Performance of MALDI-TOF MS, real-time PCR, antigen detection, and automated biochemical testing for the identification of <i>Burkholderia pseudomallei</i>

<i>Burkholderia pseudomallei</i> is the causative agent of melioidosis, a disease highly endemic to Southeast Asia and northern Australia, though the area of endemicity is expanding. Cases may occur in returning travelers or, rarely, from imported contaminated products. Identification of <i>B. pseudomallei</i> is challenging for laboratories that do not see this organism frequently, and misidentifications by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and automated biochemical testing have been reported. The <i>in vitro</i> diagnostic database for use with the Vitek MS has recently been updated to include <i>B. pseudomallei</i> and we aimed to validate the performance for identification in comparison to automated biochemical testing with the Vitek 2 GN card, quantitative real-time polymerase chain reaction (qPCR) targeting the type III secretion system, and capsular polysaccharide antigen detection using a lateral flow immunoassay (LFA). We tested a "derivation" cohort including geographically diverse <i>B. pseudomallei</i> and a range of closely related <i>Burkholderia</i> species, and a prospective "validation" cohort of <i>B. pseudomallei</i> and <i>B. cepacia</i> complex clinical isolates. MALDI-TOF MS had a sensitivity of 1.0 and specificity of 1.0 for the identification and differentiation of <i>B. pseudomallei</i> from related <i>Burkholderia</i> species when a certainty cutoff of 99.9% was used. In contrast, automated biochemical testing for <i>B. pseudomallei</i> identification had a sensitivity of 0.83 and specificity of 0.88. Both qPCR and LFA correctly identified all <i>B. pseudomallei</i> isolates with no false positives. Due to the high level of accuracy, we have now incorporated MALDI-TOF MS into our laboratory's <i>B. pseudomallei</i> identification workflow.IMPORTANCE<i>Burkholderia pseudomallei</i> causes melioidosis, a disease associated with high morbidity and mortality that disproportionately affects rural areas in Southeast Asia and northern Australia. The known area of endemicity is expanding and now includes the continental United States. Laboratory identification can be challenging which may result in missed or delayed diagnoses and poor patient outcomes. In this study, we compared mass spectrometry using an updated spectral database with multiple other methods for <i>B. pseudomallei</i> identification and found mass spectrometry highly accurate. We have therefore incorporated this fast and cost-effective method into our laboratory's workflow for <i>B. pseudomallei</i> identification.