Geostatistical modelling enables efficient safety assessment for mass drug administration with ivermectin in Loa loa endemic areas through a combined antibody and LoaScope testing strategy for elimination of onchocerciasis.

The elimination of onchocerciasis through community-based Mass Drug Administration (MDA) of ivermectin (Mectizan) is hampered by co-endemicity of Loa loa, as individuals who are highly co-infected with Loa loa parasites can suffer serious and occasionally fatal neurological reactions from the drug. The test-and-not-treat strategy of testing all individuals participating in MDA has some operational constraints including the cost and limited availability of LoaScope diagnostic tools. As a result, a Loa loa Antibody (Ab) Rapid Test was developed to offer a complementary way of determining the prevalence of loiasis. We develop a joint geostatistical modelling framework for the analysis of Ab and Loascope data to delineate whether an area is safe for MDA. Our results support the use of a two-stage strategy, in which Ab testing is used to identify areas that, with acceptably high probability, are safe or unsafe for MDA, followed by Loascope testing in areas whose safety status is uncertain. This work therefore contributes to the global effort towards the elimination of onchocerciasis as a public health problem by potentially reducing the time and cost required to establish whether an area is safe for MDA.

An Integrated District Mapping Strategy for Loiasis to Enable Safe Mass Treatment for Onchocerciasis in Gabon.

The lack of a WHO-recommended strategy for onchocerciasis treatment with ivermectin in hypo-endemic areas co-endemic with loiasis is an impediment to global onchocerciasis elimination. New loiasis diagnostics (LoaScope; Loa antibody rapid test) and risk prediction tools may enable safe mass treatment decisions in co-endemic areas. In 2017-2018, an integrated mapping strategy for onchocerciasis, lymphatic filariasis (LF), and loiasis, aimed at enabling safe ivermectin treatment decisions, was piloted in Gabon. Three ivermectin-naïve departments suspected to be hypo-endemic were selected and up to 100 adults per village across 30 villages in each of the three departments underwent testing for indicators of onchocerciasis, LF, and loiasis. An additional 67 communities in five adjoining departments were tested for loiasis to extend the prevalence and intensity predictions and possibly expand the boundaries of areas deemed safe for ivermectin treatment. Integrated testing in the three departments revealed within-department heterogeneity for all the three diseases, highlighting the value of a mapping approach that relies on cluster-based sampling rather than sentinel sites. These results suggest that safe mass treatment of onchocerciasis may be possible at the subdepartment level, even in departments where loiasis is present. Beyond valuable epidemiologic data, the study generated insight into the performance of various diagnostics and the feasibility of an integrated mapping approach utilizing new diagnostic and modeling tools. Further research should explore how programs can combine these diagnostic and risk prediction tools into a feasible programmatic strategy to enable safe treatment decisions where loiasis and onchocerciasis are co-endemic.

New promising method to assess microfilarial Loa loa load on the peripheral blood.

Loiasis is a vector-borne parasitic disease caused by the filarial Loa loa (L. loa). Definitive diagnosis can be done by identifying and counting microfilariae in the peripheral blood by microscopy and with L.loa-specific PCR. An additional diagnostic method is the detection of L.loa-specific antibodies. Accurate methods are needed to automate quantification of microfilaria (mf) in peripheral blood. Indeed, the treatment procedure depends on the microfilarial L. loa load in blood. We report the first documented use of flow cytometry as a new method to count microfilaraemia in peripheral blood from a patient with L. loa infection. The diagnosis of loiasis was strongly suspected based on clinical presentation and rapidly confirmed by identifying typical features of L. loa in the peripheral blood. This diagnosis was achieved by flow cytometry using a specific fluorescence pattern for microfilaraemia count. The current report highlights the potential of flow cytometry to assess microfilarial L. loa load from a patient with loiasis infection.

Mouse models of Loa loa.

Elimination of the helminth disease, river blindness, remains challenging due to ivermectin treatment-associated adverse reactions in loiasis co-infected patients. Here, we address a deficit in preclinical research tools for filarial translational research by developing Loa loa mouse infection models. We demonstrate that adult Loa loa worms in subcutaneous tissues, circulating microfilariae (mf) and presence of filarial biomarkers in sera occur following experimental infections of lymphopenic mice deficient in interleukin (IL)-2/7 gamma-chain signaling. A microfilaraemic infection model is also achievable, utilizing immune-competent or -deficient mice infused with purified Loa mf. Ivermectin but not benzimidazole treatments induce rapid decline (>90%) in parasitaemias in microfilaraemic mice. We identify up-regulation of inflammatory markers associated with allergic type-2 immune responses and eosinophilia post-ivermectin treatment. Thus, we provide validation of murine research models to identify loiasis biomarkers, to counter-screen candidate river blindness cures and to interrogate the inflammatory etiology of loiasis ivermectin-associated adverse reactions.

