Analysis of age-dependent trends in Ov16 IgG4 seroprevalence to onchocerciasis

BACKGROUND: Diagnostics provide a means to measure progress toward disease elimination. Many countries in Africa are approaching elimination of onchocerciasis after successful implementation of mass drug administration programs as well as vector control. An understanding of how markers for infection such as skin snip microfilaria and Onchocerca volvulus-specific seroconversion perform in near-elimination settings informs how to best use these markers. METHODS: All-age participants from 35 villages in Togo were surveyed in 2013 and 2014 for skin snip Onchocerca volvulus microfilaria and IgG4 antibody response by enzyme-linked immunosorbent assay (ELISA) to the Onchocerca volvulus-specific antigen Ov16. A Gaussian mixture model applying the expectation-maximization (EM) algorithm was used to determine seropositivity from Ov16 ELISA data. For a subset of participants (n = 434), polymerase chain reaction (PCR) was performed on the skin snips taken during surveillance. RESULTS: Within the 2,005 participants for which there was Ov16 ELISA data, O. volvulus microfilaremia prevalence and Ov16 seroprevalence were, 2.5 and 19.7 %, respectively, in the total population, and 1.6 and 3.6 % in children under 11. In the subset of 434 specimens for which ELISA, PCR, and microscopy data were generated, it was found that in children under 11 years of age, the anti-Ov16 IgG4 antibody response demonstrate a sensitivity and specificity of 80 and 97 %, respectively, against active infections as determined by combined PCR and microscopy on skin snips. Further analysis was performed in 34 of the 35 villages surveyed. These villages were stratified by all-age seroprevalence into three clusters: < 15 %; 15–20 %; and > 20 %. Age-dependence of seroprevalence for each cluster was best reflected by a two-phase force-of-infection (FOI) catalytic model. In all clusters, the lower of the two phases of FOI was associated with a younger age group, as reflected by the seroconversion rates for each phase. The age at which transition from lower to higher seroconversion, between the two phases of FOI, was found to be highest (older) for the cluster of villages with < 15 % seroprevalence and lowest (younger) for the cluster with the highest all-age seroprevalence. CONCLUSIONS: The anti-Ov16 IgG4 antibody response is an accurate marker for active infection in children under 11 years of age in this population. Applying Ov16 surveillance to a broader age range provides additional valuable information for understanding progression toward elimination and can inform where targeted augmented interventions may be needed. Clustering of villages by all-age sero-surveillance allowed application of a biphasic FOI model to differentiate seroconversion rates for different age groups within the village cluster categories. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13071-016-1623-1) contains supplementary material, which is available to authorized users

A Recombinant Positive Control for Serology Diagnostic Tests Supporting Elimination of Onchocerca volvulus.

Serological assays for human IgG4 to the Onchocerca volvulus antigen Ov16 have been used to confirm elimination of onchocerciasis in much of the Americas and parts of Africa. A standardized source of positive control antibody (human anti-Ov16 IgG4) will ensure the quality of surveillance data using these tests.A recombinant human IgG4 antibody to Ov16 was identified by screening against a synthetic human Fab phage display library and converted into human IgG4. This antibody was developed into different positive control formulations for enzyme-linked immunosorbent assay (ELISA) and rapid diagnostic test (RDT) platforms. Variation in ELISA results and utility as a positive control of the antibody were assessed from multiple laboratories. Temperature and humidity conditions were collected across seven surveillance activities from 2011-2014 to inform stability requirements for RDTs and positive controls. The feasibility of the dried positive control for RDT was evaluated during onchocerciasis surveillance activity in Togo, in 2014. When the anti-Ov16 IgG4 antibody was used as a standard dilution in horseradish peroxidase (HRP) and alkaline phosphatase (AP) ELISAs, the detection limits were approximately 1ng/mL by HRP ELISA and 10ng/mL by AP ELISA. Positive control dilutions and spiked dried blood spots (DBS) produced similar ELISA results. Used as a simple plate normalization control, the positive control antibody may improve ELISA data comparison in the context of inter-laboratory variation. The aggregate temperature and humidity monitor data informed temperature parameters under which the dried positive control was tested and are applicable inputs for testing of diagnostics tools intended for sub-Saharan Africa. As a packaged positive control for Ov16 RDTs, stability of the antibody was demonstrated for over six months at relevant temperatures in the laboratory and for over 15 weeks under field conditions.The recombinant human anti-Ov16 IgG4 antibody-based positive control will benefit inter-laboratory validation of ELISA assays and serve as quality control (QC) reagents for Ov16 RDTs at different points of the supply chain from manufacturer to field use

Additional file 1: Table S1. of Analysis of age-dependent trends in Ov16 IgG4 seroprevalence to onchocerciasis

Village name, number of study participants, Ov16 prevalence and MF prevalence values used to generate Fig. 4b. (DOCX 15 kb

Lateral flow test strip scanned images showing test line (‘T’, left) and control line (‘C’, right) results after using as samples the dried anti-Ov16 IgG4-positive control subjected to elevated temperature stress over time.

<p>Time zero shown indicates intensity of the starting signal prior to storage at 25°C, 45°C, and variable cycling temperature (20°C to 40°C, daily) for up to 19 months.</p

Concentration-dependent absorbance values of the ELISA platform for the anti-Ov16 human IgG4 serological assay.

<p>(A) Absorbance units versus concentration of recombinant anti-Ov16 IgG4-positive control antibody. (B) HRP ELISA and AP ELISA absorbance values from both plasma/serum and DBS samples derived from identical sources: anti-Ov16 IgG4-positive control-spiked negative human sera, dilutions of an Ov-positive pool from microfilaria-positive patient plasmas and sera, and the average of nine negative plasmas and sera (from donors that have not traveled to an Ov-endemic area).</p

Environmental conditions during surveillance activities in Togo 2013 and 2014.

<p>A. Daily temperature profiles (dark grey lines) in degrees Celsius during surveillance activities. The mean temperature is plotted with the bold red line. B. Daily % relative humidity (dark grey lines) cycles during surveillance activities. The mean relative humidity is plotted with the bold green line. C. Frequency of recorded temperature and relative humidity pairs experienced by monitors attached to microscopes used during the surveillance activities. The color scale represents the frequency of observations represented by intense red (least frequent) to intense yellow (most frequent).</p

(A) Combined inter-laboratory HRP ELISA signal profiles for anti-Ov16 human IgG4 antibody.

<p>Concentrations between 0.1ng/mL and 20ng/mL were assayed in replicate ELISA plates by four different laboratories using the same protocol and starting reagents. Each plate contained the concentration range run in duplicate wells. Different laboratories, identified as A-D, ran between three and five replicate plates. Shown is data collected from the laboratories (color-grouped) with each line representing the profile from a single plate of the averaged duplicate wells for each concentration tested. (B) Processed data representing the same data with the same color-coding as shown in 3A, but shown with absorbance values normalized to each plate’s respective optical absorbance at 2.5 ng/mL of control antibody.</p

Workflow for use of dried positive control with the Ov16 RDT.

<p>Workflow for use of dried positive control with the Ov16 RDT.</p

Dose-dependency of the intensity of the RDT (SD BIOLINE Onchocerciasis IgG4) test line result to increasing concentrations of the anti-Ov16 human IgG4 spiked into pooled normal human plasma.

<p>Ten microliters of the plasma is applied as sample and run according to manufacturer’s instructions.</p