic DNA in these skin biopsies are currently being explored. Mostly this is based on the detection of the O-150 repeat sequence using PCR based techniques., Methods: An isothermal, loop-mediated amplification method has been designed using the mitochondrial O. volvulus cox1 gene as a target., Results: Analysis of dilution series of synthetic DNA containing the targeted sequence show a non-linear dose-response curve, as is usually the case for isothermal amplification methods. Evaluation of cross-reactivity with the heterologous sequence from the closely related parasites Wuchereria bancrofti, Loa loa and Brugia malayi demonstrated strong specificity, as none of these sequences was amplified. The assay however amplified both O. volvulus and O. ochengi DNA, but with a different melting point that can be used to discriminate between the species. Evaluation of this assay in a set of skin snip biopsies collected in an endemic area in Ghana showed a high correlation with O-150 qPCR and also demonstrated a similar sensitivity. Compared to qPCR, LAMP had a sensitivity of 88.2% and a specificity of 99.2%., Conclusions: We have developed a sensitive and specific loop-mediated amplification method for detection of O. volvulus DNA in skin biopsies that is capable of providing results within 30 min.

Mass treatment with ivermectin for onchocerciasis was stopped in 2012 in Abu Hamed, an isolated focus on the River Nile in northern Sudan. A 3-year posttreatment surveillance (PTS) ensued, at the end of which an evaluation was conducted in 2015 following the current World Health Organization guidelines for verification of onchocerciasis elimination. Vector black flies were collected from sentinel breeding sites and finger-prick bloodspots were collected from children ≤ 10 years of age resident in 35 communities within the focus. Polymerase chain reaction (PCR) screening of 19,191 flies from four sites for the O-150 parasite-specific marker found no flies carrying Onchocerca volvulus larvae (0%, 95% upper confidence limit [UCL] = 0.16), and serological testing of 5,266 children identified only one Ov16 seropositive child (0.019%, 95% UCL = 0.074); whose skin snips were negative when tested by O-150 PCR assay. These results indicate that for the first time in Africa, onchocerciasis elimination has been verified after a successful PTS in Abu Hamed., (© The American Society of Tropical Medicine and Hygiene.)

Background: Onchocerciasis or "river blindness" is a chronic parasitic disease caused by the filarial worm Onchocerca volvulus, transmitted through infected blackflies (Simulium spp.). Bioko Island (Equatorial Guinea) used to show a high endemicity for onchocerciasis. During the last years, the disease control programmes using different larvicides and ivermectin administration have considerably reduced the prevalence and intensity of infection. Based on this new epidemiological scenario, in the present work we aimed to assess the impact of the strategies applied against onchocerciasis in Bioko Island by an evaluation of IgG4 antibodies specific for recombinant Ov-16 in ELISA., Methods: A cross-sectional study was conducted in Bioko Island from mid-January to mid-February, 2014. Twenty communities were randomly selected from rural and urban settings. A total of 140 households were chosen. In every selected household, all individuals aged 5 years and above were recruited; 544 study participants agreed to be part of this work. No previous data on onchocerciasis seroprevalence in the selected communities were available. Blood samples were collected and used in an "ELISA in-house" prepared with recombinant Ov-16, expressed and further purified. IgG4 antibodies specific for recombinant Ov-16 were evaluated by ELISA in all of the participants., Results: Based on the Ov-16 ELISA, the onchocerciasis seroprevalence was 7.9 %, mainly concentrated in rural settings; samples from community Catedral Ela Nguema (# 16) were missed during the field work. Among the rural setups, communities Inasa Maule (# 7), Ruiché (# 20) and Barrios Adyacentes Riaba (# 14), had the highest seropositivity percentages (29.2, 26.9 and 23.8 %, respectively). With respect to the urban settings, we did not find any positive case in communities Manzana Casa Bola (# 3), Colas Sesgas (# 6), Getesa (# 8), Moka Bioko (# 9), Impecsa (# 10), Baney Zona Baja (# 12) and Santo Tomás de Aquino (# 1). No onchocerciasis seropositive samples were found in 10-year-old individuals or younger. The IgG4 positive titles increased in older participants., Conclusions: A significant decline in onchocerciasis prevalence was observed in Bioko Island after years of disease-vector control and CDTI strategy. The seroprevalence increased with age, mainly in rural settings that could be due to previous exposure of population to the filarial parasite, eliminated by the control programmes introduced against onchocerciasis. A new Ov-16 serological evaluation with a larger sample size of children below 10 years of age is required to demonstrate the interruption of transmission of O. volvulus in the human population of Bioko Island (Equatorial Guinea) according to the WHO criteria.

