Comparison of diagnostic tools to assess the feasibility of programmatic use of rapid diagnostic tests for onchocerciasis: A dataset from Gabon

Due to the success of large-scale ivermectin mass drug administration (MDA), the aim of onchocerciasis intervention efforts have shifted from control of the disease to elimination of transmission. This has necessitated a greater understanding and comparison of the performance of diagnostic tools in hypoendemic (low prevalence) settings which had not been incorporated into large-scale MDA programmes before the goal switched from onchocerciasis elimination as a public health problem to elimination (interruption) of transmission (EOT). Data on age, sex and duration of residence were collected, prior to ivermectin treatment, across Gabon in 2015 from 5,829 participants in 67 communities from 14 districts. Skin-snip samples (for detection of Onchocerca volvulus microfilariae) were obtained from 4,350 (75 %) and blood samples (for detection of presence of IgG4 antibodies against the O. volvulus Ov16 antigen) from 4,257 of those skin-snip tested (98 %). Whole blood was tested in the field using the SD Ov16 Rapid Diagnostic Test Prototype (Ov16 RDT). Dried blood spots (DBS) were prepared for all blood-sampled individuals. After assessing DBS quality, 2,990 (70 %) samples underwent valid analysis in the lab using horseradish peroxidase (HRP) Ov16 enzyme-linked immunosorbent assay (Ov16 ELISA). The number of positive individuals varied between diagnostic tools with skin-snip microscopy, Ov16 RDT and Ov16 ELISA detecting 337/4,350 (8 %, 95 % CI =7 %–9 %), 383/4,257 (9 %, 8 %–10 %) and 348/2,990 (12 %, 10 %–13 %), respectively. Data were analysed to understand the age profiles of microfilarial and IgG4 antibody prevalence by diagnostic and mapped across Gabon. These data have reuse potential for policy makers, test manufacturers and country programmes when making determinations at community level of the suitability of using Ov16 RDT for conducting delineation mapping or evaluating the current stage of a community or, more generally, an evaluation unit along the EOT path. Further, these data are of use to transmission dynamics modellers who can fit models to these data to better understand the stage(s) in the O. volvulus lifecycle likely responsible for IgG4 antibody seroconversion in the presence of Ov16 antigen. This is crucial for incorporation of antibody prevalence as an output of onchocerciasis transmission models to permit evaluation of currently proposed serological thresholds to inform decisions about Start- or Stop-MDA in the context of onchocerciasis elimination mapping (OEM) and verification of EOT, respectively.

a b s t r a c t Due to the success of large-scale ivermectin mass drug administration (MDA), the aim of onchocerciasis intervention effort s have shifted from control of the disease to elimination of transmission.This has necessitated a greater understanding and comparison of the performance of diagnostic tools in hypoendemic (low prevalence) settings which had not been incorporated into large-scale MDA programmes before the goal switched from onchocerciasis elimination as a public health problem to elimination (interruption) of transmission (EOT).Data on age, sex and duration of residence were collected, prior to ivermectin treatment, across Gabon in 2015 Dataset link: Dataset of the performance of Ov16 RDTs versus skin snip microscopy and Ov16 ELISA, Gabon, 2015 (Original data) Keywords: Onchocerciasis Hypoendemicity Delineation mapping Microfilariae Ov16 RDT Ov16 ELISA from 5,829 participants in 67 communities from 14 districts.Skin-snip samples (for detection of Onchocerca volvulus microfilariae) were obtained from 4,350 (75 %) and blood samples (for detection of presence of IgG4 antibodies against the O. volvulus Ov16 antigen) from 4,257 of those skin-snip tested (98 %).Whole blood was tested in the field using the SD Ov16 Rapid Diagnostic Test Prototype (Ov16 RDT).Dried blood spots (DBS) were prepared for all blood-sampled individuals.After assessing DBS quality, 2,990 (70 %) samples underwent valid analysis in the lab using horseradish peroxidase (HRP) Ov16 enzyme-linked immunosorbent assay (Ov16 ELISA).The number of positive individuals varied between diagnostic tools with skin-snip microscopy, Ov16 RDT and Ov16 ELISA detecting 337/4,350 (8 %, 95 % CI = 7 %-9 %), 383/4,257 (9 %, 8 %-10 %) and 348/2,990 (12 %, 10 %-13 %), respectively.Data were analysed to understand the age profiles of microfilarial and IgG4 antibody prevalence by diagnostic and mapped across Gabon.These data have reuse potential for policy makers, test manufacturers and country programmes when making determinations at community level of the suitability of using Ov16 RDT for conducting delineation mapping or evaluating the current stage of a community or, more generally, an evaluation unit along the EOT path.Further, these data are of use to transmission dynamics modellers who can fit models to these data to better understand the stage(s) in the O. volvulus lifecycle likely responsible for IgG4 antibody seroconversion in the presence of Ov16 antigen.This is crucial for incorporation of antibody prevalence as an output of onchocerciasis transmission models to permit evaluation of currently proposed serological thresholds to inform decisions about Startor Stop-MDA in the context of onchocerciasis elimination mapping (OEM) and verification of EOT, respectively.
© 2024 The Author(s Beneficiaries of these data include: a) policy makers involved in the development and review of guidelines for OEM [ 2 ]; b) test manufacturers gaining further understanding of their diagnostic tools' functionality in field epidemiological settings; c) country programmes assessing the usability and acceptability of less invasive testing procedures to increase participation rates by endemic communities in onchocerciasis surveillance activities [ 3 , 4 ].

