Multiplex Detection of Three Select Agents Directly from Blood by Use of the GeneXpert System

Background: Francisella tularensis, Bacillus anthracis and Yersinia pestis are tier1 select agents with the potential to rapidly cause severe disease. Rapid detection of these bacteria from patient samples at the point of care could contribute to improved clinical outcomes in the event of a bioterrorism attack. Methods: A multiplex nested PCR assay for detection of F. tularensis , B. anthracis and Y. pestis directly from patient blood samples was developed using the GeneXpert system. The multiplex GeneXpert cartridge based assay includes all necessary sample processing and amplification reagents. Blood samples spiked with different numbers of CFU were used to measure the analytical limit of detection (LOD) and dynamic range. Sensitivity was determined by testing spiked blood samples and negative control blood in a blinded manner. Specificity was determined against non-target pathogens and blood samples from clinical patients. Results: Assay LOD was 8.5 CFU/ml for F. tularensis , 10 CFU/ml for B. anthracis and 4.5 CFU/ml for Y. pestis . Sensitivity was 100% at the LOD for all three select agent bacteria in spiked patient blood samples. Assay specificity was 100% tested against both non-target pathogens and clinical patient blood samples. Total assay time was approximately 100 minutes. Conclusions: This automated assay, which is suitable for point of care, identifies three select agents directly in blood without the need for enrichment with high sensitivity within 100 minutes. This assay may enable more rapid diagnosis and treatment of the target organisms in the event of a bioterrorism attack.