Development and evaluation of a multiplex serodiagnostic bead assay (BurkPx) for accurate melioidosis diagnosis

Abstract

Burkholderia pseudomallei, the causative agent of melioidosis, is a gram-negative soil bacterium well recognized in Southeast Asia and northern Australia. However, wider and expanding global distribution of B. pseudomallei has been elucidated. Early diagnosis is critical for commencing the specific therapy required to optimize outcome. Serological testing using the indirect hemagglutination (IHA) antibody assay has long been used to augment diagnosis of melioidosis and to monitor progress. However, cross reactivity and prior exposure may complicate the diagnosis of current clinical disease (melioidosis). The goal of our study was to develop and initially evaluate a serology assay (BurkPx) that capitalized upon host response to multiple antigens. Antigens were selected from previous studies for expression/purification and conjugation to microspheres for multiantigen analysis. Selected serum samples from non-melioidosis controls and serial samples from culture-confirmed melioidosis patients were used to characterize the diagnostic power of individual and combined antigens at two times post admission. Multiple variable models were developed to evaluate multivariate antigen reactivity, identify important antigens, and determine sensitivity and specificity for the diagnosis of melioidosis. The final multiplex assay had a diagnostic sensitivity of 90% and specificity of 93%, which was superior to any single antigen in side-by-side comparisons. The sensitivity of the assay started at >85% for the initial serum sample after admission and increased to 94% 21 days later. Weighting antigen contribution to each model indicated that certain antigen contributed to diagnosis more than others, which suggests that the number of antigens in the assay can be decreased. In summation, the BurkPx assay can facilitate the diagnosis of melioidosis and potentially improve on currently available serology assays. Further evaluation is now required in both melioidosis-endemic and non-endemic settings.

NHP BurkPx: A multiplex serodiagnostic bead assay to monitor Burkholderia pseudomallei exposures in non-human primates

“…All 21 immunoreactive antigens identified in [ 25 ] with the highest diagnostic potential for detecting melioidosis in humans were employed for detection of B . pseudomallei exposure in NHP.…”

“…A diagnostic test to detect B. pseudomallei exposures in NHP is a necessary tool to monitor sporadic outbreaks and ensure NHP enrolled in research are free of current or past infection. In the present study, we used the findings of the antigen evaluation completed by [25] to formally evaluate a diagnostic test, the BurkPx assay, for detection of B. pseudomallei exposure for NHP. Our goals were two-fold.…”

“…All 21 immunoreactive antigens identified in [25] with the highest diagnostic potential for detecting melioidosis in humans were employed for detection of B. pseudomallei exposure in NHP. Our use of xMAP technology to develop a multiplex MAGPIX assay was chosen due the ability of this technology to measure multiple analytes in a single sample, shorter turnaround time, and strong track record in sensitivity and specificity for detection of a variety of pathogens [34].…”

“…Antigen selection, purification, and conjugation have been described [25]. In short, 21 B. pseudomallei antigens, including recombinant proteins or purified carbohydrates, were conjugated to MagPlex microspheres.…”

“…Twenty-one antigens were selected for the BurkPx antigen assay, and these antigens were chosen through multiple antigen model analyses on their ability to differentiate between healthy human and melioidosis reactive individuals [25]. Here we evaluated the ability of the human targeted BurkPx antigen assay to detect exposure in NHP challenged with different B. pseudomallei strains.…”