Further evidence of the cross-reactivity of the Binax NOW® Filariasis ICT cards to non-Wuchereria bancrofti filariae: experimental studies with Loa loa and Onchocerca ochengi

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

Update on the distribution of Mansonella perstans in the southern part of Cameroon: influence of ecological factors and mass drug administration with ivermectin

Overview of gender distribution and median age in study sites. Out of 14,293 (6,746 males and 7,547 females) individuals involved in the study, 52.8 % were females. (PDF 224 kb

Why onchocerciasis transmission persists after 15 annual ivermectin mass drug administrations in South-West Cameroon

Introduction Onchocerciasis is targeted for elimination mainly with annual community-directed treatment with ivermectin (CDTI). High infection levels have been reported in South-West Cameroon, despite ≥15 years of CDTI. The aim of this study was to assess factors associated with continued onchocerciasis transmission and skin disease. Methods A large-scale cross-sectional study was conducted in 2017 in 20 communities in a loiasis-risk area in South-West Cameroon. A mixed-methods approach was used. Associations between infection levels, skin disease and adherence to CDTI were assessed using mixed regression modelling. Different community members’ perception and acceptability of the CDTI strategy was explored using semi-structured interviews. Results Onchocerciasis prevalence was 44.4% among 9456 participants. 17.5% of adults were systematic non-adherers and 5.9% participated in ≥75% of CDTI rounds. Skin disease affected 1/10 participants, including children. Increasing self-reported adherence to CDTI was associated with lower infection levels in participants aged ≥15 years but not in children. Adherence to CDTI was positively influenced by perceived health benefits, and negatively influenced by fear of adverse events linked with economic loss. Concern of lethal adverse events was a common reason for systematic non-adherence. Conclusion CDTI alone is unlikely to achieve elimination in those high transmission areas where low participation is commonly associated with the fear of adverse events, despite the current quasi absence of high-risk levels of loiasis. Such persisting historical memories and fear of ivermectin might impact adherence to CDTI also in areas with historical presence but current absence of loiasis. Because such issues are unlikely to be tackled by CDTI adaptive measures, alternative strategies are needed for onchocerciasis elimination where negative perception of ivermectin is an entrenched barrier to community participation in programmes

Implementation of test-and-treat with doxycycline and temephos ground larviciding as alternative strategies for accelerating onchocerciasis elimination in an area of loiasis co-endemicity: the COUNTDOWN consortium multi-disciplinary study protocol

Background Onchocerciasis is a priority neglected tropical disease targeted for elimination by 2025. The standard strategy to combat onchocerciasis is annual Community-Directed Treatment with ivermectin (CDTi). Yet, high prevalence rates and transmission persist following > 12 rounds in South-West Cameroon. Challenges include programme coverage, adherence to, and acceptability of ivermectin in an area of Loa loa co-endemicity. Loiasis patients harbouring heavy infections are at risk of potentially fatal serious adverse events following CDTi. Alternative strategies are therefore needed to achieve onchocerciasis elimination where CDTi effectiveness is suboptimal. Methods/design We designed an implementation study to evaluate integrating World Health Organisation-endorsed alternative strategies for the elimination of onchocerciasis, namely test-and-treat with the macrofilaricide, doxycycline (TTd), and ground larviciding for suppression of blackfly vectors with the organophosphate temephos. A community-based controlled before-after intervention study will be conducted among > 2000 participants in 20 intervention (Meme River Basin) and 10 control (Indian River Basin) communities. The primary outcome measure is O. volvulus prevalence at follow-up 18-months post-treatment. The study involves four inter-disciplinary components: parasitology, entomology, applied social sciences and health economics. Onchocerciasis skin infection will be diagnosed by skin biopsy and Loa loa infection will be diagnosed by parasitological examination of finger-prick blood samples. A simultaneous clinical skin disease assessment will be made. Eligible skin-snip-positive individuals will be offered directly-observed treatment for 5 weeks with 100 mg/day doxycycline. Transmission assessments of onchocerciasis in the communities will be collected post-human landing catch of the local biting blackfly vector prior to ground larviciding with temephos every week (0.3 l/m3) until biting rate falls below 5/person/day. Qualitative research, including in-depth interviews and focus-group discussions will be used to assess acceptability and feasibility of the implemented alternative strategies among intervention recipients and providers. Health economics will assess the cost-effectiveness of the implemented interventions. Conclusions Using a multidisciplinary approach, we aim to assess the effectiveness of TTd, alone or in combination with ground larviciding, following a single intervention round and scrutinise the acceptability and feasibility of implementing at scale in similar hotspots of onchocerciasis infection, to accelerate onchocerciasis elimination

A murine macrofilaricide pre-clinical screening model for onchocerciasis and lymphatic filariasis

