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

Yuka Hara 1 Chui-Yoke Chin 0 Rahmah Mohamed 0 Savithri D Puthucheary Sheila Nathan 0 0 School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia , Bangi, Selangor D.E 43600 , Malaysia 1 Malaysia Genome Institute , Jalan Bangi, Kajang, Selangor D.E 43000 , Malaysia Background: Burkholderia 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. Methods: The 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 nonmelioidosis controls. Results: TssD-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. Conclusions: TssD-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. - Background Melioidosis is a severe community-acquired infectious disease caused by the Gram negative bacillus Burkholderia pseudomallei. The bacterium is commonly found in soil and water in Southeast Asia and northern Australia; however, increasing cases of melioidosis have been reported in other tropical regions and the bacterium is now believed to present a serious global threat [1,2]. Clinical manifestations of melioidosis are extremely diverse and vary from acute sepsis to chronic localised pathology to latent infections which can reactivate decades later [3,4]. In all series, the lung was the most commonly affected organ, either presenting with cough and fever resulting from a primary lung abscess or pneumonia, or secondary to septicaemic spread [5]. The overall mortality rate in individuals infected with B. pseudomallei range from 30-70%, depending on whether the patient is septicaemic or not [5]. In northeast Thailand the mortality rate is reported to be 50% (35% in children) and 19% in Australia [3,6]. Melioidosis is treatable if detected early, however, early medical intervention is hindered by two major factors; firstly, the definitive diagnosis involves bacterial culture and identification which is time consuming and often leads to a critical delay in antibiotic therapy and successful management of the acute form of the disease [7]. Secondly, B. pseudomallei is resistant to diverse groups of antimicrobials including third generation cephalosporins whilst quinolones and aminoglycosides have no reliable effect [8,9]. Hence, a definitive and rapid diagnosis is critical and vital prior to the administration of ceftazidime or carbapenems as these antibiotics are generally not used as empirical treatment for septicaemia in endemic regions. Culturing of bacteria from clinical samples represents the diagnostic gold standard for melioidosis [3,10]. Whilst it is simple, reliable and economical, the long duration required to reach a definitive diagnosis is a major drawback. The most commonly used serological method, the indirect hemagglutination test (IHA), is poorly standardised worldwide. Moreover, IHA has limited clinical value in regions of endemicity due to the high background antibody titres in healthy individuals, most likely the result of repeated environmental exposure to B. pseudomallei [11]. In addition, IHA uses crude whole-cell preparation or bacterial lysates which may potentially lead to high false-positive results. A critical limitation of this assay is the lack of standardisation between laboratories with respect to the antigens used, the antigens remain poorly characterised and are likely to be variable between isolates [12]. The indirect immune-fluorescence antibody test (IFAT) using whole B. pseudomallei cells as antigen was found to be sensitive and superior to IHA and requires only a day to obtain the results [13]. The only drawback is that IFAT requires a fluorescence microscope and skilled personnel which might not be readily available in rural endemic regions of Southeast Asia. Enzyme-linked immunosorbent assay (ELISA) and related serodiagnostic strategies are being considered more favourably as rapid and reliable tools for definitive diagnosis of melioidosis [14-16]. Various antigen preparations such as crude and purified exopolysaccharide (EPS) and lipopolysaccharide (LPS) as well as recombinant flagellin and Bip proteins have been reported as potential diagnostic antigens in an ELISA format, however, sensitive and reliable antigens are yet to be identified. We have previously reported the characterisation of several immunogenic B. pseudomallei recombinant proteins including outer membrane protein A (Omp3), Omp85 and serine protease MprA (smBpF4) [17-19]. Recently, Chieng et al. [20] described the elevated induction levels of B. pseudomallei type VI secretion system HCP protein (TssD-5) over a 6 hr infection period in human macrophages and preliminary analysis of the antigenicity of this protein has indicated strong binding to antibodies in a small cohort of melioidosis-confirmed patient sera (Chieng et al. in preparation). All four recombinant proteins are unique to Burkholderia spp. and interestingly, TssD-5 is highly conserved in B. pseudomallei only whilst the other recombinant proteins are conserved in B. pseudomallei as well as in Burkholderia spp. such as B. mallei and B. thailandensis. Thus, all the selected proteins should demonstrate minimal cross reactivity with sera f (...truncated)