Encephalopathy in a patient with loiasis treated with albendazole: A case report.

BACKGROUND: Loiasis is a vector-borne parasitic infection endemic across many areas of Central and West Africa. Its treatment is tricky due to the risk of serious neurologic adverse events occurring after the administration of microfilaricidal drugs, like diethylcarbamazine or ivermectin, in subjects with high pre-treatment microfilarial load. Albendazole is currently recommended to slowly reduce microfilaremia before curative regimen is prescribed. CASE PRESENTATION: We report the case of a 25-year-old man from Guinea-Conakry who was incidentally diagnosed with highly microfilaremic Loa loa infection. A three weeks regimen of albendazole was prescribed. Minor neurologic side effects occurred after two weeks of administration, while serious encephalopathy developed one week later. Clinical and electroencephalographic features of the patient resembled those of an immune-mediated encephalitis. After exclusion of other causes of encephalopathy, treatment-related Loa loa encephalopathy induced by albendazole was suspected. Corticosteroid treatment was administered and the patient recovered. DISCUSSION: Our case confirms that Loa loa treatment-related encephalopathy may occur even during albendazole treatment. The clinical and electroencephalographic similarities between Loa loa albendazole-related encephalopathy and immune-mediated encephalitis suggest the possibility of an underlying inflammation-based pathogenesis. Although corticosteroid administration is not recommended in Loa loa ivermectin-induced encephalopathy, in this case of Loa loa albendazole-induced encephalopathy it may have played a therapeutic role.

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.

Evaluation of in vitro culture systems for the maintenance of microfilariae and infective larvae of Loa loa.

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 (T90 = 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 (T90 = 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 (T90 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 (ß = 0.490), both IMDM (ß = 0.256) and DMEM (ß = 0.198) media and the protein supplements NCS (ß = 0.052) and FBS (ß = 0.022); while for L. loa L3, in addition to feeder cells (ß = 0.259) and both IMDM (ß = 0.401) and DMEM (ß = 0.385) media, the protein supplements BSA (ß = 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.

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.

CD4+CD25hiFOXP3+ cells in cord blood of neonates born from filaria infected mother are negatively associated with CD4+Tbet+ and CD4+RORγt+ T cells.

BACKGROUND: Children who have been exposed in utero to maternal filarial infection are immunologically less responsive to filarial antigens, have less pathology, and are more susceptible to acquire infection than offspring of uninfected mothers. Moreover children from filaria infected mothers have been shown to be less responsive to vaccination as a consequence of an impairment of their immune response. However, it is not well known how in utero exposure to parasite antigens affects cellular immune responses. METHODOLOGY: Here, 30 pregnant women were examined for the presence of microfilaria of Loa loa and Mansonella perstans in peripheral blood. At delivery, cord blood mononuclear cells (CBMC) were obtained and the CD4+T cells were phenotyped by expression of the transcription factors Tbet, RORγt, and FOXP3. RESULTS: No significant difference was observed between newborns from infected versus uninfected mothers in the frequencies of total CD4+T cells and CD4+T cells subsets including CD4+Tbet+, CD4+RORγt+ T and CD4+CD25hiFOXP3+ T cells. However, there was a negative association between CD4+CD25hiFOXP3+T cells and CD4+Tbet+ as well as CD4+RORγt+ T cells in the infected group only (B = -0.242, P = 0.002; B = -0.178, P = 0.013 respectively). CONCLUSION: Our results suggest that filarial infection during pregnancy leads to an expansion of functionally active regulatory T cells that keep TH1 and TH17 in check.

The human parasite Loa loa in cytokine and cytokine receptor gene knock out BALB/c mice: survival, development and localization.

BACKGROUND: Immunological mechanisms involved in the survival and development of human filarial species in the vertebrate host are poorly known due to the lack of suitable experimental models. In order to understand the role of cytokines in the survival and development of filarial larvae in the vertebrate host, we infected different strains of BALB/c mice deficient in a number of cytokine or cytokine receptor genes with Loa loa. The survival and development of larvae were monitored. METHODS: BALB/c mice genetically deficient in IL-4R, IFN-γ, IFN-γ/IL-5, IL-5, and IL-4R/IL-5 cytokine or cytokine receptor genes were infected with a human strain of L. loa and necropsies were performed at different time intervals up to 70 days post infection to monitor the survival and development of L. loa larvae. The larvae were teased out of the skin, muscles, peritoneal and pleural cavities, heart and lung tissues. The length and width of the recovered larvae were measured to assess their growth. RESULTS: In mice deficient for IL-4R, IFN-γ, IFN-γ/IL-5, IL-5 and IL-4R/IL-5, the larvae survived up to 5, 20, 40, 50 and 70 days respectively. Worms recovered 70 days post infection in IL-4R/IL-5 DKO mice were young adults and measured 10.12 mm in length and 0.1 mm in width. Overall, 47% of larvae were recovered from subcutaneous tissues, 40% from muscles, 6% from the peritoneal cavity and 4% from the pleural cavity, lungs and heart. CONCLUSION: L. loa exhibits a differential survival and development in different strains of cytokine or cytokine receptor gene knockout mice with IL-4R and IL-5 playing critical roles in the host resistance to L. loa infection. The knock out BALB/c mouse therefore represents a useful tool to explore the key effectors of adaptive immunity involved in the killing of the L. loa parasite in a mammal host.