It was not until early 1990s that, when the Imaramagambo focus of southwest Uganda was mapped, mass treatment with a single annual dose of ivermectin for onchocerciaisis control commenced. However, comprehensive investigations on its transmission were launched after a nationwide policy for onchocerciasis elimination in 2007. Entomological surveys throughout the focus from 2007 to 2015 have yielded few or no freshwater crabs (Potamonautes aloysiisabaudiae), which serve as the obligate phoretic host of the larvae and pupae of the vector Simulium neavei No S. neavei flies have been observed or collected since 2007. Skin snips (microscopy) from 294 individuals in 2008 were negative for skin microfilariae, and of the 462 persons analyzed by polymerase chain reaction skin snip poolscreen in 2009, only five (1.08%) persons were indicated as infected with onchocerciasis. All five of the positive persons were at least 40 years old. Serosurvey results showed negative exposure among 3,332 children in 2012 and 3,108 children in 2015. Both were within the upper bound of the 95% confidence interval of the prevalence estimate of 0.06%, which confirmed the elimination of onchocerciasis. Treatment coverage in Imaramagambo was generally poor, and transmission interruption of onchocerciasis could not be attributed solely to annual mass treatment with ivermectin. There was sufficient evidence to believe that the possible disappearance of the S. neavei flies, presumed to have been the main vector, may have hastened the demise of onchocerciasis in this focus., (© The American Society of Tropical Medicine and Hygiene.)

Background: The reason for the high prevalence of epilepsy in onchocerciasis endemic areas remains unknown. The aim of this study was to detect risk factors associated with epilepsy in a region endemic for onchocerciasis., Methods: In June 2014, a case-control study was performed in Titule, Bas-Uélé Province in the Democratic Republic of the Congo. Individuals with unprovoked convulsive epilepsy of unknown aetiology were enrolled as cases (n=59). Healthy members of families without cases of epilepsy in the same village were recruited as controls (n=61). A multivariate binomial logistic regression analysis was performed to identify potential risk factors associated with epilepsy. To evaluate the potential protective effect of ivermectin treatment on the development of epilepsy, a nested age-matched case-control study was performed including only those who were eligible for ivermectin treatment in the year before they developed epilepsy., Results: Suspected onchocerciasis skin lesions were more often present in cases than in controls: 12/41 (29%) vs. 1/56 (2%), respectively (odds ratio (OR) 20.26, 95% confidence interval (CI) 2.42-170; p<0.01). Ivermectin had been taken 7 months earlier in 29/59 (49%) cases and 29/61 (48%) controls. Onchocerca volvulus (OV) DNA was detected by PCR in skin snips in 26/34 cases (76%) and 10/14 controls (71%) (p=0.7), and there was presence of OV IgG4 antibodies in 35/48 (73%) cases and 15/18 (83%) controls (p=0.5). OV DNA was not detected in the cerebrospinal fluid of cases (controls not tested). Both cases and controls reported frequent bites by blackflies (Diptera, Simuliidae). Bathing daily as opposed to less often (OR 16.7, 95% CI 2.2-125.8; p<0.01), bathing between 11 a.m. and 4 p.m. (OR 12.7, 95% CI 1.6-103.7; p=0.02), and washing clothes between 11 a.m. and 4 p.m. (OR 10.9, 95% CI 1.5-77.3; p=0.02) were all independently associated with epilepsy. Blood screening by specific PCR tests for Toxoplasma and Wuchereria bancrofti was negative in all cases and controls. A Loa loa infestation was found in only one case and one control by PCR and Giemsa smear. Antibodies to Taenia solium, Toxocara, and Trypanosoma sp were not detected in any of the participants. In an age-matched case-control analysis, 16/18 (89%) cases had not taken ivermectin the year before they developed epilepsy, compared to 7/18 (39%) controls that same year (p=0.002)., Conclusions: These data suggest that frequent activities at rivers known to be blackfly breeding sites and a historical lack of ivermectin treatment were risk factors for epilepsy in this onchocerciasis endemic area., (Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Onchocerciasis or "river blindness" is a chronic parasitic neglected tropical disease which is endemic both in mainland and insular Equatorial Guinea. We aim to estimate the current epidemiological situation of onchocerciasis in Bioko Island after vector elimination in 2005 and more than sixteen years of Community Directed Treatment with Ivermectin (CDTI) by using molecular and serological approaches for onchocerciasis diagnosis. A community-based cross-sectional study was carried out in Bioko Island from mid-January to mid-February 2014. A total of 544 study participants were recruited. A complete dermatological examination was performed and three skin snips were performed in every participant for parasitological and molecular assessments. Blood spots were also taken for determination of Ov16 IgG4 antibodies trough an "in-house" ELISA assay. Overall, we found 15 out of 522 individuals suffering any onchocerciasis specific cutaneous lesions and 16 out of 528 (3.0%) with onchocercal nodules in the skin. Nodules were significantly associated with age, being more common in subjects older than 10 years than in younger people (3.9% vs. 0%, p = 0.029). Regarding the onchocerciasis laboratory assessment, no positive parasitological test for microfilaria detection was found in the skin snips. The calculated seroprevalence through IgG4 serology was 7.9%. No children less than 10 years old were found to be positive for this test. Only one case was positive for Onchocerca volvulus (O. volvulus) after skin PCR. The present study points out that the on-going mass ivermectin treatment has been effective in reducing the prevalence of onchocerciasis and corroborates the interruption of transmission in Bioko Island. To our knowledge, this is the first time that accurate information through molecular and serological techniques is generated to estimate the onchocerciasis prevalence in this zone. Sustained support from the national program and appropriate communication and health education strategies to reinforce participation in CDTI activities are essential to ensure progress towards onchocerciasis elimination in the country.

