Validation of the Brugia Test Plus to detect IgG4 antibodies in individuals from Belitung Timur, a Brugia malayi endemic area in Indonesia

Abstract

The Brugia Test Plus (BT+) is a new rapid diagnostic test for Brugia species which detects human IgG4 antibodies specific for the immunogenic Brugia protein BmR1. The aim of this study was to evaluate the BT+ assay with several types of sample-matrices: whole blood, plasma, and dried blood spots (DBS) from individuals living in Belitung Timur, a Brugia malayi endemic area in Indonesia. Night blood was collected from residents living in four presumed endemic villages, while DBS were collected from schoolchildren living in those four villages. The sensitivity of BT+ was measured by comparing the BT+ results to microscopic examination for microfilaria (Mf). The sensitivity of BT+ with whole blood under field conditions was 84.9% (95% CI 68.1-94.9, n = 33), and with EDTA plasma under laboratory conditions was 95.9% (95% CI 88.5-99.1, n = 73). Specificity was not assessed as it is impossible do so for an antibody test in endemic areas. In Mf-negative individuals, BT+ detected IgG4 antibodies in 204 out of 1,547 adult plasma samples (13.2%) and 7 out of 146 plasma samples collected from children aged 10-16 (4.8%). Detection of IgG4 antibodies in DBS collected from first and second grade schoolchildren (age 6-8) showed that only 1 out of 244 schoolchildren was positive (0.1%). Three individual readers were responsible for reading the BT+ . Statistical analysis showed high agreement among those readers (Kappa agreement value above 0.9). Laboratory technicians found the BT+ is simple to perform, easy to interpret the results, and appreciated the small volume of blood required (5 µL). This study has demonstrated that the novel BT+ is feasible for teams to implement and achieves good sensitivity, making it well suited for monitoring and evaluating the progress of the brugian- filariasis elimination program.

Fig 1. Map of the study area in Belitung Timur District, Indonesia.

Belitung Island is divided into two districts: Belitung District (left) and Belitung Timur District (right). The study area, Belitung Timur District, includes sixteen labeled study villages. Among these, fourteen were classified as endemic (highlighted in pink) and two were non-endemic (in yellow). From the endemic villages, four high-endemicity villages were chosen for the Brugia Test Plus diagnostic assessment (red dots). Base map layer sourced from GADM for Indonesia (https://gadm.org/download_country.html, select “Indonesia”) and used under the GADM license (https://gadm.org/license.html). Map created using QGIS.

Fig 2. The flow of study participants in Belitung Timur.

Fig 3. Comparison of Fingerstick blood with and without anticoagulant on the same subject.

Row A and B show BT+ from blood samples without anticoagulant in duplicates while row C shows BT+ from EDTA-treated blood.

Fig 4. Appearance of the test line when run with fresh blood, without anticoagulants, compared to Mf density.

Fig 5. Comparison of 3 matrices from the same patient with EDTA whole blood (A), EDTA plasma (B), and DBS (C).

Whole blood and DBS give very tenuous test lines, while plasma gives a more easily visible test line.