Abstract
Burkholderia pseudomallei, the causative agent of melioidosis, is an environmental bacillus found in northeast Thailand. The mortality rate of melioidosis is ∼40%. An indirect hemagglutination assay (IHA) is used as a reference serodiagnostic test; however, it has low specificity in areas where the background seropositivity of healthy people is high. To improve assay specificity and reduce the time for diagnosis, four rapid enzyme-linked immunosorbent assays (ELISAs) were developed using two purified polysaccharide antigens (O-polysaccharide [OPS] and 6-deoxyheptan capsular polysaccharide [CPS]) and two crude antigens (whole-cell [WC] antigen and culture filtrate [CF] antigen) of B. pseudomallei. The ELISAs were evaluated using serum samples from 141 culture-confirmed melioidosis patients from Thailand along with 188 healthy donors from Thailand and 90 healthy donors from the United States as controls. The areas under receiver operator characteristic curves (AUROCC) using Thai controls were high for the OPS-ELISA (0.91), CF-ELISA (0.91), and WC-ELISA (0.90), while those of CPS-ELISA (0.84) and IHA (0.72) were lower. AUROCC values using U.S. controls were comparable to those of the Thai controls for all ELISAs except IHA (0.93). Using a cutoff optical density (OD) of 0.87, the OPS-ELISA had a sensitivity of 71.6% and a specificity of 95.7% for Thai controls; for U.S. controls, specificity was 96.7%. An additional 120 serum samples from tuberculosis, scrub typhus, or leptospirosis patients were evaluated in all ELISAs and resulted in comparable or higher specificities than using Thai healthy donors. Our findings suggest that antigen-specific ELISAs, particularly the OPS-ELISA, may be useful for serodiagnosis of melioidosis in areas where it is endemic and nonendemic.
Highlights
Melioidosis is a severe illness caused by Burkholderia pseudomallei, a Gram-negative bacterium that is naturally found in moist soils and surface waters in tropical and subtropical regions
We have recently developed two rapid latex agglutination assays based on B. pseudomallei surface polysaccharides, the O-polysaccharide (OPS) component of lipopolysaccharide (LPS) or capsular polysaccharide (CPS), and demonstrated that OPS is a promising antigen for serodiagnosis of melioidosis in areas where the disease is nonendemic [24]
Since our results showed a strong correlation between the OPS-enzyme-linked immunosorbent assays (ELISAs) and the two ELISAs based on crude antigens (WC-ELISA and Culture filtrate (CF)-ELISA), we investigated the possibility that OPS might be the predominant antigen recognized by human antibodies in the crude preparations
Summary
Melioidosis is a severe illness caused by Burkholderia pseudomallei, a Gram-negative bacterium that is naturally found in moist soils and surface waters in tropical and subtropical regions. Isolation of B. pseudomallei from clinical specimens followed by biochemical identification is routinely used in hospital laboratories This technique is specific and relatively low cost, definitive identification of B. pseudomallei requires experience and can be time-consuming (2 to 7 days). Culture is not a perfect gold standard because it has only 60% sensitivity [15] Possible explanations for this may be the low B. pseudomallei numbers in clinical samples [16] or the presence of unculturable forms of the organism that have been associated with previous antibiotic treatment in some patients. While these tests are highly specific (Ͼ95%), the sensitivities are only 48% for IFA [17], 34% to 61% for PCR based on TTS1 and 16S rRNA genes, respectively, and 44% for LAMP based on the TTS1 gene [18, 19]. A lateral flow immunoassay (LFI) for the detection of the B. pseudomallei 6-deoxyheptan capsular polysaccharide (CPS) antigen has been developed and shown to be highly specific but had low sensitivity (40%) when used with wholeblood specimens [22, 23]
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