Quantitative Pathology of Inhalational Anthrax I: Quantitative Microscopic Findings Lev M

Home , Fibrinoid necrosis

Quantitative Pathology of Inhalational Anthrax I: Quantitative Microscopic Findings Lev M. Grinberg, M.D., Faina A. Abramova, M.D., Olga V. Yampolskaya, M.D., David H. Walker, M.D., Jerome H. Smith, M.D. Department of Pathology, Tuberculosis and Pulmonary Diseases Unit (LMG, FAA), Hospital 40, Ekaterinburg, Russia; Department of Infectious Diseases, Central Postgraduate Institute, Botkin Hospital (OVY), Moscow, Russia; and Department of Pathology, University of Texas Medical Branch (DHW, JHS), Galveston, Texas

culitis, hemorrhages, and edema. These pathologic Forty-one cases of documented inhalational an- findings are consistent with previous experimental thrax from the Sverdlovsk epidemic of 1979 traced studies showing transport of inhaled spores to me- to release of aerosols of Bacillus anthracis at a se- diastinal lymph nodes, where germination and cret biologic-agent production facility were evalu- growth lead to local lesions and systemic spread, ated by semiquantitative histopathologic analysis of with resulting edema and cell death, owing to the tissue concentrations of organisms, inflammation, effects of edema toxin and lethal toxin. The identi- hemorrhage, and other lesions in the mediastinum, fication of the vascular lesions as a basis for the mediastinal lymph nodes, bronchi, lungs, heart, prominent hemorrhages is a novel observation for spleen, liver, intestines, kidneys, adrenal glands, human inhalational anthrax. and central nervous system. These data were correlated with clinical, epidemiologic, and demographic KEY WORDS: Bacillus anthracis, Edema, Hemor- data. rhage, Inhalational anthrax, Mediastinum, Respira- The patients’ courses, with a variable incubation tory failure, Vasculitis. period and short nonspecific course (4 days before Mod Pathol 2001;14(5):482–495 hospitalization) with rapid demise (1 day of hospitalization before death), correlated with systemic An epidemic of inhalational anthrax occurred in bacterial infection and lesions. Bacillus anthracis Sverdlovsk (now Ekaterinburg), Russia in April and were identified in all cases in which there was no May of 1979 (1–3). This outbreak was initially re- antibiotic treatment or there was treatment for ported to be intestinal anthrax (4, 5), but lay press fewer than 21 hours. The lesions that were the most investigation raised questions (6–8) that culmi- severe and apparently of longest duration were in nated in an international study, which established the mediastinal lymph nodes and mediastinum. that the outbreak was caused by at least four strains There and elsewhere, peripheral transudate sur- of Bacillus anthracis (3), documented that the rounded fibrin-rich edema; necrosis of arteries and pathologic findings were those of inhalational an- veins was the most likely source of large hemor- thrax (1), and uncovered by an epidemiological in- rhages displacing tissue or infiltrating tissue, re- vestigation a high likelihood that the origin was a spectively; and apoptosis of lymphocytes was ob- military base, Compound 19, in southern Sverd- served. Respiratory function was compromised by lovsk (2). Forty-two deceased patients were autop- mediastinal expansion, large pleural effusions, and sied during that epidemic (1). This is the largest hematogenous and retrograde lymphatic vessel series of human inhalational anthrax that has un- spread of B. anthracis to the lung with consequent dergone postmortem analysis; the next largest se- pneumonia. The central nervous system and intes- ries was a cluster of three deaths (9, 10), and other tines manifested similar hematogenous spread, vas- individual case autopsies have also been reported (11–15). The pathogenesis of inhalational anthrax has Copyright © 2001 by The United States and Canadian Academy of Pathology, Inc. been studied in experimental animals (16–21). In- VOL. 14, NO. 5, P. 482, 2001 Printed in the U.S.A. Date of acceptance: January 22, 2001. haled spores are phagocytosed by alveolar macro- Address reprint requests to: David H. Walker, M.D., WHO Collaborating phages and carried to regional lymph nodes in the Center for Tropical Diseases, University of Texas Medical Branch, 301 Uni- versity Boulevard, Galveston, TX 77555-0609; e-mail: [email protected]; fax: mediastinum within 6–18 hours in guinea pigs (18), 409-772-2500. rhesus monkeys (19), and chimpanzees (20). Ger-

482 mination of spores and multiplication of bacilli oc- After initial examination of microscopic slides cut cur in these lymph nodes, and vegetative bacteria from preserved paraffin blocks and formalin fixed spread via lymphohematogenous route to dissemi- tissues, the slides were reviewed by two of the au- nated loci (21). A similar pattern was found in a thors (JHS and LG) on a double-headed micro- human series (9, 21). More recently, B. anthracis scope, and the microscopic features itemized in has been shown to elaborate three proteins that Tables 4–10 were semiquantified (scale of 1–4; see comprise two toxins: protective antigen (PA), which below) and tabulated. binds to cell surfaces and mediates internalization of the proteins; edema factor (EF), and lethal factor ϩ Definition of Terms (LF; 22). EF acts as an Ca 2- and calmodulin- dependent adenylate cyclase which induces edema Hemorrhage was differentiated into high- (22). LF is a metalloprotease that cleaves the amino pressure hemorrhage and low-pressure hemor- terminus of MAP kinase kinases 1 and 2 and thus rhage based on whether aggregates of “pure” red inhibits the intracellular MAPK signal transduction cells displaced and compressed adjacent tissue pathway (23), which may favor apoptosis. A suble- (high-pressure hemorrhage) or whether red blood thal concentration of LF also causes macrophages cells diffusely permeated parenchyma without dis- to produce TNF-␣ and IL-1 (24), which might in- torting its architecture (low-pressure hemorrhage). duce pathologic effects. The Russian literature on pathology of anthrax Previous reports of this series focused upon the (which is more extensive than that of Western med- gross (macroscopic) findings (1, 24); the micro- ical literature) differentiates macroscopically be- scopic findings reported here have been published tween carbuncles and diffuse hemorrhage in the in abbreviated form in the Russian literature (25– gastrointestinal tract; carbuncles are discreet, often 27) and in abstracts of presentations (28–30). The circular areas of mucosa elevated by high-pressure present report summarizes the quantitated micro- hemorrhage. The term carbuncle has a quite differ- scopic findings in human inhalational anthrax and ent usage in the modern English medical literature, correlates these with results of experimental studies but its meaning did include hemorrhage in the (16–21) and previous reports of human inhalational archaic English medical literature, We prefer the (9–15) and intestinal anthrax (31, 32). term submucosal hemorrhage to the translated Rus- sian term carbuncle for these lesions. Diffuse gastrointestinal hemorrhage is flat, but hemorrhagi- cally discolored, mucosa and correlates with low- MATERIALS AND METHODS pressure hemorrhage in the submucosa, as Gross and microscopic specimens, as well as opposed to superficial hemorrhagic necrosis, which some clinical information, were available for 42 is hemorrhage into the lamina propria, resulting patients who underwent postmortem examination from reperfusion. during the epidemic and were thought clinically to The tissue concentrations of B. anthracis, in other ϫ have inhalational anthrax, as reported earlier (1). In words, Gram-positive bacilli/25 objective field, one long survivor (Case 24), clinical evidence of B. were semiquantified as in Table 1. The tissue con- anthracis infection was not available, and there was centration of inflammatory cells was semiquanti- no objective evidence of B. anthracis infection at fied as described in Table 2. Other parameters, such autopsy after a full 8 days of antibiotic therapy and as congestion and transudate were quantified as clinical recovery from the apparent anthrax illness defined in Table 3. (she presumably died of complications from the Additionally, selected blocks were stained with anthrax). In view of the tenuous nature of the evi- periodic acid–Schiff/Luxol fast blue, Mallory’s dence that her illness was indeed anthrax, we have trichrome, phosphotungstic acid–hematoxylin, pe- excluded this patient’s case from this series. riodic acid–methenamine silver, Perl’s (iron), Each of the following epidemiological variables Brown-Brenn, Brown-Hopps, Dieterle, Gridley’s was extracted from available records and tabulated: methenamine silver, hematoxylin–azure II–eosin, name, age, sex, duration of known antibiotic ther- Giemsa, Ziehl–Neelsen, Verhoef–Van Giesen, Movat apy (in hours), estimated date of exposure, date of onset of illness, date of admission to Hospital 40, date of death, incubation period (in days), duration TABLE 1. Criteria for Quantification of Tissue of illness (in days), duration of hospitalization (in Concentrations of Bacillus anthracis days), overall duration of infection from exposure 0 Bacillus anthracis/25 ϫ objective field ϭ0 Ͻ1 Bacillus anthracis/25 ϫ objective field ϭ1 to death (in days), postmortem interval (in hours), 1–10 Bacillus anthracis/25 ϫ objective field ϭ2 culture results for B. anthracis, and histological de- 11–100 Bacillus anthracis/25 ϫ objective field ϭ3 tection of large Gram-positive bacilli morphologi- 101–500 Bacillus anthracis/25 ϫ objective field ϭ4 ϫ ϭ cally consistent with B. anthracis. 501 or more Bacillus anthracis/25 objective field 5