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.

Encephalopathy after ivermectin treatment in a patient infected with Loa loa and Plasmodium spp.

Despite over 350 million people being safely treated with ivermectin, there have been rare cases of death post-treatment; these events are most often associated with high Loa loa microfilaremia. This first autopsy description of an encephalopathy case following the administration of ivermectin involves a 45-year-old male who became comatose 3 days after treatment. He slowly deteriorated over 5 weeks and died at 54 days after the anthelminthic treatment, probably as a result of a secondary skin or pulmonary infection exacerbated by malnutrition. The major pre- and post-autopsy findings included the presence of high loads of Loa loa, positivity for Plasmodium, the presence of a longstanding respiratory condition, and vascular pathology in the brain. The central nervous system lesions have similarities with those described in previously reported cases of Loa loa-associated death following diethylcarbamazine treatment.

Paciente procedente de la República del Congo con dolor ocular intermitente / Patient from the Republic of Congo with intermittent ocular pain

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Microfilarial distribution of Loa loa in the human host: population dynamics and epidemiological implications.

Severe adverse events (SAEs) following ivermectin treatment may occur in people harbouring high Loa loa microfilarial (mf) densities. In the context of mass ivermectin distribution for onchocerciasis control in Africa, it is crucial to define precisely the geographical distribution of L. loa in relation to that of Onchocerca volvulus and predict the prevalence of heavy infections. To this end, we analysed the distribution of mf loads in 4183 individuals living in 36 villages of central Cameroon. Mf loads were assessed quantitatively by calibrated blood smears, collected prior to ivermectin distribution. We explored the pattern of L. loa mf aggregation by fitting the (zero-truncated) negative binomial distribution and estimating its overdispersion parameter k by maximum likelihood. The value of k varied around 0.3 independently of mf intensity, host age, village and endemicity level. Based on these results, we developed a semi-empirical model to predict the prevalence of heavy L. loa mf loads in a community given its overall mf prevalence. If validated at the continental scale and linked to predictive spatial models of loiasis distribution, this approach would be particularly useful for optimizing the identification of areas at risk of SAEs and providing estimates of populations at risk in localities where L. loa and O. volvulus are co-endemic.

Loiasis.

Loiasis affects millions of individuals living in the forest and savannah regions of Central Africa. In some areas, this disease constitutes one of the most common reasons for medical consultation. The burden posed by loiasis is probably under-estimated and, in addition, individuals harbouring high Loa microfilarial loads are at risk of developing serious neurological reactions after treatment with diethylcarbamazine or ivermectin. These events are currently significantly hampering the development of the African Programme for Onchocerciasis Control, and operational research is required to address the issue. The results of recent studies, involving either human populations from endemic areas or monkey models, have provided much more detail of the mechanisms associated with amicrofilaraemic or so-called 'occult' loiasis. New diagnostic tools have also been developed in the last decade, and various protocols are now available for the risk-free treatment of loiasis cases.

Loa loa in the anterior chamber of the eye: a case report.

An unusual case of loiasis from Assam is reported here. Loa loa is a subcutaneous filarial parasite of man and is transmitted to humans by chrysops flies. The patient presented with foreign body sensation and visual disturbances of the right eye. Examination revealed a white coiled structure in the cornea. Routine blood and other investigations were within normal limits. A live adult worm was extracted and identity was confirmed by microscopy to be Loa loa. Patient was treated with diethylcarbamazine and steroid. We found this case interesting as the worm was present in the anterior chamber--an unusual site and there were no other positive findings besides the lone worm.

Human loiasis in a Cameroonian village: a double-blind, placebo-controlled, crossover clinical trial of a three-day albendazole regimen.

