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Updated: Jun 14, 2025
Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS
Published on: July 11, 2016
Performance of MALDI-TOF MS, real-time PCR, antigen detection, and automated biochemical testing for the
Stuart Campbell1, Brooke Taylor2, Dimitrios Menouhos2
1Department of Infectious Diseases, Division of Medicine, Royal Darwin Hospital, Darwin, Australia.
Abstract:
Burkholderia pseudomallei 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 B. pseudomallei 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 in vitro diagnostic database for use with the Vitek MS has recently been updated to include B. pseudomallei 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 B. pseudomallei and a range of closely related Burkholderia species, and a prospective "validation" cohort of B. pseudomallei and B. cepacia complex clinical isolates. MALDI-TOF MS had a sensitivity of 1.0 and specificity of 1.0 for the identification and differentiation of B. pseudomallei from related Burkholderia species when a certainty cutoff of 99.9% was used. In contrast, automated biochemical testing for B. pseudomallei identification had a sensitivity of 0.83 and specificity of 0.88. Both qPCR and LFA correctly identified all B. pseudomallei isolates with no false positives. Due to the high level of accuracy, we have now incorporated MALDI-TOF MS into our laboratory's B. pseudomallei identification workflow.IMPORTANCEBurkholderia pseudomallei 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 B. pseudomallei identification and found mass spectrometry highly accurate. We have therefore incorporated this fast and cost-effective method into our laboratory's workflow for B. pseudomallei identification.
Insights
Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) accurately identifies Burkholderia pseudomallei, the cause of melioidosis. This method is now integrated into laboratory workflows for reliable B. pseudomallei detection.
Area of Science:
- Clinical Microbiology
- Infectious Diseases
- Diagnostic Technology
Background:
- Burkholderia pseudomallei is the causative agent of melioidosis, a serious disease endemic to Southeast Asia and Australia, with expanding geographical reach.
- Accurate and timely laboratory identification of B. pseudomallei is crucial for patient management, yet can be challenging, leading to potential misdiagnoses.
- Existing diagnostic methods like automated biochemical testing and MALDI-TOF MS have reported identification inaccuracies.
Purpose of the Study:
- To validate the performance of an updated MALDI-TOF MS database for identifying Burkholderia pseudomallei.
- To compare the accuracy of MALDI-TOF MS against automated biochemical testing (Vitek 2 GN card), quantitative real-time PCR (qPCR), and lateral flow immunoassay (LFA).
- To assess the utility of MALDI-TOF MS for differentiating B. pseudomallei from closely related Burkholderia species.
Main Methods:
- A derivation cohort of geographically diverse B. pseudomallei and related Burkholderia species was tested.
- A prospective validation cohort of clinical isolates, including B. pseudomallei and B. cepacia complex, was analyzed.
- Identification performance was evaluated using MALDI-TOF MS with a 99.9% certainty cutoff, Vitek 2 GN card, qPCR targeting the type III secretion system, and LFA for capsular polysaccharide antigen.
Main Results:
- MALDI-TOF MS demonstrated a sensitivity of 1.0 and specificity of 1.0 for B. pseudomallei identification and differentiation when using a 99.9% certainty threshold.
- Automated biochemical testing showed lower performance with a sensitivity of 0.83 and specificity of 0.88.
- Both qPCR and LFA achieved perfect identification of all B. pseudomallei isolates without any false positives.
Conclusions:
- The updated MALDI-TOF MS database provides highly accurate identification and differentiation of Burkholderia pseudomallei.
- MALDI-TOF MS is a reliable, fast, and cost-effective method for B. pseudomallei identification, surpassing automated biochemical testing.
- The laboratory has incorporated MALDI-TOF MS into its routine workflow for B. pseudomallei identification due to its high accuracy.