Background: The African Programme for Onchocerciasis Control has proposed provisional thresholds for the prevalence of microfilariae in humans and of L3 larvae in blackflies, below which mass drug administration (MDA) with ivermectin can be stopped and surveillance started. Skin snips are currently the gold standard test for detecting patent Onchocerca volvulus infection, and the World Health Organization recommends their use to monitor progress of treatment programmes (but not to verify elimination). However, if they are used (in transition and in parallel to Ov-16 serology), sampling protocols should be designed to demonstrate that programmatic goals have been reached. The sensitivity of skin snips is key to the design of such protocols., Methods: We develop a mathematical model for the number of microfilariae in a skin snip and parameterise it using data from Guatemala, Venezuela, Ghana and Cameroon collected before the start of ivermectin treatment programmes. We use the model to estimate sensitivity as a function of time since last treatment, number of snips taken, microfilarial aggregation and female worm fertility after exposure to 10 annual rounds of ivermectin treatment., Results: The sensitivity of the skin snip method increases with time after treatment, with most of the increase occurring between 0 and 5 years. One year after the last treatment, the sensitivity of two skin snips taken from an individual infected with a single fertile female worm is 31 % if there is no permanent effect of multiple ivermectin treatments on fertility; 18 % if there is a 7 % reduction per treatment, and 0.6 % if there is a 35 % reduction. At 5 years, the corresponding sensitivities are 76 %, 62 % and 4.7 %. The sensitivity improves significantly if 4 skin snips are taken: in the absence of a permanent effect of ivermectin, the sensitivity of 4 skin snips is 53 % 1 year and 94 % 5 years after the last treatment., Conclusions: Our model supports the timelines proposed by APOC for post-MDA follow-up and surveillance surveys every 3-5 years. Two skin snips from the iliac region have reasonable sensitivity to detect residual infection, but the sensitivity can be significantly improved by taking 4 snips. The costs and benefits of using four versus two snips should be evaluated.

Background: Onchocerciasis, also known as river blindness is one of the neglected tropical diseases affecting millions of people, mainly in sub-Saharan Africa and is caused by the filarial nematode Onchocerca volvulus. Efforts to eliminate this disease are ongoing and are based on mass drug administration programs with the microfilaricide ivermectin. In order to monitor the efficacy of these programs, there is an unmet need for diagnostic tools capable of identifying infected patients. We have investigated the diagnostic potential of urinary N-acetyltyramine-O,β-glucuronide (NATOG), which is a promising O. volvulus specific biomarker previously identified by urine metabolome analysis., Methods: A liquid chromatography tandem mass spectrometry (LC-MS/MS) method was used to assess the stability characteristics of NATOG and to evaluate the levels of NATOG in study samples. An LC-fluorescence method was also developed., Results: Stability characteristics of NATOG were investigated and shown to be ideally suited for use in tropical settings. Also, an easy and more accessible method based on liquid chromatography coupled to fluorescence detection was developed and shown to have the necessary sensitivity (limit of quantification 1 μM). Furthermore, we have evaluated the levels of NATOG in a population of 98 nodule-positive individuals from Ghana with no or low levels of microfilaria in the skin and compared them with the levels observed in different control groups (endemic controls (n = 50), non-endemic controls (n = 18) and lymphatic filariasis (n = 51). Only a few (5 %) of nodule-positive individuals showed an increased level (> 10 μM) of NATOG and there was no statistical difference between the nodule-positive individuals and the control groups (P > 0.05)., Conclusions: Results of the present study indicate the limited potential of NATOG as a diagnostic biomarker for O. volvulus infection in amicrofilaridermic individuals.

Background: Onchocerciasis control for years has been based on mass drug administration (MDA) with ivermectin (IVM). Adherence to IVM repeated treatment has recently been shown to be a confounding factor for onchocerciasis elimination precisely in rain forest areas where transmission continues and Loa loa co-exists with Onchocerca volvulus. In this study, participants' oral declarations were used as proxy to determine the relationship between adherence to IVM treatment and parasitological indicators of onchocerciasis in the rain forest area of Cameroon with more than a decade of MDA., Methods: Participants were recruited based on their IVM intake profile with the aid of a semi-structured questionnaire. Parasitological examinations (skin sniping and nodule palpation) were done on eligible candidates. Parasitological indicators were calculated and correlated to IVM intake profile., Results: Of 2,364 people examined, 15.5% had never taken IVM. The majority (40.4%) had taken the drug 1-3 times while only 18% had taken ≥ 7 times. Mf and nodule prevalence rates were still high at 47%, 95% CI [44.9-49.0%] and 36.4%, 95% CI [34.4-38.3%] respectively. There was a treatment-dependent reduction in microfilaria prevalence (rs =-0.986, P = 0.01) and intensity (rs =-0.96, P = 0.01). The highest mf prevalence (59.7%) was found in the zero treatment group and the lowest (33.9%) in the ≥ 7 times treatment group (OR = 2.8; 95% CI [2.09-3.74]; P < 0.001). Adults with ≥ 7 times IVM intake were 2.99 times more likely to have individuals with no microfilaria compared to the zero treatment group (OR = 2.99; 95% CI [2.19-4.08], P < 0.0001). There was no clear correlation between treatment and nodule prevalence and intensity., Conclusion: Adherence to ivermectin treatment is not adequate in this rain forest area where L. loa co-exists with O. volvulus. The prevalence and intensity of onchocerciasis remained high in individuals with zero IVM intake after more than a decade of MDA. Our findings show that using parasitological indicators, reduction in prevalence is IVM intake-dependent and that participants' oral declaration of treatment adherence could be relied upon for impact studies. The findings are discussed in the context of challenges for the elimination of onchocerciasis in this rain forest area.

