Procedure for Handling and Storage of Onchocerca volvulus Microfilariae Obtained from Skin Snips for Downstream Genetic Work.

WHO and endemic countries target elimination of transmission of Onchocerca volvulus, the parasite causing onchocerciasis. Population genetic analysis of O. volvulus may provide data to improve the evidence base for decisions on when, where, and for how long to deploy which interventions and post-intervention surveillance to achieve elimination. Development of necessary methods and tools requires parasites suitable for genetic analysis. Based on our experience with microfilariae obtained from different collaborators, we developed a microfilariae transfer procedure for large-scale studies in the Democratic Republic of Congo (DRC) comparing safety and efficacy of ivermectin, the mainstay of current onchocerciasis elimination strategies, and moxidectin, a new drug. This procedure is designed to increase the percentage of microfilariae in skin snips suitable for genetic analysis, improve assignment to metadata, and minimize time and materials needed by the researchers collecting the microfilariae. Among 664 microfilariae from South Sudan, 35.7% and 39.5% failed the mitochondrial and nuclear qPCR assay. Among the 576 microfilariae from DRC, 16.0% and 16.7% failed these assays, respectively. This difference may not only be related to the microfilariae transfer procedure but also to other factors, notably the ethanol concentration in the tubes in which microfilariae were stored (64% vs. ≥75%).

Cost and community acceptability of enhanced antibiotic distribution approaches for trachoma in the Republic of South Sudan: enhancing the A in SAFE (ETAS) study protocol.

BACKGROUND: The World Health Organization targeted trachoma for global elimination as a public health problem by 2030. Reaching elimination thresholds by the year 2030 in the Republic of South Sudan will be a considerable challenge, as the country currently has many counties considered hyper-endemic (> 30% trachomatous inflammation-follicular [TF]) that have yet to receive interventions. Evidence from randomized trials, modeling, and population-based surveys suggests that enhancements may be needed to the standard-of-care annual mass drug administration (MDA) to reach elimination thresholds in a timely manner within highly endemic areas. We describe a protocol for a study to determine the cost and community acceptability of enhanced antibiotic strategies for trachoma in South Sudan. METHODS: The Enhancing the A in SAFE (ETAS) study is a community randomized intervention costing and community acceptability study. Following a population-based trachoma prevalence survey in 1 county, 30 communities will be randomized 1:1 to receive 1 of 2 enhanced MDA interventions, with the remaining communities receiving standard-of-care annual MDA. The first intervention strategy will consist of a community-wide MDA followed by 2 rounds of targeted treatment to children ages 6 months to 9 years, 2 weeks and 4 weeks after the community MDA. The second strategy will consist of a community-wide biannual MDA approximately 6 to 8 months apart. The costing analysis will use a payer perspective and identify the total cost of the enhanced interventions and annual MDA. Community acceptability will be assessed through MDA coverage monitoring and mixed-methods research involving community stakeholders. A second trachoma-specific survey will be conducted 12 months following the original survey. DISCUSSION: ETAS has received ethical clearance and is expected to be conducted between 2022 and 2023. Results will be shared through subsequent manuscripts. The study's results will provide information to trachoma programs on whether enhanced interventions are affordable and acceptable to communities. These results will further help in the design of future trachoma-specific antibiotic efficacy trials. Enhanced MDA approaches could help countries recover from delays caused by conflict or humanitarian emergencies and could also assist countries such as South Sudan in reaching trachoma elimination as a public health problem by 2030. TRIAL REGISTRATION: This trial was registered on December 1st, 2022 (clinicaltrails.org: NCT05634759).

Onchocerciasis-associated epilepsy: an explorative case-control study with viral metagenomic analyses on Onchocerca volvulus.

Background: A high prevalence of onchocerciasis-associated epilepsy (OAE) has been observed in onchocerciasis-endemic areas with high ongoing Onchocerca volvulus transmission. However, the pathogenesis of OAE remains to be elucidated. We hypothesise that the O. volvulus virome could be involved in inducing epilepsy. With this study, we aim to describe the O. volvulus virome and identify potential neurotropic viruses linked to OAE. Methods: In Maridi County, an onchocerciasis endemic area in South Sudan with a high prevalence of OAE, we will conduct an exploratory case-control study enrolling 40 persons aged 12 years and above with palpable onchocerciasis nodules. Cases will be participants with OAE (n=20), who will be age- and village-matched with controls without epilepsy (n=20). For each study participant, two skin snips at the iliac crest will be obtained to collect O. volvulus microfilariae, and one nodulectomy will be performed to obtain adult worms. A viral metagenomic study will be conducted on microfilariae and adult worms, and the O. volvulus virome of persons with and without OAE will be compared. The number, size, and localisation of onchocerciasis nodules in persons with and without OAE will be described. Moreover, the pre- and post-nodulectomy frequency of seizures in persons with OAE will be compared. Ethics and dissemination: The protocol has been approved by the Ethics Committee of the University of Antwerp and the Ministry of Health of South Sudan. Findings will be disseminated nationally and internationally via meetings and peer-reviewed publications. Registration: ClinicalTrials.gov registration NCT05868551 ( https://clinicaltrials.gov/study/NCT05868551). Protocol version: 1.1, dated 09/05/2023.

