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(American Journal of Pathology. 2003;163:701-709.)
© 2003 American Society for Investigative Pathology

Pathology and Pathogenesis of Bioterrorism-Related Inhalational Anthrax

Jeannette Guarner^*, John A. Jernigan, Wun-Ju Shieh^*, Kathleen Tatti^*,, Lisa M. Flannagan, David S. Stephens, Tanja Popovic, David A. Ashford, Bradley A. Perkins and Sherif R. Zaki^* and the Inhalational Anthrax Pathology Working Group

From the Infectious Diseases Pathology Activity,^* Division of Viral Rickettsial Diseases; Intervention and Evaluation Section, Prevention and Evaluation Branch, Division of Healthcare and Quality Promotion; and the Meningitis and Special Pathogens Branch, Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia; and the Medical Examiner Office, Palm Beach County, Palm Beach, Florida

	Abstract

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During October and November 2001, public health authorities investigated 11 patients with inhalational anthrax related to a bioterrorism attack in the United States. Formalin-fixed samples from 8 patients were available for pathological and immunohistochemical (IHC) study using monoclonal antibodies against the Bacillus anthracis cell wall and capsule. Prominent serosanguinous pleural effusions and hemorrhagic mediastinitis were found in 5 patients who died. Pulmonary infiltrates seen on chest radiographs corresponded to intraalveolar edema and hyaline membranes. IHC assays demonstrated abundant intra- and extracellular bacilli, bacillary fragments, and granular antigen-staining in mediastinal lymph nodes, surrounding soft tissues, and pleura. IHC staining in lung, liver, spleen, and intestine was present primarily inside blood vessels and sinusoids. Gram’s staining of tissues was not consistently positive. In 3 surviving patients, IHC of pleural samples demonstrated abundant granular antigen-staining and rare bacilli while transbronchial biopsies showed granular antigen-staining in interstitial cells. In surviving patients, bacilli were not observed with gram’s stains. Pathological and IHC studies of patients who died of bioterrorism-related inhalational anthrax confirmed the route of infection. IHC was indispensable for diagnosis of surviving anthrax cases. The presence of B. anthracis antigens in the pleurae could explain the prominent and persistent hemorrhagic pleural effusions.

Previous pathological studies of fatal human cases of inhalational anthrax have described the presence and distribution of B. anthracis in formalin-fixed tissues by using special stains.^7-12 During the bioterrorism outbreak, special stains and novel B. anthracis cell wall and capsule-specific IHC assays were used to study tissue samples from surviving and non-surviving patients who had received multiple antibiotics. Knowledge of the consequences of effective treatment for B. anthracis on tissues has been limited. This report describes the pathology observed in 8 patients with bioterrorism-related inhalational anthrax and demonstrates how B. anthracis IHC assays can be successfully used for diagnosis and for better understanding the pathogenesis of this disease.

	Materials and Methods

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Epidemiological, demographic, and clinical information for the 8 patients who contracted inhalational anthrax during the 2001 US bioterrorism attack has been reported previously.^1-6,13-17 The tissues received for IHC study from each case are listed in Table 1 . Sections from all tissues were stained by using hematoxylin and eosin (H&E) and IHC. Gram’s staining (Brown-Hopps and Brown and Brenn stains) and Steiner silver staining were done on selected blocks from the mediastinum, lungs, liver, and spleen of the 5 patients who died, and on all tissues from the 3 patients who survived.

Positive controls included tissue sections of formalin-fixed, paraffin-embedded cultures of B. anthracis minced with normal human tissues and tissue samples from veterinary and other human confirmed cases. Negative controls consisted of a sequential tissue section from each block incubated with an IgM isotype antibody (a monoclonal anti-Rhizomucor antibody, catalog number M3565; DAKO Corporation). Controls also included formalin-fixed tissue specimens from autopsies and pleural effusion cell blocks of non-anthrax patients received during the 2001 investigation. Other negative tissue controls tested with the cell-wall and capsule anthrax antibodies included cases of Group A streptococcus, Streptocococcus pneumoniae, Francisella tularensis, Legionellla pneumophila, Yersinia pestis, Clostridium novyi, Clostridium perfringens, Rickettsia rickettsii, Rickettsia akari, lassa fever virus, yellow fever virus, Ebola hemorrhagic fever virus, and human herpesvirus type 1 and 3.