• Programmatic applications:
Diagnostic performance of Ov16 RDT could be used to assess whether RDT can replace both skin-snip microscopy and ELISA in the assessment of onchocerciasis prevalence and in turn be suitable for both OEM and Stop-MDA decisions on the path to verification of EOT.

• Transmission modelling applications:
Testing various hypotheses on which O. volvulus lifecycle stage(s) is/are responsible for IgG4 seroconversion in response to Ov16 in humans and occurrence of seroreversion.Integrating and validating IgG4 antibody prevalence model outputs [5][6][7][8].Simulating IgG4 antibody prevalence age profiles under different pre-and post-MDA epidemiological scenarios to examine the suitability of proposed age groups and serological thresholds to inform Start-MDA and Stop-MDA decisions.

Onchocerciasis interventions initially aimed to control
Onchocerca volvulus infection to the extent it would no longer be a public health problem, focusing on regions with high levels of transmission and morbidity arising from ocular and skin disease.Given the success of ivermectin mass drug administration (MDA) programmes, the paradigm shifted from disease control to elimination (interruption) of transmission (EOT) [ 9 ].In endemic countries, nationwide EOT requires that interruption of transmission be reached in all foci, including those previously nonprioritised because of their low baseline prevalence.This necessitates mapping and assessing hypoendemic areas for their integration into ivermectin treatment programmes (onchocerciasis elimination mapping, OEM).Parasitological diagnosis relies on skin-snip microscopy, a mildly painful and relatively invasive procedure which has low sensitivity in low-prevalence areas [ 10 ].Tests that use finger-prick blood sampling to detect IgG4 antibodies to the O. volvulus Ov16 antigen represent a less invasive option.Conducting ELISA tests from these blood samples can be time-consuming and resource-intensive in endemic settings, with point-of-care rapid diagnostic tests (RDTs) offering a more practical alternative.We describe a dataset from delineation map-ping in Gabon that includes skin-snip, Ov16 RDT and Ov16 ELISA data from the same individuals in hypoendemic communities prior to ivermectin treatment.

Data Description
All data were gathered through the analysis of skin-snip microscopy and blood samples collected for onchocerciasis delineation mapping across Gabon as part of a multi-country study to compare diagnostic tools, endorsed by the African Programme for Onchocerciasis Control (APOC) [ 11 , 12 ].The raw data were entered into an .xlsx.file [ 1 ].

