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Background: The mass drug administration of ivermectin for onchocerciasis control has contributed to a significant drop in Loa loa microfilaria loads in humans that has, in turn, led to reduction of infection levels in Chrysops vectors. Accurate parasite detection is essential for assessing loiasis transmission as it provides a potential alternative or indirect strategy for addressing the problem of co-endemic loiasis and lymphatic filariasis through the Onchocerciasis Elimination Programme and it further reflects the true magnitude of the loiasis problem as excess human mortality has been reported to be associated with the disease. Although microscopy is the gold standard for detecting the infection, the sensitivity of this method is compromised when the intensity of infection is low. The loop-mediated isothermal amplification (LAMP) assay of parasite DNA is an alternative method for detecting infection which offers operational simplicity, rapidity and versatility of visual readout options. The aim of this study was to validate the Loa loa LAMP assay for the detection of infected Chrysops spp. under experimental and natural field conditions., Methods: Two sets of 18 flies were fed on volunteers with either a low (< 10 mf/ml) or high (> 30,000mf/ml) microfilarial load. The fed flies were maintained under laboratory conditions for 14 days and then analysed using LAMP for the detection of L. loa infection. In addition, a total of 9270 flies were collected from the north-west, east, and south-west regions (SW 1 and 2) of Cameroon using sweep nets and subjected to microscopy (7841 flies) and LAMP (1291 flies plus 138 nulliparous flies) analyses., Results: The LAMP assay successfully detected parasites in Chrysops fed on volunteers with both low and high microfilariaemic loads. Field validation and surveillance studies revealed LAMP-based infection rates ranging from 0.5 to 31.6%, with the lowest levels in SW 2 and the highest infection rates in SW 1. The LAMP assay detected significantly higher infection rates than microscopy in four of the five study sites., Conclusion: This study demonstrated the potential of LAMP as a simple surveillance tool. It was found to be more sensitive than microscopy for the detection of experimental and natural L. loa infections in Chrysops vectors.

Basic and translational research on loiasis, a filarial nematode infection of medical importance, is impeded by a lack of suitable Loa loa infection models and techniques of obtaining and culturing life cycle stages. We describe the development of a new method for routine production of infective third-stage larvae (L3) of L. loa from the natural intermediate arthropod vector host, Chrysops silacea, following experimental infection with purified microfilariae. At 14-days post-infection of C. silacea, the fly survival rate was 43%. Survival was significantly higher in flies injected with 50 mf (55.2%) than those that received 100 mf (31.0%). However, yield per surviving fly and total yield of L3 was markedly higher in the group of flies inoculated with 100 mf (3474 vs 2462 L3 produced). The abdominal segment hosted the highest percentage recovery of L3 (47.7%) followed by head (34.5%) and thorax (17.9%). L. loa larval survival was higher than 90% after 30 days of in vitro culture. The in vitro moulting success rate to the L4 larval stage was 59.1%. After experimental infection of RAG2-/-IL-2γc-/-mice, the average L. loa juvenile adult worm recovery rate was 10.5% at 62 dpi. More than 87% of the worms were recovered from the muscles and subcutaneous tissues. Worms recovered measured an average 24.3 mm and 11.4 mm in length for females (n = 5) and males (n = 5), respectively. In conclusion, L. loa mf injected into C. silacea intrathoracically develop into infective larvae that remain viable and infective comparable to L3 obtained through natural feeding on the human host. This technique further advances the development of a full laboratory life cycle of L. loa where mf derived from experimentally-infected animals may be utilized to passage life cycle generations via intrathoracic injections of wild-caught vector hosts., Competing Interests: The authors have declared that no competing interests exist.