	Results

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Table 2 summarizes the demographic and clinical characteristics of the 8 inhalational anthrax patients and indicates the primary laboratory confirmation of B. anthracis. Most patients had occupations that involved handling of mail and presented with a febrile disease that included malaise, cough, and gastrointestinal symptoms. Pleural effusions were demonstrated on the initial chest radiograph of 7 patients. CT scans of 5 patients showed pleural effusions (including the patient with normal initial chest radiograph) and 4 of 5 demonstrated mediastinal lymphadenopathy. Cerebrospinal fluid was obtained only from the index case and showed abundant neutrophils and bacilli. In all fatal cases, anthrax sepsis was confirmed either before (patients 1, 10, and 11) or after death (patients 5 and 6) by culture of blood samples obtained at the time of hospitalization. Although the diagnosis of anthrax was already known in fatal cases, autopsies and IHC assays were performed primarily to establish or confirm the route of entry of B. anthracis. In comparison, pathology and IHC assays of pleural samples from the 3 surviving patients played a pivotal role in diagnosis and clinical management.

View larger version (107K): [in this window] [in a new window]   Figure 1. Pathology and immunohistochemisty of mediastinal lymph nodes. A: Photograph of mediastinal compartment showing an enlarged, hemorrhagic lymph node and edematous, hemorrhagic soft tissues. B: Photomicrograph of mediastinal soft tissues showing hemorrhage. C: Photomicrograph of mediastinal lymph node showing necrosis and pyknotic nuclei. D: Photomicrograph of mediastinal lymph node showing hemorrhage, immunoblasts, and pyknotic nuclei. E: Photomicrograph of mediastinal lymph node showing abundant granular antigen-staining and bacillary fragments (B. anthracis cell-wall antibody). F: Photomicrograph of the same mediastinal lymph node, note the different pattern of granular antigen-staining when using the B. anthracis capsule antibody. H&E stain (B, C, D); immunohistochemical assay using naphthol/fast red substrate with hematoxylin counterstain (E, F). Original magnifications: B, x5; C, D, E, and F x40.

Samples from all patients who died showed hemorrhage (Figure 1B) and various amounts of necrosis of mediastinal lymph nodes (Figure 1C) . In 3 of these 5 patients, mediastinal lymph nodes also showed infiltration by neutrophils and prominent immunoblasts (Figure 1D) . Focal hemorrhage and edema of pleura and interhilar septa were found in all 5 non-surviving patients but were prominent in 3. In 2 of the 5 patients, these findings were accompanied by various degrees of mononuclear inflammation (Figure 2A) . Other findings in the lungs included variable intraalveolar edema and focal areas of hyaline membrane formation (Figure 2B) ; interstitial mononuclear inflammation was present in 1 patient (Figure 2B) . Intraalveolar inflammatory infiltrate was not observed in any of the non-surviving inhalational anthrax patients.

View larger version (101K): [in this window] [in a new window]   Figure 2. Photomicrograph showing pathology and immunohistochemistry of lung. A: Lung showing thickened pleura with inflammation and fibrin. B: Lung showing intraalveolar edema and inflammation in the alveolar septa. C: Pleura showing gram-positive bacilli. D: Pleura showing silver staining bacilli. E: Lung showing abundant granular antigen-staining in the pleura, interalveolar septa, and intra alveolar macrophages. F: Lung showing granular antigen-staining of intraalveolar macrophages. H&E stain (A and B); gram’s stain (C); Steiner stain (D); immunohistochemical assay using the B. anthracis capsule antibody and naphthol/fast red substrate with hematoxylin counterstain (E and F). Original magnifications: A, x10; B, x20; C, x100; D, E, and F, x40.

All 3 surviving inhalational anthrax patients showed reactive mesothelial cells admixed with inflammatory elements in pleural samples (Figure 3A) . Transbronchial biopsies of 2 patients demonstrated soft tissue edema, various amounts of hemorrhage, and minimal inflammatory infiltrate.

