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Onchocerciasis is a filarial vector borne disease which affects several million people mostly in Africa. The therapeutic approach of its control was based on a succession of drugs which always showed limits. The last one: ivermectin is not the least. It was shown to be only microfilaricidal and induced resistance to the human parasite Onchocerca volvulus. The approach using medicinal plants used in traditional medicine is a possible alternative method to cure onchocerciasis. Onchocerca ochengi and Onchocerca gutturosa are the parasite models used to assess anthelmintic activity of potentially anthelmintic plants. Numerous studies assessed the in vitro and/or in vivo anthelmintic activity of medicinal plants. Online electronic databases were consulted to gather publications on in vitro and in vivo studies of anti-Onchocerca activity of plants from 1990 to 2017. Globally, 13 plant families were investigated for anti-Onchocerca activity in 13 studies. The most active species were Anacardium occidentale, Euphorbia hirta and Acacia nilotica each with an LC 50 value of 2.76, 6.25 and 1.2 μg/mL, respectively. Polycarpol, voacamine, voacangine, ellagic acid, gallic acid, gentisic acid, 3-O-acetyl aleuritolic acid and (-)-epigallocatechin 3-O-gallate were the isolated plant compounds with anti-Onchocerca activity. Most of the assessed extract/compounds showed a good safety after in vivo acute toxicity assays and/or in vitro cytotoxicity test. The exception was the ethanol extract of Trichilia emetica, which killed completely and drastically mice at a dose of 3000 mg/kg. Several plant groups of compounds were shown active against Onchocerca sp. such as tannins, alkaloids, triterpenoids and essential oils. Nevertheless, none of the active compounds was subjected to clinical trial, to assessment of its diffusibility through nodular wall or its capability to induce genetic resistance of Onchocerca sp.

River blindness, caused by infection with the filarial nematode Onchocerca volvulus, is a neglected tropical disease affecting millions of people. There is a clear need for diagnostic tools capable of identifying infected patients, but that can also be used for monitoring disease progression and treatment efficacy. Plasma-derived parasitic microRNAs have been suggested as potential candidates for such diagnostic tools. We have investigated whether these parasitic microRNAs are present in sufficient quantity in plasma of Onchocerca-infected patients to be used as a diagnostic biomarker for detection of O. volvulus infection or treatment monitoring. Plasma samples were collected from different sources (23 nodule-positive individuals and 20 microfilaridermic individuals), microRNAs (miRNAs) were extracted using Qiagen miRNeasy kit, and a set of 17 parasitic miRNAs was evaluated on these miRNA extracts using miRCURY Locked Nucleic Acid (LNA) Universal RT microRNA PCR system. Of the 17 miRNAs evaluated, only 7 miRNAs were found to show detectable signal in a number of samples: bma-miR-236-1, bma-miR-71, ov-miR71-22nt, ov-miR-71-23nt, ov-miR-100d, ov-bantam-a, and ov-miR-87-3p. Subsequent melting curve analysis, however, indicated that the signals observed for ov-miR-71 variants and ov-miR-87-3p are non-specific. The other miRNAs only showed positive signal in one or few samples with Cq values just below the cutoff. Our data indicate that parasitic miRNAs are not present in circulation at a sufficiently high level to be used as biomarker for O. volvulus infection or treatment monitoring using LNA-based RT-qPCR analysis.

The posttreatment entomological surveillance (ES) of onchocerciasis in Latin America requires quite large numbers of flies to be examined for parasite infection to prove that the control strategies have worked and that the infection is on the path of elimination. Here, we report a high-throughput automated DNA isolation of Onchocerca volvulus for PCR using a major Latin American black fly vector of onchocerciasis. The sensitivity and relative effectiveness of silica-coated paramagnetic beads was evaluated in comparison with phenol chloroform (PC) method which is known as the gold standard of DNA extraction for ES in Latin America. The automated method was optimized in the laboratory and validated in the field to detect parasite DNA in Simulium ochraceum sensu lato flies in comparison with PC. The optimization of the automated method showed that it is sensitive to detect O. volvulus with a pool size of 100 flies as compared with PC which utilizes 50 flies pool size. The validation of the automated method in comparison with PC in an endemic community showed that 5/67 and 3/134 heads pools were positive for the two methods, respectively. There was no statistical variation (P < 0.05) in the estimation of transmission indices generated by automated method when compared with PC method. The fact that the automated method is sensitive to pool size up to 100 confers advantage over PC method and can, therefore, be employed in large-scale ES of onchocerciasis transmission in endemic areas of Latin America.

