Table 1.
Characteristics of the Burkholderia pseudomallei antigens used in this study.
Table 2.
Characteristics of the sera and plasmas used in this study.
Fig 1.
Construction of a B. pseudomallei protein microarray.
The protein array was spotted with 20 different B. pseudomallei proteins and further internal positive and negative controls, for a total of 445 protein spots. All proteins were applied in triplicate and at five dilutions (0.01 mg/ml to 0.45 mg/ml) on glass slides, including human IgG and IgM controls and further other internal controls, i.e., murine IgG and IgM, bovine IgG, porcine IgG, caprine IgG and ovine IgG controls. All protein arrays were incubated A only with anti-human-IgG antibodies (empty control), B with melioidosis-positive or C melioidosis-negative blood sera or blood plasmas at 1:1000 dilutions (A, B and C are representative images). IgG antibodies bound to B. pseudomallei antigens were detected using horseradish-peroxidase (HRP) linked secondary anti-human-IgG antibodies and 3,3’,5,5’-tetramethyl-benzidine (TMB), which caused a blue precipitate. Protein arrays were read out by the ArrayMate (Alere Technologies GmbH, Germany). Highlighted are human IgG controls (green rectangle) and horseradish-peroxidase controls (red rectangle); all other controls are not shown.
Fig 2.
Heatmap of probing a collection of melioidosis-positive sera and negative control sera.
Protein arrays containing 20 B. pseudomallei recombinant proteins were probed with 260 melioidosis and nonmelioidosis sera. The melioidosis positive sera (n = 75) were drawn at week 0 (p.a.) from patients with B. pseudomallei infections. All positive sera were sampled in Ubon Ratchathani, Thailand. Negative control sera of healthy persons (n = 125) were sampled in the endemic regions of Thailand ((Ubon Ratchathani (U.R.) and Bangkok (B.)), Thailand and non-endemic region of Greifswald, Germany. Additionally, further negative control sera of patients with other bacteremia or fungaemia (n = 60) were used from the non-endemic region of Greifswald. Not shown are the results of incubations with meliodosis-positive sera obtained 12 and 52 weeks after admission. The antigens are shown in rows with five increasing concentrations per protein, and the patient samples are represented in columns. Array signals are reflected by the intensities of the color (white to blue) inside the boxes. The heatmap was created using Multi experiment Viewer (MeV 4.9.0) from TM4 suite, USA.
Fig 3.
Average signal intensities of IgG antibodies bound to B. pseudomallei proteins probed with melioidosis-positive and negative control samples.
The diagram shows the average signal intensity of each antigen (spotted protein solution of 0.45 mg/ml) incubated with sera from melioidosis-positive groups (week 0, 12 and 52 p.a.), healthy control individuals from endemic and non-endemic areas, as well as samples from patients with other bacteremia or fungaemia obtained in the non-endemic area of Greifswald. Not shown are values for antigens with His-tag. Error bars indicate standard error of the mean (SEM).
Fig 4.
Experimental timeline of probing a collection of positive sera drawn from single melioidosis patients (n = 36) upon admission (week 0) and after 12 and 52 weeks p.a.
All sera were sampled in Ubon Ratchathani, Thailand. The antigens are shown in rows with five increasing concentrations per protein, and the patient samples are represented in columns. Array signals are reflected by the intensities of the color (white to blue) inside the boxes. The heatmap was created using Multi experiment Viewer (MeV 4.9.0) from TM4 suite, USA.
Fig 5.
Development of signal intensities of grouped antigens inducing a long-term antibody response (A) and a short-term antibody response (B).
Sera of individual patients (n = 36) were drawn upon admission (week 0 p.a.), 12 and 52 weeks p.a. Two graphs per antigen are shown. Left: the mean signal intensity per serum. Right: number of sera recognizing the respective antigen. Figures include only data of antigens found to be significantly recognized by melioidosis-positive sera. Statistical analyses were performed using repeated-measures ANOVA followed by Bonferroni's Multiple Comparison test comparing signal intensities measured in sera of week 0, 12 and 52 p.a. (*p<0.05; **p<0.01; ***p<0.001)
Fig 6.
Measured IHA titers (A) and protein-array–derived average signal intensities (B) per serum.
Melioidosis-positive and -negative samples were drawn in endemic areas of Thailand. Additionally, means with standard error of the mean (SEM) are shown for each group. The IHA titer was determined using the indirect hemagglutionation assay described elsewhere (http://www.melioidosis.info/home.aspx). Statistical analyses were performed using the Kruskal-Wallis test followed by Dunn's Multiple Comparison test, comparing titers or signal intensities measured in sera of weeks 0 (n = 75), 12 (n = 50), and 52 (n = 46), as well as from healthy individuals (n = 100) (*p<0.05; **p<0.01; ***p<0.001).
Fig 7.
Comparison of false-negative signals between IHA and the protein array.
VENN diagrams show the false-negative signals of IHA and the protein array using the melioidosis-positive sera of weeks 0 (n = 75), 12 (n = 50), and 52 (n = 46). Green shows the false-negative signals only for the IHA and blue only for the protein array. The overlaps are false-negative signals obtained by both methods. The cut-off IHA titer used was ≥160.
Table 3.
Comparative calculation of IHA and protein array sensitivity.#