Integrating Multiple Biomarkers to Increase Sensitivity for the Detection of Onchocerca volvulus Infection

Abstract

Background: Serological assessments for human onchocerciasis are based on IgG4 reactivity against the OV-16 antigen, with sensitivities of 60-80%. We have previously identified 7 novel proteins that could improve serodiagnosis.

Methods: IgG4 responses to these 7 proteins were assessed by luciferase immunoprecipitation (LIPS) and enzyme-linked immunosorbent (ELISA) immunoassays.

Results: OVOC10469 and OVOC3261 were identified as the most promising candidates by IgG4-based immunoassays with sensitivities of 53% for rOVOC10469 and 78% for rOVOC3261 while specificity for each was >99%. These 2 antigens in combination with OV-16 increased the sensitivity for patent infections to 94%. The kinetics of appearance of these IgG4 responses based on experimentally infected non-human primates indicated that they were microfilarial- driven. Further, the IgG4 responses to both OVOC10469 and OVOC3261 (as well as to OV-16) drop significantly (p<0.05) following successful treatment for onchocerciasis. A prototype lateral flow rapid diagnostic test to detect IgG4 to both Ov-16 and OVOC3261 was developed and tested demonstrating an overall 94% sensitivity.

Conclusion: The combined use of rOVOC3261 with OV-16 improved serologic assessment of O. volvulus infection, a current unmet need toward the goal of elimination of transmission of O. volvulus.

Keywords: OV-16; OVOC3261; biomarkers; diagnostics; onchocerciasis.

Figure 1.

Screening of OVOC biomarkers by luciferase immunoprecipitation system (LIPS). The dot plots show the IgG4 net reactivity (value + 100) of the individual sera from Onchocerca volvulus-infected and microfilariae-positive (INF), endemic normal (EN), healthy US blood bank volunteers (BB), and Wuchereria bancrofti/ Loa loa/ Strongyloides stercoralis infections (other helminth) in a LIPS assay to OV-16 (A), OVOC10469 (B), OVOC3261 (C), and OVOC5127 (D). Dotted red lines denote the receiver operating characteristic (ROC) cutoff values (based on 100% specificity using the other helminth group as control). Sensitivity values are based on ROC cutoff with 100% specificity. The horizontal red bars denote geometric means. Negative values were assigned zero prior to adding 100 for all values. The values denote the net relative light units (RLU).

Figure 2.

Validation of combinations of biomarkers by luciferase immunoprecipitation system (LIPS). The dot plots show the IgG4 net reactivity (value + 100) of the individual sera from Onchocerca volvulus-infected and microfilariae-positive (INF), endemic normal (EN), healthy US blood bank volunteers (BB), and Wuchereria bancrofti/ Loa loa/ Strongyloides stercoralis infections (other helminth) in a LIPS assay to OV-16 in combination with OVOC10469 (A), OVOC3261 (B), and OVOC10469 and OVOC3261 (C). The dotted red lines denote the receiver operating characteristic (ROC) cutoff values (based on 100% specificity using the other helminth group as control). Sensitivity values are based on ROC cutoff with 100% specificity. The horizontal red bars denote the geometric means. Negative values were assigned zero prior to adding 100 for all values. The values denote the net relative light units (RLU). D, The Venn diagram shows the differential reactivity of the OV-16–negative individuals that were positive to OVOC10469, OVOC3261, and/or OVOC5127. E, The heatmap depicts the samples classified as positive (red) or negative (blue) for OV-16, OVOC10469, OVOC3261, or combinations. Blank spaces denote that the sample was not tested.

Figure 3.

Comparison of IgG4 responses to OV-16, rOVOC10469, and rOVOC3261 by enzyme-linked immunosorbent assay (ELISA). The scatter plots depict the signal to noise values (S/N) of OV-16 (A), rOVOC10469 (B), and rOVOC3261 (C) reactivity in sera of Onchocerca volvulus-infected (INF), endemic normal (EN), healthy US blood bank volunteers (BB), and Wuchereria bancrofti, Loa loa, Strongyloides stercoralis (other helminth) infections by ELISA. The dotted lines denote the receiver operating characteristic (ROC) cutoff values using the other helminth group as control. D, Heat map depicts the samples classified as positive (red) or negative (blue) for OV-16, rOVOC10469, and rOVOC3261 based on ROC. E, Signal to noise ratio of OV-16–negative sera (blue segment from heat map shown in Figure 3D) showing the differential reactivity to rOVOC10469 and rOVOC3261.

Figure 4.

Kinetics of development of antibody responses to OV-16, OVOC3261, and OVOC10469 in nonhuman primates (NHP). The kinetics of IgG (dotted) and IgG4 (solid) responses to OV-16 (red), OVOC3261 (blue), and OVOC10469 (green) in serum of 4 experimentally infected NHPs (A–D) by luciferase immunoprecipitation system. The values on the y-axis denote the net relative light units (RLU). The vertical violet dotted line denotes the time of appearance of skin microfilariae.

Figure 5.

IgG4 responses pre- and posttreatment. The paired IgG4 responses by enzyme-linked immunosorbent assay to OV-16 (A), rOVCO10469 (B), rOVOC3261 (C), and combination of OV-16, rOVOC10469, and rOVOC3261 (D) in patients that were treated and followed-up longitudinally for over 10 years (red) or between 5 and 7 years (blue). Signal to noise values are plotted on the y-axis.

Figure 6.

Testing of prototype rapid diagnostic test. Prototype rapid diagnostic test strips with OV-16 and rOVOC3261 were developed (A) and tested with infected and uninfected or control sera. B, The top panel shows representative images of strips from Onchocerca volvulus microfilariae (mf)-positive sera that were positive for both OV-16 and rOVOC3261 (+/+), or positive only for OV-16 (+/−), or positive only for rOVOC3261 (−/+). The bottom panel shows representative images from reactivity against serum from healthy blood bank volunteers (BB) or people infected with Loa loa (Ll), Wuchereria bancrofti (Wb), or Strongyloides stercoralis (Ss). C, The Venn diagrams shows the number of samples positive for OV-16 and/or rOVOC3261 in the test strips tested at National Institutes of Health (solid lines) and at Centers for Disease Control and Prevention (dotted lines).