Rapid Detection of Bacillus anthracis Bloodstream Infections by Use of a Novel Assay in the GeneXpert System

Abstract

Bacillus anthracis is a tier 1 select agent with the potential to quickly cause severe disease. Rapid identification of this pathogen may accelerate treatment and reduce mortality in the event of a bioterrorism attack. We developed a rapid and sensitive assay to detect B. anthracis bacteremia using a system that is suitable for point-of-care testing. A filter-based cartridge that included both sample processing and PCR amplification functions was loaded with all reagents needed for sample processing and multiplex nested PCR. The assay limit of detection (LOD) and dynamic range were determined by spiking B. anthracis DNA into individual PCR mixtures and B. anthracis CFU into human blood. One-milliliter blood samples were added to the filter-based detection cartridge and tested for B. anthracis on a GeneXpert instrument. Assay specificity was determined by testing blood spiked with non-anthrax bacterial isolates or by testing blood samples drawn from patients with concurrent non-B. anthracis bacteremia or nonbacteremic controls. The assay LODs were 5 genome equivalents per reaction and 10 CFU/ml blood for both the B. anthracis Sterne and V1B strains. There was a 6-log₁₀ dynamic range. Assay specificity was 100% for tests of non-B. anthracis bacterial isolates and patient blood samples. Assay time was less than 90 min. This automated system suitable for point-of-care detection rapidly identifies B. anthracis directly from blood with high sensitivity. This assay might lead to early detection and more rapid therapy in the event of a bioterrorism attack.

Keywords: Bacillus anthracis; GeneXpert; diagnostic; diagnostics; whole blood.

FIG 1

Limit of detection of B. anthracis assay. (A) Positivity rate for assay cartridges spiked with the indicated numbers of B. anthracis Ames35 genomic DNA equivalents. Ten to 22 reaction mixtures were tested for each concentration shown. (B and C) Positivity rate for blood samples spiked with the indicated numbers of CFU of B. anthracis Sterne (B) and B. anthracis V1B (C). Between 10 and 12 replicates were tested for each concentration. The Gompertz fit curve (f = a × exp{−exp[−(x − x)]/b}) and 95% confidence band were generated in SigmaPlot v.13. Cart, filter-based cartridge.

FIG 2

Dynamic range of B. anthracis assay. Cartridge-based assay showing the cycle threshold values for different numbers of B. anthracis Sterne CFU spiked into human blood. Three biological replicates of 3 (n = 9) were tested at each concentration.