PCR-Based Detection of Bacillus anthracis in Formalin-Fixed Tissue from a Patient Receiving Ciprofloxacin

Abstract

We demonstrate that Bacillus anthracis may be detected from a formalin-fixed, paraffin-embedded biopsy specimen, even after the patient has received antibiotic treatment. Although traditional PCR methods may not be sufficiently sensitive for anthrax detection in such patients, cycle numbers can be increased or PCR can be repeated by using an aliquot from a previous PCR as the template.Bacillus anthracis, the causative agent of anthrax, has been postulated to be a likely agent of biological warfare or terrorism because of its physical properties and its virulence factors (8). Nonetheless, anthrax had not been encountered in the United States as a weapon of terror until the fall of 2001 (2). Current reports have documented 22 cases of anthrax that have met the Centers for Disease Control and Prevention (CDC) case definition; 12 cases (7 confirmed and 5 suspected) were of the cutaneous form (3, 10). Due to variation in symptoms and findings in cases of cutaneous anthrax, a definitive diagnosis of infection requires identification of the organism within the tissue. However, many pathological specimens are routinely fixed in formalin and embedded in paraffin prior to histological examination. Such samples are easy to store, transport, and section for histological staining. Tissue architecture and proteins are well maintained with this method. Nonetheless, extraction of nucleic acids is difficult, often yielding only degraded DNA (4). Obtaining usable bacterial DNA is made more difficult if a patient has already been started on antibiotic therapy.We report our ability to amplify B. anthracis DNA from a formalin-fixed, paraffin-embedded skin biopsy specimen from a 34-year-old patient who had received ciprofloxacin for 3 days before the biopsy was performed. The major clinical features of this case have been previously reported (6; H. Yee, T. C. Gallagher, B. Strober, M. Pomerantz, M. Sanchez, S. Levine, M. J. Blaser, R. Hoffman, and B. A. Hanna, Abstr. 41st Intersci. Conf. Antimicrob. Agents Chemother., abstr. UL-7, 2001). In brief, the patient, an employee in the mailroom of a New York City newspaper, presented with a rapidly growing nodule on his left forearm. Within 24 h, the nodule developed a central black eschar (6). Notably, histological sections from the biopsy specimen obtained after 3 days of antibiotic treatment showed an edematous lesion (with pronounced superficial papillary dermal edema with near-vesicle formation) with diffuse interstitial dermal collagen fibrin deposition, diffuse scattered acute inflammation, vascular congestion, and vasculitis. A tissue Gram stain revealed scattered gram-positive, rod-shaped organisms, some of which were fragmented forms. Serological results for anthrax were negative, as were Gram-stained smears and cultures of the unfixed tissue. The CDC reported positive identification of anthrax in the tissue biopsy specimen by immunohistochemical staining.Formalin-fixed, paraffin-embedded tissue samples obtained by punch biopsies of skin from three different patients were studied....

Detection of the bacterium Flavobacterium psychrophilum from a natural infection in rainbow trout, Oncorhynchus mykiss (Walbaum), using formalin‐fixed, wax‐embedded fish tissues

“…DNA was extracted from each of the tissue samples using a Dneasy Tissue Kit (Qiagen) and purified as described by Levine et al. (2002).…”

“…A recent study by Levine, Perez‐Perez, Olivares, Yee, Hanna & Blaser (2002) detected the presence of the bacterium Bacillus anthracis from formalin‐fixed wax‐embedded biopsy specimens in man. These samples came from clinical cases of natural infections and amplification of the bacterial DNA was possible even after the patient had started antibiotic treatment.…”

Recent advances in the rapid detection of Bacillus anthracis

“…The ideal assay would produce results within minutes. Recent developments in immunological methods for rapid testing focus on the improvement of reporter systems that would provide test results in real-time [42,47,48,50].…”

“…Both genes are amplified in multiplex using Roche's LightCycler machine. Modified protocols using commercially available kits have been shown to detect B. anthracis from human tissue, even subsequent to patient antibiotic prophylaxis [42]. In the real-time PCR assay developed by CDC for B. anthracis, a chromosomal marker is included in addition to the two plasmid markers [http://www.bt.cdc.gov/agent/anthrax/ faq/labtesting.asp].…”

Pathology, Diagnosis, and Treatment of Anthrax in Humans

“…In addition, several methods which detect B. anthracis nucleic acids from isolates, blood, or nasal swabs have been published and may have future applications within the LRN laboratories (Hoffmaster et al, 2002 ;Oggioni et al, 2002 ;Sacchi et al, 2002 ;Tomioka et al, 2005 ;Uhl et al, 2002 ).Some of the PCR -based detection methods have been validated in formalin -fi xed, paraffi n -embedded samples, providing the possibility of using tissue samples that are noninfectious (Levine et al, 2002 ;Tatti et al, 2006 ).…”