Evaluation and Validation of a Real-Time Polymerase Chain Reaction Assay for Rapid Identification of Bacillus anthracis Supplement

During the recent outbreak of bioterrorism-associated anthrax in the United States, 11 patients were diagnosed with inhalational anthrax and 7 with cutaneous anthrax (Table 1 and 2) (1–6). During the extensive epidemiologic investigation, >125,000 clinical and environmental specimens were collected and analyzed for Bacillus anthracis, the causative agent of anthrax. We used the Laboratory Response Network (LRN) polymerase chain reaction (PCR) assay (real-time PCR assay) during the anthrax outbreak to detect B. anthracis DNA in environmental samples and clinical specimens. This assay provided 100% sensitivity and specificity when evaluated and validated on our panel of diverse bacterial isolates. On clinical specimens, this assay was one of three used to confirm anthrax cases when isolation of B. anthracis failed after antimicrobial drug treatment was initiated. In these culture-negative cases, laboratory confirmation was based on at least two supportive laboratory tests including this PCR, immunohistochemical stain (IHC), or anti-protective antigen (PA) titer (immunoglobulin [Ig]G enzyme-linked immunosorbent assay [ELISA]). PCR assays have not been previously used in an outbreak setting to detect B. anthracis directly in clinical specimens in a real-time manner. We evaluated the use of this assay on the clinical specimens and environmental samples received during the outbreak.