Macro and microfilaricidal activities of extracts of Annona senegalensis and Milletia comosa against Onchocerca ochengi and Loa loa.

Despite the efforts employed for the control of onchocerciasis, the latter has remained a significant public health problem, due mainly to the lack of safe and effective adult worm drugs and/or microfilaricides that do not kill Loa loa microfilariae (mf). Serious adverse events have been encountered after administering ivermectin to some onchocerciasis patients coinfected with Loa loa. There is therefore, an urgent need for a macro and/or microfilaricidal drug which kills Onchocerca but not L. loa microfilariae. A total of 12 crude extracts from Milletia comosa and Annona senegalensis were prepared and screened in vitro against the bovine species of Onchocerca, O. ochengi, and L. loa mf from humans. Mf and male worm viabilities were determined by motility scoring using microscopy at 120 h of incubation with drug, while adult female worm viability and cytotoxicity were determined biochemically by MTT/formazan colorimetry after 120 h of incubation with drug. Out of the 12 extracts, all 6 from M. comosa and 4 from A. senegalensis were active against male, female and mf of O. ochengi. The hexane extract from M. comosa leaves (MCL hex) was the most active with IC50 values of 1.38, 0.86 and 17.74 µg/mL for O. ochengi adult males, adult female and the mf, respectively. About 58% of the extracts were more active against O. ochengi than L. loa mf. These results demonstrate that these extracts contain active principles that kill Onchocerca parasite and to a lesser extent L. loa, and suggest that they can be fractionated for isolation of lead molecules for the safe treatment of onchocerciasis.

Effect of flubendazole on developing stages of Loa loa in vitro and in vivo: a new approach for screening filaricidal agents.

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.

Comparison of different drug regimens for the treatment of loiasis-A TropNet retrospective study.

BACKGROUND: Loa loa infection is endemic in limited areas of West-Central Africa. Loiasis has been associated with excess mortality, but clinical studies on its treatment are scant, particularly outside endemic areas, due to the rarity of cases diagnosed. METHODOLOGY/PRINCIPAL FINDINGS: With this retrospective TropNet (European Network for Tropical Medicine and Travel Health) study, we aimed at outlining the treatment schedules followed by different reference centers for tropical medicine across Europe. We gathered information about 238 cases of loiasis, 165 of which had follow up data. The regimens followed by the different centers were heterogeneous. The drugs most frequently administered were: diethylcarbamazine alone (74/165, 45.1%), ivermectin alone (41/165, 25%), albendazole + ivermectin (21/164, 11.6%), ivermectin + diethylcarbamazine (16/165, 9.7%). CONCLUSIONS/SIGNIFICANCE: The management of loiasis substantially differs across specialized travel clinics in Europe. These discrepancies could be due to different local protocols as well as to (un)availability of the drugs. An harmonization of clinical protocols for the treatment of loiasis would be suggested across reference centers for tropical medicine in Europe.

Case Report: Monoclonal Gammopathy of Undetermined Significance is Associated with Loa loa Infection.

A 63-year-old woman who migrated from Nigeria to the United States was found to have an elevated total serum protein, anemia, and eosinophilia. Serum protein electrophoresis (SPEP) and serum protein immunofixation electrophoresis (SPIFE) demonstrated monoclonal immunoglobulin G (IgG) κ restricted bands (IgG 3,820 mg/dL; κ/λ ratio 4.47), indicative of monoclonal gammopathy of unknown significance (MGUS). A rapid diagnostic test (RDT) for malaria was positive for Plasmodium falciparum (BinaxNOW®; Alere Scarborough Inc., Scarborough, ME). Giemsa-stained blood smears were negative for malarial parasites, however, Loa loa microfilariae were identified. Reverse transcription polymerase chain reaction for P. falciparum, Plasmodium ovale, Plasmodium malariae, and Plasmodium vivax yielded a negative result. She was treated for loiasis with diethylcarbamazine and received no malaria medication. Treatment resulted in a resolution of the microfilaremia and eosinophilia, a negative RDT for malaria, and marked reduction in the monoclonal gammopathy. This is the first reported human case of MGUS associated with loiasis and its resolution after antiparasitic treatment.

Case Report: Probable Case of Spontaneous Encephalopathy Due to Loiasis and Dramatic Reduction of Loa loa Microfilariaemia with Prolonged Repeated Courses of Albendazole.