Inhalational Anthrax Histopathology (L. M. Grinberg et al.) 483 TABLE 2. Criteria for Quantification of Tissue RESULTS Concentrations of Inflammatory Cells

0 cells/25 ϫ objective field ϭ0 Bacillus anthracis organisms were observed in Ͼ0–10 cells/25 ϫ objective field ϭ1 most organs examined microscopically in cases 11–25 cells/25 ϫ objective field ϭ2 26–100 cells/25 ϫ objective field ϭ3 treated with antibiotics for fewer than 21 hours or Ͼ100 cells/25 ϫ objective field ϭ4 not treated with antibiotics at all. The bacilli (Fig. 1) were large (1 to 2 ϫ 8to12␮m), often curved, in sausage-like chains, and usually Gram-positive (on Brown-Brenn or Brown-Hopps stains), without pentachrome, Rebiger’s and Mekhin stains for bac- spores (Schaeffer-Fulton modification of Wirtz terial capsules, and Schaeffer-Fulton modification spore stains and electron microscopy) or capsules of Wirtz spore stains. detected by light microscopy (Rebiger’s and Immunohistochemical assessment of hepatitis B Mekhin stains). The bacilli stained well with Diet- viral core and surface antigens, fibrinogen, IgG, erle’s stain, weakly with Gridley’s methenamine sil- IgM, C3 complement, B. anthracis antigens, CD45 ver, and blue with hematoxylin–azure II–eosin, Gi- (LCA), S-100, L26, CD45 RO, and KP1 was per- emsa and Ziehl-Neelsen stains. In some specimens, formed on selected blocks, using positive and neg- nearly all of the bacilli appeared Gram-negative or ative controls from University of Texas Medical partially Gram-negative. Bacilli from cases in which Branch archival tissues. bacilli stained Gram-negative or Gram-variable, as For the immunocytochemical assessment of pu- well as cases in which bacilli were typical Gram- tative anthrax bacilli in mediastinal lymph nodes positive bacilli, stained immunohistochemically and cerebral meninges, rabbit antibody (primary with rabbit antibody to B. anthracis (Vollum strain) antibody) against B. anthracis (Vollum strain) spores. Bacilli pervaded soft tissues with predilec- spores (provided by Dr. Martin Hugh-Jones, Loui- tion for lymphangioles. In the lungs of some cases, siana State University, Baton Rouge, LA) was incu- aggregates of bacilli appeared to obstruct capillaries bated with deparaffinized, rehydrated, protease- and venules. In the brain, bacilli often surrounded digested 7-␮m sections; after washing, horseradish blood vessels, imparting a felt-work zone inter- peroxidase–conjugated goat anti-rabbit antibody posed between the vascular adventitia and the ad- (secondary antibody) was applied, and after incu- jacent neuropil. Bacilli were often observed at the bation, unbound antibody was rinsed off with APK highest concentration in the middle of lesions pro- wash buffer using standard protocols for the Ven- gressively diminishing in concentration toward the tana 320 immunohistologic automated stainer. The periphery, but high-pressure hemorrhage disrupted positive control was a 3-day-old injection site of an such central concentrations. anthrax immunization in the skin and subcutane- Epidemiological, clinical, and general pathologic ous tissue of the arm in one of the cases (polymer- data and microscopic findings for all cases are ase chain reaction testing [PCR] [3]on this lesion shown in Tables 4–10. Many of the values exhibited showed a single vaccine strain of B. anthracis); neg- large variances, which appeared to be due to differ- ative control was the section of colon and luminal ences between those patients with known antibi- feces containing a variety of bacteria from another otic treatment and those without. patient in this series. All data were entered into a Microsoft Excel 5.0 for Windows spreadsheet program on one of sev- Epidemiological Data eral IBM-clone desktop computers and analyzed The age was available in 98% of patients; the with the standard statistical packages available in mean age was 46 years old (range, 25 to 71 years). the Data Analysis section of the program’s Tools There were 33 men and 8 women. Twenty-two of 41 menu. Quantitative data presented in the text and (54%) of patients were known to have received an- Ϯ tables are the mean SEM unless otherwise tibiotic therapy. The incubation period could be specified. determined in 30 of 41 (73%) of patients; the mean incubation period was 16 days. Duration of clinical illness and duration of hospitalization were known TABLE 3. Criteria for Quantification of Other Parameters in 73% and 90.2% of cases, respectively; the average

0 ϭ Absent duration of clinical illness and duration of hospital- 1/2 ϭ Minimal ϭ Present, but barely evident ization were 3.85 days and 1 day, respectively. The 1 ϭ Mild ϭ Clearly present, but probably mean overall duration of the infection from puta- pathophysiologically insignificant 2 ϭ Moderate ϭ Present and significant tive exposure to death was known in all cases and 3 ϭ Marked ϭ Prominent, but without destruction of the organ was 19.5 days, but there was a wide range from 6 architecture days to 69 days. The mean postmortem interval ϭ ϭ 4 Severe Prominent with organ architecture destruction (which bears upon the validity of the microscopic

484 Modern Pathology FIGURE 1. Photomicrograph of Bacillus anthracis in tissue of short FIGURE 4. Photomicrograph of capillaritis (arrows) and fibrin-rich survivor (death occured 2 days after onset of illness). Brown-Hopps edema in interstitium of mediastinum in patient with short survival stain, 500ϫ. (death occurred 3 days after onset of illness). Hematoxylin and eosin staining; 300ϫ.

FIGURE 2. Photomicrograph of mediastinal arterial branch in a short-surviving patient (death occurred 3 days after onset of illness) FIGURE 5. Photomicrograph of microaneurysm (arrows) in a small with acute vasculitis characterized by fibrinoid necrosis and mixed, artery with acute vasculitis in a short-surviving patient (death occurred predominantly neutrophil infiltrates. Hematoxylin and eosin staining; 3 days after onset of illness). Brown-Hopps staining; 300ϫ. 100ϫ.

