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

Abstract

We conducted a comprehensive, multiphase laboratory evaluation of the Anthrax BioThreat Alert Ò 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.

Rapid and Sensitive Multiplex Assay for the Detection of B. anthracis Spores from Environmental Samples

“…We, therefore, propose that the newly developed methodology described in this work allows the detection of spore concentrations that are 3-4 orders of magnitude lower than published immuno-affinity assays [11,36], being comparable to sensitive PCR-based assays [7,8,20], while being performed in under 3.5 h for the total assay. This time frame could potentially be shortened even further by modifying the multiplex assay step to be performed as a one-step assay (all in one) by using direct labeled secondary antibodies or a biotin-streptavidin complex for signal enhancement.…”

“…Additional identification assays have low sensitivities for environmental samples, suffering a 3-4 orders of magnitude decrease in sensitivity compared to pure samples [10]. This was demonstrated using specific phage [35] or other commercial assays [36]. The detection of B. anthracis spores from environmental samples in various immune affinity-based assays has been shown to be effective for the detection of 1 × 10 5 -1 × 10 6 spores [11,36].…”

“…This was demonstrated using specific phage [35] or other commercial assays [36]. The detection of B. anthracis spores from environmental samples in various immune affinity-based assays has been shown to be effective for the detection of 1 × 10 5 -1 × 10 6 spores [11,36]. In addition, using specific reporter phage enables the detection of 1 × 10 5 CFU in assays, taking several hours to completion [35].…”

Comprehensive Laboratory Evaluation of a Specific Lateral Flow Assay for the Presumptive Identification of Francisella tularensis in Suspicious White Powders and Aerosol Samples

“…They were able to detect F. tularensis in both lemming carcasses and the well water in which the carcasses were found; however, this assay was less sensitive than PCR. Rapid BTA assays have previously been evaluated for the detection of biothreat agents including orthopoxviruses, 36 ricin, 37 abrin, 38 Bacillus anthracis, 32,39 and Yersinia pestis. 40 Limited evaluations have also been conducted with assays for the detection of Yersinia pestis, 41 botulinum neurotoxins, 42,43 and staphylococcal enterotoxins.…”

“…Table 3 shows the information about the 61 strains of diverse environmental background organisms used in the study. 32 Each of the microorganisms was inoculated onto optimal medium and incubated under appropriate conditions for 24 to 48 hours. A single, isolated colony was selected and inoculated onto a second agar plate and incubated for 1 to 6 days, depending on the organism and its growth rate.…”