Relationship between PFGE and MLST analysis of soil isolates.

<p>Relationship between PFGE and MLST analysis of soil isolates.</p

Genotyping results for 200 colonies of <i>B. pseudomallei</i> from each of three independent sampling points.

<p>Genotyping results for 200 colonies of <i>B. pseudomallei</i> from each of three independent sampling points.</p

Venn diagram showing culture (blue), lateral flow immunoassay (LFI; yellow) and qPCR assay (red) results of 105 soil samples.

<p>Culture was performed according to the consensus guidelines for environmental sampling of <i>B</i>. <i>pseudomallei</i> developed by DEBWorP [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005204#pntd.0005204.ref011" target="_blank">11</a>], in which soil specimens were enriched in the enrichment broth for 2 days and the upper layer of enrichment broth on day 2 was streaked on agar plates and observed daily. For the LFI and qPCR assay, soil specimens were enriched in the broth for 7 days and the broth on day 7 was used for the tests.</p

Utility of a Lateral Flow Immunoassay (LFI) to Detect <i>Burkholderia pseudomallei</i> in Soil Samples

<div><p>Background</p><p>Culture is the gold standard for the detection of environmental <i>B</i>. <i>pseudomallei</i>. In general, soil specimens are cultured in enrichment broth for 2 days, and then the culture broth is streaked on an agar plate and incubated further for 7 days. However, identifying <i>B</i>. <i>pseudomallei</i> on the agar plates among other soil microbes requires expertise and experience. Here, we evaluate a lateral flow immunoassay (LFI) developed to detect <i>B</i>. <i>pseudomallei</i> capsular polysaccharide (CPS) in clinical samples as a tool to detect <i>B</i>. <i>pseudomallei</i> in environmental samples.</p><p>Methodology/Principal Findings</p><p>First, we determined the limit of detection (LOD) of LFI for enrichment broth of the soil specimens. Soil specimens (10 grams/specimen) culture negative for <i>B</i>. <i>pseudomallei</i> were spiked with <i>B</i>. <i>pseudomallei</i> ranging from 10 to 10⁵ CFU, and incubated in 10 ml of enrichment broth in air at 40°C. Then, on day 2, 4 and 7 of incubation, 50 μL of the upper layer of the broth were tested on the LFI, and colony counts to determine quantity of <i>B</i>. <i>pseudomallei</i> in the broth were performed. We found that all five soil specimens inoculated at 10 CFU were negative by LFI on day 2, but four of those five specimens were LFI positive on day 7. The LOD of the LFI was estimated to be roughly 3.8x10⁶ CFU/ml, and culture broth on day 7 was selected as the optimal sample for LFI testing. Second, we evaluated the utility of the LFI by testing 105 soil samples from Northeast Thailand. All samples were also tested by standard culture and quantitative PCR (qPCR) targeting <i>orf2</i>. Of 105 soil samples, 35 (33%) were LFI positive, 25 (24%) were culture positive for <i>B</i>. <i>pseudomallei</i>, and 79 (75%) were qPCR positive. Of 11 LFI positive but standard culture negative specimens, six were confirmed by having the enrichment broth on day 7 culture positive for <i>B</i>. <i>pseudomallei</i>, and an additional three by qPCR. The LFI had 97% (30/31) sensitivity to detect soil specimens culture positive for <i>B</i>. <i>pseudomallei</i>.</p><p>Conclusions/Significance</p><p>The LFI can be used to detect <i>B</i>. <i>pseudomallei</i> in soil samples, and to select which samples should be sent to reference laboratories or proceed further for bacterial isolation and confirmation. This could considerably decrease laboratory workload and assist the development of a risk map for melioidosis in resource-limited settings.</p></div

Graph of the proportion of all pairwise comparisons showing 0, 1, 2 … 7 allelic mismatches for each of 200 primary colonies (strains) examined at three independent sampling points.

<p>The “trials” data represents mean values for 100 random samples of 200 strains drawn from the combined data set of 600 strains from all three sampling points (with replacement). Error bars are based on the 5^th and 95^th percentiles of the 100 random samples. No pw comparisons (real or trial data) differ at all seven loci since the locus <i>ndh</i> is monomorphic (invariant).</p

Diagnostic sensitivity and specificity of real-time PCR assays targeting <i>rrs</i> or <i>lipL32.</i>

<p>Diagnostic sensitivity and specificity of real-time PCR assays targeting <i>rrs</i> or <i>lipL32.</i></p

Summary data for antimicrobial susceptibility profiles of 17 CA-MRSA isolates.

<p>Summary data for antimicrobial susceptibility profiles of 17 CA-MRSA isolates.</p

The presence of <i>B. pseudomallei</i> in 100 spaced sampling points within an area of disused land in northeast Thailand.

<p>The presence of <i>B. pseudomallei</i> in 100 spaced sampling points within an area of disused land in northeast Thailand.</p

Map of Cambodia showing location of residence for 17 children with CA-MRSA infection presenting to the Angkor Hospital for Children in Siem Reap (A).

<p>Zoomed image of the three affected provinces. Numbers in brackets denote the number of children affected in each village (B).</p

<i>Leptospira</i> spp. used during this study.

@<p>WHO/FOA/OIE/ Collaborating Center for Reference and Research on Leptospirosis, Australia;</p><p>*Bureau of Emerging Infection Disease, Ministry of Public Health, Thailand;</p>#<p>American Type Culture Collection, USA;</p>$<p>Royal Tropical Institute (KIT), Netherland;</p>a<p>Dr Thareerat Kalambaheti, Mahidol University, Thailand.</p><p>+ and − indicate a positive or negative result in the relevant PCR assay, respectively. (+) indicates a positive PCR result at a high Cq.</p