FIGURE 3. Photomicrograph of arterial branch in a longer surviving FIGURE 6. Photomicrograph of high-pressure hemorrhage patient (death occurred 8 days after onset of illness) with vasculitis compressing adjacent lung tissue and distorting its architecture. characterized by mononuclear infiltrates of intima, media and Hematoxylin and eosin staining; 20ϫ. adventitia; thrombosis in lumen is noted (upper portion of figure). Hematoxylin and eosin staining; 50ϫ.

present in all cases, but its focality predisposed to data) was known in 88% and was approximately 19 sampling error. Both arteries and veins as well as hours. See Table 6. intervening smaller vessels were involved. Larger vessels in patients dying early in the course of the disease presented vasculitis, which was character- General Pathologic Data ized by loss of nuclei in the smooth muscle cells of Vasculitis (Table 4 and Figs. 2–4) was very evident the media, necrosis associated with fibrin (Biebrich in some specimens and some cases; it was probably scarlet staining in Mallory’s trichrome; dark purple

Inhalational Anthrax Histopathology (L. M. Grinberg et al.) 485 TABLE 4. Quantitative Microscopic Findings in inflammation was present and consisted of neutro- Inhalational Anthrax: Epidemiologic, Clinical and General Pathologic Observations phils, macrophages, and fibrin-rich edema, which is the microscopic counterpart of grossly observed Mean Ϯ SD % with Lesion “gelatinous” edema (Fig. 9a). Fibrin-rich edema Age (years) 46.13 Ϯ 1.99 Gender (males ϭ 1; females ϭ 2) 1.195 Ϯ 0.062 (Fig. 9b) appeared as strands of fibrin (Biebrich Antibiotic therapy (h) 16.61 Ϯ 5.85 scarlet staining by Mallory’s trichrome; phospho- Incubation period (d) 16.07 Ϯ 1.80 tungstic acid–hematoxylin positive; red with Mo- Duration of clinical illness (d) 3.85 Ϯ 0.41 Duration of hospitalization (d) 0.96 Ϯ 0.27 vat’s pentachrome; positive for fibrinogen in im- Overall interval from exposure to death (d) 19.54 Ϯ 1.93 munohistochemical method) coursing through Postmortem interval (h) 18.82 Ϯ 2.94 eosinophilic homogeneous “fluid.” The density and Volume of bilateral pleural fluid (mL) 1776 Ϯ 177 Vasculitis and capillaritis (0 to 5 scale) 3.448 Ϯ 0.49 81 eosinophilia of the continuous phase progressively Vasculitis only (0 to 5 scale) 2.45 Ϯ 0.41 73 diminished from the center to periphery of lesions. High pressure hemorrhage (0 to 5 scale) 3.46 Ϯ 0.55 78 There was low-pressure hemorrhage into mediasti- Hemorrhagic diathesis 0.73 Ϯ 0.07 71 Bacillus anthracis burden (sum of all 11.68 Ϯ 1.81 nal lymph nodes in a higher proportion of patients organs) and to a more severe degree than high-pressure hemorrhage. Hyperplasia of what appeared to be large lymphocytes was observed mostly in patients who succumbed later in the disease’s course. These by phosphotungstic acid-hematoxylin; red with round to ovoid cells were up to 20 ␮m in diameter, Movat’s pentachrome; positive for fibrinogen by with basophilic cytoplasm and hyperchromatic nu- immunohistochemical method), and variable neutrophil infiltrates; in patients surviving longer, mononuclear cells predominated in the intima, ad- TABLE 5. Quantitative Microscopic Findings in Heart, ventitia, and perivascular space. At one site, a small Mediastinal Lymph Nodes, Mediastinum, and Peribronchial Soft Tissue in Inhalational Anthrax artery with vasculitis had a saccular aneurysm (Fig. 5), and in another vessel, a rupture of a similar Severity % with Feature “microaneurysm” was noted. The latter lesions may Heart Bacillus anthracis burden 0.56 Ϯ 0.20 36 be the origin of the high-pressure hemorrhage Mediastinal lymph nodes noted, but these hemorrhages were much larger B. anthracis burden 1.493 Ϯ 0.274 60 and more frequent than observed foci of vasculitis Lymphocytolysis Follicular 2.936 Ϯ 0.188 94 or aneurysms (Table 4). High-pressure hemorrhage Paracortical 3.186 Ϯ 0.224 89 (Fig. 6) was seen in 32 of 41 cases; low-pressure Low-pressure hemorrhage 2.671 Ϯ 0.180 94 hemorrhages were seen in all cases. Hemorrhagic High-pressure 1.586 Ϯ 0.255 69 hemorrhage diathesis was characterized by numerous pete- Emperiopolesis 0.444 Ϯ 0.099 45 chiae, and ecchymoses in skin and serosal surfaces Hyperplasia 54 seen macroscopically (both clinically and at Sinusoidal 0.118 Ϯ 0.067 Follicular 0.059 Ϯ 0.059 autopsy). Paracortical 0.059 Ϯ 0.059 Atrophy 0.147 Ϯ 0.058 Edema 0.485 Ϯ 0.134 48 Quantitative Microscopic Findings by Organ Inflammation Neutrophils 1.570 Ϯ 0.202 82 Heart Mononuclear cells 0.770 Ϯ 0.160 60 Minimal concentrations of Bacillus anthracis Fibrin-rich edema 1.100 Ϯ 0.166 69 Large lymphocyte 0.999 Ϯ 0.212 were identified in cardiac sections of 9 of 25 pa- hyperplasia tients (Table 5), and there were no specific cardiac Vasculitis 0.600 Ϯ 0.159 36 microscopic findings, although occasional cases Fibrin 1.962 Ϯ 0.237 92 Neutrophils 1.077 Ϯ 0.193 92 showed individual myocyte hypereosinophilia and Mononuclear cells 1.115 Ϯ 0.378 69 rare focal-contraction band necroses, attributed to Mediastinum and agonal hypotension and hypoxemia. peribronchial soft tissue B. anthracis burden 1.375 Ϯ 0.269 54 Fibrin-rich edema 2.146 Ϯ 0.179 92 Mediastinal Lymph Nodes Low-pressure hemorrhage 2.274 Ϯ 0.170 95 See Table 5. These contained identified B. an- High-pressure 1.368 Ϯ 0.244 59 hemorrhage thracis in 21 of 35 cases (Fig. 7). There was marked Neutrophils 1.061 Ϯ 0.153 81 lymphocytolysis (Fig. 8), which was morphologi- Mononuclear cells 0.702 Ϯ 0.112 76 cally apoptotic, in both follicular and paracortical Lymphangitis 1.472 Ϯ 0.236 65 Vasculitis 0.681 Ϯ 0.168 46 areas. There was prominent phagocytosis of lym- Bacterial obstruction 0.259 Ϯ 0.165 11 phocytes by macrophages (emperipolesis) in 13 of Fibrin 2.000 Ϯ 0.272 87 29 cases and/or edema (12 of 25 patients), but the Neutrophils 0.983 Ϯ 0.185 87 Ϯ average severity of these lesions was minimal. Mild Mononuclear cells 0.960 0.310 67

486 Modern Pathology extensive and/or older than in other sites in the body, but this was not quantifiable.

Mediastinum and Peribronchial Soft Tissues See Table 5. These contained identifiable B. anthracis in 20 of 37 cases. Bacilli were identified in lymphangioles in 24 of 37 cases with a decreasing gradient extending from heavily burdened lymph nodes to lesser quantities of organisms in the pulmonary hilum and lungs (retrograde extension) and downstream toward the thoracic duct. There was moderate fibrin-rich edema and low-pressure hemorrhage, mild and less frequent high-pressure hem- FIGURE 7. Gross photograph of hemorrhagic mediastinal lymphadenitis and mediastinitis and lungs of fatal inhalational anthrax orrhage, and mild neutrophilic and mononuclear patient. infiltrates and mild-to-moderate lymphangitis. There was only a minimal degree of vasculitis characterized by fibrinoid necrosis, neutrophils, and mononuclear cells (predominantly lymphocytes and plasma cells). Vasculitis in 3 of 28 short survivors produced obstruction of blood vessels by aggregates of B. anthracis with preobstructive dilatation and postobstructive collapse.

