Identification and Validation of Loa loa Microfilaria-Specific Biomarkers: a Rational Design Approach Using Proteomics and Novel Immunoassays.

UNLABELLED: Immunoassays are currently needed to quantify Loa loa microfilariae (mf). To address this need, we have conducted proteomic and bioinformatic analyses of proteins present in the urine of a Loa mf-infected patient and used this information to identify putative biomarkers produced by L. loa mf. In total, 70 of the 15,444 described putative L. loa proteins were identified. Of these 70, 18 were L. loa mf specific, and 2 of these 18 (LOAG_16297 and LOAG_17808) were biologically immunogenic. We developed novel reverse luciferase immunoprecipitation system (LIPS) immunoassays to quantify these 2 proteins in individual plasma samples. Levels of these 2 proteins in microfilaremic L. loa-infected patients were positively correlated to mf densities in the corresponding blood samples (r = 0.71 and P < 0.0001 for LOAG_16297 and r = 0.61 and P = 0.0002 for LOAG_17808). For LOAG_16297, the levels in plasma were significantly higher in Loa-infected (geometric mean [GM], 0.045 µg/ml) than in uninfected (P < 0.0001), Wuchereria bancrofti-infected (P = 0.0005), and Onchocerca volvulus-infected (P < 0.0001) individuals, whereas for LOAG_17808 protein, they were not significantly different between Loa-infected (GM, 0.123 µg/ml) and uninfected (P = 0.06) and W. bancrofti-infected (P = 0.32) individuals. Moreover, only LOAG_16297 showed clear discriminative ability between L. loa and the other potentially coendemic filariae. Indeed, the specificity of the LOAG_16297 reverse LIPS assay was 96% (with a sensitivity of 77%). Thus, LOAG_16297 is a very promising biomarker that will be exploited in a quantitative point-of-care immunoassay for determination of L. loa mf densities. IMPORTANCE: Loa loa, the causative agent of loiasis, is a parasitic nematode transmitted to humans by the tabanid Chrysops fly. Some individuals infected with L. loa microfilariae (mf) in high densities are known to experience post-ivermectin severe adverse events (SAEs [encephalopathy, coma, or death]). Thus, ivermectin-based mass drug administration (MDA) programs for onchocerciasis and for lymphatic filariasis control have been interrupted in parts of Africa where these filarial infections coexist with L. loa. To allow for implementation of MDA for onchocerciasis and lymphatic filariasis, tools that can accurately identify people at risk of developing post-ivermectin SAEs are needed. Our study, using host-based proteomics in combination with novel immunoassays, identified a single Loa-specific antigen (LOAG_16297) that can be used as a biomarker for the prediction of L. loa mf levels in the blood of infected patients. Therefore, the use of such biomarker could be important in the point-of-care assessment of L. loa mf densities.

Ecdysteroid-like compounds in the serum and urine of African patients infected with Loa loa and Mansonella perstans microfilariae.

Ecdysteroids are compounds related to 20-hydroxyecdysone, the insect moulting hormone. Surprisingly, they have been found in serum and urine of patients infected with helminths. In these cases, the substances are assumed to be produced by the parasites and, therefore, might be used as a marker of parasitic infection. Thus, we need to know exactly which species, at which developmental stage, can release ecdysteroids in such large quantities that they could be detected in the biological fluids of the host. Large-scale investigations must, accordingly, be devoted to the major species of helminths. In the present study, we examined 100 African patients with Loa loa and/or Mansonella perstans microfilaraemia. About 70 of them had high levels of ecdysteroid-like materials in serum or urine. In contrast, uninfected patients and European controls had much lower concentrations. However, the ecdysteroid titres did not reflect the concentration of microfilariae actually present in the blood, and some heavily infected patients were even negative. Nevertheless, the most important point was that high ecdysteroid levels in man were always associated with a pathological condition. The precise significance of the phenomenon should be determined.