Fig 1.
Serum reactivity of melioidosis-confirmed patients and non-melioidosis individuals to B. pseudomallei whole cell lysate ELISA.
Serum IgG (A) or IgM (B) reactivity as determined by B. pseudomallei 1026b whole cell lysate ELISA. Triangles represent ELISA analyzed serum (n = 585) from 436 individuals (36 melioidosis and 400 non-melioidosis individuals) and squares represent selected serum samples to evaluate potential diagnostic antigens. The overlap between the negative and the positive samples are shown in grey.
Table 1.
Purified antigens coupled to bead regions and the associated purpose.
Fig 2.
Heat map of MAGPIX multiplexed assay for IgG (A) and IgM (B).
Each row represents an individual antigen, and each column represents an individual serum sample (n = 188). The sera are stratified by non-melioidosis negative and melioidosis samples. The negative group is further stratified by the 1026b whole cell lysate (WCL) reactivity (overlap and non-reactive IgG and IgM). The melioidosis serum is grouped by the sample’s collection time after initial sample collection. For the heat map, blue indicates 0 median fluorescent intensity (MFI) for both panels. For IgG panel A, yellow indicates 5,000 MFI and red indicates >20,000 MFI. For IgM panel B, yellow indicates 4,000 MFI and red indicates >8,000 MFI. Black indicates samples where data was not collected.
Fig 3.
ROC analysis of B. pseudomallei lysate (multiple antigens—A) and single antigens (B-D) selected by day 21 area under the curve (AUC) analysis performance.
Responses to 1026b WCL (A), CPS (B), GroEL (C), and LPS B (D) antigens are shown using non-melioidosis human sera samples compared with day 0 (black), day 21 (purple), or all melioidosis-confirmed patient samples (dashed). The area under the curve (AUC) value for each data set is shown.
Table 2.
AUC Summary statistics of the top 10 individual or multiple antigens as determined by day 21 samples1.
Table 3.
Sensitivity and specificity of the top 10 individual antigens or WCL conjugated to Luminex microspheres1.
Fig 4.
p-hat distribution using the LASSO (A and C) and Ridge Regression (RR, B and D) models.
Samples were stratified based on the patient status and collection time. The models were developed using IgG (A and B) and IgM (C and D) reactivity. A (p-hat) value of 0 is unlikely to be melioidosis and a value of 1 is likely to be melioidosis. A cutoff to call sensitivity and specificity is shown by the dotted line and corresponds to Table 4.
value significance was determined by a Kruskal-Wallis test followed by a Dunns pairwise comparison. The Day 0 vs Day 21 was not significant for all antibody isotype models.
Fig 5.
ROC analysis of the LASSO and Ridge Regression (RR) IgG and IgM models using human patient samples.
A (p-hat) score were generated from the multiple antigen reactivity (median fluorescent intensity shown in Fig 2) for IgG or IgM. n = 188 patient samples with 112 paired samples from 56 individuals.
Table 4.
Summary information for each multivariate model.
Table 5.
Antigens and the associated antibody response that contributed to a binomial or LASSO logistic regression models1.
Fig 6.
Comparison of the IHA results to the multivariate serology assay.
IHA results (X-axis) and the multivariate p-hat probability score (Y-axis) are shown for the same sample collected from culture confirmed melioidosis patients. The different multivariate models are shown in separate panels (A-D). The day 0 (red circle) and day 21 (white squares) are shown. The cutoff for the IHA assay is 1:40 and the multivariate model cut-offs are consistent as shown in Table 5. The interpretation of the cutoff panels and which assay would correctly diagnose melioidosis is shown in panel A. A total of 40 samples that have both IHA and multivariate model values were compared.