Bronchi See Table 6. These contained B. anthracis in 10 of 28 cases. Ulceration, pseudomembrane formation, and squamous metaplasia were observed infrequently with mild acute bronchitis (neutrophil infiltrates, congestion, and edema) and mild chronic FIGURE 8. Photomicrograph of lymphocytolysis (and low-pressure bronchitis (bronchial basement membrane thick- hemorrhage) in mediastinal lymph node of patient succumbing to fatal ening, mononuclear infiltrates, increased goblet inhalational anthrax. Hematoxylin and eosin staining; 200ϫ. cells, and bronchial gland hyperplasia occurring in most of the cases). Squamous metaplastia and chronic bronchitis were presumably underlying clei, and stained immunocytochemically with CD45 conditions. There was mild fibrin-rich edema and (LCA) and S-100. Vasculitis within mediastinal low-pressure hemorrhage in bronchi and minimal lymph nodes consisted of a minimal degree of fi- high-pressure hemorrhage. Bronchial vasculitis brinoid necrosis, neutrophils, and macrophages. In noted infrequently was characterized by fibrinoid all cases, the lesions in the mediastinal lymph necrosis, neutrophils, and mononuclear cells (pre- nodes and mediastinal soft tissues appeared most dominantly lymphocytes and plasma cells).

FIGURE 9. Gelatinous or fibrin-rich edema. A, Macroscopic appearance of gelatinous edema spreading from mediastinum along dorsal costal parietal pleura. B, Photomicrograph of fibrin-rich edema in mediastinum of patient with fatal inhalational anthrax. Strands of positively staining material comprise a network within the edema-expanded interstitium. Immunohistochemical stain with antifibrinogen antibodies; 100ϫ.

Inhalational Anthrax Histopathology (L. M. Grinberg et al.) 487 Lungs congested. There was mild capillaritis in the majority See Table 7. These contained B. anthracis in 17 of of cases characterized by minimal fibrinoid necrosis, 35 cases. Over half of the bacilli were in alveolar mild to moderate neutrophilia, and minimal-to-mild septal capillaries and venules; less than a fifth were infiltration by macrophages. Pulmonary vasculitis in alveolar air spaces, and a quarter were in the was identified in the majority of cases but was mini- extravascular interstitium of the alveolar septa. mal in severity, composed of infrequent foci of fibrin- Thus, there appeared to be a gradient from blood to oid necrosis, mild neutrophil infiltration, and moder- air space, suggesting that pulmonary involvement ate infiltration by macrophages. Lymphangioles in by vegetative bacilli was of hematogenous origin, the interstitium were mildly to moderately dilated in rather than endobronchial origin. There was peri- most cases, with mild acute lymphangitis and mini- hilar interstitial pneumonia in all lungs, presum- mal hemorrhage; in a few patients, lymphatic chan- ably an extension of the process originating in the nels were obstructed by aggregates of bacilli, and the mediastinal lymph nodes and mediastinal soft tis- average degree of involvement by this feature was sues; this perihilar interstitial pneumonia extended minimal. into the air spaces in some, but not all, patients. Independent of the perihilar process, there was Spleen acute bronchopneumonia in nearly half of the pa- See Table 8. The spleen contained B. anthracis in tients. Acute bronchopneumonia involved all five 18 of 35 patients. White pulp: lymphoid follicles lobes of lung in three patients, the entire left lung in (B-cell areas) contained moderate to marked lym- one case, left upper lobe only in two patients, left phocytolysis, consistent with apoptosis, whereas lower lobe only in 3 patients, right middle and lower periarteriolar lymphatic sheaths (T-cell areas) lobes only in 2 patients, right upper and lower lobes showed marked lymphocytolysis. Follicles exhibited only in 1 case, right lower lobe only in 2 patients, right upper lobe only in 3 cases and right middle lobe only TABLE 7. Quantitative Microscopic Findings in Lung in in 1 patient. In the other 8 patients, acute broncho- Inhalational Anthrax pneumonia was only identified microscopically, and % with Severity the specific lobe involved was not identified. Micro- Feature scopic characteristics of the bronchopneumonia are Bacillus anthracis burden 1.39 Ϯ 0.28 49 best described according to each of four microana- % B. anthracis, intravascular 56.88 Ϯ 10.18 (rangeϭ0–100; tomic compartments: intra-alveolar exudate, intersti- median ϭ 80) % B. anthracis, intraalveolar 16.75 Ϯ 6.67 (range ϭ 0–95; tial exudate, capillaries, and lymphangioles. Alveolar median ϭ 6) spaces contained mild transudate, mild fibrin exu- Anthrax pneumonia, 0.515 Ϯ 0.113 49 date, mild hemorrhage, and mild macrophage infil- severity Intraalveolar exudate: tration; there was minimal neutrophil infiltration, Transudate 0.986 Ϯ 0.187 60 minimal lymphocyte infiltration, minimal plasma cell Fibrin 0.950 Ϯ 0.202 46 infiltration, and minimal nuclear debris. Alveolar in- Hemorrhage 1.300 Ϯ 0.218 66 Neutrophils 0.297 Ϯ 0.096 37 terstitium was expanded by mild transudation, mild Macrophages 1.136 Ϯ 0.161 71 fibrin deposition, mild hemorrhage, and mild infiltra- Lymphocytes 0.314 Ϯ 0.145 17 tion by macrophages with minimal neutrophil infil- Plasma cells 0.121 Ϯ 0.080 9 Nuclear debris 0.066 Ϯ 0.034 14 tration, minimal lymphocyte infiltration, and mini- Interstitial exudate: mal plasma cell infiltration but negligible nuclear Transudate 1.243 Ϯ 0.132 86 debris. Capillaries (and venules) were moderately Fibrin 0.772 Ϯ 0.142 57 Hemorrhage 0.943 Ϯ 0.153 66 Neutrophils 0.315 Ϯ 0.090 43 Macrophages 0.657 Ϯ 0.126 54 TABLE 6. Quantitative Microscopic Findings in Bronchi Lymphocytes 0.449 Ϯ 0.161 29 in Inhalational Anthrax Plasma cells 0.300 Ϯ 0.125 17 Nuclear debris 0.057 Ϯ 0.033 11 % with Severity Capillaries: Feature Congestion 2.043 Ϯ 0.200 91 Bacillus anthracis burden 0.917 Ϯ 0.239 36 Capillaritis 0.889 Ϯ 0.174 57 Ulceration 0.036 Ϯ 0.036 4 Fibrin 0.637 Ϯ 0.123 75 Pseudomembranous tracheobronchitis 0.0185 Ϯ 0.0185 4 Neutrophils 1.663 Ϯ 0.207 90 Squamous metaplasia 0.098 Ϯ 0.052 14 Macrophages 0.763 Ϯ 0.223 50 Chronic bronchitis 1.389 Ϯ 0.165 81 Vasculitis 0.514 Ϯ 0.154 66 Acute bronchitis 1.223 Ϯ 0.186 81 Fibrin 1.125 Ϯ 0.289 91 Fibrin-rich edema 1.134 Ϯ 0.192 64 Neutrophils 0.875 Ϯ 0.223 87 Low-pressure hemorrhage 1.375 Ϯ 0.231 71 Mononuclear 1.792 Ϯ 0.356 91 High-pressure hemorrhage 0.304 Ϯ 0.198 11 Lymphangioles Vasculitis 0.185 Ϯ 0.120 11 Dilatation 1.386 Ϯ 0.178 74 Fibrin 1.333 Ϯ 0.441 Lymphangitis 0.890 Ϯ 0.220 37 Neutrophils 0.833 Ϯ 0.583 Obstruction 0.414 Ϯ 0.149 23 Mononuclear 1.667 Ϯ 0.882 Hemorrhage 0.540 Ϯ 0.180 43

