Multiple-antigen ELISA for melioidosis - a novel approach to the improved serodiagnosis of melioidosis

Abstract

BackgroundBurkholderia pseudomallei, the causative agent of melioidosis, is endemic to Southeast Asia and northern Australia. Clinical manifestations of disease are diverse, ranging from chronic infection to acute septicaemia. The current gold standard of diagnosis involves bacterial culture and identification which is time consuming and often too late for early medical intervention. Hence, rapid diagnosis of melioidosis is crucial for the successful management of melioidosis.MethodsThe study evaluated 4 purified B. pseudomallei recombinant proteins (TssD-5, Omp3, smBpF4 and Omp85) as potential diagnostic agents for melioidosis. A total of 68 sera samples from Malaysian melioidosis patients were screened for the presence of specific antibodies towards these proteins using enzyme-linked immunosorbent assay (ELISA). Sera from patients with various bacterial and viral infections but negative for B. pseudomallei, as well as sera from healthy individuals, were also included as non-melioidosis controls. The Mann Whitney test was performed to compare the statistical differences between melioidosis patients and the non-melioidosis controls.ResultsTssD-5 demonstrated the highest sensitivity of 71% followed by Omp3 (59%), smBpF4 (41%) and Omp85 (19%). All 4 antigens showed equally high specificity (89-96%). A cocktail of all 4 antigens resulted in slightly reduced sensitivity of 65% but improved specificity (99%). Multiple-antigen ELISA provided improved sensitivity of 88.2% whilst retaining good specificity (96%). There was minimal reactivity with sera from healthy individuals proposing the utility of these antigens to demarcate diseased from non-symptomatic individuals in an endemic country.ConclusionsTssD-5 demonstrated high detection sensitivity and specificity and the results were obtained within a few hours compared to time consuming culture and IFAT methods commonly used in a clinical setting. The use of multiple-antigens resulted in improved sensitivity (88.2%) whilst maintaining superior specificity. These data highlight the use of TssD-5 and other recombinant antigens tested in this study as potential serodiagnostic agents for melioidosis.

Efficacy of indirect ELISA for serodiagnosis of melioidosis using immunodominant antigens from non-pathogenic Burkholderia thailandensis

“…Several previous studies demonstrate that truncated flagellin fragment and OmpA protein of B. pseudomallei serve as the diagnostic potential of melioidosis (Allwood et al 2008 ; Arora et al 2015 ; Chen et al 2003 ; Hara et al 2013 ). In this study, the recombinant proteins, OmpABT and FLAG300, from the closely related species, B. thailandensis , were evaluated as antigens which specifically react to melioidosis antibodies using indirect ELISA and we calculated the serodiagnostic indices.…”

“…A number of serodiagnostic antigens of B. pseudomallei have been revealed using protein microarray (Felgner et al 2009 ) and indirect enzyme-linked immunosorbent assays (ELISA) (Allwood et al 2008 ; Arora et al 2015 ; Wajanarogana et al 2013 ; Hara et al 2013 ). The majority of such antigens are cell surface-associated proteins such as outer membrane proteins (Omps), and secreted molecules.…”

“…The FLAG300 indicates as the potential antigen for diagnosis of melioidosis (Wajanarogana et al 2013 ); however, culture-confirmed melioidosis serum samples have not yet been studied. As well as a flagellin fragment, OmpA of B. pseudomallei has immunogenic properties which has been used for serodiagnosis of melioidosis (Allwood et al 2008 ; Arora et al 2015 ; Hara et al 2013 ). Moreover, both of these antigens have been suggested as potential candidate vaccines (Brett and Woods 1996 ; Hara et al 2009 ; Ye and Gan 2007 ).…”

Development of repeatable arrays of proteins using immobilized DNA microplate (RAPID-M) technology

“…B. pseudomallei is the causative agent of melioidosis and generally, diagnosis is fully dependent on bacterial culture. Many diagnostic tests have been developed to detect B. pseudomallei including real time polymerase chain reaction (PCR) detecting type III secretion systems [ 18 ], loop-mediated isothermal amplification (LAMP) method [ 19 ], multiple-antigen enzyme-linked immunosorbent assay (ELISA) [ 20 ] and a protein microarray [ 17 ]. However, none of these tests have been translated into a standard form of diagnosis.…”

Evolving serodiagnostics by rationally designed peptide arrays: the Burkholderia paradigm in Cystic Fibrosis

“…proteins and lipopolysaccharides) adsorbed on a rigid support as baits to capture antibodies. ELISA is reasonably quick but limited in the number of probes and the use of complete antigens (such as recombinant proteins or complex saccharides) sets severe limitations in terms of cost and versatility 1 2 .…”