Background: New drugs effective against adult filariae (macrofilaricides) would accelerate the elimination of lymphatic filariasis and onchocerciasis. Anti-Onchocerca drug development is hampered by the lack of a facile model. We postulated that SCID mice could be developed as a fmacrofilaricide screening model. Methods: The filaricides: albendazole (ABZ), diethylcarbamazine (DEC), flubendazole (FBZ), ivermectin (IVM) and the anti-Wolbachia macrofilaricide, minocycline (MIN) were tested in Brugia malayi (Bm)-parasitized BALB/c SCID mice vs vehicle control (VC). Responses were compared to BALB/c wild type (WT). Onchocerca ochengi male worms or onchocercomata were surgically implanted into BALB/c SCID, CB.17 SCID, BALB/c WT mice or Meriones gerbils. Survival was evaluated at 7–15 days. BALB/c SCID were tested to evaluate the responsiveness of pre-clinical macrofilaricides FBZ and rifapentine (RIFAP) against male Onchocerca. Results: WT and SCID responded with >95% efficacy following ABZ or DEC treatments against Bm larvae (P < 0.0001). IVM was partially filaricidal against Bm larvae in WT and SCID (WT; 39.8%, P = 0.0356 and SCID; 56.7%, P = 0.026). SCID responded similarly to WT following IVM treatment of microfilaraemias (WT; 79%, P = 0.0194. SCID; 76%, P = 0.0473). FBZ induced a total macrofilaricidal response against adult Bm in WT and SCID (WT; P = 0.0067, SCID; P = 0.0071). MIN induced a >90% reduction in Bm Wolbachia burdens (P < 0.0001) and a blockade of microfilarial release (P = 0.0215) in SCID. Male Onchocerca survival was significantly higher in SCID vs WT mice, but not gerbils, after +15 days (60% vs 22% vs 39% P = 0.0475). Onchocercoma implants had engrafted into host tissues, with evidence of neovascularisation, after +7 days and yielded viable macro/microfilariae ex vivo. FBZ induced a macrofilaricidal effect in Onchocerca male implanted SCID at +5 weeks (FBZ; 1.67% vs VC; 43.81%, P = 0.0089). Wolbachia loads within male Onchocerca were reduced by 99% in implanted SCID receiving RIFAP for +2 weeks. Conclusions: We have developed a ‘pan-filarial’ small animal research model that is sufficiently robust, with adequate capacity and throughput, to screen existing and future pre-clinical candidate macrofilaricides. Pilot data suggests a murine onchocercoma xenograft model is achievable

Impact of repeated annual community directed treatment with ivermectin on loiasis parasitological indicators in Cameroon: Implications for onchocerciasis and lymphatic filariasis elimination in areas co-endemic with Loa loa in Africa.

BACKGROUND Loiasis is a filarial infection endemic in the rainforest zone of west and central Africa particularly in Cameroon, Gabon, Republic of Congo, and Democratic Republic of the Congo. Repeated treatments with ivermectin have been delivered using the annual community directed treatment with ivermectin (CDTI) approach for several years to control onchocerciasis in some Loa loa-Onchocerca volvulus co-endemic areas. The impact of CDTI on loiasis parasitological indicators is not known. We, therefore, designed this cross sectional study to explore the effects of several rounds of CDTI on parasitological indicators of loiasis. METHODOLOGY/PRINCIPAL FINDINGS The study was conducted in the East, Northwest and Southwest 2 CDTI projects of Cameroon. Individuals who consented to participate were interviewed for ivermectin treatment history and enrolled for parasitological screening using thick smears. Ivermectin treatment history was correlated with loiasis prevalence/intensity. A total of 3,684 individuals were recruited from 36 communities of the 3 CDTI projects and 900 individuals from 9 villages in a non-CDTI district. In the East, loiasis prevalence was 29.3% (range = 24.2%-34.6%) in the non-CDTI district but 16.0% (3.3%-26.6%) in the CDTI district with 10 ivermectin rounds (there were no baseline data for the latter). In the Northwest and Southwest 2 districts, reductions from 30.5% to 17.9% (after 9 ivermectin rounds) but from 8.1% to 7.8% (not significantly different after 14 rounds) were registered post CDTI, respectively. Similar trends in infection intensity were observed in all sites. There was a negative relationship between adherence to ivermectin treatment and prevalence/intensity of infection in all sites. None of the children (aged 10-14 years) examined in the East CDTI project harboured high (8,000-30,000 mf/ml) or very high (>30,000 mf/ml) microfilarial loads. Individuals who had taken >5 ivermectin treatments were 2.1 times more likely to present with no microfilaraemia than those with less treatments. CONCLUSION In areas where onchocerciasis and loiasis are co-endemic, CDTI reduces the number of, and microfilaraemia in L. loa-infected individuals, and this, in turn, will help to prevent non-neurological and neurological complications post-ivermectin treatment among CDTI adherents