488 Modern Pathology minimal atrophy, and phagocytosis of lymphocytes TABLE 8. Quantitative Microscopic Findings in Spleen and Liver in Inhalational Anthrax by macrophages was observed in a few cases. Red pulp: Billroth cords were markedly thickened with Severity % with Feature moderate neutrophil infiltrates and moderate-to- Spleen Bacillus anthracis burden 1.664 Ϯ 0.307 51 marked mononuclear cell infiltrates and moderate White pulp numbers of B. anthracis–laden macrophages. There Follicular was mild endophlebitis, minimal low-pressure hem- Atrophy 0.471 Ϯ 0.089 46 Emperiopolesis 0.393 Ϯ 0.142 29 orrhage, and rare foci of high-pressure hemorrhage. Lymphocytolysis 2.521 Ϯ 0.243 89 Red-pulp sinusoids were mildly to moderately con- Periarteriolar lymphatic sheath gested, with mild infiltration of neutrophils and Lymphocytolysis 2.886 Ϯ 0.180 100 Large lymphocyte hyperplasia 2.371 Ϯ 0.183 mononuclear cells. Vasculitis (fibrinoid necrosis and Red pulp neutrophils) was seen in only one case and was min- Billroth cords imal in severity. Thickening 2.578 Ϯ 0.106 100 Neutrophils 1.793 Ϯ 0.138 100 Mononuclear cells 2.590 Ϯ 0.120 100 Liver B. anthracis–laden macrophages 1.816 Ϯ 0.224 83 See Table 8. Liver contained B. anthracis in 15 of Endophlebitis 0.882 Ϯ 0.092 83 Low-pressure hemorrhage 0.338 Ϯ 0.109 28 31 cases; all but one of these cases had bacilli in the High-pressure hemorrhage 0.029 Ϯ 0.029 3 sinusoids (nearly a third of the intrahepatic bacilli Sinusoids were in the sinusoids). All of them had bacilli in Von Congestion 1.424 Ϯ 0.149 86 Neutrophils 0.912 Ϯ 0.144 71 Kupffer cells (over half of the intrahepatic bacilli Mononuclear cells 1.064 Ϯ 0.162 71 were in Von Kupffer cells). Thirteen of 15 cases had Vasculitis 0.007 Ϯ 0.007 3 bacilli in the space of Disse, but only 10% of intra- Liver B. anthracis burden 1.258 Ϯ 0.269 48 hepatic bacilli were localized there. Intrasinusoidal % B. anthracis in inflammation was present in all but two cases and Sinusoids 29.93 Ϯ 7.71 consisted of mild to moderate neutrophilia, mild von Kupffer cells 55.57 Ϯ 9.12 Spaces of Disse 13.43 Ϯ 6.63 neutrophilic aggregates, and minimal mononuclear Intrasinusoidal inflammation infiltrates. Kupffer cells were mildly to moderately Neutrophils 1.315 Ϯ 0.140 94 hypertrophic and hyperplastic. Parenchymal cells Neutrophil aggregates 1.177 Ϯ 0.174 81 Mononuclear cells 0.516 Ϯ 0.136 35 had mild to moderate fatty metamorphosis in about von Kupffer cells half of the cases. Apoptotic hepatocytes were occa- Hypertrophy 1.605 Ϯ 0.178 87 sionally observed. Minimal to mild centrilobular Hyperplasia 1.326 Ϯ 0.153 87 Parenchymal cells coagulation necrosis was noted infrequently, and Fatty metamorphosis 0.798 Ϯ 0.177 52 one case had acute viral hepatitis, and another had Apoptotic hepatocytes 0.242 Ϯ 0.103 23 chronic viral hepatitis. Centrilobular coagulation necrosis 0.194 Ϯ 0.089 16 Viral hepatitis Acute 0.032 Ϯ 0.032 3 Intestines Chronic 0.032 Ϯ 0.032 3 The walls of the intestines (Table 9) contained B. anthracis in 20 of 33 patients. Grossly apprised submucosal hematomas correlated with high-pressure hemorrhages, and diffuse enteric hemorrhage cor- rhage was surrounded by a zone of fibrin-rich related with low-pressure hemorrhage (Figs. 10– edema (grossly, gelatinous edema) that varied in 12). Submucosal hematomas were numerous, of extent, and in turn, this was encompassed by a zone relatively uniform size, and small relative to those of simple edema. Mature submucosal hematomas described in primary intestinal anthrax (33). Mini- had high-pressure hemorrhage that disrupted the mal vasculitis was seen in several cases and was central collection of bacilli. characterized by mild-to-moderate fibrinoid necrosis and mild-to-moderate neutrophilic vasculitis. Kidneys Mild-to-moderate cellulitis (diffuse neutrophil infil- See Table 9. The kidneys harbored B. anthracis in tration of the interstitium, usually in the submu- 7 of 25 cases. Glomeruli had minimal capillaritis cosa) was noted in about half of the cases. In cases (neutrophil clusters in capillary loops) in a few with early submucosal hematomas, the center of cases, and mild mesangial cell proliferation was the lesion was usually in the area of the submucosa observed occasionally, as were intratubular red where the highest concentration of bacilli could be blood cell casts and evidence of early acute tubular seen, surrounded by low-pressure hemorrhage. necrosis (mononuclear cells in the vasa recta). This hemorrhage often encroached upon the lamina propria above and the muscularis propria below Adrenal Glands to some extent but was most conspicuous in its See Table 9. These contained B. anthracis in7of lateral spread though the submucosa. This hemor- 16 cases. There was minimal cortical atrophy and

Inhalational Anthrax Histopathology (L. M. Grinberg et al.) 489 cerebral). Meninges contained B. anthracis in 23 of 29 cases (Figs. 13–16). Meninges frequently exhibited minimal-to-mild transudates, mild fibrin exu- dates, mild-to-moderate low-pressure hemorrhage, minimal neutrophilic and macrophage infiltrates, and less often, mild high-pressure hemorrhage. In many cases, a few lymphocytes were identified in the meninges; rare plasma cells were seen in one case, and minimal nuclear debris was noted infrequently. Moderate vasculitis was identified in several cases and was characterized by mild-to- moderate foci of fibrinoid necrosis, neutrophils, and minimal mononuclear cells. In the meninges, a similar pattern of a central focal bacillary concen- FIGURE 10. Gross photograph of intestine with diffuse enteric tration and hemorrhage surrounded by zones of hemorrhage and submucosal hematomas (arrows). fibrin-rich edema and, in turn, surrounded by simple edema was observed and appeared to have expanded progressively through the leptomeninges. In all cases in which both meninges and mediastinal lymph nodes could be examined microscopically, the stage and severity of the lesions in the mediastinum was older and more intense than in the meninges, suggesting that the mediastinal lesions preceded those in the meninges. Cerebral parenchyma had modest mean bacillary burden. In 12 of 13 cases in which bacilli were detected in the cerebral parenchyma, the bacilli were in perivascular (Virchow-Robin) spaces. Vas- culitis was composed of mild focal fibrinoid necro- FIGURE 11. Low-magnification photomicrograph of margin of sis, neutrophils, and, less often, mononuclear cells. submucosal hematoma showing high-pressure hemorrhage. Minimal low-pressure hemorrhage was seen more Hematoxylin and eosin staining, 7ϫ. frequently than high-pressure hemorrhage. Isch- emic necroses, in other words, infarcts, were present in several cases, but mild diffuse acute neuronal necrosis was observed in the majority, and hypotensive sulcal necrosis was noted occasionally.