View larger version (122K): [in this window] [in a new window]   Figure 3. Pathology and immunohistochemistry of biopsy specimens obtained from surviving patients. A: Photomicrograph of parietal pleural biopsy specimen showing reactive mesothelium and mixed inflammation. B: Photomicrograph of parietal pleural biopsy specimen showing abundant granular antigen-staining. C: Photomicrograph of pleural effusion cell block showing bacillary fragments and granular antigen-staining. D: Photomicrograph of transbronchial biopsy showing granular antigen-staining in histiocytes of the submucosa. H&E stain (A); immunohistochemical assay using the B. anthracis capsule antibody (B) or the B. anthracis cell-wall antibody (C, D) naphthol/fast red substrate with hematoxylin counterstain. Original magnifications: A and B, x40; C and D, x63.

IHC Assays

Overall, IHC using cell-wall antibody demonstrated bacilli, bacillary fragments, and lesser amounts of granular antigen-staining. Using the B. anthracis capsule antibody, the IHC pattern showed mostly bacterial granular antigens, while intact bacilli were less frequently identified. Bacilli were observed most often in extracellular spaces, while bacterial granular antigen-staining and bacillary fragments were seen intracellularly (in mononuclear inflammatory cells and interstitial fibroblastic cells) and extracellularly. Positive tissue sections always showed staining with both the capsule and cell-wall antibody; however, the amounts varied in each tissue. Negative tissue controls did not react with the cell-wall or capsule anthrax antibodies.

Abundant granular antigen-staining and bacillary fragments were present in necrotic areas of mediastinal lymph nodes and surrounding soft tissues of all 5 patients who died (Figure 1, E and F) and in the pleurae (Figure 2E) and interhilar lung septae of 4 of the 5 patients. Granular antigen-staining was present but in lower amounts in intraalveolar macrophages (3 of 5 patients) (Figure 2F) , and interalveolar septa (3 of 5), around small bronchi (2 of 5), and on the surface of bronchial epithelial cells (1 of 5). Cell blocks prepared from pleural fluid from 1 of the non-surviving patients showed granular antigen-staining in mononuclear inflammatory cells. In the abdominal cavity, granular antigen-staining and bacillary fragments were present in Kupffer’s cells of the liver (3 of 4 patients) (Figure 4A) , spleen (3 of 4) (Figure 4B) , necrotic hepatocytes (1 of 4), and focally in the serosa (Figure 4C) and blood vessels (Figure 4D) of the intestine (2 of 3). Sections from the nasal mucosa were available from 1 patient and showed granular antigen-staining in the submucosa. Sections from a sample of the dermal hematoma showed granular antigen-staining in macrophages of dermis. Other tissues available for study (conjunctiva, oral mucosa, central nervous system) did not demonstrate IHC staining.

View larger version (129K): [in this window] [in a new window]   Figure 4. Photomicrographs showing immunohistochemistry of abdominal organs from patients who died. A: Liver showing bacilli in the sinusoids and bacillary fragments in Kupffer’s cells. B: Spleen showing bacillary fragments and granular antigen-staining. C: Intestinal serosa showing granular antigen-staining. D: Intestinal submocosal blood vessel showing granular antigen-staining. Immunohistochemical assay using the B. anthracis capsule antibody (B, C, and D) or the B. anthracis cell wall antibody (A) naphthol/fast red substrate with hematoxylin counterstain. Original magnifications: A and B, x100; C, x63; D, x40.

IHC staining of tissues from surviving patients demonstrated abundant granular antigen-staining and bacillary fragments in pleural specimens (pleural tissue fragments, Figure 3B ; pleural effusion cell blocks, Figure 3C ). Bacilli were abundant in the patient who received less than 55 hours of antibiotic treatment, while the patients with more than 72 hours of treatment showed rare bacillary fragments. The transbronchial biopsies of 2 patients who survived showed rare interstitial histiocytes with granular antigen-staining (Figure 3D) .