Onchocerciasis is a severe parasitic infestation caused by Onchocerca volvulus which causes disabling skin and subcutaneous tissue changes and ultimately leads to blindness. It has a huge public health impact due to its socioeconomic burden and the vast number of people it affects in developing countries. In this case, a 60 years old woman was encountered with leopard skin like changes, rashes and pruritus on the left leg; which had been managed as cutaneous mycosis for over a period of 8 years. A diagnosis of onchocerciasis was finally made after a skin snip identified onchocercal microfilariae. The above case shows that onchocerciasis is still a neglected tropical disease (NTD) in Cameroon. This emphasizes the need for more expansive outreach programs in remote areas in Cameroon, a change in health policies to ensure the eradication of this disabling disease and health promotion amongst vulnerable populations.

Elimination programs for Wuchereria bancrofti and Onchocerca volvulus are in critical need of sensitive, specific, and point-of-contact (POC) tools that can be used for surveillance years beyond cessation of mass drug administration when infection intensities are low. Previously, Wb123 and Ov16 were identified individually as potential filarial antigens for an antibody-based POC test. The present study compares single-antigen Wb123- and Ov16-based POC tests with an integrated configuration to detect antibodies to Wb123 and Ov16 simultaneously. Wb123 and Ov16 isolates were striped onto lateral flow strips containing anti-IgG4. Sera from W. bancrofti-, O. volvulus-, and other helminth-infected or -uninfected individuals were added to the strips with buffer. Strips were read for the appearance of a positive or negative test line for both antigens at 20 min and following drying. Sensitivity, specificity, and predictive values were calculated for the single-antigen and biplex strips. Single and biplex lateral flow strips showed nearly identical results, with >90% sensitivity for Ov16 and >92% sensitivity for Wb123. Overall specificities for the single and biplex tests were 98% and 96% for Ov16 and Wb123, respectively. Biplex tests performed as well as the single-antigen tests regardless of the intensity of patient IgG4 response. The high sensitivity and specificity make these new biplex tests extremely useful for POC long-term surveillance following mass drug administration in Africa that should reduce time and cost in areas where bancroftian filariasis and onchocerciasis are coendemic., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Background: Despite being certified guinea worm free in 2007, Cameroon continues surveillance efforts to ensure rapid verification of any suspected reoccurrence. This includes the investigation of every rumor and confirmation of each suspicious expulsed worm. This paper presents fieldwork carried out to investigate a guinea worm rumor in Cameroon which turned out to be an Onchocerca volvulus mimicking Dracunculus medinensis., Methods: The investigation included a field visit to the subsistence farming community where the rumor was reported. During the visit, interviews were conducted with health staff who managed the case and the elderly farmer from whom the worm was retrieved. An investigation of any potential missed guinea worm cases was also conducted through interviews with community residents and reviews of the health facility's medical records. This was combined with laboratory analyses of water samples from the community's water sources and the retrieved worm which was removed from the patient via wrapping it around a stick., Results: Microscopy and molecular analyses of the retrieved worm revealed a female Onchocerca volvulus whose expulsion strongly mimicked guinea worm. In addition to presenting findings of our investigation, this paper discusses distinguishing elements between the two parasites and gives an overview of guinea worm eradication efforts in Cameroon as well as current challenges to the worm's eradication globally., Conclusions: The investigation findings suggest the evolving Onchocerca volvulus worm tropisms' adaptive survival behavior worth further investigation. Strategies used to successfully control guinea worm in Cameroon could be adapted for Onchocerca volvulus control.

Background: Mexico is one of the six countries formerly endemic for onchocerciasis in Latin America. Transmission has been interrupted in the three endemic foci of that country and mass drug distribution has ceased. Three years after mass drug distribution ended, post-treatment surveillance (PTS) surveys were undertaken which employed entomological indicators to check for transmission recrudescence., Methodology/principal Findings: In-depth entomologic assessments were performed in 18 communities in the three endemic foci of Mexico. None of the 108,212 Simulium ochraceum s.l. collected from the three foci were found to contain parasite DNA when tested by polymerase chain reaction-enzyme-linked immunosorbent assay (PCR-ELISA), resulting in a maximum upper bound of the 95% confidence interval (95%-ULCI) of the infective rate in the vectors of 0.035/2,000 flies examined. This is an order of magnitude below the threshold of a 95%-ULCI of less than one infective fly per 2,000 flies tested, the current entomological criterion for interruption of transmission developed by the international community. The point estimate of seasonal transmission potential (STP) was zero, and the upper bound of the 95% confidence interval for the STP ranged from 1.2 to 1.7 L3/person/season in the different foci. This value is below all previous estimates for the minimum transmission potential required to maintain the parasite population., Conclusions/significance: The results from the in-depth entomological post treatment surveillance surveys strongly suggest that transmission has not resumed in the three foci of Mexico during the three years since the last distribution of ivermectin occurred; it was concluded that transmission remains undetectable without intervention, and Onchocerca volvulus has been eliminated from Mexico.