Experimental design
Study sites were selected to conduct onchocerciasis delineation mapping in Gabon.A previous mapping exercise, based on prevalence of palpable nodules (where a proportion of adult O. volvulus worms reside) termed REMO (rapid epidemiological mapping of onchocerciasis), had determined that most districts in Gabon were hypoendemic (low prevalence) [ 15 ].Another mapping exercise, based on the history of eye-worm ( Loa loa ) had also determined that most of the country was co-endemic with loiasis [ 16 ].Therefore, Gabon had not received ivermectin treatment.
Biological samples were collected from community members who consented to participate, with the sampling frame determined by the total number of communities selected by APOC for OEM during 2015 [ 11 ].
In each of the communities, convenience sampling was conducted to select a total of up to 300 individuals.Individuals aged 5 years and older, who consented themselves to be examined (or via their parents/guardians) were included, with blood samples only taken from those who agreed to be skin snipped.Sample collectors used a census form which listed the identification (ID) numbers assigned to each consenting participant.

Skin-snipping
Both right and left iliac crests of the participants ( n = 4350) were cleaned using an alcoholbased swab.Two bloodless skin-snip samples were collected from the crests using a 2-mm Holth corneoscleral punch (Holth, New York, NY, USA) with each biopsy placed into a well of a microtitration plate.Following 24-hour incubation of the snips in saline solution, the medium, into which microfilariae emerged, was spread onto microscope slides and examined under (low magnification) light microscopy to detect the presence of and enumerate O. volvulus microfilariae in the skin snips [ 17 ].All Holth punches were sterilised after every use to maintain aseptic conditions.

Blood sampling 4.3.1. Detection of IgG4 antibodies to Ov16 antigen using rapid diagnostic test (Ov16 RDT)
Blood samples were collected ( n = 4257) from consenting participants (who had been skinsnipped) by finger-prick and 15 μL were used for Ov16 RDT.The test applied was the SD Ov16 Rapid Diagnostic Test Prototype (Standard Diagnostics, Inc., Suwon city, Republic of Korea).This is an immuno-chromatographic strip test that detects human IgG4 antibodies to the O. volvulus Ov16 antigen present in the blood sample.The test strip is composed of an absorbent pad, a nitrocellulose membrane striped with a control line reagent, a test reagent and a conjugate pad which contains dried colloidal gold detector reagent.The test line is a dried preparation of a recombinant fusion protein of glutathione-S-transferase and the Ov16 protein from O. volvulus , produced at PATH (Program for Appropriate Technology in Health, Seattle, WA, USA).The control line is a dried preparation of a commercially available goat anti-mouse IgG antibody.The detection reagent is a mouse anti-human IgG4 and colloidal gold nanoparticle conjugate, dried onto a glass-fibre conjugate pad.The sample and rehydrated detection reagent flow up the strip via capillary action through the nitrocellulose membrane when Ov16 buffer is added to the test.The immunoglobulins in the sample may bind the Ov16 antigen line in the nitrocellulose and the detection reagent may subsequently bind to the human immunoglobulins and will also bind to the control line antibody in the nitrocellulose.Results are visually interpreted at 20 min.If the sample contains IgG4 antibodies to the O. volvulus Ov16 antigen, the complex binds to the antigen in the test line, producing a red line indicating a positive result.In the absence of IgG4 antibodies to O. volvulus Ov16, no red line will form in the test line area, indicating a negative result.Absence of the control line indicates an invalid result [ 18 ].

Preparation of dried blood spots
Blood samples ( n = 4257) were collected by finger prick (100 μL) and placed onto Whatman 903 filter paper to prepare dried blood spots (DBS).Four aliquots of 6 drops of blood (25 μL for each aliquot, for approximately 4 μL per drop) were collected from each individual and absorbed onto the filter paper.Blood spots were labelled with their corresponding ID, dried, individually wrapped and stored at 4 °C, and subsequently at −20 °C until shipped to the testing laboratory.Only DBS of suitable quality, determined by examining the front and the back of the filter paper to ensure complete saturation ( n = 3012), were tested for Ov16 ELISA at Smith College, Northampton, MA , USA .For the ELISA , blood spots were eluted from two 6-mm punches (see below).Each eluate (the product of two punches) was run in duplicate in the Ov16 ELISA to detect anti-Ov16 IgG4 antibodies.