	Discussion

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The patients presented here represent the first human cases of fatal and non-fatal inhalation anthrax related to a bioterrorism attack that have been studied using newly developed IHC assays. Pleural effusions that were small on presentation progressively enlarged and persisted even with effective antibiotic treatment were the most significant clinicopathologic feature.⁶ The compromised respiratory function present in inhalational anthrax cases has been attributed, in part, to pleural effusions.⁷ Bacilli and abundant B. anthracis cell wall and capsule antigens were demonstrated in the pleura of these patients by use of IHC. In addition, PCR amplification of B. anthracis was most successful from the pleural fluid of these patients.²⁰ Bacilli were abundant in the non-surviving patients who received less than 55 hours of antibiotic treatment. Granular cell wall and capsule antigen-staining and bacillary fragments were prominent in cell blocks prepared from pleural effusions of non-surviving and surviving patients who had received antibiotics for more than 72 hours. Our findings suggest that during the initial phase of inhalational anthrax, the pleura became infected by large numbers of B. anthracis, leading to the development of pleural effusions. In later phases of the infection, the large, persistent pleural effusions that occurred even after effective treatment may be explained by a continued inflammatory reaction to residual bacterial antigens and possibly the effect of toxins. The prominent finding of B. anthracis bacilli, bacillary fragments, and antigens in pleural effusions emphasizes the importance of IHC diagnostic assays in pleural fluid specimens of suspected anthrax patients.

The fatal inhalational anthrax cases related to this bioterrorism attack demonstrated hemorrhagic mediastinitis without pneumonia; the pulmonary infiltrates that were noted on chest radiographs corresponded to pulmonary edema and hyaline membrane formation. Animal models have demonstrated that inhaled B. anthracis spores are taken up by the alveolar macrophages in the lungs and transported to the mediastinal lymph nodes within hours, leaving no specific pulmonary lesions.^21-23 In the lymph nodes, the anthrax spores germinate, forming vegetative bacilli, which multiply intra- and extracellularly and release toxins. The edema and lethal-factor anthrax toxins bind to the protective anthrax antigen, allowing entry to the cell cytosol.²⁴ The edema toxin induces cyclic AMP, which results in damage to water homeostasis. The lethal toxin cleaves MAP kinases, releasing oxygen radicals and proinflammatory cytokines that result in cell death. Our IHC findings are consistent with these animal models; all patients who died had large numbers of bacilli, bacillary fragments, and B. anthracis cell wall and capsule antigens in the mediastinal lymph nodes, surrounding soft tissues, and pleura, with lesser numbers found in the lung parenchyma and organs in the abdominal cavity. Since the highest burden of B. anthracis bacilli and antigens was found in the mediastinal tissues and pleura, B. anthracis bacilli in the mediastinal lymph nodes may have directly infected the adjoining pleural space and rapidly spread in the thoracic cavity covering the lungs. Alternatively, some spore-containing macrophages may have disseminated to the pleural spaces where they germinated forming bacilli.

Non-human primate inhalational anthrax models have shown that hematogenous spread to abdominal organs and central nervous system follows the initial mediastinal lymph node seeding.²⁵ In a pathological study of human inhalational anthrax after a large outbreak in Sverdlovsk, Russia, hematogenous spread of B. anthracis was found to be associated with capillary and vascular lesions that consist of fibrin deposition and various amounts of neutrophilic infiltrate surrounding the vessel wall.⁷ The capillaritis and vasculitis weakened the vessel wall and produced high- and low-pressure hemorrhages.⁷ We observed hemorrhages in the 2001 bioterrorism-related cases, but the small vessels and capillaries showed minimal inflammation. The reasons for the difference in pathological findings between these two groups of inhalational anthrax cases are unclear, but potential explanations include earlier recognition and initiation of treatment, treatment with newer antibiotic combinations and better supportive care, dose and characteristics of B. anthracis spores used in the attack, or a combination of the above.

Meningeal spread of B. anthracis has been described in up to 80% of inhalational anthrax cases.⁷ The initial patient with inhalational anthrax related to this bioterrorism attack presented with meningitis, and B. anthracis was cultured from cerebrospinal fluid. Other patients did not show meningitis clinically and the 2 patients who had postmortem examination of the central nervous system had no meningeal involvement.^14,16 The decreased meningeal spread in the bioterrorism-related anthrax cases can be explained by the early recognition and effective treatment regimens, and correlates with mortality rates lower than those previously associated with inhalational anthrax.¹⁷