Despite successful mass drug administration and vector control programs, the nematode Onchocerca volvulus is far from being eradicated. Therefore, new long-term sterilizing or macrofilaricidal drugs are needed. The depletion of Wolbachia endobacteria using doxycycline leads to long-term sterilizing effects and macrofilaricidal activity against female filariae of more than 60%. The worms die or degenerate 18-27 months after doxycycline. However, during this time patients may be exposed to new infections. We evaluated these newly acquired worms in onchocercomas of doxycycline-treated patients in relation to transmission using morphology, histochemistry, and immunohistology. On an average, 10% of the female filariae had been newly acquired per year in the treated groups. Our observations showed: (a) Sixty-three of 68 newly acquired worms harbored many intact Wolbachia, whereas none of the other surviving worms contained many bacteria. (b) Higher percentages of dead filariae than originally reported were calculated, when the new worms were excluded, indicating a stronger macrofilaricidal activity than previously reported. The difference was significant for female filariae after doxycycline treatment for 6 weeks. (c) Only newly acquired worms presented normal embryogenesis and microfilariae production after sufficient treatment. We conclude that newly acquired filariae have to be considered when evaluating drug efficacy in onchocerciasis.

Drugs exist that show long-lasting inhibition of embryogenesis and microfilaria production or macrofilaricidal activity against Onchocerca volvulus. Therefore, the patients have to be followed-up for several years. Clinical drug trials have to be performed in areas with ongoing transmission to assess the efficacy on younger worms. In addition, future vaccine trials may also require demonstrating efficacy against establishment of new worms. For the evaluation of the efficacy, it is necessary to differentiate between older worms, which were exposed to the drug, and younger worms newly acquired after drug treatment or vaccination. Here, we describe criteria for the differentiation between young and old filariae based on histological studies of worms with a known age from travellers, or from children, or patients living in areas with interrupted transmission in Burkina Faso, Ghana or Uganda. Older worms were larger and presented degenerated tissues. Gomori's iron stain showed that the worms accumulated more iron with increasing age, first in the gut and later in other organs. Using an antibody against O. volvulus lysosomal aspartic protease, the gut of young worms was stained only weakly; whereas, it was stronger labelled in older worms, accompanied by additional staining of hypodermis and epithelia. Using morphological and immunohistological criteria, it was possible to differentiate young (1-3 years old) from older females and to identify young males.

Onchocercomata with a single live or dead worm were analyzed to elucidate the infiltration of eosinophils. Females were classified according to the presence or absence of microfilariae in their uteri and in the nodular tissues. Immunohistochemical staining was performed using antibodies against eosinophil cationic protein, peroxidase, and major basic protein. Very few eosinophils were detected in nodules containing females without microfilariae or male or dead worms only, whereas eosinophils were abundant in all nodules with females producing microfilariae. The occurrence of eosinophils was not related to the age of the worm. Occasionally, degenerated or dead microfilariae attacked by activated eosinophils were found. By examination of onchocercomata with or without microfilariae from the same patient, it was excluded that the occurrence of eosinophils was dependent mainly on the host's immune status. In conclusion, live adult Onchocerca volvulus do not elicit an invasion of eosinophils as long as they do not produce microfilariae. The absence of eosinophilia does not exclude onchocerciasis.

A polymerase chain reaction (PCR) of a 150-bp tandem repeat of Onchocerca volvulus (O-150) combined with Southern-blot hybridization to species-specific DNA probes was employed for DNA detection. O-150 was amplified from parasites originating from Uganda, Benin, Cameroon, Liberia, Ghana, Burkina Faso, Mali, and Zaire and was successfully hybridized to digoxigenin-labeled oligonucleotides. To investigate the sensitivity of the PCR, 2 skin biopsies were taken from each of 227 persons from Uganda with proven O. volvulus infections but with low microfilaria (mf) densities due to ivermectin treatment. One biopsy was tested by PCR and the other was digested using collagenase to assess the total number of mf. The PCR revealed 76.2% of the samples to be positive, and the collagenase method showed that 78.9% were positive, indicating similar sensitivity for the two methods. It is probable that for both techniques the biopsy must contain at least one live mf or fragments of a dead mf. In this study, no free or circulating O. volvulus DNA could be detected in skin biopsies by PCR.

The characteristics and kinetic properties of an arylalkylamine N-acetyltransferase were studied in partially purified preparations of the human filarial parasite Onchocerca volvulus. The enzyme, which had a relative molecular mass (M(r)) of 37-38 kDa, catalyzed the acetylation of arylalkylamines but did not accept arylamines or polyamines as substrates. The optimal pH for enzyme activity was found to be 8.5 in TRIS-HCI. The apparent Michaelis constant (K(m)) and maximum velocity (Vmax) determined from Lineweaver-Burk plots for tryptamine were 1.8 microM and 29 nmol min-1 mg protein-1, respectively. Except for the catecholamines, the other arylalkylamines such as 5-hydroxytryptamine (5-HT), tyramine, and octopamine similarly exhibited high affinities and reaction rates. Whereas the enzyme is inhibited by metals and p-chloro-mercuribenzoate, it is inactivated neither by amethopterin nor by cystamine and is thereby distinguished from the mammalian arylamine N-acetyltransferase. Like other N-acetyltransferases whose function is the regulation of intracellular amine levels, the enzyme may have a role in the inactivation of excess biogenic amine in this parasite.