Successful long-term maintenance of Mansonella perstans in an in vitro culture system

Abstract Background Approximately 114 million people are infected with Mansonella perstans in large proportions of Africa. In contrast to other filariae that infect humans, M. perstans-infected individuals show no distinct pathology or specific clinical picture, indicating a well-tuned adaptation to the host. In addition, since M. perstans adult worms reside in serous cavities which are difficult to access, research has been hindered and there is a paucity of knowledge about the biology of M. perstans, especially the development of the different life stages as well as M. perstans-driven immune responses. Thus in this study, an in vitro culture system was developed which allows an in-depth analysis of M. perstans. Results Culicoides species were caught in Ediki (Kumba), Southwest Region within Cameroon following a blood meal on a microfilaremic donor that had 1500 microfilariae/ml of peripheral blood and kept in captivity for 12 days at 23 °C. In a pilot experiment, 15 infective larvae were obtained from the midges and co-cultured with a confluent monolayer of monkey kidney epithelial cells (LLC-MK2) in DMEM medium supplemented with 10% FBS for up to 77 days. The resulting survival rates of 33% revealed that the cell-conditioned medium was suitable for long-term maintenance of M. perstans worms. To confirm these preliminary observations, 249 infective larvae were cultured for 50 days and their development was monitored daily and microscopically graded for motility. In total, 170 (68.3%) filariae survived and 124 (49.8%) larvae moulted between days 21–30 to become L5 stage larvae which were motile and showed continuous vigorous movement. Conclusion We have established an in vitro culture system for the generation and long-term maintenance of viable M. perstans worms. This technique will be an important tool to study parasite biology and development, the role in host immunity, and might be helpful to discover novel treatment strategies against this filariae

Dataset on in vitro maintenance of Mansonella perstans microfilariae and drug testing

The data presented here were collected from in vitro culture of M. perstans microfilariae, while asssessing the ability of two basic culture media; Dulbecco’s Modified Eagle’s Medium (DMEM) and Roswell Park Memorial Institute (RPMI-1640) supplemented with 10% fetal bovine serum (FBS) and a monkey kidney epithelial cell line (LLC-MK2) to support the survival of this parasite stage; and their sussceptibility to chemotherapeutic agents. Parasites motlity were scored daily on a 4 scales system, and the average motility computed, as well as the area under the motility curve. Mortility was determined as the percentges of imotiled worms

In vitro maintenance of Mansonella perstans microfilariae and its relevance for drug screening

Background: Mansonellosis arises from infections with threadlike filarial nematodes in millions of individuals, especially in sub-Saharan Africa. Since infections present no overt clinical symptoms but attenuate immune responses that might lead to increased susceptibility and worsened disease course of concomitant infections, it is truly a neglected tropical disease. Nevertheless, only few studies focus on identifying suitable safe drugs for its control and little is known about the requirements for in vitro maintenance of the Mansonella perstans transmission stage. This study, therefore, evaluated the survival of M. perstans microfilariae (mf) using in vitro conditions that have been shown to promote survival of Loa loa, a closely related filarial nematode. Furthermore, the in vitro microfilaricidal effect of 15 agents was assessed on this helminth. Methods: The ability of two basic culture media; Dulbecco's Modified Eagle's Medium (DMEM) and Roswell Park Memorial Institute (RPMI-1640) supplemented with 10% fetal bovine serum (FBS) and a monkey kidney epithelial cell line (LLC-MK2) to support the survival of M. perstans microfilariae was investigated. Subsequently, 6 anti-helminthics, 5 anti-malarials, 1 anti-microbacterial, 2 trypanocidals and 1 anti-cancer agent were tested in vitro against mf. The suitability of the culture media as well as the effect of the anti-infective agents on mf survival was assessed by scoring their motility. Results: FBS supplement and additional LLC-MK2 cells significantly improved the survival of mf in DMEM and RPMI-1640 culture. In detail, RPMI-1640 supplemented with 10% FBS and LLC-MK2 cells sustained the maintenance of mf for at least 20 days (100.00 ± 0.00% survival). In co-cultures with LLC-MK2 cells without serum, M. perstans mf were maintained in DMEM and RPMI-1640 medium with a motility above 99% by day 5. Mefloquine displayed the highest microfilaricidal effect in vitro followed by artesunate. Conclusion: Both RPMI and DMEM in the presence of LLC-MK2 cells are suitable for the maintenance of M. perstans mf in vitro. In absence of the feeder cells, the addition of 10% FBS to RPMI-1640 medium improved the parasite survival rate and motility. The microfilaricidal activity of mefloquine and artesunate on M. perstans m