Before it was evident that these anthrax cases were part of a bioterrorism attack, the first autopsy was performed to definitively determine how the patient had acquired anthrax (cutaneous versus gastrointestinal versus inhalational).^1,6 The pathological and IHC features that indicated a respiratory route included mediastinal enlargement and hemorrhage with abundant IHC staining of mediastinal tissues, persistent pleural effusions, IHC evidence of vascular spread to other organs, and lack of an obvious primary cutaneous or gastrointestinal focus. Once it was established that B. anthracis had been acquired through the respiratory tract, public health officials were able to better define potential sources of inhalational anthrax. As new suspect cases appeared in this bioterrorism attack, postmortem and IHC studies were an indispensable factor in determining that the infections were acquired through the respiratory route and thus increased awareness of precautions necessary when performing autopsies on patients with infectious diseases.^1-5 In anthrax patients, most of the bacteria present in tissues during the first hours after death are in the vegetative stage; thus, the risk to personnel performing autopsies is through splashes and splatters to mucous membranes and percutaneous injuries. Procedures such as opening the skull and embalming should be avoided if possible since they can create aerosols with microscopic droplets containing bacilli, which may eventually sporulate. To prevent persistence of B. anthracis (in either vegetative or spore forms) in the environment and the consequent potential for occupational risk of cutaneous anthrax, the autopsy instruments and suite should be decontaminated with 0.5% hypochlorite solution. CDC advises that all clinicians, pathologists, and laboratorians need to be aware of the clinicopathologic features of diseases potentially caused by bioterrorism agents (eg, anthrax, tularemia, plague, botulism, hemorrhagic fevers, smallpox) and precautions necessary to obtain specimens that would aid in the prompt diagnosis so that public health measures are instituted to contain possible casualties.^26-29

IHC performed in formalin-fixed, paraffin-embedded tissues has traditionally offered several advantages, including the specific diagnosis of an agent, use of specimens that pose minimal biohazard for laboratory personnel, provision of a permanent record, and preservation of tissue morphology, which permits localization of the microorganism in specific structures. Interpretation of IHC assays should be done with caution, taking into consideration the clinical and exposure histories, morphological and technical aspects (eg, previously reported cross reactivity of the cell wall antibody with occasional Bacillus cereus strains), and results of other confirmatory tests, such as culture, PCR, and serology.^17,19 In our experience, the IHC diagnosis of anthrax infections should include interpretation of assays with both the cell wall and capsule antibodies. The cell wall antibody stained the bacilli present in tissues of patients who briefly received treatment, while the capsule antibody demonstrated bacillary fragments and granular antigens prominently in tissues of patients who had been treated for up to 14 days. IHC proved extremely valuable in 3 surviving patients who received treatment before collection of specimens and from whom gram’s stains, culture, and PCR failed to detect B. anthracis.

In summary, pathological and B. anthracis-specific IHC studies were performed in 8 inhalational anthrax cases related to the bioterrorism attack of 2001 in the United States. The pathological and IHC studies were indispensable for the investigation since they confirmed the route of infection for the 5 patients who died and contributed to the diagnosis in 3 surviving patients. IHC for B. anthracis is a powerful diagnostic modality since it demonstrates bacilli, bacillary fragments, and granular antigen-staining. We demonstrated for the first time by IHC that large numbers of bacilli and large amounts of cell wall and capsule antigens were present in pleural tissues. IHC was an important diagnostic tool and provided insights into the pathogenesis of the large, persistent pleural effusions present in inhalational anthrax.

	Acknowledgements

	Footnotes

 
Address reprint requests to Sherif R. Zaki, MD, Ph.D., Infectious Disease Pathology Activity, Centers for Disease Control and Prevention, Mailstop G32, 1600 Clifton Rd, NE, Atlanta, GA 30333. E-mail: szaki{at}cdc.gov

The research was carried out at the National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA.

The members of the Inhalational Anthrax Pathology Working Group are Jeanine Bartlett, Patricia Greer, Tara Ferebee-Harris, Jeltley Montague, Tim Morken, Christopher Paddock, and Chalanda Smith (from Infectious Diseases Pathology Activity); Conrad Quinn, Marc Fischer, and Rob Weyant (from the Division of Bacterial and Mycotic Diseases); Richard F. Meyer (from Bioterrorism Preparedness and Response); Michael Bell (from Division of Healthcare and Quality Promotion); and State Health Epidemiologist Steven Wiersma.

Accepted for publication May 5, 2003.

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