Rapid and Sensitive Multiplex Detection of Burkholderia pseudomallei-Specific Antibodies in Melioidosis Patients Based on a Protein Microarray Approach

Abstract

BackgroundThe environmental bacterium Burkholderia pseudomallei causes the infectious disease melioidosis with a high case-fatality rate in tropical and subtropical regions. Direct pathogen detection can be difficult, and therefore an indirect serological test which might aid early diagnosis is desirable. However, current tests for antibodies against B. pseudomallei, including the reference indirect haemagglutination assay (IHA), lack sensitivity, specificity and standardization. Consequently, serological tests currently do not play a role in the diagnosis of melioidosis in endemic areas. Recently, a number of promising diagnostic antigens have been identified, but a standardized, easy-to-perform clinical laboratory test for sensitive multiplex detection of antibodies against B. pseudomallei is still lacking.Methods and Principal FindingsIn this study, we developed and validated a protein microarray which can be used in a standard 96-well format. Our array contains 20 recombinant and purified B. pseudomallei proteins, previously identified as serodiagnostic candidates in melioidosis. In total, we analyzed 196 sera and plasmas from melioidosis patients from northeast Thailand and 210 negative controls from melioidosis-endemic and non-endemic regions. Our protein array clearly discriminated between sera from melioidosis patients and controls with a specificity of 97%. Importantly, the array showed a higher sensitivity than did the IHA in melioidosis patients upon admission (cut-off IHA titer ≥1:160: IHA 57.3%, protein array: 86.7%; p = 0.0001). Testing of sera from single patients at 0, 12 and 52 weeks post-admission revealed that protein antigens induce either a short- or long-term antibody response.ConclusionsOur protein array provides a standardized, rapid, easy-to-perform test for the detection of B. pseudomallei-specific antibody patterns. Thus, this system has the potential to improve the serodiagnosis of melioidosis in clinical settings. Moreover, our high-throughput assay might be useful for the detection of anti-B. pseudomallei antibodies in epidemiological studies. Further studies are needed to elucidate the clinical and diagnostic significance of the different antibody kinetics observed during melioidosis.

Co-evolutionary signals fromBurkholderia pseudomalleigenomics identify its survival strategies and highlight improving environmental health as prevention policy

“…This genetic mobility possibly contributes to the presence of multiple BPSL1661 alleles observed in our study. We detected 6 major alleles of BPSL1661 (n ≥ 5 isolates, see methods) consistent with previous reports on multiple protein epitopes 31,32 . All BPSL1661 alleles share a conserved hemolysin-type calcium binding domain which is common in proteins secreted through a type I secretion system, and two copies of the VCBS domain (a repeat domain found in Vibrio, Colwellia, Bradyrhizobium and Shewanella) known to be involved in cell adhesion.…”

“…Interestingly, previous studies reported heterogeneity in human immune responses to polypeptides generated from different BPSL1661 alleles, ranging from null to strong antibody responses [31][32][33] . Such disparity in host recognition of different BPSL1661 alleles potentially suggests that the protein may not principally function in host cell invasion but play other significant roles in B. pseudomallei survival.…”

Antibodies in Melioidosis: The Role of the Indirect Hemagglutination Assay in Evaluating Patients and Exposed Populations

“…Enzyme-linked immunosorbent assay has the advantage of allowing focus on IgG subclasses and use of reader machines to reduce interlaboratory variation. Other approaches based on pathogen detection strategy including PCR, 58 – 60 microarray, 61 , 62 lateral flow immunoassay 63 – 66 and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry 67 , 68 are also in development to confirm melioidosis diagnosis.…”

A novel synthetic peptide microarray assay detects Chlamydia species-specific antibodies in animal and human sera

“…Compared to currently employed ELISA technology, several studies have demonstrated that the present ArrayTube platform works faster (approx. 2 h per run) and requires only minute amounts of serum, usually 1 μl of a 1:100 dilution 42 – 44 . More important, the information content of the experimental output is far higher, because reactive antibodies are characterized through their binding to a number of peptide antigens derived from multiple proteins.…”