Ov16 IgG4 detection using ELISA
Blood spots were eluted if their quality allowed for two full 6-mm punches to be taken from the saturated area.These two punches were then transferred to a single well within a 96-well elution plate (Corning® 3788), with the sample ID recorded in an elution plate map.100μL of DBS elution buffer (phosphate buffered saline (PBS) + 0.05 % Tween + 5 % bovine serum albumin (BSA)) was added to each well containing the two DBS punches making sure that the punches were completely submerged in the buffer, with no air bubbles surrounding them.Plates were covered and incubated overnight (12-24 h) in a refrigerator at 2-8 °C [ 18 ].
Immulon 2HB plates (Thermo Scientific, Waltham, MA, USA) were coated with Ov16 antigen.For coating, stock antigen at a concentration of 5 μg/mL was diluted 1:100 with PBS.Once plated, the coating solution was refrigerated at 4 °C overnight and subsequently removed via inversion of the plates.Plates were then blocked with 200μL/well of blocking solution (2.5 mL of foetal bovine serum (FBS), 25 mL of PBS-tween (PBST) and 22.5 mL of ddH 2 O) for 30 min in a 37 °C incubator.The wells were then washed three times with 300μL of PBST.
Control sera were subsequently prepared.The negative control consisted of 50μl of PBST + 5 % FBS, with the positive control being a dilution panel of AbD_32 antibody (PATH, Seattle, WA, USA) (20 0 0 ng/mL stock) diluted into PBST + 5 % FBS at concentrations: 20 ng/mL, 10 ng/mL, 5 ng/mL, 2.5 ng/mL, 1 ng/mL, 0.5 ng/mL, 0.25 ng/mL, 0.1 ng/mL.The remainder of the wells contained 50μL of the DBS eluates, with each eluate (collected from two DBS punches) run in duplicate.An eluted known positive DBS sample labelled 'DBS Positive 250 ng/mL' was also run in duplicate.
Once added into the designated wells, the plates were incubated for a further hour at 37 °C and washed three times with 300μL of PBST.Mouse anti-human IgG4 antibody (HP6025) (Hybridoma Reagent Laboratory, Baltimore, MD, USA) was added to each well at a ratio of 1:50 0 0. The antibody was diluted in PBST + 5 % FBS and 50 μL was added to each well.The plates were further incubated for 1 hour at 37 °C and washed three additional times with 300μL of PBST.and blood samples from 4257 participants (98 % of those skin-snip tested; solid horizontal arrow).All 4257 were tested using Ov16 RDT with whole blood and dried blood spots (DBS) were prepared.After assessing DBS quality, 2990 (70 %) had a valid test using Ov16 ELISA (dashed horizontal arrow).A total of 337 participants (8 %) were positive for skinsnip microscopy, 383 (9 %) were seropositive for Ov16 RDT and 348 (12 %) for Ov16 ELISA.Of the 4257 participants, 174 individuals tested positive for both skin-snip microscopy and RDT.Of the 2990 DBS samples which underwent valid testing with Ov16 ELISA, 156 were positive for both skin-snip and ELISA, and 192 were negative for skin-snip but positive for ELISA (for a total of 348 ELISA-positive).When calculated against skin-snip microscopy, the sensitivity and specificity of the Ov16 RDT were, respectively, 52 % and 95 %.The sensitivity and specificity of the Ov16 ELISA were, respectively, 59 % and 93 %.50 μL of a goat anti-mouse secondary antibody conjugated to horseradish peroxidase (G αM-HRP) (Jackson Immuno, West Grove, PA, USA), diluted in PBST + 5 % FBS, was added to each well at a ratio of 1:10,0 0 0. The plates were further incubated for 1 hour at 37 °C and washed three additional times using 300μL of PBST.
3,3,5,5-Tetramethyl Benzidine (TMB) (Sigma, Mumbai, India) was equilibrated to room temperature.100 μL of TMB was added per well and incubated at room temperature for a further 15 min.The enzymatic reaction was stopped by adding 50 μL of 1 N HCl to each well.The plates were placed in the plate reader (SpectraMax M2, Molecular Devices, San Jose, CA, USA), with the endpoint ELISA absorbance (optical density, OD) read at 450 nm.
The cut-off values were determined as the mean absorbance of the negative control wells plus three times the standard deviation.If the OD of the sample was above the cut-off, the sample was designated as a positive result.
Standard curves were generated for each plate by plotting known concentrations of the positive control (AbD_32) dilution series (described above).A 4-parameter logistic regression model was used to fit the standard curve, where x is the inferred concentration, y is the measured OD, a and d are, respectively, the minimum and maximum responses, c represents the point of inflection and b the gradient of the curve at the inflection point.The standard curve was used to generate inferred concentrations from the OD readings of the positive samples.Samples identified to have OD readings inconsistent with the standard curve, and which could not be rectified by re-assaying, were deemed invalid ( n = 22) and excluded, leaving 2990 valid samples for analysis [ 18 ].

