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BACKGROUND: The human parasite Onchocerca volvulus harbours Wolbachia endosymbionts essential for worm embryogenesis, larval development and adult survival. In this study, the development of Wolbachia-depleted microfilariae (first stage larvae) to infective third stage larvae (L3) in the insect vector Simulium damnosum was analysed. METHODS: Infected volunteers in Cameroon were randomly and blindly allocated into doxycycline (200 mg/day for 6 weeks) or placebo treatment groups. After treatment, blackflies were allowed to take a blood meal on the volunteers, captured and dissected for larval counting and DNA extraction for quantitative real-time PCR analysis. RESULTS: PCR results showed a clear reduction in Wolbachia DNA after doxycycline treatment in microfilariae from human skin biopsies with > 50% reduction at one month post-treatment, eventually reaching a reduction of > 80%. Larval stages recovered from the insect vector had similar levels of reduction of endosymbiotic bacteria. Larval recoveries were analysed longitudinally after treatment to follow the kinetics of larval development. Beginning at three months post-treatment, significantly fewer L3 were seen in the blackflies that had fed on doxycycline treated volunteers. Concomitant with this, the proportion of second stage larvae (L2) was significantly increased in this group. CONCLUSIONS: Doxycycline treatment and the resulting decline of Wolbachia endobacteria from the microfilaria resulted in retarded development of larvae in the insect vector. Thus, anti-wolbachial treatment could have an additive effect for interrupting transmission by reducing the number of L3 that can be transmitted by blackflies.

Great strides have been made toward onchocerciasis elimination by mass drug administration (MDA) of ivermectin. Focusing on MDA-eligible areas, we investigated where the elimination goal can be achieved by 2025 by continuation of current practice (annual MDA with ivermectin) and where intensification or additional vector control is required. We did not consider areas hypoendemic for onchocerciasis with loiasis coendemicity where MDA is contraindicated. We used 2 previously published mathematical models, ONCHOSIM and EPIONCHO, to simulate future trends in microfilarial prevalence for 80 different settings (defined by precontrol endemicity and past MDA frequency and coverage) under different future treatment scenarios (annual, biannual, or quarterly MDA with different treatment coverage through 2025, with or without vector control strategies), assessing for each strategy whether it eventually leads to elimination. Areas with 40%-50% precontrol microfilarial prevalence and ≥10 years of annual MDA may achieve elimination with a further 7 years of annual MDA, if not achieved already, according to both models. For most areas with 70%-80% precontrol prevalence, ONCHOSIM predicts that either annual or biannual MDA is sufficient to achieve elimination by 2025, whereas EPIONCHO predicts that elimination will not be achieved even with complementary vector control. Whether elimination will be reached by 2025 depends on precontrol endemicity, control history, and strategies chosen from now until 2025. Biannual or quarterly MDA will accelerate progress toward elimination but cannot guarantee it by 2025 in high-endemicity areas. Long-term concomitant MDA and vector control for high-endemicity areas might be useful.

Background The onchocerciasis focus surrounding the lower Mbam and Sanaga rivers, where Onchocerca volvulus is transmitted by Simulium damnosum s.l. (Diptera: Simuliidae), was historically the largest in the southern regions of Cameroon. Annual community-directed treatment with ivermectin (CDTI) has been taking place since 2000, but recent studies have shown that new infections are occurring in children. We aimed to investigate blackfly biting and O. volvulus transmission rates along the lower Mbam river 16 years after the formal onset of annual CDTI. Methods Black flies were collected for three consecutive days each month between July 2016 and June 2017 at two riverside villages and two inland sites situated 4.9 km and 7.9 km from the riverside. Specimens collected at each site were dissected on one of the three collection days each month to estimate parity rates and O. volvulus infection rates, while the remaining samples were preserved for pool screening. Results In total, 93,573 S. damnosum s.l. black flies were recorded biting humans and 9281 were dissected. Annual biting rates of up to 606,370 were estimated at the riverside, decreasing to 20,540 at 7.9 km, while, based on dissections, annual transmission potentials of up to 4488 were estimated at the riverside, decreasing to 102 and 0 at 4.9 km and 7.9 km, respectively. However, pool screening showed evidence of infection in black flies at the furthest distance from the river. Results of both methods demonstrated the percentage of infective flies to be relatively low (0.10–0.36%), but above the WHO threshold for interruption of transmission. In addition, a small number of larvae collected during the dry season revealed the presence of Simulium squamosum E. This is the first time S. squamosum E has been found east of Lake Volta in Ghana, but our material was chromosomally distinctive, and we call it S. squamosum E2. Conclusions Relatively low O. volvulus infection rates appear to be offset by extremely high densities of biting black flies which are sustaining transmission along the banks of the lower Mbam river. Graphical Abstract