Background: 37 million individuals are currently infected with Onchocerca volvulus (O. volvulus), a parasitic nematode that elicits various dermal manifestations and eye damage in man. Disease control is primarily based on distributing ivermectin in mass drug administration (MDA) programmes which aim at breaking transmission by eliminating microfilariae (MF), the worm's offspring. The majority of infected individuals present generalized onchocerciasis, which is characterized by hyporesponsive immune responses and high parasite burden including MF. Recently, in areas that have been part of MDA programmes, individuals have been identified that present nodules but are amicrofilaridermic (a-MF) and our previous study showed that this group has a distinct immune profile. Expanding on those findings we determined the immune responses of O. volvulus-infected individuals to a Plasmodium-derived antigen MSP-1 (merozoite surface protein-1), which is required by the parasite to enter erythrocytes., Methods: Isolated PBMCs from O. volvulus-infected individuals (164 MF(+) and 46 a-MF) and non-infected volunteers from the same region (NEN), were stimulated with MSP-1 and the resulting supernatant screened for the presence of IL-5, IL-13, IFN-γ, TNF-α, IL-6, IL-17A and IL-10. These findings were then further analyzed following regression analysis using the covariates MF, ivermectin (IVM) and region. The latter referred to the Central or Ashanti regions of Ghana, which, at the time sampling, had received 8 or 1 round of MDA respectively., Results: IL-5, IL-13 and IFN-γ responses to MSP-1 were not altered between NEN and O. volvulus-infected individuals nor were any associations revealed in the regression analysis. IL-10, IL-6 and TNF-α MSP-1 responses were, however, significantly elevated in cultures from infected individuals. Interestingly, when compared to a-MF individuals, MSP-induced IL-17A responses were significantly higher in MF(+) patients. Following multivariable regression analysis these IL-10, IL-6, TNF-α and IL-17A responses were all dominantly associated with the regional covariate., Conclusions: Consequently, areas with a lowered infection pressure due to IVM MDA appear to influence bystander responses to Plasmodium-derived antigens in community members even if they have not regularly participated in the therapy.

Background: Loiasis is currently receiving attention as a disease of public health importance because of the possibility of increased risk of developing neurologic serious adverse event following mass ivermectin treatment against onchocerciasis in individual co-infected with Onchocerca volvulus and Loa loa., Methodology/principal Findings: Rapid assessment procedure for loiasis (RAPLOA) was conducted in 12 communities covering the 3 senatorial districts of Osun State, Nigeria. A total of 960 people were interviewed for history of eye worm using the WHO guidelines for rapid assessment. The survey confirmed the presence of loiasis in all the 12 communities with 4 in Osun East/Ife south senatorial district being at high risk with a prevalence of over 40%. Based on the RAPLOA results, communities within Osun East/Ife south senatorial district were selected for microfilaraemic assessment of L. loa and O. volvulus. A total of 1115 and 1091 individuals were screened for L. loa and O. volvulus microfilaria worms respectively. 160 (14.3%) had L. loa microfilaria detected in their blood with 8 (5.0%) individuals having L. loa loads above 8000 mf/ml. 166 (15.2%) subjects had O. volvulus microfilaria (range 4-504 mf/ml) detected in their skin snip. 30 (2.69%) subjects were co-infected with both L. loa and O. volvulus. There was a significant variation in the prevalence (2.1% to 33.3%) of onchocerciasis in the communities studied (p = 0.001). Five (41.7%) of the studied communities had a prevalence that is equal to or greater than 20%., Conclusions/significance: Low prevalence of onchocerciasis and loiasis co-infection in this study suggests that loiasis may not pose a serious epidemiological threat to the continuous distribution and sustainability of ivermectin for the treatment of onchocerciasis. Evaluation of the interruption of onchocerciasis transmissions in this region using all the indicators set forth by WHO is therefore suggested.

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.

Background: Onchocerciasis is caused by Onchocerca volvulus and transmitted by Simulium species (black flies). In the Americas, the infection has been previously described in 13 discrete regional foci distributed among six countries (Brazil, Colombia, Ecuador, Guatemala, Mexico and Venezuela) where more than 370,000 people are currently considered at risk. Since 2001, disease control in Venezuela has relied on the mass drug administration to the at-risk communities. This report provides empirical evidence of interruption of Onchocerca volvulus transmission by Simulium metallicum in 510 endemic communities from two Northern foci of Venezuela, after 10-12 years of 6-monthly Mectizan (ivermectin) treatment to all the eligible residents., Methods: In-depth entomologic and epidemiologic surveys were serially conducted from 2001-2012 in selected (sentinel and extra-sentinel) communities from the North-central (NC) and North-east (NE) onchocerciasis foci of Venezuela in order to monitor the impact of ivermectin treatment., Results: From 2007-2009, entomological indicators in both foci confirmed that 0 out of 112,637 S. metallicum females examined by PCR contained L3 infection in insect heads. The upper bound of the 95% confidence intervals of the infective rate of the vector reached values below 1% by 2009 (NC) and 2012 (NE). Additionally, after 14 (NC) and 22 (NE) rounds of treatment, the seasonal transmission potential (±UL CIs) of S. metallicum was under the critical threshold of 20 L3 per person per season. Serological analysis in school children < 15 years-old demonstrated that 0 out of 6,590 individuals were harboring antibodies to Ov-16. Finally, epidemiological surveys made during 2010 (NC) and 2012 (NE) showed no evidence of microfilariae in the skin and eyes of the population., Conclusions: These results meet the WHO criteria for absence of parasite transmission and disease morbidity in these endemic areas which represent 91% of the population previously at-risk in the country. Consequently, the two Northern foci are currently under post-treatment onchocerciasis surveillance status in Venezuela.