Loiasis is a vector-borne parasitic disease caused by the filarial nematode Loa loa and transmitted by the tabanid vectors from the genus Chrysops. Loa loa infection is associated with clinical manifestations such as pruritus, migratory transient edema, passage of adult worm in the bulbar conjunctiva, retinal damage, glomerular damage, albuminuria, pleural effusion, hydrocele, and endomyocardial fibrosis. Data reporting the occurrence of spontaneous encephalopathy associated with loiasis are very scanty. Severe adverse events occurring post-ivermectin administered in the framework of the fight against onchocerciasis and/or lymphatic filariasis in loiasis co-endemic areas have been closely associated with very high L. loa microfilariaemia. Different regimens have been used to lower L. loa microfilariaemia before definitive treatment, and many discrepancies have been reported. We report the case of a patient who was admitted to a health facility and hospitalized for 34 days for altered consciousness, blurred vision, headache, and chills. After other potential diagnoses were eliminated, the patient was confirmed with encephalopathy due to loiasis and referred to the Centre for Research on Filariasis and other Tropical Diseases (CRFilMT). On admission at CRFilMT, the patient was harboring 28,700 microfilariae per milliliter of blood (mf/mL), and after four 21-day courses of 400 mg daily albendazole, the L. loa microfilariaemia lowered to 5,060 mf/mL. The patient was then treated with ivermectin 3 mg and a total clearance of microfilariae was observed, with satisfactory clinical evolution and no adverse event. This case study further confirmed that albendazole is effective against L. loa, but might necessitate a longer course.

Heterogeneity in the in vitro susceptibility of Loa loa microfilariae to drugs commonly used in parasitological infections.

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 (CR50 = 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 (CR50 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.

Potential Role for Flubendazole in Limiting Filariasis Transmission: Observations of Microfilarial Sensitivity.

Flubendazole (FLBZ) is a potent and efficacious macrofilaricide after parenteral administration. Studies in animal models and one trial in patients infected with Onchocerca volvulus revealed that FLBZ elicits minimal effects on microfilariae (mf). Severe complications after ivermectin (IVM) treatment of patients with high Loa loa microfilaraemia are of great concern. We examined the potential of FLBZ to rapidly kill L. loa mf, the phenomenon proposed to underlie the complications. Mf of L. loa were exposed to FLBZ, its reduced metabolite, albendazole, or IVM in vitro. Viability of L. loa mf was unaffected by FLBZ (10 µM, 72 hours); similar results were obtained with mf of Brugia malayi. We also measured the effects of FLBZ on transmission of mf. Aedes aegypti were fed FLBZ-exposed B. malayi mf and dissected 24 hours or 14 days postfeeding to count mf that crossed the midgut and developed to infective L3. FLBZ impaired the ability of mf to cross the midgut, regardless of duration of exposure (≥ 2 hours). FLBZ also prevented the development of mf to L3s, irrespective of duration of exposure or concentration. FLBZ is not microfilaricidal under these conditions; however, it blocks transmission. These results support the possibility that FLBZ may be a useful macrofilaricide in loiasis regions and may limit transmission from treated individuals.

Loiasis in US Traveler Returning from Bioko Island, Equatorial Guinea, 2016.

The filarial parasite Loa loa overlaps geographically with Onchocera volvulus and Wuchereria bancrofti filariae in central Africa. Accurate information regarding this overlap is critical to elimination programs targeting O. volvulus and W. bancrofti. We describe a case of loiasis in a traveler returning from Bioko Island, Equatorial Guinea, a location heretofore unknown for L. loa transmission.

Protein Translation Enzyme lysyl-tRNA Synthetase Presents a New Target for Drug Development against Causative Agents of Loiasis and Schistosomiasis.

Helminth parasites are an assemblage of two major phyla of nematodes (also known as roundworms) and platyhelminths (also called flatworms). These parasites are a major human health burden, and infections caused by helminths are considered under neglected tropical diseases (NTDs). These infections are typified by limited clinical treatment options and threat of drug resistance. Aminoacyl-tRNA synthetases (aaRSs) are vital enzymes that decode genetic information and enable protein translation. The specific inhibition of pathogen aaRSs bores well for development of next generation anti-parasitics. Here, we have identified and annotated aaRSs and accessory proteins from Loa loa (nematode) and Schistosoma mansoni (flatworm) to provide a glimpse of these protein translation enzymes within these parasites. Using purified parasitic lysyl-tRNA synthetases (KRSs), we developed series of assays that address KRS enzymatic activity, oligomeric states, crystal structure and inhibition profiles. We show that L. loa and S. mansoni KRSs are potently inhibited by the fungal metabolite cladosporin. Our co-crystal structure of Loa loa KRS-cladosporin complex reveals key interacting residues and provides a platform for structure-based drug development. This work hence provides a new direction for both novel target discovery and inhibitor development against eukaryotic pathogens that include L. loa and S. mansoni.