TABLE 9. Quantitative Microscopic Findings in Intestines, Kidneys, and Adrenal Glands in Inhalational Anthrax

Severity % with Feature Intestines Bacillus anthracis burden 1.879 Ϯ 0.350 61 Vasculitis 0.324 Ϯ 0.101 27 Fibrin 1.528 Ϯ 0.369 89 Neutrophils 1.611 Ϯ 0.380 89 Ϯ FIGURE 12. Photomicrograph of vasculitis and low-pressure Mononuclear cells 0.111 0.111 11 Ϯ hemorrhage in intestine with macroscopically diffuse enteric Cellulitis 0.742 0.168 52 hemorrhage with 7-day survival. Hematoxylin and eosin staining; 100ϫ. Kidneys B. anthracis burden 0.720 Ϯ 0.262 28 Glomerulus Capillaritis 0.390 Ϯ 0.147 36 minimal lipid depletion. There was mild-to- Mesangial proliferation 0.104 Ϯ 0.060 12 Tubules moderate pseudotubular change (glandlike separa- RBC casts 0.090 Ϯ 0.056 9 tion of trabeculae in the zona glomerulosa) and Acute tubular necrosis 0.273 Ϯ 0.188 20 occasionally minimal cortical hemorrhage. Adrenal Glands B. anthracis burden 0.875 Ϯ 0.301 44 Cortical atrophy 0.772 Ϯ 0.104 81 Brain Lipid depletion 0.524 Ϯ 0.080 88 See Table 10. These samples showed microscopic Pseudotubular change 1.625 Ϯ 0.268 88 Ϯ lesions in the meninges and parenchyma (usually Hemorrhage 0.234 0.111 31

490 Modern Pathology TABLE 10. Quantitative Microscopic Findings in the these data to medical scientists would very likely Central Nervous System in Inhalational Anthrax permit innovation of better therapies for inhala- Severity % with Feature tional anthrax. Brain The bacilli observed in histologic sections in this Meninges Bacillus anthracis burden 2.297 Ϯ 0.344 79 study are morphologically consistent with B. an- Transudate 0.759 Ϯ 0.152 59 thracis by light microscopy, and the identification Fibrin 0.901 Ϯ 0.159 83 was confirmed by immunohistochemical methods Low-pressure 1.239 Ϯ 0.221 90 hemorrhage and culture in most of the cases. PCR done on DNA High-pressure 0.603 Ϯ 0.240 21 extracted from paraffin blocks from this study de- hemorrhage tected four of the five known strains of Bacillus Neutrophils 0.524 Ϯ 0.142 55 Macrophages 0.478 Ϯ 0.104 55 anthracis (3). There were no quantitative bacterial Lymphocytes 0.181 Ϯ 0.062 72 cultures of the tissues and organs to corroborate Plasmacytes 0.017 Ϯ 0.017 3 the validity of the microscopic estimation of bacil- Nuclear debris 0.078 Ϯ 0.048 10 Vasculitis 0.375 Ϯ 0.179 24 lary burden. Therefore, this estimate of bacterial Fibrin 1.571 Ϯ 0.468 71 involvement of organs is the only measured param- Neutrophils 1.500 Ϯ 0.450 71 eter of dissemination from the portal of entry or Mononuclear cells 0.429 Ϯ 0.229 43 Parenchyma point of initial spore germination. Moreover, not all B. anthracis burden 1.200 Ϯ 0.312 42 bacteria seen were strongly Gram-positive, suggest- Vasculitis 0.342 Ϯ 0.107 32 ing that many may not have been viable; alterna- Fibrin 1.150 Ϯ 0.259 90 Neutrophils 0.950 Ϯ 0.337 60 tively, the staining characteristics may have been Mononuclear cells 0.150 Ϯ 0.107 20 altered by prolonged storage of the samples. Perivascular B. anthracis 0.683 Ϯ 0.203 39 The most severe and advanced lesions in each burden Low-pressure 0.567 Ϯ 0.162 29 case were in the mediastinal lymph nodes, suggest- hemorrhage ing that this involvement was the earliest in the High-pressure 0.333 Ϯ 0.140 19 course of the disease. This pattern is consistent hemorrhage Ischemic necrosis 0.227 Ϯ 0.091 16 with animal experiments of inhalational anthrax (infarct) (15–20) in which inhaled spores are phagocytosed Acute neuronal necrosis 0.705 Ϯ 0.072 77 by alveolar macrophages and transported by these Hypotensive sulcal 1.000 Ϯ 0.000 10 necrosis cells via pulmonary lymphangioles to the pulmonary hilar and mediastinal lymph nodes, where their germination results in dissemination of vege- DISCUSSION tative bacilli. This early involvement of mediastinal lymph nodes also indicates that these infections The conditions under which these autopsies were were acquired by inhalation, rather than ingestion, performed were comparable to those of forensic as originally reported (1, 2, 27). The morphologic pathologists studying casualties from a mass disas- lymphangitis, spreading from the mediastinal ter. The raw data came from the autopsy records lymph nodes, further supports a lymphohematog- and notes made by two of the authors (LG and FAA; enous centrifugal spread from the site of initial clinical records were confiscated by the KGB), and spore germination. museum specimens and samples preserved for microscopic examination in the autopsy suite of Hos- pital 40. These two factors, the work overload and Epidemiological and Clinical Variables the confiscation of materials, account for the in- Patients’ ages averaged in the mid-forties, with a completeness of the microscopic and gross data on moderate preponderance of men. The youngest each case in this set. man was 25 years old, and the youngest woman was The rationale for this detailed study of this in- the same age; thus, the age and gender demo- complete material was to separate the patients’ graphic variables in this set of autopsied fatalities other medical conditions from the “anthrax were not significantly different from that of all changes” by statistical analysis. Such analysis is deaths reported or all cases, fatal or surviving (2). warranted because this is the largest single autopsy Surprisingly, no children or adolescents were au- series of documented human inhalational anthrax. topsied, nor were there any children or adolescents This analysis is intended to provide the opportunity with recorded nonfatal infections (2). It is tempting for correlation of the microscopy of human inhala- to speculate that in some manner, children are tional anthrax with experimental animal models. excluded from contracting or dying of inhalational The present study emphasizes the need for more anthrax. However, there are no data available on clinical and premortem laboratory information on the demographic composition of the population at these patients to correlate with the autopsy data, risk in the putative area of exposure at the time of but these data remain sequestered. The opening of exposure, so age- or gender-specific incidence and