Because of the life-threatening, post-treatment reactions that have occurred in patients with loiasis treated with ivermectin, evaluation of a short-course albendazole regimen was undertaken in a Loa-endemic region of Cameroon. In a placebo-controlled, double-blinded, crossover study, 99 subjects with microfilaremia (100-3,3837/mL) were assigned to receive albendazole (400 mg; n = 48) or placebo (n = 51) for three days and were followed for 180 days; at day 180, the groups were crossed over and followed for an additional six months. In those initially receiving albendazole (ALB/PLAC), microfilarial levels decreased significantly by day 90 (P < 0.043), but returned to baseline by day 180. In those receiving albendazole at day 180 (PLAC/ALB), microfilarial levels also decreased following albendazole (P = 0.005). Blood eosinophil and antifilarial IgG levels did not change significantly for either group, although antifilarial IgG4 levels did in the ALB/PLAC group at day 180. Most subjects continued to have elevations in microfilaremia, suggesting that more intensive regimens of albendazole will be necessary to reduce Loa microfilaremia to levels safe enough to allow for ivermectin use.

Identification and specificity of a 38 kDa Loa loa antigenic fraction in sera from high-microfilaraemic Gabonese patients.

Circulating antigens isolated from sera of three high-microfilaraemic ( Loa loa) Gabonese patients were fractionated by gel filtration. A major component (38 kDa) was identified after SDS-PAGE and immunoblotting using sera of amicrofilaraemic patients with high level of antimicrofilariae Loa loa antibodies. The 38 kDa fraction was not found in the sera of parasitised patients or healthy controls. We looked for the 38 kDa antigen in the various stages of the filarial life cycle and found it in extracts of Loa loa microfilariae but not in somatic extracts of Loa loa male and female adult worms. This fraction could be used as a diagnostic marker in loiasis for amicrofilaraemic patients.

Parasitological and immunological effects induced by immunization of Mandrillus sphinx against the human filarial Loa loa using infective stage larvae irradiated at 40 Krad.

Six mandrills were immunized with 150 Loa loa infective stage larvae (L3) irradiated with 40 Krad, and challenged with 100 L3, 60 days after initial vaccination. The parasitological outcome of this immunization was compared to results from six mandrills infected with normal L3. No clear association was seen between vaccination and microfilaremia until day 245 when a significant drop in the level of microfilaria occurred in vaccinated compared to infected animals (5 vs 10 mf/ml; p = 0.012). A one-year follow-up of the humoral immune response showed a strong adult, microfilariae (Mf) and L3 specific IgG response, with distinct profiles for each extract. In immunized animal a significant decrease in antibody level was systematically observed between days 90-145 for the anti-L3 and anti-adult IgG. However, in the same group anti-Mf antibody levels that peaked around 160-175 days post-challenge, were inversely correlated with the decrease in Mf density between day 200 and day 386. These results suggest that immunization with irradiated L3 using these specific conditions may affect the appearance of Mf.

Expression of filarial-specific IgG subclasses under different transmission intensities in a region endemic for loiasis.

Specific IgG subclasses were investigated in two villages (Okoumbi and Ndjokaye) in southeast Gabon with different Loa loa transmission intensities of approximately 9,000 and 1,300 infective larvae (L3) per person per year, respectively. IgG subclasses were measured by an enzyme-linked immunosorbent assay (ELISA) using extracts of L. loa L3, microfilariae (MF), or adult worms. Levels of L3-specific IgG3 were significantly higher in the village with low transmission (Ndjokaye) (P = 0.006). In contrast, MF-specific IgG2 was significantly higher in Okoumbi than in Ndjokaye (P = 0.0009). In the high-transmission village (Okoumbi), levels of both MF- and adult-specific IgG4 were significantly increased in MF carriers compared with amicrofilaremic subjects (P = 0.0015 and P = 0.003, respectively), while levels of L3- and MF-specific IgG1 were significantly higher in amicrofilaremic individuals compared with MF carriers (P = 0.04 and P = 0.03, respectively). Furthermore, among microfilaremic individuals, the level of the specific IgG1 subclass was much lower in Okoumbi than in Ndjokaye (P = 0.036). These results suggest that the expression of antigen-specific IgG3 and IgG2 is more likely to vary with transmission intensity, whereas antigen-specific IgG4 and IgG1 varies with adult worm and MF burden.

Subconjunctival Loa Loa worm: case report.

A 38-year old Ghanaian suddenly had the sensation of a foreign body in his right eye. Slit-lamp examination revealed a transparent worm underneath the conjunctiva. With topical anesthesia, the complete worm, 3.5 cm long, was removed surgically. A microbiological analysis at the Institute of Tropical Medicine confirmed the diagnosis of Loa Loa. Laboratory tests showed negative blood eosinophilia, positive blood film examination for microfilariae and positive results for filarial serology. The postoperative treatment consisted of progressive doses of di-ethyl-carbamazine (50-->100-->200 mg/d). A subconjunctival Loa Loa worm is rare in Belgium and usually occurs in immigrants or travellers returning from Tropical (Equatorial) West and Central Africa. Our patient visited Nigeria in 1985 and Ivory Coast in 1986. Those regions are highly endemic for Loa Loa.