Urine metabolites for the identification of Onchocerca volvulus infections in patients from Cameroon.

BACKGROUND: The tropical disease onchocerciasis (river blindness), caused by Onchocerca volvulus filarial nematodes, is targeted for elimination by mass treatment with nematocidal and antimicrobial drugs. Diagnosis of O. volvulus infections is based on counts of skin-borne microfilariae, but additional diagnostic tools, e.g. worm- or host-derived small RNAs, proteins or metabolites, are required for high-throughput screening. N-acetyltyramine-O,ß-glucuronide (NATOG) was suggested as a biomarker for onchocerciasis but its viability as diagnostic tool has been challenged. METHODS: We performed a screening program of urine samples from individuals from Cameroon infected with O. volvulus, Loa loa, Mansonella perstans or a combination thereof. Urine metabolites were measured by liquid chromatography-mass spectrometry (LC-MS). Principle component analysis (PCA) revealed that onchocerciasis causes complex changes of the urine metabolome. RESULTS: The mean NATOG content was elevated in urine of O. volvulus-infected compared with non-infected individuals, but NATOG levels showed considerable variation. However, 13.8% of all O. volvulus-infected individuals had high NATOG levels never reached by individuals without filarial infections or only infected with L. loa or M. perstans. Therefore, the identification of individuals with high NATOG levels might be used to screen for the elimination of onchocerciasis after mass drug application. Additional metabolites, including a compound identified as cinnamoylglycine, had high PC1/PC2 loadings in the data set. Mean levels of cinnamoylglycine were increased in O. volvulus-infected individuals, and 17.2% of all O. volvulus individuals had elevated cinnamoylglycine levels not reached by the controls. CONCLUSIONS: On an individual level, NATOG alone had poor discriminative power distinguishing infected from non-infected individuals. However, 13.8% of all O. volvulus-infected individuals had NATOG levels never reached by individuals without filarial infections or infected with only L. loa or M. perstans. Discrimination of O. volvulus infections from controls or individuals suffering from multiple infections was improved by the measurement of additional metabolites, e.g. cinnamoylglycine. Thus, measuring a combination of urine metabolites may provide a way to assess onchocerciasis on the population level. This provides the possibility to design a strategy for large-scale onchocerciasis epidemiological screening programs based on urine rather than invasive techniques.

Onchocerca volvulus-neurotransmitter tyramine is a biomarker for river blindness.

Onchocerciasis, also known as "river blindness", is a neglected tropical disease infecting millions of people mainly in Africa and the Middle East but also in South America and Central America. Disease infectivity initiates from the filarial parasitic nematode Onchocerca volvulus, which is transmitted by the blackfly vector Simulium sp. carrying infectious third-stage larvae. Ivermectin has controlled transmission of microfilariae, with an African Program elimination target date of 2025. However, there is currently no point-of-care diagnostic that can distinguish the burden of infection--including active and/or past infection--and enable the elimination program to be effectively monitored. Here, we describe how liquid chromatography-MS-based urine metabolome analysis can be exploited for the identification of a unique biomarker, N-acetyltyramine-O,ß-glucuronide (NATOG), a neurotransmitter-derived secretion metabolite from O. volvulus. The regulation of this tyramine neurotransmitter was found to be linked to patient disease infection, including the controversial antibiotic doxycycline treatment that has been shown to both sterilize and kill adult female worms. Further clues to its regulation have been elucidated through biosynthetic pathway determination within the nematode and its human host. Our results demonstrate that NATOG tracks O. volvulus metabolism in both worms and humans, and thus can be considered a host-specific biomarker for onchocerciasis progression. Liquid chromatography-MS-based urine metabolome analysis discovery of NATOG not only has broad implications for a noninvasive host-specific onchocerciasis diagnostic but provides a basis for the metabolome mining of other neglected tropical diseases for the discovery of distinct biomarkers and monitoring of disease progression.

Novel, sensitive and low-cost diagnostic tests for 'river blindness'--detection of specific antigens in tears, urine and dermal fluid.

Sensitive, specific and low-cost diagnostic tests for onchocerciasis are indispensable for monitoring the efficacy of control programs, as well as for preventing blindness (when the tests are combined with efficacious chemotherapy. Three new tests to detect Onchocerca-specific antigens in tears, dermal fluid and urine employ antibodies to O. volvulus-specific recombinant proteins, Oncho-C27 and OvD3B, encoded by genes within the immunodominant Onchocerca OV 33-3 gene family, and expressed in yeast and in E. coli, respectively. In these assays, Onchocerca-specific antigens in test samples are bound onto a solid surface and revealed using appropriate enzyme-labelled antibodies. Proteins in the samples are first transferred to Hybond-N + membrane disks or nitrocellulose paper using either a transblot or a dotblot machine, and then reacted with specific O. volvulus antibodies. Bound antibodies are revealed with species-specific peroxidase-labelled antibodies and peroxidase substrate. Positive tests give a brown colour. In one of the two assays developed to detect Onchocerca antigens in tears, the sensitivity was enhanced by first adsorbing the specific antibodies onto the membrane surface in order to immobilize and concentrate the Onchocerca-specific antigen molecules on the membrane. The specificity of the recombinant proteins for Onchocerca volvulus had been verified by ELISA, classical Western blot and modified DSIA. The tests are a dipstick immunobinding assay for ocular microfilariae (DSIA), a transblot immunobinding assay for the detection of skin microfilariae (TADA) and a dot-blot immunobinding assay for detecting urinary microfilariae and their antigens (DIA). Their specificity and sensitivity were evaluated in the field on 110 subjects with proven ocular microfilariae, 130 subjects with clinical and parasitological evidence of onchocerciasis, 25 subjects infected with other helminths and 120 normal controls. The minimal detection limits of Oncho-C27 protein by DSIA, TADA and DIA were 500 ng/ml, 154 ng/ml and 508 ng/ml, respectively By contrast, their sensitivities were: 100% for DSIA and 82.5% for TADA employed on samples of tears; 97% for TADA skin test and 96% for DIA used on urine samples.