Inhalational Anthrax Histopathology (L. M. Grinberg et al.) 491 case-fatality rates cannot be determined. This area to the serous inflammation of earlier Russian his- is known to be highly industrialized, and children topathologic descriptions (31). and adolescents might indeed be underrepresented That death of cells in the path of the diffusing in the exposed population in factories at particular toxins began as fibrin-rich edema was established. periods of the day or night. Conversely, several Both B and T lymphocytes, as well as endothelium, large apartment complexes were in the area of ex- were highly sensitive to injury, but columnar epi- posure, and children would have been expected to thelium and glandular cells of varying types ap- have been exposed there. peared relatively intact. Lymphocyte death mor- The mean incubation period was 16 days. The phologically resembled apoptosis and resulted in mean duration of illness before admission and du- faintly basophilic debris. It involved both B-cell and ration of hospitalization (of those patients who T-cell zones of lymph nodes and spleen similarly died) was nearly 4 days and 1 day, respectively. but was also prominent near clusters of bacilli. Le- Overall duration from date of putative exposure to thal toxin interferes with intracellular signaling (23) death was 19.5 days; the standard errors of the and may induce apoptosis. Inflammatory response means were quite wide, reflecting the great range in was scant compared with the magnitude of necro- overall duration among the patients. Indeed, many sis/apoptosis, but neutrophils were prominent in parameters measured exhibited wide variances. It patients with shorter survival, whereas mononu- could be hypothesized that these variations result clear cells (large lymphocytes, immunoblasts, and from variations in age, sex, duration, and intensity macrophages) were significantly associated with of exposure, as well as variation in the amount, longer survival. Previous reports of both human timeliness, and duration of antibiotic therapy, or (11–15) and experimental (16–21) inhalational and perhaps the heterogeneity of the strains of B. an- intestinal (31) anthrax variably report neutrophilic thracis encountered (3). However, there is also infiltrates associated with the hemorrhagic, ne- marked variation in the incubation period in exper- crotic tissue injury in anthrax; most descriptions imental animals attributed to variation in duration include some, even significant, neutrophil infil- of the period before germination of the inhaled trates. Whereas the average magnitude of the neu- spores (19). The mean postmortem interval was trophil exudate in this series was low in most af- almost 19 hours. There was wide variation, but fected tissues, it was prominent in some locations postmortem interval had no relationship to any in some cases and prominent in some organs, and other measured variable in this study, so the results not others, in other cases. We conclude that a neu- presented are not simply artifacts of autolysis. trophilic infiltrate is generally present but is scant The earliest tissue reaction to the presence of in comparison to the amount of cell death. This bacilli was edema, which apparently resulted from would correlate with an apoptotic mechanism of the adenyl cyclase action (22) of diffusing edema cell death. Most of the lesions implicate toxin injury toxin (ET) upon adjacent endothelium; its histo- and meager neutrophilic response; patients surviv- logic appearance is a watery expansion of the inter- ing longer appear to shift from neutrophils to stitial spaces around the vegetative bacilli. This was mononuclear leukocytes, especially in the vasculitis supplanted by fibrin-rich edema, in which fibrin (vide infra). varying in appearance from congealed amorphous Vasculitis consisted of endothelial adherence by masses to irregularly curvilinear interwoven strands inflammatory cells, endothelial necrosis with intra- to short straight branching and anastomosing pat- vascular fibrin deposition, inflammatory cell infil- terns in a continuous phase of slightly eosinophilic trate into the vascular intima and media, and fibrin- edema was observed. The different morphologic oid necrosis of vessel walls. Frequency and severity types did not conform to any particular distribu- of vasculitis in all sites were averaged to give an tion. Fibrin-rich edema corresponds to the macro- overall estimate of the phenomenon in each case. scopically described gelatinous edema, which ap- Vasculitis, including capillaritis, was prominent in pears as an amber gel expanding soft tissues (1). all cases. Vasculitis, excluding capillaritis, was mod- Clearly, the leakage of fibrinogen and other protein erate to marked. Analysis of the vasculitis in various macromolecules into the interstitium represents organs (Tables 4–10) revealed that vascular fibrin- greater endothelial permeability than simple oid necrosis was seen with equal frequency and edema. It may represent the early direct effect of severity in shorter and longer durations of illness, lethal toxin (LT; 22) upon endothelium or the re- whereas neutrophils were predominant in patients lease of inflammatory mediators by macrophages with shorter survival, and mononuclear cells dom- triggered by LT (24). Certainly, protective antigen inated in longer survivors. The pathogenic mecha- (PA) must diffuse more rapidly than either LF or EF nism of this vasculitis is not known. It may be a because it must first bind to cells before either EF or direct or indirect effect of the toxins produced by LT can be internalized within the cell. It is probable the bacilli and could be related to release of proin- that this fibrin-rich (gelatinous) edema corresponds flammatory cytokines (24). Immunohistochemical

492 Modern Pathology FIGURE 13. Gross photograph of hemorrhagic meningitis in situ in a FIGURE 16. Photomicrograph of B. anthracis filling Virchow-Robin case of fatal inhalational anthrax. space in cerebral cortex. Brown-Hopps staining; 200ϫ.

hemorrhage observed (31). Derizhanov’s (31) histopathologic observations of the lesions in autopsies of patients dying of intestinal anthrax were remarkably coincident with our observations, ex- cept that the primary site was distal small intestine and cecum with initial spread to mesenteric lymph nodes rather than the lungs and mediastinal lymph nodes, respectively, in these inhalational-anthrax cases. High pressure hemorrhage, hemorrhage which

FIGURE 14. Photomicrograph of B. anthracis in the subarachnoid compressed and displaced the adjacent tissues, was space of the meninges. Brown-Hopps stain; 200ϫ. prominent in all cases. Although these hemorrhages probably result from the vascular rupture on the arterial side of the microcirculation, perhaps due to infection-induced aneurysms, as speculated by Derizhanov (31), demonstrating such small focal phenomena is more difficult than finding the larger hemorrhages, and the hemorrhage would very likely obliterate the small vascular lesion of origin. Such hemorrhages would be expected to occur in loci with more dense concentrations of bacilli, where high toxin concentrations and tissue damage would be maximal. Low-pressure hemorrhages, those in which erythrocytes permeate interstitial tissues, clearly re- FIGURE 15. Gross photograph of hemorrhagic encephalitis in case of sult from increased endothelial permeability fatal inhalational anthrax. and/or necrosis, probably on the venular side of the microcirculation. The injury involves vascular basement membranes and mural components of vessels study revealed some IgM and/or IgG, but no com- with relatively low intraluminal pressure; it is most plement, within some vessel walls; therefore, the probable that this damage results from bacillary distribution of these proteins may be a result of toxin injury to the smallest, thinnest-walled vessels, immunoglobulin leakage into damaged vessels, the capillaries and venules. rather than causing the vasculitis. Vasculitis has not Hemorrhagic diatheses were assessed macro- been reported in previous studies of human inha- scopically by the presence of multifocal petechiae lational anthrax (11–15), nor was it identified in and ecchymoses in the skin, serosal surfaces, and experimental studies (16–21), but vasculitis and visceral parenchyma and were present in 29 of the even microaneurysms were recognized by Russian 40 cases in which data were available. Although workers, as significant constituents in the patho- hemorrhage was probably in part a result of the genesis of intestinal anthrax; vascular injury was toxic vascular injury, one of the authors (OY), who even postulated to be responsible for some of the was active in treatment of these patients and others

