Date Published: July 18, 2016
Publisher: Public Library of Science
Author(s): Christian Kohler, Susanna J. Dunachie, Elke Müller, Anne Kohler, Kemajittra Jenjaroen, Prapit Teparrukkul, Vico Baier, Ralf Ehricht, Ivo Steinmetz, Pamela L. C. Small. http://doi.org/10.1371/journal.pntd.0004847
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.
Partial Text: Melioidosis is an often fatal tropical infectious disease caused by the Gram-negative environmental bacterium Burkholderia pseudomallei [1, 2]. The disease is known to be highly endemic in Southeast Asia and northern Australia. However, an increasing number of melioidosis case reports or environmental isolation of B. pseudomallei from other parts of Asia, Africa, the Caribbean, and Central and South America suggest a worldwide, but grossly underreported distribution of B. pseudomallei between latitudes 20° N and 20° S [3–9]. Recently, Limmathurotsakul and coworkers predicted about 165,000 cases of human melioidosis per year worldwide, from which 89,000 people die . Farmers and indigenous inhabitants of rural tropical areas are population groups at greatest risk of infection, especially in times of heavy rains [1, 2, 5]. Melioidosis usually has an incubation period of 1 to 21 days (mean: 9 days) and causes a wide range of acute or chronic clinical manifestations, including pneumonia, abscesses in various organs, neurological manifestations, or severe septicemia [1, 2, 11–13]. Since B. pseudomallei is intrinsically resistant to many antibiotics, it requires an immediate diagnosis followed by specific and prolonged antibiotic therapy. Melioidosis has a case fatality rate of around 40% in northeast Thailand . In acute forms, death can occur within 24–48 hours of the onset of symptoms [15, 16].
The alarmingly large number of predicted melioidosis cases worldwide with a high mortality  emphasizes the need to improve the current diagnostic tools to detect B. pseudomallei infections. The methods for the analysis of immune responses during infection have recently been expanded by including protein microarrays that target pathogen-specific antigens [36–38]. Protein microarrays have the potential advantage of overcoming the limitations of a more or less monoplex antibody detection when single antigens are used [20, 39]. The application of single antigens and thus the restriction to certain epitopes might limit the sensitivity and specificity of diagnostic serological assays. Recently, a protein array approach was used as an antigen discovery platform, and a significant number of serodiagnostic marker proteins of B. pseudomallei were identified that were more reactive in melioidosis patients compared to controls [25, 40].