Background Leucocytozoonosis, a parasitic disease of birds, is caused by haemosporidian protozoan parasites of the genus Leucocytozoon , which infect diverse avian species, including poultry. These parasites are transmitted by several black fly species, but knowledge of the factors determining the diversity and prevalence in these vectors, which is crucial for fully understanding disease epidemiology, is largely unexplored. In this study, we investigated factors associated with the prevalence and diversity of Leucocytozoon species in black flies from Thailand. Methods Adults of two black fly taxa ( Simulium asakoae Takaoka and Davies complex and S. khelangense Takaoka, Srisuka and Saeung) were collected using sweep nets at nine locations in northern and northeastern regions of Thailand. Specimens were identified morphologically and the results corroborated by DNA barcoding. Molecular methods using specific primers for amplification of the mitochondrial cytochrome b (cyt b ) gene of Leucocytozoon were used to detect the parasite in black flies. Species and lineages of Leucocytozoon were determined using the MalAvi database of malaria parasites and related haemosporidians in avian hosts. Regression analysis was used to examine relationships between Leucocytozoon diversity and prevalence, black fly abundance and habitat characteristics. Results A total of 11,718 adult black flies were collected, of which 4367 were members of the S. asakoae complex and 7351 were S. khelangense . For molecular detection of Leucocytozoon , we randomly selected 300 individual female black flies of the S . asakoae complex and 850 females of S . khelangense pooled into groups of five individuals (= 170 pools). A total of 34 of the 300 specimens of the S. asakoae complex and 118 of the 170 pools of S . khelangense were positive for Leucocytozoon . Fifty-four lineages (haplotypes) were identified, all of which belonged to those reported in domestic chickens, Gallus gallus , with one exception that was identified in S. khelangense and found to be closely related to the Leucocytozoon lineages reported in owls; this is the first record of the latter lineage in Asian black flies. Among these haplotypes, nine and 45 were exclusively found in the S. asakoae complex and S. khelangense , respectively. No lineage was shared between these black fly taxa. Analysis of similarity (ANOSIM) revealed significant Leucocytozoon lineage composition between the two black flies. Phylogenetic analysis found that Leucocytozoon lineages in the S. asakoae complex and S. khelangense are largely isolated, agreeing with the ANOSIM result. The overall prevalence of Leucocytozoon in the S. asakoae complex was 11.3% and ranged from 9% to 13% in each collection. Leucocytozoon prevalence in S. khelangense was 21%, varying from 13% to 37% in each collection. The Shannon H′ index indicated greater Leucocytozoon diversity in S. khelangense (H′ = 3.044) than in the S. asakoae complex (H′ = 1.920). Regression analysis revealed that Leucocytozoon diversity was positively related to black fly abundance and negatively related to maximum air temperature. Conclusions The results of this study show that the prevalence and diversity of Leucocytozoon lineages in the S . asakoae complex and S. khelangense from Thailand were associated with the abundance of these black flies and with air temperature. The Leucocytozoon lineages identified also showed some degree of black fly taxon specificity, possibly related to different abundance peaks of these vectors. The environmental conditions that favor the development of black flies are possibly a driver of Leucocytozoon prevalence, diversity and vector–parasite co-evolution. Graphical Abstract