Inhalational Anthrax Histopathology (L. M. Grinberg et al.) 493 who survived, believes that the hemorrhagic dia- displaced by large lakes of erythrocytes. We hypoth- theses seen at autopsy were, in part, the result of esize that low-pressure hemorrhages occur by com- disseminated intravascular coagulation. She recol- promise of endothelium of low-pressure vessels, such lects that the patients generally had decreased fi- as capillaries and venules, whereas high-pressure brinogen, increased bleeding time, marked prolon- hemorrhages resulted from rupture of high-pressure gation of clotting time, and decreased platelets. vessels, such as small arteries and arterioles. The apparent clinical well-being of patients with The composition of lesions observed in these in- systemic anthrax during most of the course of the halational anthrax cases (peripheral simple edema disease and then the sudden onset of septic shock surrounding a zone of gelatinous edema, in turn has been a consistent observation in both experi- encompassing a central zone of necrosis and hem- mental and human anthrax (2, 9–15, 19–21, 31). orrhage) resembles other forms of anthrax, such as The usual stigmata of septic hypotension and cutaneous anthrax, where a desiccated central splanchnic vasoconstriction (centrilobular necrosis of hemorrhagic necrotic lesion is surrounded by rings the liver, acute tubular necrosis of the kidneys, ARDS, of gelatinous edema and then simple edema. This superficial hemorrhagic necrosis of the intestines and pattern corresponds with the pathophysiologic hy- cerebral watershed injuries) were encountered rather pothesis of differential diffusion of three proteins: infrequently among these cases. This suggests that first PA, then EF and LF, from foci of bacillary septic hypotension, which was seen clinically, was so proliferation. In such a model, EF diffuses more precipitous and profound that its sequelae had no rapidly than LF. LF interrupts intracellular commu- time to develop. Indeed, short hospital courses were nication, resulting in cell death, but also (at lower the rule in these fatal cases. concentrations) causes release of inflammatory These human data are consistent with a portal of mediators (IL-1 and TNF-␣), which may produce entry in the lower respiratory tract, with phagocy- the profound septic hypotension seen terminally. tosis and transport of spores by macrophages to We have relearned and refined the basic histopa- pulmonary hilar and mediastinal lymph nodes, thology and pathogenesis of anthrax, initially dis- where germination and vegetative bacillary prolif- cerned from observations on humans (31, 32) and eration ensued. Subsequent transport of proliferat- in experimentally infected animals (11–15). This ing bacilli via the lymphatics would be enhanced by large series confirms previous reports of the patho- increased lymph flow and would disseminate the logic changes in human inhalational anthrax (16– bacilli through the thoracic duct into the blood, as 21) but reveals the greater range of variation in the well as via retrograde lymphangitic spread back disease. This information would be particularly im- into the perihilar regions of the lungs. Hematoge- portant to attending pathologists if and when the nous spread to all other organs followed. In each first cases of deliberate bioterrorist dispersal of an- disseminated site, bacilli proliferated first within thrax come to pass. vessels and then extravascularly and spread in con- centrically enlarging spheres through the intersti- Acknowledgments: The authors thank Kelly Cassity tium and within draining lymphatic channels. This for expert secretarial assistance in the preparation of spread appeared to be facilitated by the edema that the manuscript and the advice of Dr. Benjamin accompanied bacillary proliferation. These findings Gelman in interpretation of the central nervous sys- corroborate the validity of experimental models tem microscopic lesions. (16–21) for the human disease. The composition of the lesions in the mediastinal REFERENCES lymph nodes and soft tissues, as well as in “metastat- ic” sites was similar. Initial bacillary proliferation oc- 1. Abramova FA, Grinberg LM, Yampolskaya OV, Walker DH. curs, followed by transudation of edema, implying a Pathology of inhalational anthrax in 42 cases from the Sver- dlovsk outbreak of 1979. Proc Natl Acad SciUSA1993;90: loss of endothelial integrity; further endothelial com- 2291–4. promise results in leakage of fibrinogen and its poly- 2. Meselson M, Guillemin J, Hugh-Jones M, Langmuir A, Pop- merization to form the fibrin-rich or “gelatinous” ova I, Shelokov A, et al. The Sverdlovsk anthrax outbreak of edema, an amber gelatinous expansion of the soft 1979. Science 1994;266:1202–08. tissue central to the more fluid edema. Finally, cellu- 3. Jackson PJ, Hugh-Jones ME, Adair DM, Green G, Hill KK, Kuske CR, et al. PCR analysis of tissue samples from 1979 lar death, probably apoptotic rather than necrotic, Sverdlovsk anthrax victims: the presence of multiple Bacillus judging by the sparse neutrophilic response, ensues anthracis strains in different victims. Proc Natl Acad Sci U S with loss of vascular integrity and either low-pressure A 1997;95:1224–29. or high-pressure hemorrhage. In low-pressure hem- 4. Meselson M. The biologic weapons convention and the Sver- orrhage, erythrocytes infiltrate parenchyma (often al- dlovsk anthrax outbreak of 1979. J Fed Am Scientists 1988; 41:1–6. ready necrotic) in a diffuse manner without displac- 5. Bezdenzhnykh IS, Nikiforov VN. Epidemiologic analysis of ing tissue architecture, whereas in high-pressure anthrax in Sverdlovsk. Zh Mikrobiol Epidemiol Immunobiol hemorrhage, these reticular architectures are clearly 1980;5:111–13.

494 Modern Pathology 6. Gumbel P. The anthrax mystery. Wall Street J, October 21, 21. Albrink WS. Pathogenesis of inhalation anthrax. Bacteriol 1991. Rev 1961;25:268–73. 7. Gumbel P. Death in the air. Wall Street J, October 22, 1991. 22. Aroura N, Leppla SH. Residues 1–254 of anthrax toxin lethal 8. Gumbel P. Anthrax: The survivors speak. Wall Street J, Oc- factor are sufficient to cause cellular uptake of fused tober 23, 1991. polypeptides. J Biol Chem 1993;268:3334–41. 9. Albrink WS, Brooks SM, Biron RE, Kopel M. Human inhala- 23. Duesbery NS, Webb CP, Leppla SH, Gordon VM, Klimpel tion anthrax: A report of three fatal cases. Am J Pathol 1960; KR, Copeland TD, et al. Proteolytic inactivation of MAP 61:457–71. kinase-kinase by anthrax lethal factor. Science 10. Brachman PS, Pagano JS, Albrink WS. Two cases of fatal 1998;280:734–7. pulmonary anthrax, one associated with sarcoidosis. N Engl 24. Hanna PC, Acosta D, Collier RJ. On, the role of macrophages J Med 1961;265:203–8. in anthrax. PNAS 1993;90:10198–201. 11. Entiknap JB, Galbratih NS, Tomlinson AJH, Elias-Jones TF. 25. Abramova AA, Grinberg LM. [Pathology of anthrax sepsis Pulmonary anthrax caused by contaminated sacks. Br J Ind according to materials of the infectious outbreak in 1979 in Med 1968;25:72–4. Sverdlovsk (macroscopic changes).] Arkh Patol 1993;55:12– 12. La Force FM, Bumford FH, Feeley JC, Stokes SL, Snow DB. 17. Epidemiologic study of a fatal case of inhalation anthrax. 26. Abramova AA, Grinberg LM. [Pathology of anthrax sepsis Arch Environ Health 1969;18:798–805. according to materials of the infectious outbreak in 1979 in 13. Vessal K, Yeganehdoust J, Dutz W, Kohout E. Radiologic Sverdlovsk (microscopic changes).] Arkh Patol 1993;55:18– changes in inhalation anthrax. A report of radiological and 23. pathological correlation in two cases. Clin Radiol 1975;26: 27. Grinberg LM, Abramova AA. [Pathology of anthrax sepsis 471–4. according to materials of the infectious outbreak in 1979 in 14. Severn M. A fatal case of pulmonary anthrax. Br Med J Sverdlovsk (various aspects of morpho-, patho- and thanato- 1976;1:748. genesis).] Arkh Patol 1993;55:23–6. 15. Suffin SC, Carnes WH, Kaufman AF. Inhalation anthrax in a 28. Grinberg LM, Abramova FA, Yampolskaya OV, Smith JH, home craftsman. Hum Pathol 1978;9:594–7. Walker DH. Pathology of human inhalation anthrax [Ab- 16. Young GA, Zelle MR, Lincoln RE. Respiratory pathogenicity stract]. Mod Pathol 1998;11:144A. of Bacillus anthracis spores. I. Methods of study and obser- 29. Grinberg LM, Abramova FA, Yampolskaya OV, Smith JH, vation on pathogenesis. J Infect Dis 1946;79:233–46. Walker DH. Quantitative microscopic findings in inhala- 17. Barnes JM. The development of anthrax following adminis- tional anthrax [Abstract]. In: Program and abstracts of the tration of spores by inhalation. Br J Exp Pathol 1947;28:385– 46th Annual Meeting of the American Society of Tropical 94. Medicine and Hygiene 1997;57:128. 18. Ross JM. The pathogenesis of anthrax following the admin- 30. Grinberg LM, Abramova FA, Yampolskaya OV, Smith istration of spores by the respiratory route. J Pathol Bacteriol JH, Walker DH. Quantitative microscopic findings in 1957;73:485–94. inhalational anthrax [Abstract] Am J Clin Pathol 1998;109: 19. Henderson DW, Peacock S, Belton FC. Observations on the 480A. prophylaxis of experimental pulmonary anthrax in the mon- 31. Derizhanov SM, Pathologic anatomy and pathogenesis of key. J Hyg 1956;54:28–36. the intestinal form of anthrax. Smolensk, Russia: Western 20. Albrink WS, Goodlow RJ. Experimental inhalation anthrax in Oblast Governmental Printing House; 1935. the chimpanzee. Am J Pathol 1959;35:1055–65. 32. Dutz W, Kahout E. Anthrax. Pathol Annu 1971;6:209–48.

Inhalational Anthrax Histopathology (L. M. Grinberg et al.) 495