Evaluation of Polysaccharide-Based Latex Agglutination Assays for the Rapid Detection of Antibodies to Burkholderia pseudomallei.

Melioidosis is a severe disease caused by the Gram-negative bacterium Burkholderia pseudomallei. Diagnosis of melioidosis currently relies on the isolation of B. pseudomallei from clinical samples, which can take several days. An indirect hemagglutination assay (IHA) is widely used for serodiagnosis, but it has a short shelf life, is poorly standardized, and requires a viable bacteria culture performed in a biosafety level 3 (BSL-3) laboratory. To improve the diagnostic methods, we have developed two rapid latex agglutination tests based on purified B. pseudomallei O-polysaccharide (OPS) and capsular polysaccharide (CPS) antigens. The immunodiagnostic potential of these tests was evaluated using serum from culture-confirmed melioidosis patients (N = 143) and healthy donors from either endemic (N = 199) or non-endemic areas (N = 90). The sensitivity of the OPS-based latex agglutination assay (OPS-latex; 84.4%) was significantly higher than both the CPS-latex (69.5%) (P \u3c 0.001) and IHA (69.5%) (P = 0.001). When evaluated with Thai donor serum, the OPS-latex had comparable specificity (56.9%) to the CPS-latex (63.8%) (P = 0.053), but was significantly lower than the IHA (67.6%) (P = 0.002). In contrast, all tests with U.S. donor serum were highly specific (≥ 97.8%). These results suggest that polysaccharide-based latex agglutination assays may be useful for serodiagnosis of melioidosis in non-endemic areas

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the identification of Burkholderia pseudomallei from Asia and Australia and differentiation between Burkholderia species.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is increasingly used for rapid bacterial identification. Studies of Burkholderia pseudomallei identification have involved small isolate numbers drawn from a restricted geographic region. There is a need to expand the reference database and evaluate B. pseudomallei from a wider geographic distribution that more fully captures the extensive genetic diversity of this species. Here, we describe the evaluation of over 650 isolates. Main spectral profiles (MSP) for 26 isolates of B. pseudomallei (N = 5) and other Burkholderia species (N = 21) were added to the Biotyper database. MALDI-TOF MS was then performed on 581 B. pseudomallei, 19 B. mallei, 6 B. thailandensis and 23 isolates representing a range of other bacterial species. B. pseudomallei originated from northeast and east Thailand (N = 524), Laos (N = 12), Cambodia (N = 14), Hong Kong (N = 4) and Australia (N = 27). All 581 B. pseudomallei were correctly identified, with 100% sensitivity and specificity. Accurate identification required a minimum inoculum of 5 x 107 CFU/ml, and identification could be performed on spiked blood cultures after 24 hours of incubation. Comparison between a dendrogram constructed from MALDI-TOF MS main spectrum profiles and a phylogenetic tree based on recA gene sequencing demonstrated that MALDI-TOF MS distinguished between B. pseudomallei and B. mallei, while the recA tree did not. MALDI-TOF MS is an accurate method for the identification of B. pseudomallei, and discriminates between this and other related Burkholderia species

MALDI-TOF mass protein spectra of <i>B</i>. <i>pseudomallei</i> and 8 other genetically related <i>Burkholderia</i> species.

<p>The vertical axis shows relative intensities of ions and the horizontal axis shows mass to charge ratio (m/z) or masses of ions (Da).</p

<i>B</i>. <i>pseudomallei</i> and other <i>Burkholderia</i> species used in this study.

<p><i>B</i>. <i>pseudomallei</i> and other <i>Burkholderia</i> species used in this study.</p

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the identification of <i>Burkholderia pseudomallei</i> from Asia and Australia and differentiation between <i>Burkholderia</i> species

<div><p>Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is increasingly used for rapid bacterial identification. Studies of <i>Burkholderia pseudomallei</i> identification have involved small isolate numbers drawn from a restricted geographic region. There is a need to expand the reference database and evaluate <i>B</i>. <i>pseudomallei</i> from a wider geographic distribution that more fully captures the extensive genetic diversity of this species. Here, we describe the evaluation of over 650 isolates. Main spectral profiles (MSP) for 26 isolates of <i>B</i>. <i>pseudomallei</i> (N = 5) and other <i>Burkholderia</i> species (N = 21) were added to the Biotyper database. MALDI-TOF MS was then performed on 581 <i>B</i>. <i>pseudomallei</i>, 19 <i>B</i>. <i>mallei</i>, 6 <i>B</i>. <i>thailandensis</i> and 23 isolates representing a range of other bacterial species. <i>B</i>. <i>pseudomallei</i> originated from northeast and east Thailand (N = 524), Laos (N = 12), Cambodia (N = 14), Hong Kong (N = 4) and Australia (N = 27). All 581 <i>B</i>. <i>pseudomallei</i> were correctly identified, with 100% sensitivity and specificity. Accurate identification required a minimum inoculum of 5 x 10⁷ CFU/ml, and identification could be performed on spiked blood cultures after 24 hours of incubation. Comparison between a dendrogram constructed from MALDI-TOF MS main spectrum profiles and a phylogenetic tree based on <i>recA</i> gene sequencing demonstrated that MALDI-TOF MS distinguished between <i>B</i>. <i>pseudomallei</i> and <i>B</i>. <i>mallei</i>, while the <i>recA</i> tree did not. MALDI-TOF MS is an accurate method for the identification of <i>B</i>. <i>pseudomallei</i>, and discriminates between this and other related <i>Burkholderia</i> species.</p></div

MALDI-TOF scores of <i>B</i>. <i>pseudomallei</i> from different geographical origins.

<p>MALDI-TOF scores of <i>B</i>. <i>pseudomallei</i> from different geographical origins.</p

Phylogenetic tree of protein profiles of <i>B</i>. <i>pseudomallei</i> and 8 other genetically related <i>Burkholderia</i> species.

<p>Distance is displayed in relative units. Annotated with MLST sequence type (ST) where known; each color represents a ST. The latex agglutination test was based on a monoclonal that recognizes <i>B</i>. <i>pseudomallei</i> capsular polysaccharide.</p