Onchocerciasis caused by the filarial parasite Onchocerca volvulus and transmitted by Simulium damnosum s.l. remains a public health concern in Nigeria. Infestation of S. damnosum s.l. along rivers Gurara and Kaduna; and heavy intensity of O. volvulus infection in Kaduna were documented in 1956. Control of onchocerciasis in Kaduna started 1954 with larviciding using dichlorodiphenyltrichloroethane reduced S. damnosum s.l. population by 94% in 1966. Diethylcarbamazine used for human treatment was discontinued due to toxicity. Kaduna State Ministry of Health and its partners built on this achievement; used annual mass administration of ivermectin consistently between 1989 and 2017 which led to interruption of onchocerciasis transmission in 2018. This study investigated possibility of recrudescence of onchocerciasis with the hypothesis that insecurity-induced migration could cause recrudescence of onchocerciasis. Six out of the 23 Local Government Areas (LGAs) in Kaduna State were selected for evaluation. Adult S. damnosum s.l. were captured across seven breeding sites using human landing collectors from July to October 2023. Pooled screen assays of 72 pools heads of black flies were conducted using quantitative polymerase chain reaction (qPCR) for O. volvulus detection. Dried blood samples from 3107 children aged 5-9 years were collected; with 1502 samples analysed using alkaline phosphatase enzyme-linked immunosorbent assay due to financial constraints. Twenty pools of the 72 pools of heads (27.8%) of S. damnosum s.l. analysed were positive for O. volvulus in Kagarko and Kachia LGAs (> 1/2000 infective flies; 95% upper confidence limit 0.49) with qPCR prevalence of 0.32%. Two children from security compromised communities tested seropositive (prevalence 0.31%; 95% upper confidence limit 0.317). The findings demonstrated ongoing onchocerciasis transmission in Kaduna despite the interruption in 2018. This calls for evaluation of the extent of recrudescence and identification of key drivers such as human migration, fly movement and insecurity.

Despite several rounds of ivermectin treatment, onchocerciasis transmission persists in Mvolo and Mundri West Counties (Western Equatoria State, South Sudan). A community-based \"Slash and clear\" (S&C) vector control method was recently introduced, but its effectiveness remains unclear. Between October 2023 and November 2024, entomological studies were conducted to map blackfly breeding sites along River Naam (Mvolo County) and River Yei (Mundri West County). Following baseline assessment of daily biting rates at multiple catching sites, one round of S&C was implemented at selected intervention sites. Monthly biting rates (MBR) were monitored over a follow-up period of 8-14 months and compared between intervention and control sites. Thirteen breeding sites of Simulium damnosum, sensu lato. were found on the Naam and Yei Rivers. Biting rates were consistently higher during the rainy season than the dry season across both Counties. On the Naam River (14 months of follow-up), Mann-Kendall trend tests showed non-significant reductions in MBRs at both intervention (tau = -0.038, p-value = 0.881) and control sites (tau = -0.135, p-value = 0.313). Similar non-significant changes were observed on the Yei River (8 months follow-up), with tau = 0.046 (p-value = 0.820) and tau = 0.163 (p-value = 0.363) for intervention and control sites, respectively. Generalized additive models (GAM) regression analysis indicated that seasonality was the only significant predictor of MBR, with increased biting rates during the rainy season (p < 0.001). A single round of S&C at baseline did not result in significant reductions in MBRs (p-value = 0.651 in Mvolo and p-value = 0.531 in Mundri West). Blackfly biting rates in Mvolo and Mundri West Counties are strongly influenced by seasonal variations, peaking during the rainy season. Our findings indicate that a single round of S&C is insufficient to reduce blackfly biting in the medium term. Repeated and strategically timed annual implementation of S&C is likely required to achieve significant and lasting vector control impacts.

