Development of a prototype Lateral Flow Immunoassay (LFI) for the rapid diagnosis of melioidosis

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

Altered Growth, Pigmentation, and Antimicrobial Susceptibility Properties of Staphylococcus aureus Due to Loss of the Major Cold Shock Gene cspB▿

An insertional mutation made in the major cold shock gene cspB in Staphylococcus aureus strain COL, a methicillin-resistant clinical isolate, yielded a mutant that displayed a reduced capacity to respond to cold shock and many phenotypic characteristics of S. aureus small-colony variants: a growth defect at 37°C, a reduction in pigmentation, and altered levels of susceptibility to many antimicrobials. In particular, a cspB null mutant displayed increased resistance to aminoglycosides, trimethoprim-sulfamethoxazole, and paraquat and increased susceptibility to daptomycin, teicoplanin, and methicillin. With the exception of the increased susceptibility to methicillin, which was due to a complete loss of the type I staphylococcal cassette chromosome mec element, these properties were restored to wild-type levels by complementation when cspB was expressed in trans. Taken together, our results link a stress response protein (CspB) of S. aureus to important phenotypic properties that include resistance to certain antimicrobials

Prototype AMD LFI for detection of <i>B. pseudomallei</i> CPS in melioidosis patient samples.

<p>(A) Preliminary testing of a variety of archived patient samples from Australia and Thailand. (B) Detection of CPS in melioidosis patient urine samples (filtered) listed in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002727#pntd-0002727-t002" target="_blank">Table 2</a>. Urine (50 µl) was combined with 100 µl of chase buffer and applied to the sample pad. Note that samples that were positive by antigen-capture immunoassay (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002727#pntd-0002727-t002" target="_blank">Table 2</a>) were also positive by LFI and the levels of CPS detected between both assays are congruent.</p

Detection of purified CPS by antigen-capture ELISA.

<p>mAb 3C5 was used in the capture phase of the ELISA at the concentrations listed. Following a wash and blocking step, purified CPS was serially diluted across the microtiter plate at the concentrations listed. The wells were then washed and HRP-labeled mAb 3C5 was used in the indicator phase to detect captured CPS. The ELISA was performed in triplicate and mean values are plotted.</p

Prototype Active Melioidosis Detect (AMD) LFI.

<p>(A) Schematic of LFI components. (B) <i>B. pseudomallei</i> strain Bp82 colony grown on an agar plate was picked and suspended in 2 drops of lysis buffer. The lysate was added to the sample pad followed by three drops of LFI chase buffer (top LFI). The LFI was imaged following a 15 min run time. The same test condition were used with a colony of <i>E. coli</i> (bottom LFI).</p

Calculation of mAb 3C5 affinity for CPS.

<p>A BIAcore X100 instrument was used to determine the affinity of mAb 3C5 for CPS. Biotinylated CPS was immobilized on the surface of a streptavidin sensor chip. Samples (two-fold serial dilution of mAb 3C5 [333–5.2 nM]) were injected over the sensor surface for 60 s, after which the mAb was allowed to passively dissociate for 120 s (left panel). The dissociation constant (K_D) was determined using the steady-state model in BIAevaluation software (right panel).</p

Active Melioidosis Detect analytical reactivity and specificity.

<p>*Indicates strains that were tested for reactivity against mAb 3C5 via western blot.</p