"Slash and Clear", a Community-Based Vector Control Method to Reduce Onchocerciasis Transmission by Simulium sirbanum in Maridi, South Sudan: A Prospective Study.

BACKGROUND: High ongoing Onchocerca volvulus transmission was recently documented in Maridi County, South Sudan. To complement community-directed treatment with ivermectin (CDTI) as the main onchocerciasis control strategy, we initiated a community-based vector control method "slash and clear" at the Maridi dam, a Simulium damnosum s.l. breeding site, to reduce O. volvulus transmission. METHODS: Simulium damnosum s.l. biting rates were collected before and during the twenty months following the "slash and clear" intervention using the human landing catches. Black flies were dissected to measure parity rates before and twelve months after the intervention. Larvae and pupae of S. damnosum s.l. were collected from the dam for morphological and chromosomal characterization to identify the cytospecies involved. RESULTS: Biting rates of S. damnosum s.l. close to the Maridi dam spillway decreased by >90% post-"slash and clear" for more than six months. Twelve months after the "slash and clear" intervention, the reduction in biting rates was still at <50% (p = 0.0007). Parity rates reduced from 13% pre-"slash and clear" (November 2019) to 5.6% post-"slash and clear" (November 2020). Larvae collected from the dam were identified as Simulium sirbanum. CONCLUSION: The "slash and clear" method was found to be an effective and cheap community-based method to reduce black fly biting rates caused by S. sirbanum. When repeated at least annually together with a high CDTI coverage, this intervention has the potential to considerably accelerate onchocerciasis elimination.

Persons with onchocerciasis-associated epilepsy and nodding seizures have a more severe form of epilepsy with more cognitive impairment and higher levels of Onchocerca volvulus infection.

Following previous reports of very high epilepsy prevalence in the onchocerciasis-endemic villages in Maridi County, South Sudan, a study was conducted to investigate the association between the level of Onchocerca volvulus infection, epilepsy, and related outcomes. In December 2018, persons with epilepsy (PWE) were recruited from villages where an epilepsy prevalence of 4.4% (range: 3.5-11.9%) was documented. We enrolled 318 participants from whom two skin snips were taken for microscopic detection of O. volvulus microfilariae (mf). Seizure history was obtained for all PWE and their degree of disability assessed using the modified Rankin scale. Almost all (84.9%) PWE had detectable mf in their skin snips. Onchocerciasis-infected PWE experienced nodding seizures more often than uninfected PWE (p=0.034). Moreover, persons with nodding seizures had more frequent seizures (p<0.001) and higher disability scores (p<0.001), and were more often cognitively impaired and younger at the time of their first epileptic seizure (nine years vs 12 years, p<0.001) compared to PWE without nodding seizures. Based on multivariate models, nodding seizures were associated with higher mf densities (aOR: 1.022; 95% CI: 1.005-1.041). Epilepsy onset at a younger age was associated with a worse outcome. Higher frequency of seizures, longer duration of epilepsy and younger age were associated with increased disability. Regular antiepileptic drug use was associated with better cognitive and disability outcomes. PWE with nodding seizures have a more severe form of onchocerciasis-associated epilepsy, with earlier seizure onset and higher levels of O. volvulus infection. Younger PWE were prone to worse epilepsy outcomes, which would be prevented with regular antiepileptic treatment.

Onchocerca volvulus is not detected in the cerebrospinal fluid of persons with onchocerciasis-associated epilepsy.

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.

Knowledge, attitudes and practices about human African trypanosomiasis and their implications in designing intervention strategies for Yei county, South Sudan.

BACKGROUND: A clear understanding of the knowledge, attitudes and practices (KAP) of a particular community is necessary in order to improve control of human African trypanosomiasis (HAT).New screening and diagnostic tools and strategies were introduced into South Sudan, as part of integrated delivery of primary healthcare. Knowledge and awareness on HAT, its new/improved screening and diagnostic tools, the places and processes of getting a confirmatory diagnosis and treatment are crucial to the success of this strategy. METHODOLOGY: A KAP survey was carried out in Yei County, South Sudan, to identify gaps in community KAP and determine the preferred channels and sources of information on the disease. The cross-sectional KAP survey utilized questionnaires, complemented with key informant interviews and a focus group discussion to elicit communal as well as individual KAP on HAT. FINDINGS: Most (90%) of the respondents had general knowledge on HAT. Lower levels of education, gender and geographic locations without a history of HAT interventions were associated with incorrect knowledge and/or negative perceptions about the treatability of HAT. Symptoms appearing in the late stage were best known. A majority (97.2%) would seek treatment for HAT only in a health centre. However, qualitative data indicates that existing myths circulating in the popular imagination could influence people's practices. Seventy-one percent of the respondents said they would offer social support to patients with HAT but qualitative data highlights that stigma still exists. Misconceptions and stigma can negatively influence the health seeking behaviour of HAT cases. In relation to communication, the top preferred and effective source of communication was radio (24%). CONCLUSION: Gaps in relation to KAP on HAT still exist in the community. Perceptions on HAT, specifically myths and stigma, were key gaps that need to be bridged through effective education and communication strategies for HAT control alongside other interventions.