A Quadruplex Real-Time PCR Assay for the Rapid Detection and Differentiation of the Most Relevant Members of the B. pseudomallei Complex: B. mallei, B. pseudomallei, and B. thailandensis.

The Burkholderia pseudomallei complex classically consisted of B. mallei, B. pseudomallei, and B. thailandensis, but has now expanded to include B. oklahomensis, B. humptydooensis, and three unassigned Burkholderia clades. Methods for detecting and differentiating the B. pseudomallei complex has been the topic of recent research due to phenotypic and genotypic similarities of these species. B. mallei and B. pseudomallei are recognized as CDC Tier 1 select agents, and are the causative agents of glanders and melioidosis, respectively. Although B. thailandensis and B. oklahomensis are generally avirulent, both display similar phenotypic characteristics to that of B. pseudomallei. B. humptydooensis and the Burkholderia clades are genetically similar to the B. pseudomallei complex, and are not associated with disease. Optimal identification of these species remains problematic, and PCR-based methods can resolve issues with B. pseudomallei complex detection and differentiation. Currently, no PCR assay is available that detects the major species of the B. pseudomallei complex. A real-time PCR assay in a multiplex single-tube format was developed to simultaneously detect and differentiate B. mallei, B. pseudomallei, and B. thailandensis, and a common sequence found in B. pseudomallei, B. mallei, B. thailandensis, and B. oklahomensis. A total of 309 Burkholderia isolates and 5 other bacterial species were evaluated. The assay was 100% sensitive and specific, demonstrated sensitivity beyond culture and GC methods for the isolates tested, and is completed in about an hour with a detection limit between 2.6pg and 48.9pg of gDNA. Bioinformatic analyses also showed the assay is likely 100% specific and sensitive for all 84 fully sequenced B. pseudomallei, B. mallei, B. thailandensis, and B. oklahomensis strains currently available in GenBank. For these reasons, this assay could be a rapid and sensitive tool in the detection and differentiation for those species of the B. pseudomallei complex with recognized clinical and practical significance.

PCR-based Methodologies Used to Detect and Differentiate the Burkholderia pseudomallei complex: B. pseudomallei, B. mallei, and B. thailandensis.

Methods for the rapid detection and differentiation of the Burkholderia pseudomallei complex comprising B. pseudomallei, B. mallei, and B. thailandensis, have been the topic of recent research due to the high degree of phenotypic and genotypic similarities of these species. B. pseudomallei and B. mallei are recognized by the CDC as tier 1 select agents. The high mortality rates of glanders and melioidosis, their potential use as bioweapons, and their low infectious dose, necessitate the need for rapid and accurate detection methods. Although B. thailandensis is generally avirulent in mammals, this species displays very similar phenotypic characteristics to that of B. pseudomallei. Optimal identification of these species remains problematic, due to the difficulty in developing a sensitive, selective, and accurate assay. The development of PCR technologies has revolutionized diagnostic testing and these detection methods have become popular due to their speed, sensitivity, and accuracy. The purpose of this review is to provide a comprehensive overview and evaluation of the advancements in PCR-based detection and differentiation methodologies for the B. pseudomallei complex, and examine their potential uses in diagnostic and environmental testing.

The differential susceptibility of spores from virulent and attenuated Bacillus anthracis strains to aldehyde- and hypochlorite-based disinfectants.

This study compared the sensitivity of spores from virulent and attenuated Bacillus anthracis strains in suspension to inactivation by various chemical disinfectants. Spore suspensions from two virulent strains (A0256 and A0372) and two attenuated strains (Sterne and A0141) of B. anthracis were tested against two aldehyde-based disinfectants and one hypochlorite-based disinfectant. A novel statistical model was used to estimate 4-log(10) reduction times for each disinfectant/strain combination. Reduction times were compared statistically using approximate Z and χ(2) tests. Although there was no consistent correlation between virulence and increased sporicidal resistance across all three disinfectants, spores from the two virulent and two attenuated strains did display significantly different susceptibilities to different disinfectants. Significant disinfectant-strain interactions were observed for two of the three disinfectants evaluated. The comparative results suggest that the use of surrogate organisms to model the inactivation kinetics of virulent B. anthracis spores may be misleading. The accuracy of such extrapolations is disinfectant dependent and must be used with caution.