Background The behaviour of blood-sucking arthropods is a crucial determinant of blood protozoan distribution and hence of host-parasite coevolution, but it is very challenging to study in the wild. The molecular identification of parasite lineages in vectors can be a useful key to understand the behaviour and transmission patterns realised by these vectors. Methods In this study, we collected blackflies around nests of three raptor species in the upper forest canopy in central Europe and examined the presence of vertebrate DNA and haemosporidian parasites in them. We molecularly analysed 156 blackfly individuals, their vertebrate blood meals, and the haemosporidian parasite lineages they carried. Results We identified nine species of Simulium blackflies, largely belonging to the subgenera Nevermannia and Eusimulium . Only 1% of the collected specimens was visibly engorged, and only 4% contained remains of host DNA. However, in 29% of the blackflies Leucocytozoon lineages were identified, which is evidence of a previous blood meal on an avian host. Based on the known vertebrate hosts of the recorded Leucocytozoon lineages, we can infer that large and/or abundant birds, such as thrushes, crows, pigeons, birds of prey, owls and tits are the main targets of ornithophilic blackflies in the canopy. Blackfly species contained similar proportions of host group-specific parasite lineages and thus do not appear to be associated with particular host groups. Conclusions The Leucocytozoon clade infecting thrushes, crows, and pigeons present in most represented blackfly species suggests a lack of association between hosts and blackflies, which can increase the probability of host switches of blood parasites. However, the composition of the simuliid species differed between nests of common buzzards, goshawks and red kites. This segregation can be explained by coinciding habitat preferences between host and vector, and may lead to the fast speciation of Leucocytozoon parasites. Thus, subtle ecological preferences and lack of host preference of vectors in the canopy may enable both parasite diversification and host switches, and enforce a habitat-dependent evolution of avian malaria parasites and related haemosporidia.

Accurate detection of filarial parasites in humans is essential for the implementation and evaluation of mass drug administration programs to control onchocerciasis and lymphatic filariasis. Determining the infection levels in vector populations is also important for assessing transmission, deciding when drug treatments may be terminated and for monitoring recrudescence. Immunological methods to detect infection in humans are available, however, cross-reactivity issues have been reported. Nucleic acid-based molecular assays offer high levels of specificity and sensitivity, and can be used to detect infection in both humans and vectors. In this study we developed loop-mediated isothermal amplification (LAMP) tests to detect three different filarial DNAs in human and insect samples using pH sensitive dyes for enhanced visual detection of amplification. Furthermore, reactions were performed in a portable, non-instrumented nucleic acid amplification (NINA) device that provides a stable heat source for LAMP. The efficacy of several strand displacing DNA polymerases were evaluated in combination with neutral red or phenol red dyes. Colorimetric NINA-LAMP assays targeting Brugia Hha I repeat, Onchocerca volvulus GST1a and Wuchereria bancrofti LDR each exhibit species-specificity and are also highly sensitive, detecting DNA equivalent to 1/10-1/5000th of one microfilaria. Reaction times varied depending on whether a single copy gene (70 minutes, O. volvulus) or repetitive DNA (40 min, B. malayi and W. bancrofti) was employed as a biomarker. The NINA heater can be used to detect multiple infections simultaneously. The accuracy, simplicity and versatility of the technology suggests that colorimetric NINA-LAMP assays are ideally suited for monitoring the success of filariasis control programs.

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. 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. 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.

Retrospective cohort studies in Cameroon found an association between Onchocerca volvulus microfilarial load in childhood (measured in 1991–1993) and risk of developing epilepsy later in life (measured in 2017). We parameterised and integrated this relationship (across children aged 3–15 years) into the previously published, stochastic transmission model, EPIONCHO-IBM, for Simulium damnosum sensu lato-transmitted onchocerciasis. We simulated 19 years (1998–2017) of annual ivermectin mass drug administration (MDA) reflecting coverage in the study area, and modelled epilepsy prevalence and incidence. Scenario-based simulations of 25 years of (annual and biannual) MDA in hyper- and holoendemic settings, with 65% and 80% therapeutic coverage, were also conducted. EPIONCHO-IBM predicted 7.6% epilepsy prevalence (compared to 8.2% in the Cameroon study) and incidence of 317 cases/100,000 person-years (compared to 350). In hyperendemic areas, 25 years of biannual MDA (80% coverage) eliminated onchocerciasis-associated epilepsy (OAE) and protected untreated under-fives from its development. Strengthening onchocerciasis programmes, implementing alternative strategies, and evaluating treatment for under-fives and school-age children are crucial to prevent OAE in highly-endemic settings. Onchocerciasis is a vector-borne disease endemic to parts of sub-Saharan Africa and associated with substantial morbidity including reports of onchocerciasis-associated epilepsy. Here, the authors use mathematical modelling to assess the impact of community-directed treatment with ivermectin on onchocerciasis-associated epilepsy.