Filaricidal activities on Onchocerca ochengi and Loa loa, toxicity and phytochemical screening of extracts of Tragia benthami and Piper umbellatum.

BACKGROUND: Onchocerciasis is the world's second leading infectious cause of blindness. Its control is currently hampered by the lack of a macrofilaricidal drug and by severe adverse events observed when the lone recommended microfilaricide, ivermectin is administered to individuals co-infected with Loa loa. Therefore, there is the need for a safe and effective macrofilaricidal drug that will be able to cure the infection and break transmission cycles, or at least, an alternative microfilaricide that does not kill L. loa microfilariae (mf). METHODS: Fourteen extracts from two medicinal plants, Tragia benthami and Piper umbellatum were screened in vitro against Onchocerca ochengi parasite and L. loa mf. Activities of extracts on male worms and microfilariae were assessed by motility reduction, while MTT/Formazan assay was used to assess biochemically the death of female worms. Cytotoxicity and acute toxicity of active extracts were tested on monkey kidney cells and Balb/c mice, respectively. RESULTS: At 500 µg/mL, all extracts showed 100 % activity on Onchocerca ochengi males and microfilariae, while 9 showed 100 % activity on female worms. The methylene chloride extract of Piper umbellatum leaves was the most active on adult male and female worms (IC50s: 16.63 µg/mL and 35.65 µg/mL, respectively). The three most active extracts on Onchocerca ochengi females were also highly active on Loa loa microfilariae, with IC50s of 35.12 - 13.9 µg/mL. Active extracts were generally more toxic to the worms than to cells and showed no acute toxicity to Balb/c mice. Phytochemical screening revealed the presence of saponins, steroids, tannins and flavanoids in the promising extracts. CONCLUSIONS: These results unfold potential sources of novel anti-Onchocerca lead compounds and validate the traditional use of the plants in onchocerciasis treatment.

Repurposing auranofin as a lead candidate for treatment of lymphatic filariasis and onchocerciasis.

Two major human diseases caused by filariid nematodes are onchocerciasis, or river blindness, and lymphatic filariasis, which can lead to elephantiasis. The drugs ivermectin, diethylcarbamazine (DEC), and albendazole are used in control programs for these diseases, but are mainly effective against the microfilarial stage and have minimal or no effect on adult worms. Adult Onchocerca volvulus and Brugia malayi worms (macrofilariae) can live for up to 15 years, reproducing and allowing the infection to persist in a population. Therefore, to support control or elimination of these two diseases, effective macrofilaricidal drugs are necessary, in addition to current drugs. In an effort to identify macrofilaricidal drugs, we screened an FDA-approved library with adult worms of Brugia spp. and Onchocerca ochengi, third-stage larvae (L3s) of Onchocerca volvulus, and the microfilariae of both O. ochengi and Loa loa. We found that auranofin, a gold-containing drug used for rheumatoid arthritis, was effective in vitro in killing both Brugia spp. and O. ochengi adult worms and in inhibiting the molting of L3s of O. volvulus with IC50 values in the low micromolar to nanomolar range. Auranofin had an approximately 43-fold higher IC50 against the microfilariae of L. loa compared with the IC50 for adult female O. ochengi, which may be beneficial if used in areas where Onchocerca and Brugia are co-endemic with L. loa, to prevent severe adverse reactions to the drug-induced death of L. loa microfilariae. Further testing indicated that auranofin is also effective in reducing Brugia adult worm burden in infected gerbils and that auranofin may be targeting the thioredoxin reductase in this nematode.

Absence of an association between Plasmodium falciparum infection and post-ivermectin Loa-related non-neurologic serious adverse events.

Although ivermectin treatment can induce serious adverse events (SAEs) in individuals harboring high Loa loa microfilaremia (mf), not all patients with high mf levels develop such reactions, suggesting that cofactors may be involved. A study was conducted in Cameroon to investigate the possible role of Plasmodium coinfection at the time of ivermectin treatment in the development of SAEs. Before their first ivermectin treatment, thick smears were obtained from 4,175 individuals to determine the burden of Plasmodium sp., L. loa, and Mansonella perstans. After treatment, 18 (4.3 per 1,000) patients developed a non-neurologic SAE. Logistic regression analysis, adjusting for age, sex, P. falciparum infection, and M. perstans infection intensities, confirmed that L. loa mf was the main risk factor for SAEs. We found no evidence that coinfection with P. falciparum at the time of ivermectin treatment was associated with the occurrence of Loa-related SAEs in this population.

