Development of a Prototype Lateral Flow Immunoassay (LFI) for the Rapid Diagnosis of Melioidosis

Raymond L Houghton,Paul J Brett,Sharon J Peacock,David P Aucoin,Direk Limmathurotsakul,Vanessa Theobald,Michael J Dillon,Gumphol Wongsuvan,Alex R Hoffmaster,Hongjing Chen,Dana E Reed,Derek S Sarovich,Brea Duval,Mary N Burtnick,Mark Mayo,Bart J Currie,Narisara Chantratita,Mark A Hubbard

doi:10.1371/journal.pntd.0002727

Abstract

Burkholderia pseudomallei is a soil-dwelling bacterium and the causative agent of melioidosis. Isolation of B. pseudomallei from clinical samples is the “gold standard” for the diagnosis of melioidosis; results can take 3–7 days to produce. Alternatively, antibody-based tests have low specificity due to a high percentage of seropositive individuals in endemic areas. There is a clear need to develop a rapid point-of-care antigen detection assay for the diagnosis of melioidosis. Previously, we employed In vivo Microbial Antigen Discovery (InMAD) to identify potential B. pseudomallei diagnostic biomarkers. The B. pseudomallei capsular polysaccharide (CPS) and numerous protein antigens were identified as potential candidates. Here, we describe the development of a diagnostic immunoassay based on the detection of CPS. Following production of a CPS-specific monoclonal antibody (mAb), an antigen-capture immunoassay was developed to determine the concentration of CPS within a panel of melioidosis patient serum and urine samples. The same mAb was used to produce a prototype Active Melioidosis Detect Lateral Flow Immunoassay (AMD LFI); the limit of detection of the LFI for CPS is comparable to the antigen-capture immunoassay (∼0.2 ng/ml). The analytical reactivity (inclusivity) of the AMD LFI was 98.7% (76/77) when tested against a large panel of B. pseudomallei isolates. Analytical specificity (cross-reactivity) testing determined that 97.2% of B. pseudomallei near neighbor species (35/36) were not reactive. The non-reactive B. pseudomallei strain and the reactive near neighbor strain can be explained through genetic sequence analysis. Importantly, we show the AMD LFI is capable of detecting CPS in a variety of patient samples. The LFI is currently being evaluated in Thailand and Australia; the focus is to optimize and validate testing procedures on melioidosis patient samples prior to initiation of a large, multisite pre-clinical evaluation.

Highlights

Burkholderia pseudomallei is an environmental Gram-negative bacillus and the cause of melioidosis
We have developed a lateral flow immunoassay that can be used in the clinical setting to diagnose melioidosis in 15 minutes
Our previous report described the ability of monoclonal antibody (mAb) 3C5 to detect B. pseudomallei capsular polysaccharide (CPS) in urine from patients with melioidosis [10]

Summary

Introduction

Burkholderia pseudomallei is an environmental Gram-negative bacillus and the cause of melioidosis. The clinical manifestations of melioidosis are broad and include disseminated disease with organ abscesses, severe sepsis, and mild infection of the skin and soft tissue [1]. Most patients have risk factors for infection, which include diabetes, heavy alcohol use, and chronic pulmonary or kidney disease [1,2,3]. Rising incidence rates have been recorded in northeast Thailand between. 1997–2006, during which the average mortality rate was 42.6% [3]. In 2006, melioidosis and tuberculosis mortality rates in northeast Thailand were equivalent and second only to HIV/ AIDS for infectious disease deaths [3]. In northern Australia the mortality rate over the last five years of the Darwin prospective melioidosis study was calculated at 9% [2]. The authors attributed the low mortality rate to early diagnosis and treatment, and access to and improvements in intensive care management [2]

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Conclusion