Onchocerciasis is a neglected tropical disease caused by infection with the parasite Onchocerca volvulus (Ov). An estimated 180 million people are at risk for Ov infection, and 37 million people are infected, mostly in Africa. A lateral flow-based assay to detect human IgG4 antibodies to the Ov-specific antigen Ov-16 was developed as a rapid tool to detect exposure to Ov. The test, when performed on 449 sera specimens from patients with microfiladermia and Ov-negative patients, has a sensitivity of 89.1% (95% confidence interval: 86.2%-92.0%), and specificity of 97% (95% confidence interval: 95.4%-98.6%). Because the intended use of the test is for surveillance, it is highly desirable to have a stable, long-lasting result. An extended read window is thus desirable for a high-volume, busy workflow and facilitates post-surveillance quality assurance. The main restriction on achieving an extended read window for this assay was the erythrocyte lysis that can alter the signal-to-noise ratio, especially in those with low IgG4 levels (weak positives). We describe a test housing that incorporates a user-independent feature driven by assay fluid and an expanding wick that detaches the blood separation membrane from the nitrocellulose used in the assay, but before hemolysis occurs. We demonstrated material functionality at extreme operational conditions (37°C, 80% relative humidity) and a read window of a minimum of 70 days. The fluid-driven assay device performs equally as well with whole blood as with plasma, as demonstrated with 100 spiked clinical specimens (with a correlation coefficient of 0.96). We show a novel, inexpensive, and simple approach to actuating the detachment of the blood separation membrane from the nitrocellulose test with no impact on the performance characteristics of the test.

Background: Onchocerciasis has been implicated in the pathogenesis of epilepsy. The debate on a potential causal relationship between Onchocerca volvulus and epilepsy has taken a new direction in the light of the most recent epidemic of nodding syndrome., Objective: To document MRI changes in people with different types of epilepsy and investigate whether there is an association with O. volvulus infection., Methods: In a prospective study in southern Tanzania, an area endemic for O. volvulus with a high prevalence of epilepsy and nodding syndrome, we performed MRI on 32 people with epilepsy, 12 of which suffered from nodding syndrome. Polymerase chain reaction (PCR) of O. volvulus was performed in skin and CSF., Results: The most frequent abnormalities seen on MRI was atrophy (twelve patients (37.5%)) followed by intraparenchymal pathologies such as changes in the hippocampus (nine patients (28.1%)), gliotic lesions (six patients (18.8%)) and subcortical signal abnormalities (three patients (9.4%)). There was an overall trend towards an association of intraparenchymal cerebral pathologies and infection with O. volvulus based on skin PCR (Fisher's Exact Test p=0.067) which was most pronounced in children and adolescents with nodding syndrome compared to those with other types of epilepsy (Fisher's Exact Test, p=0.083). Contrary to skin PCR results, PCR of CSF was negative in all patients., Conclusion: The observed trend towards an association of intraparenchymal cerebral pathological results on MRI and a positive skin PCR for O. volvulus despite negative PCR of CSF is intriguing and deserves further attention.

Background: The Southern Chiapas focus of onchocerciasis in Southern Mexico represents one of the major onchocerciasis foci in Latin America. All 559 endemic communities of this focus have undergone semi-annual mass treatment with ivermectin since 1998. In 50 communities of this focus, ivermectin frequency shifted from twice to four times a year in 2003; an additional 113 communities were added to the quarterly treatment regimen in 2009 to achieve a rapid suppression of transmission., Methodology/principal Findings: In-depth epidemiologic and entomologic assessments were performed in six sentinel communities (which had undergone 2 rounds of ivermectin treatment per year) and three extra-sentinel communities (which had undergone 4 rounds of ivermectin treatment per year). None of the 67,924 Simulium ochraceum s.l. collected from this focus during the dry season of 2011 were found to contain parasite DNA when tested by polymerase chain reaction-enzyme-linked immunosorbent assay (PCR-ELISA), resulting in an upper bound of the 95% confidence interval (95%-ULCI) of the infective rate in the vectors of 0.06/2,000 flies examined. Serological assays testing for Onchocerca volvulus exposure conducted on 4,230 children 5 years of age and under (of a total population of 10,280 in this age group) revealed that 2/4,230 individuals were exposed to O. volvulus (0.05%; one sided 95% confidence interval = 0.08%)., Conclusions/significance: The in-depth epidemiological and entomological findings from the Southern Chiapas focus meet the criteria for interruption of transmission developed by the international community.