Assessment of loiasis and outcomes of ivermectin masstreatment in Ijebu-North, Nigeria.

A total of 286 individuals from 3 selected communities (Areedi-Aje, Ipakodo/Ojokodo, and Ijebu-Igbo) of Ijebu-North, southwestern Nigeria were examined for Loa loa microfilaremia using finger prick blood smear, between December 2008 and March 2009. Rapid assessment procedure for loiasis (RAPLOA) was used to obtain information, from 187 Ijebu-Igbo residents, on adverse reactions experienced from retrospective treatments with ivermectin and history of eye worm. Only 33.9% of the respondents reported having had a history of eye worm while 33.2% had microfilaremia. The demographic factor of gender was not significant determinants of the prevalence (P>0.05) while age was significant (P<0.05). The highest prevalence of eye worm history and microfilaremia were recorded in 61-70 and 15-20 years of age categories, respectively. Ijebu-Igbo had 27.3% eye worm history, 32.1% microfilaremia, and the highest intensity of 140 microfilariae (mf)/ml. Ipakodo area had the highest eye worm history of 54.4% and the highest intensity of 420 mf/ml. Areedi-Aje had the highest occurrence of 45.2% microfilaremia and the highest intensity of 460 mf/ml. Predictably, Areedi-Aje and Ipakodo areas were high risk communities. The low intensity of L. loa infection with an insignificant (2.1%; P>0.05) adverse reactions from 187 subjects involved in the retrospective ivermectin administration confirmed that ivermectin delivery may be considered safe. The community-directed treatment with ivermectin (CDTI) programme was most probably responsible for the low prevalence and intensity.

Analysis of the mdr-1 gene in patients co-infected with Onchocerca volvulus and Loa loa who experienced a post-ivermectin serious adverse event.

Ivermectin (IVM) is exceptionally safe in humans, and is used for mass treatment of onchocerciasis and lymphatic filariasis. However, cases of encephalopathy, sometimes fatal, have been reported in a small number of individuals who harbored large numbers of Loa loa microfilariae (mf). A loss-of-function mutation in the mdr-1 gene in some dog breeds and in mice leads to accumulation of the drug in the brain, causing coma and death. This hypothesis was tested in four individuals from Cameroon who experienced a post-IVM serious adverse event (SAE) and in nine non-SAE matched controls. No loss-of-function mutation was detected in mdr-1 in any subject. However, haplotypes, associated with altered drug disposition, were present as homozygotes in two of the SAE patients (50%), but absent as homozygotes in the controls (0%). An association of high Loa mf load and a genetic predisposition to altered IVM distribution could be involved in IVM SAEs.

In vitro activities of plant extracts on human Loa loa isolates and cytotoxicity for eukaryotic cells.

Loa loa, a filarial worm, can cause fatal encephalitis in humans. In an attempt to find alternatives to the standard treatments (ivermectin and diethylcarbamazine citrate), we tested 12 methanolic extracts of nine traditional plant remedies. The extracts (100-0.09 microg/ml) were incubated with 20 Loa loa microfilariae isolated from patients at 37 degrees C with 5% CO(2) in modified Eagle's medium supplemented with 10% fetal serum and antibiotics. Activity was evaluated 120 h later by counting live microfilariae under a microscope. Cytotoxicity for eukaryotic cells was estimated by measuring 3-[4,5-dimethylthiazol-2-yl]-2-5 diphenyl tetrazolium bromide transformation to formazan at 450 nM in a spectrophotometer. The plants tested were Lophira alata, Greenwayodendron suaveolens, Uapaca togoensis, Zanthoxylum heitzii, Peperomia pellucida, Piptadeniastrum africanum, Petersianthus macrocarpus, Vernonia conferta, and Vernonia hymenolepis. Chemical screening showed that most of the extracts contained reducing sugars, tannin or polyphenols, sterols or triterpenes, saponosides, and alkaloids. None contained carotinoids and few contained flavonoids. The 50% lethal concentration ranged from 0.22 to 70.28 microg/ml, while the 50% inhibitory concentration for eukaryotic cells (IC(50)) ranged from 8.52 to 119.52 microg/ml. Extracts of P. macrocarpus (selectivity index = 72.16), P. africanum (13.69), Z. heitzii (12.11), and L. alata (9.26) were highly selective for L. loa.