Tandem Use of OvMANE1 and Ov-16 ELISA Tests Increases the Sensitivity for the Diagnosis of Human Onchocerciasis

Abstract

The current serological test for human onchocerciasis relies on IgG4 reactivity against the parasite Ov-16 antigen, with reported sensitivities of only 60-80%. As control programs move from control to elimination, it is imperative to identify novel molecules that could improve the serodiagnosis reliability of this disease. In this study we compared the sensitivity of total IgG against OvMANE1-a chimeric antigen previously identified as a potential biomarker of human onchocerciasis-with that of an Ov-16 antibody test to detect an Onchocerca volvulus infection in persons presenting with microfilaria in skin snips. One hundred and ninety serum samples were obtained from persons with epilepsy in an onchocerciasis-endemic area at Ituri in the Democratic Republic of Congo where ivermectin has never been distributed. Fifty-nine (31.1%) samples were from individuals with a positive skin snip test; 41 (69.5%) of these 59 samples were positive with the OvMANE1 test and 41 (69.5%) with the Ov-16 test; 30 (50.8%) samples were positive for both tests and in 52 (88.1%) at least one of the tests was positive. Testing the 131 sera from persons with a negative skin snip result revealed that 63 (48.1%) were positive exclusively with the OvMANE1 test, 13 (9.9%) exclusively with the Ov-16 test and 25 (19.1%) with both tests. Nine European samples from individuals without past travel history in onchocerciasis endemic zones and 15 samples from Rwanda, a hypoendemic country for onchocerciasis were all negative for the OvMANE1 and Ov-16 tests. However, the specificity of both tests was difficult to determine due to the lack of a gold standard for antibody tests. In conclusion, the tandem use of OvMANE1 and Ov-16 tests improves the sensitivity of detecting Onchocerca volvulus seropositive individuals but, the OvMANE1 test needs to be further evaluated on samples from a population infected with other helminths to cautiously address its specificity.

Keywords: ELISA; Onchocerca volvulus; Ov-16; OvMANE1; antibodies; diagnostic; sensitivity.

Figure 1

Analysis of humoral immune response to OvMANE1 and Ov-16 antigens using sera from O. volvulus infected and non-infected individuals. In separate reactions, OvMANE1_MBP and Ov-16 antigens were used to coat microtiter plates. The plates were blocked and incubated with different serum samples followed by incubation with secondary antibodies, namely goat anti-human IgG (for plates coated with OvMANE1_MBP chimeric antigen) or mouse monoclonal anti-Human IgG4 (for plates coated with Ov-16 antigen), all peroxidase conjugated. The plates were revealed using TMB, the optical density (OD) was read at 450 nm and OD values were plotted against the different serum types. OVS = O. volvulus serum (n = 59), HES = Hypo-endemic serum (n = 15), ECS = European control serum (n = 09). The groups were compared using Kruskal Wallis test followed by Dunn’s test for multiple comparisons. **** indicates a significant difference with p < 0.0001.

Figure 2

Analysis of humoral immune response to OvMANE1 and Ov-16 antigens using O. volvulus serum (OVS, n = 22), which were either positive according to OvMANE1 test (n = 11) but not Ov-16 test (n = 11) and vice versa. (A) optical densities (OD) at 450 nm were plotted against OvMANE1 and Ov-16 tests using color codes and shapes to match the specific serum samples in both tests. The average microfilaria (mf) load per skin snip was plotted against (B) the OD of OVS in OvMANE1 test and (C) the OD of OVS in Ov-16 test using color codes and shapes that matches the specific serum sample. Color codes were assigned to each sample to clearly differentiate the OD of the sample in the different tests.

Figure 2

Analysis of humoral immune response to OvMANE1 and Ov-16 antigens using O. volvulus serum (OVS, n = 22), which were either positive according to OvMANE1 test (n = 11) but not Ov-16 test (n = 11) and vice versa. (A) optical densities (OD) at 450 nm were plotted against OvMANE1 and Ov-16 tests using color codes and shapes to match the specific serum samples in both tests. The average microfilaria (mf) load per skin snip was plotted against (B) the OD of OVS in OvMANE1 test and (C) the OD of OVS in Ov-16 test using color codes and shapes that matches the specific serum sample. Color codes were assigned to each sample to clearly differentiate the OD of the sample in the different tests.