Background: Different immune mechanisms are capable of killing developmental stages of filarial nematodes and these mechanisms are also likely to vary between the primary and a challenge infection. However, the lack of a detailed analysis of cytokine, chemokine and immunoglobulin levels in human loiasis is still evident. Therefore, detailed analysis of immune responses induced by the different developmental stages of Loa loa in immune-competent BALB/c mice will aid in the characterization of distinct immune responses that are important for the immunity against loiasis., Methods: Different developmental stages of L. loa were obtained from human peripheral blood (microfilariae, MF), the transmitting vector, Chrysops (larval stage 3, L3) and infected immune-deficient BALB/cRAG2γc -/- mice (L4, L5, adult worms). Groups of wildtype BALB/c mice were then injected with the isolated stages and after 42 days post-infection (pi), systemic cytokine, chemokine and immunoglobulin levels were determined. These were then compared to L. loa-specific responses from in vitro re-stimulated splenocytes from individual mice. All parameters were determined using Luminex technology., Results: In a pilot study, BALB/c mice cleared the different life stages of L. loa within 42 days pi and systemic cytokine, chemokine and immunoglobulin levels were equal between infected and naive mice. Nevertheless, L. loa-specific re-stimulation of splenocytes from mice infected with L5, MF or adult worms led to induction of Th2, Th17 and chemokine secretion patterns., Conclusions: This study shows that although host immunity remains comparable to naive mice, clearance of L. loa life-cycle development stages can induce immune cell memory leading to cytokine, chemokine and immunoglobulins secretion patterns which might contribute to immunity and protection against reinfection.

Background: Loiasis, an often-neglected tropical disease, is a threat to the success of lymphatic filariasis and onchocerciasis elimination programmes in rainforest areas of the central and western Africa. Its control and even its elimination might be possible through the use of a safe macrofilaricide, a prophylactic drug, or perhaps a vaccine. This present study evaluated the effect of flubendazole (FLBZ) on the development of Loa loa L3 in vitro and in vivo., Methods: Infective stages of L. loa were isolated and co-cultured in Dulbecco's Modified Eagle's Medium in the presence of monkey kidney epithelial cells (LLC-MK2) feeder cells. FLBZ and its principal metabolites, reduced flubendazole (RFLBZ) and hydrolyzed flubendazole (HFLBZ), were screened in vitro at concentrations 0.05, 0.1, 0.5, 1 and 10 μg/ml. The viability of the parasites was assessed microscopically daily for 15 days. For in vivo study, a total of 48 CcR3 KO mice were infected subcutaneously with 200 L. loa L3 and treated with 10 mg/kg FLBZ once daily for 5 consecutive days. Twenty-four animals were used as control and received L3 and vehicle. They were dissected at 5, 10, 15 and 20 days post-treatment for worm recovery., Results: The motility of L3 larvae in vitro was reduced from the second day of incubation with drugs at in vivo plasma concentration levels, with a strong correlation found between reduced motility and increased drug concentration (Spearman's rho = -0.9, P < 0.0001). Except for HFLBZ (0.05 μg/ml and 0.01 μg/ml), all concentrations of FLBZ, HFLBZ and RFLBZ interrupted the moulting of L. loa infective larvae to L4. In vivo, regardless of the experimental group, there was a decrease in parasite recovery with time. However, at each time point this reduction was more pronounced in the group of animals treated with FLBZ compared to equivalent control. Parasites were recovered from the flubendazole-treated groups only on day 5 post-inoculation at an average rate of 2.1%, a value significantly lower (Mann-Whitney U-test, U = 28, P = 0.0156) than the average of 31.1% recovered from the control group., Conclusions: This study reveals the ability of flubendazole to inhibit the development of L. loa L3 both in vitro and in vivo, and in addition validates the importance of in vitro and animal models of L. loa as tools for the development of drugs against loiasis.