Background/objective: Ivermectin has been the keystone of onchocerciasis control for the last 25 years. Sub-optimal responses to the drug have been reported in Ghanaian communities under long-term treatment. We assessed, in two Cameroonian foci, whether the microfilaricidal and/or embryostatic effects of ivermectin on Onchocerca volvulus have been altered after several years of drug pressure., Methods: We compared the dynamics of O. volvulus skin microfilarial densities after ivermectin treatment in two cohorts with contrasting exposure to this drug: one received repeated treatment for 13 years whereas the other had no history of large-scale treatments (referred to as controls). Microfilarial densities were assessed 15, 80 and 180 days after ivermectin in 122 multiply treated and 127 ivermectin-naïve individuals. Comparisons were adjusted for individual factors related to microfilarial density: age and number of nodules., Findings: Two weeks post ivermectin, microfilarial density dropped equally (98% reduction) in the ivermectin-naïve and multiply treated groups. Between 15 and 180 days post ivermectin, the proportion of individuals with skin microfilariae doubled (from 30.8% to 67.8%) in controls and quadrupled (from 19.8% to 76.9%) in multiply treated individuals but the mean densities remained low in both sites. In fact, between 15 and 80 days, the repopulation rate was significantly higher in the multiply treated individuals than in the controls but no such difference was demonstrated when extending the follow-up to 180 days. The repopulation rate by microfilariae was associated with host factors: negatively with age and positively with the number of nodules., Conclusion: These observations may indicate that the worms from the multi-treated area recover mf productivity earlier but would be less productive than the worms from the ivermectin-naïve area between 80 and 180 days after ivermectin. Moreover, they do not support the operation of a strong cumulative effect of repeated treatments on the fecundity of female worms as previously described.

Background: Nodding syndrome (repetitive nodding and progressive generalized seizures) is assuming epidemic proportions in South Sudan, Tanzania and Uganda., Objective: To describe clinical and epidemiological features of nodding syndrome in southern Sudan based on preliminary investigations conducted in 2001 and 2002., Method: Household surveys, clinical, electrophysiological (EEG) assessments, informant interviews and case-control studies were conducted in the town of Lui and the village of Amadi in southern Sudan., Results: Nodding syndrome is characterized by involuntary repetitive nodding of the head, progressing to generalized seizures; mental and physical deterioration. The EEGs were consistent with progressive epileptic encephalopathy. Prevalence of Nodding syndrome in Lui and Amadi was 2.3% and 6.7% respectively. All case control studies showed a positive association between cases and Onchocerca volvulus. A history of measles was negatively associated with being a case: 2/13 of cases and 11/19 of controls had had measles: odds ratio 0.13 (95% CI 0.02, 0.76). Environmental assessment did not reveal any naturally occurring or manmade neurotoxic factors to explain Nodding Syndrome, although fungal contamination of food could not be ruled out., Conclusion: Nodding Syndrome was strongly associated with Onchocerca volvulus. There was no evidence to suggest an environmental pollutant, chemical agent, or other toxic factor.

Repeated ivermectin treatment will clear microfilaria (Mf) of Onchocerca volvulus from skin and eyes of onchocerciasis patients while adult filaria remains alive and reproductive, and such occult O. volvulus infection may persist for years. To investigate the effect of residual adult filaria on the immune response profile, chemokines and cytokines were quantified 1) in onchocerciasis patients who developed an occult O. volvulus infection (Mf-negative) due to repeated ivermectin treatments, 2) patients who became Mf-negative without ivermectin treatments due to missing re-infection, and 3) endemic and non-endemic O. volvulus Mf-negative controls. With occult O. volvulus infection, serum levels of pro-inflammatory chemokines MCP-1/CCL2, MIP-1α/CCL3, MIP-1β/CCL4, MPIF-1/CCL23 and CXCL8/IL-8 enhanced and approached higher concentrations as determined in infection-free controls, whilst regulatory and Th2-type cytokines and chemokines MCP-4/CCL13, MIP-1δ/CCL15, TARC/CCL17 and IL-13 lessened. Levels of Eotaxin-2/CCL24, MCP-3/CCL7 and BCA-1/CXCL13 remained unchanged. At 3 days post-initial ivermectin treatment, MCP-1/CCL2, MCP-4/CCL13, MPIF-1/CCL23 and Eotaxin-2/CCL24 were strongly enhanced, suggesting that monocytes and eosinophil granulocytes have mediated Mf clearance. In summary, with occult and expiring O. volvulus infections the serum levels of inflammatory chemokines enhanced over time while regulatory and Th2-type-promoting cytokines and chemokines lessened; these changes may reflect a decreasing effector cell activation against Mf of O. volvulus, and in parallel, an enhancing inflammatory immune responsiveness., (Copyright © 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

