Comprehensive Laboratory Evaluation of a Highly Specific Lateral Flow Assay for the Presumptive Identification of Bacillus anthracis Spores in Suspicious White Powders and Environmental Samples

Abstract

We conducted a comprehensive, multiphase laboratory evaluation of the Anthrax BioThreat Alert(®) test strip, a lateral flow immunoassay (LFA) for the rapid detection of Bacillus anthracis spores. The study, conducted at 2 sites, evaluated this assay for the detection of spores from the Ames and Sterne strains of B. anthracis, as well as those from an additional 22 strains. Phylogenetic near neighbors, environmental background organisms, white powders, and environmental samples were also tested. The Anthrax LFA demonstrated a limit of detection of about 10(6) spores/mL (ca. 1.5 × 10(5) spores/assay). In this study, overall sensitivity of the LFA was 99.3%, and the specificity was 98.6%. The results indicated that the specificity, sensitivity, limit of detection, dynamic range, and repeatability of the assay support its use in the field for the purpose of qualitatively evaluating suspicious white powders and environmental samples for the presumptive presence of B. anthracis spores.

Figure 1.

Dot density diagram that summarizes the testing performed in this validation. It provides a visual representation of the BTA value distribution in each phase. The number of tests, including positive and negative controls, for each phase is displayed at the top of each phase's cluster. The cut-off value of 200 is shown as a solid line. Color images available at www.liebertonline.com/hs

Figure 2.

The titration curves depict BTA reader value with respect to the log₁₀ concentration of anthrax spores from the Ames strain as well as the Sterne strain. The curves were generated using the average of at least 5 tests, and the error bars are the standard deviations. The cut-off value of 200 is shown as a solid line. For both strains, the first test concentration that is above the cut-off value is 10⁶ cfu/mL. Color images available at www.liebertonline.com/hs

Figure 3.

Probit regressions for the B. anthracis Sterne and Ames strain spores. The curves are calculated probability of detection as a function of spore concentration. The estimated limit of detection is calculated by finding the spore concentration with a probability of detection at 0.95. For Sterne spores, the LOD is 4.3 × 10⁵ cfu/mL (6.4 × 10⁴ cfu/assay), and for Ames spores the LOD is 1.4 × 10⁶ cfu/mL (2.1 × 10⁵ cfu/assay). Color images available at www.liebertonline.com/hs

Figure 4.

Receiver operator characteristic (ROC) curve provides a visual representation of the sensitivity and specificity of this assay. Each point on the curve is a possible cut-off value, and its place on the curve is determined by its specificity and sensitivity. The calculated assay sensitivity is 99.3%, and the specificity is 98.6%. Color images available at www.liebertonline.com/hs

Figure 5.

A dot density diagram that shows all 1,246 tests performed, grouped as designated positive and designated negative by the BTA reader. The cut-off value of 200 is shown as a solid line. The number of tests performed in each group is shown in parentheses. Any data points in the designated negative group that were above the cut-off value are false positive, while any data points in the designated positive group that were below the cut-off value are false negative. Color images available at www.liebertonline.com/hs