A four-antigen mixture for rapid assessment of Onchocerca volvulus infection

Abstract

Background: Onchocerciasis, an infection caused by the filarial nematode Onchocerca volvulus, is a major public health concern. Given the debilitating symptoms associated with onchocerciasis and concerns about recrudescence in areas of previous onchocerciasis control, more efficient tools are needed for diagnosis and monitoring of control measures. We investigated whether luciferase immunoprecipitation systems (LIPS) may be used as a more rapid, specific, and standardized diagnostic assay for Onchocerca volvulus infection.

Methods: Four recombinantly produced Onchocerca volvulus antigens (Ov-FAR-1, Ov-API-1, Ov-MSA-1 and Ov-CPI-1) were tested by LIPS on a large cohort of blinded sera comprised of both uninfected controls and patients with a proven parasitic infection including Onchocerca volvulus (Ov), Wuchereria bancrofti (Wb), Loa loa (Ll), Strongyloides stercoralis (Ss), and with other potentially cross-reactive infections. In addition to testing all four Ov antigens separately, a mixture that tested all four antigens simultaneously was evaluated in the standard 2-hour incubation format as well as in a 15-minute rapid LIPS format.

Findings: Antibody responses to the four different Ov antigens allowed for unequivocal differentiation between Ov-infected and uninfected control sera with 100% sensitivity and 100% specificity. Analysis of the antibody titers to each of these four antigens in individual Ov-infected sera revealed that they were markedly different and did not correlate (r(S) = -0.11 to 0.58; P = 0.001 to 0.89) to each other. Compared to Ov-infected sera, patients infected with Wb, Ll, Ss, and other conditions had markedly lower geometric mean antibody titers to each of the Ov 4 antigens (P<0.0002 for each antigen). The simplified method of using a mixture of the 4 Ov antigens simultaneously in the standard format or a quick 15-minute format (QLIPS) showed 100% sensitivity and 100% specificity in distinguishing the Ov-infected sera from the uninfected control sera. Finally, the QLIPS format had the best performance with 100% sensitivity and specificity values of 76%, 84% and 93% for distinguishing Ov from Wb, Ll and Ss-infected sera.

Conclusions: The multi-antigen LIPS assay can be used as a rapid, high throughput, and specific tool to not only to diagnose individual Ov infections but also as a sensitive and potentially point-of-care method for early detection of recrudescent infections in areas under control and for mapping new areas of transmission of Ov infection.

Figure 1. Heat map representation of patient antibody profiles to the 4 Ov antigens.

The antibody levels for each serum were log₁₀ transformed and then the levels were color-coded as indicated by the log₁₀ scale on the left, in which signal intensities range from red to green indicating high (red) and low (green) titers. The samples were rank ordered from highest to lowest based on the sum of the antibody titers to the 4 antigen panel. The samples on the left are from uninfected, while the samples on the right are Ov-infected sera.

Figure 2. LIPS detection of antibodies to 4 different Ov antigens.

Each symbol represents individual samples from the 38 Ov-infected, 90 Wb, 90 Ll, 27 Ss, 72 control uninfected samples and 12 other control patients. Antibody levels in LU are plotted on the Y-axis using a log₁₀ scale and short solid horizontal lines indicate the geometric mean titer (GMT) for each antibody per group. The diagnostic performance related to cross-reactivity with other filarial infections was also evaluated. As described in the text, the long solid line represents the cut-off level corresponding to 100% sensitivity, while the long stippled line corresponds to the cut-off for 100% specificity with sera from the Wb cohort.

Figure 3. A four Ov antigen panel used in the standard 2 hour or QLIPS format shows 100% sensitivity and 100% specificity.

Each symbol represents individual samples from the 38 Ov-infected, 90 Wb, 90 Ll, 27 Ss, 72 control uninfected samples and 12 other control patients. These LIPS tests were either evaluated as a mixture in the standard format (A) or with QLIPS (B). As shown, both tests showed 100% sensitivity and 100% specificity in distinguishing the uninfected from the Ov-infected sera. The diagnostic performance related to cross-reactivity with other filarial infections was also evaluated. As described in the text, the long solid line represents the cut-off level corresponding to 100% sensitivity, while the long stippled line corresponds to the cut-off for 100% specificity with sera from the Wb cohort.