In November 2010, the Ministry of Health of the proposed nation of South Sudan requested CDC assistance in investigating a recent increase and geographic clustering of an illness resulting in head nodding and seizures. The outbreak was suspected to be nodding syndrome, an unexplained neurologic condition characterized by episodes of repetitive dropping forward of the head, often accompanied by other seizure-like activity, such as convulsions or staring spells. The condition predominantly affects children aged 5-15 years and has been reported in South Sudan from the states of Western and Central Equatoria and in Northern Uganda and southern Tanzania. Because of visa and security concerns, CDC investigators did not travel to South Sudan until May 2011. On arrival, a case-control study was conducted that included collecting exposure information and biologic specimens to assess the association of nodding syndrome with suspected risk factors. A total of 38 matched case-control pairs were enrolled from two different communities: Maridi and Witto. Overall, current infection with Onchocerca volvulus diagnosed by skin snip was more prevalent among the 38 case-patients (76.3%) than the controls (47.4%) (matched odds ratio [mOR] = 3.2). This difference was driven by the 25 pairs in Maridi (88.0% among case-patients, 44.0% among controls, mOR=9.3); among the 13 pairs in Witto, no significant association with onchocerciasis (known as river blindness) was observed. Although onchocerciasis was more prevalent among case-patients, whether infection preceded or followed nodding syndrome onset was unknown. Priorities for nodding syndrome investigations include improving surveillance to monitor the number of cases and their geographic distribution and continued work to determine the etiology of the syndrome.

Background: Entomological surveys of Simulium vectors are an important component in the criteria used to determine if Onchocerca volvulus transmission has been interrupted and if focal elimination of the parasite has been achieved. However, because infection in the vector population is quite rare in areas where control has succeeded, large numbers of flies need to be examined to certify transmission interruption. Currently, this is accomplished through PCR pool screening of large numbers of flies. The efficiency of this process is limited by the size of the pools that may be screened, which is in turn determined by the constraints imposed by the biochemistry of the assay. The current method of DNA purification from pools of vector black flies relies upon silica adsorption. This method can be applied to screen pools containing a maximum of 50 individuals (from the Latin American vectors) or 100 individuals (from the African vectors)., Methodology/principal Findings: We have evaluated an alternative method of DNA purification for pool screening of black flies which relies upon oligonucleotide capture of Onchocerca volvulus genomic DNA from homogenates prepared from pools of Latin American and African vectors. The oligonucleotide capture assay was shown to reliably detect one O. volvulus infective larva in pools containing 200 African or Latin American flies, representing a two-four fold improvement over the conventional assay. The capture assay requires an equivalent amount of technical time to conduct as the conventional assay, resulting in a two-four fold reduction in labor costs per insect assayed and reduces reagent costs to $3.81 per pool of 200 flies, or less than $0.02 per insect assayed., Conclusions/significance: The oligonucleotide capture assay represents a substantial improvement in the procedure used to detect parasite prevalence in the vector population, a major metric employed in the process of certifying the elimination of onchocerciasis.

Background: The parasite Onchocerca volvulus has, until recently, been regarded as the cause of a chronic yet non-fatal condition. Recent analyses, however, have indicated that in addition to blindness, the parasite can also be directly associated with human mortality. Such analyses also suggested that the relationship between microfilarial load and excess mortality might be non-linear. Determining the functional form of such relationship would contribute to quantify the population impact of mass microfilaricidal treatment., Methodology/principal Findings: Data from the Onchocerciasis Control Programme in West Africa (OCP) collected from 1974 through 2001 were used to determine functional relationships between microfilarial load and excess mortality of the human host. The goodness-of-fit of three candidate functional forms (a (log-) linear model and two saturating functions) were explored and a saturating (log-) sigmoid function was deemed to be statistically the best fit. The excess mortality associated with microfilarial load was also found to be greater in younger hosts. The attributable mortality risk due to onchocerciasis was estimated to be 5.9%., Conclusions/significance: Incorporation of this non-linear functional relationship between microfilarial load and excess mortality into mathematical models for the transmission and control of onchocerciasis will have important implications for our understanding of the population biology of O. volvulus, its impact on human populations, the global burden of disease due to onchocerciasis, and the projected benefits of control programmes in both human and economic terms.

Background: Mass treatment with ivermectin controls onchocerciasis as a public health problem, but it was not known if it could also interrupt transmission and eliminate the parasite in endemic foci in Africa where vectors are highly efficient. A longitudinal study was undertaken in three hyperendemic foci in Mali and Senegal with 15 to 17 years of annual or six-monthly ivermectin treatment in order to assess residual levels of infection and transmission, and test whether treatment could be safely stopped. This article reports the results of the final evaluations up to 5 years after the last treatment., Methodology/principal Findings: Skin snip surveys were undertaken in 131 villages where 29,753 people were examined and 492,600 blackflies were analyzed for the presence of Onchocerca volvulus larva using a specific DNA probe. There was a declining trend in infection and transmission levels after the last treatment. In two sites the prevalence of microfilaria and vector infectivity rate were zero 3 to 4 years after the last treatment. In the third site, where infection levels were comparatively high before stopping treatment, there was also a consistent decline in infection and transmission to very low levels 3 to 5 years after stopping treatment. All infection and transmission indicators were below postulated thresholds for elimination., Conclusion/significance: The study has established the proof of principle that onchocerciasis elimination with ivermectin treatment is feasible in at least some endemic foci in Africa. The study results have been instrumental for the current evolution from onchocerciasis control to elimination in Africa.