Data analysis
Raw data were collated using MS Excel, being cleaned and analysed using R, version 4.33 [ 19 ].Ninety-five percent confidence intervals (95 % CI) were calculated using the Wilson score method [ 13 ].Analysed data were visualised using R packages: ggplot2, patchwork (plotting and arrangement) and sf (mapping) using shape files for implementation units of Gabon, sourced from the ESPEN portal [ 14 ].Fig. 1 , Fig. 2 , Fig. 3 , Table 1 Limitations Ov16 ELISAs were only conducted on DBS samples deemed to be of sufficient quality.As such, the sample size for Ov16 ELISA is smaller compared to the other two diagnostics.Caution must therefore be applied when interpreting the diagnostic performance of the Ov16 ELISA.Further, the Ov16 ELISA data do not include the sex of the participants.

Ethics Statement
All surveys and sample collections were conducted after receiving informed consent from the participants.If the participant was aged 15 years or younger, the permission of a legal parent or guardian was obtained.Ethical approval was granted prior to the commencement of the study from the Ministry of Health and Social Affairs of the Republic of Gabon, Reference: APOC/EVE/030/15/HZ.In addition, we confirm that all authors have abided by the ethical re-  quirements for publication in Data in Brief and that our work did not involve the use of animal experiments or the collection of data from social media platforms.

Fig. 1 .
Fig. 1.Participant testing flowchart.Of an initial 5829 participants, skin-snip samples were collected from 4350 (75 %) and blood samples from 4257 participants (98 % of those skin-snip tested; solid horizontal arrow).All 4257 were tested using Ov16 RDT with whole blood and dried blood spots (DBS) were prepared.After assessing DBS quality, 2990 (70 %) had a valid test using Ov16 ELISA (dashed horizontal arrow).A total of 337 participants (8 %) were positive for skinsnip microscopy, 383 (9 %) were seropositive for Ov16 RDT and 348 (12 %) for Ov16 ELISA.Of the 4257 participants, 174 individuals tested positive for both skin-snip microscopy and RDT.Of the 2990 DBS samples which underwent valid testing with Ov16 ELISA, 156 were positive for both skin-snip and ELISA, and 192 were negative for skin-snip but positive for ELISA (for a total of 348 ELISA-positive).When calculated against skin-snip microscopy, the sensitivity and specificity of the Ov16 RDT were, respectively, 52 % and 95 %.The sensitivity and specificity of the Ov16 ELISA were, respectively, 59 % and 93 %.

Fig. 3 .
Fig. 3. Geographical comparison of Onchocerca volvulus microfilarial prevalence and Ov16 seroprevalence in Gabon, 2015 .Of 51 implementation units (IUs), sampling took place in 31, with 20 (grey-shaded) not sampled.Yellow-shaded IUs show the lowest recorded prevalence and darkest red-shaded IUs show the highest prevalence.Panel A: microfilarial prevalence measured by skin-snip microscopy; Panel B: Ov16 seroprevalence measured by rapid diagnostic test (Ov16 RDT) as described in the text.Samples were collected prior to implementation of ivermectin treatment.Districts where sampling was undertaken were located within IUs as mapped in the Expanded Special Project for Elimination of Neglected Tropical Diseases (ESPEN) portal for Gabon [ 14 ].

Table 1
). Published by Elsevier Inc.This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ ) ; Fig.s 1-3 Data collection Skin-snip microscopy: Bloodless skin-snip samples were collected from the right and left iliac crests using a 2-mm Holth corneoscleral punch.Following 24-hour incubation of the snips in saline solution in a microtitration plate's wells, the medium was spread onto microscope slides and examined under light microscopy to detect O. volvulus microfilariae in the skin.