Background: Suitable and scalable in vitro culture conditions for parasite maintenance are needed to foster drug research for loiasis, one of the neglected tropical diseases which has attracted only limited attention over recent years, despite having important public health impacts. The present work aims to develop adequate in vitro culture systems for drug screening against both microfilariae (mf) and infective third-stage larvae (L3) of Loa loa., Methods: In vitro culture conditions were evaluated by varying three basic culture media: Roswell Park Memorial Institute (RPMI-1640), Dulbecco's modified Eagle's medium (DMEM) and Iscove's modified Dulbecco's medium (IMDM); four sera/proteins: newborn calf serum (NCS), foetal bovine serum (FBS), bovine serum albumin (BSA) and the lipid-enriched BSA (AlbuMax® II, ALB); and co-culture with the Monkey Kidney Epithelial Cell line (LLC-MK2) as a feeder layer. The various culture systems were tested on both mf and L3, using survival (% motile), motility (T 90 = mean duration (days) at which at least 90% of parasites were fully active) and moulting rates of L3 as the major criteria. The general linear model regression analysis was performed to assess the contribution of each variable on the viability of Loa loa L3 and microfilarie. All statistical tests were performed at 95% confidence interval., Results: Of the three different media tested, DMEM and IMDM were the most suitable sustaining the maintenance of both L. loa L3 and mf. IMDM alone could sustain L3 for more than 5 days (T 90 = 6.5 ± 1.1 day). Serum supplements and LLC-MK2 co-cultures significantly improved the survival of parasites in DMEM and IMDM. In co-cultures with LLC-MK2 cells, L. loa mf were maintained in each of the three basic media (T 90 of 16.4-19.5 days) without any serum supplement. The most effective culture systems promoting significant moulting rate of L3 into L4 (at least 25%) with substantial maintenance time were: DMEM + BSA, DMEM + NCS, DMEM-AlbuMax®II, DMEM + FBS all in co-culture with LLC-MK2, and IMDM + BSA (1.5%), DMEM + FBS (10%) and DMEM + NCS (5%) without feeder cells. DMEM + 1% BSA in co-culture scored the highest moulting rate of 57 of 81 (70.37%). The factors that promoted L. loa mf viability included feeder cells (β = 0.490), both IMDM (β = 0.256) and DMEM (β = 0.198) media and the protein supplements NCS (β = 0.052) and FBS (β = 0.022); while for L. loa L3, in addition to feeder cells (β = 0.259) and both IMDM (β = 0.401) and DMEM (β = 0.385) media, the protein supplements BSA (β = 0.029) were found important in maintaining the worm motility., Conclusions: The findings from this work display a range of culture requirements for the maintenance of Loa loa stages, which are suitable for developing an effective platform for drug screening.

Background: Co-infection with loiasis remains a potential problem in control programs targeting filarial infections. The effects of many anti-parasitic drugs often administered to Loa loa infected people are not well documented. This study compared the in vitro activity of several of these drugs on the viability of L. loa microfilariae (mf)., Methods: Human strain L. loa mf were isolated from baboon blood using iso-osmotic Percoll gradient, and cultured in RPMI 1640/10% FBS with antimalarial drugs (mefloquine, amodiaquine, artesunate, chloroquine and quinine), anthelmintics (ivermectin, praziquantel, flubendazole and its reduced and hydrolyzed metabolites), two potential trypanocidal agents (fexinidazole and Scynexis-7158) and the anticancer drug imatinib. The drug concentrations used varied between 0.156 μg/ml and 10 μg/ml. Mf motility (CR 50 = 50% immotility) and a metabolic viability assay (MTT) were used to assess the effects of these drugs on the parasites., Results: Mf in control cultures showed only a slight reduction in motility after 5 days of culture. Active inhibition of Loa loa motility was seen with mefloquine and amodiaquine (CR 50 values of 3.87 and 4.05 μg/ml, respectively), immobilizing > 90% mf within the first 24 hours: mefloquine killed the mf after 24 hours of culture at concentrations ≥ 5 μg/ml. SCYX-7158 also induced a concentration-dependent reduction in mf motility, with > 50% reduction in mf motility seen after 5 days at 10 μg/ml. The anticancer drug imatinib reduced mf motility at 10 μg/ml from the first day of incubation to 55% by day 5, and the reduction in motility was concentration-dependent. Praziquantel and fexinidazole were inactive, and FLBZ and its metabolites, as well as ivermectin at concentrations > 5 μg/ml, had very minimal effects on mf motility over the first 4 days of culture., Conclusions: The considerable action of the anti-malarial drugs mefloquine and amodiaquine on Loa mf in vitro highlights the possibility of repurposing the existing anti-infectious agents for the development of drugs against loiasis. The heterogeneity in the activity of anti-parasitic agents on Loa loa mf supports the need for further investigation using animal models of loiasis.

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.