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Overview

Various techniques are used for the direct identification of B. anthracis in clinical material. Firstly, specimens may be Gram stained. Bacillus spp. are quite large in size (3 to 4 μm long), they grow in long chains, and they stain Gram-positive. To confirm the organism is B. anthracis, rapid diagnostic techniques such as polymerase chain reaction-based assays and immunofluorescence microscopy may be used.

All Bacillus species grow well on 5% sheep blood agar and other routine culture media. Polymyxin-lysozyme-EDTA-thallous acetate can be used to isolate B. anthracis from contaminated specimens, and bicarbonate agar is used as an identification method to induce capsule formation. Bacillus spp. usually grow within 24 hours of incubation at 35°C, in ambient air (room temperature) or in 5% CO₂. If bicarbonate agar is used for identification, then the medium must be incubated in 5% CO₂. B. anthracis colonies are medium-large, gray, flat, and irregular with swirling projections, often referred to as having a "medusa head" appearance, and are not hemolytic on 5% sheep blood agar. The bacteria are not motile, susceptible to penicillin, and produce a wide zone of lecithinase on egg yolk agar. Confirmatory testing to identify B. anthracis includes gamma bacteriophage testing, indirect hemagglutination, and enzyme linked immunosorbent assay to detect antibodies. The best confirmatory precipitation test for anthrax is the Ascoli test.

Diagnostic Studies

Other studies to diagnose Anthrax infection include:

Laboratory findings

Test	Initial Findings	Serial Monitoring
EKG	Atrial fibrillation with rapid ventricular response
Lumbar Puncture	At admission unless contraindicated	Headache confusion Other neurologic symptom If meningitis, meningeal signs will only be present at a later stage
Liver Enzymes Serum Albumin	Elevated transaminase levels hypoalbuminemia
PT PTT D-dimer Fibrinogen	Normal PT/PTT does not exclude DIC or coagulopathy	Low threshold for hypercoagulability workup: Haptoglobin LDH Fibrin split products ADAMTS 13 if hemolytic anemia
C-Reactive Protein	Characterization of Inflammatory Response Typically low CRP in injection anthrax
Gram stain Cultures Toxic Assays	Blood Serum CSF Pleural fluid Ascites Wound exudate Bronchial exudate	Cultures usually negative after antibiotics Toxins may be detected
Cardiac Enzymes BNP	Troponin leak caused by increased cardiac demand from infection (particularly if atrial fibrillation with rapid ventricular response

Hemolysis

B. anthracis was shown to be haemolytic on blood agar made with sheep red cells that had been washed with buffered saline containing calcium and magnesium. Similarly, the anthrolysin is presumably haemolytic. Reports are also occasionally encountered of haemolysis in blood or on agar containing blood of certain species, including human.

Lecithinase

B. anthracis either produces lecithinase to a lesser extent than its close relatives, B. cereus and B. thuringiensis, or produces a lecithinase with a lower activity. In the lecithinase test on egg yolk agar, the zone of opalescence or “halo” almost always seen around colonies or areas of growth of B. cereus and B. thuringiensis is only sometimes visible around B. anthracis, probably only becoming apparent at 48 hours (35-37 °C) and usually in a narrow band when present. Opalescence should be looked for under the colony/area of growth by scraping away some of the colony material. It can be seen here after 24 hours incubation at 35–37 °C. While B. anthracis grows well on conventional egg yolk agar, it grows less well than B. cereus on Kendall’s BC egg yolk-mannitol agar; the growth of B. anthracis on this medium (24–48 hours) is greyish as compared to the deep purple of B. cereus and, in contrast to B. cereus, a zone of opalescence does not form around the growth of B. anthracis. once again, colony material must be scraped away to see the underlying LV (lecithovitellin) reaction.

Motility

Although examination for motility is always listed as one of the primary identification tests for B. anthracis, it is doubtful that the test is often done on new isolates or checked more than rudimentarily and occasionally on culture collection cultures. Certainly, the first obvious appearance for diagnostic test purposes is lack of motility, but in view of the existence of genes associated with motility, and even one recent report that includes electron micrographs showing flagella, perhaps the “absoluteness” of non-motility in B. anthracis should be revisited.

PCR

PCR is becoming more widely available as a means of confirming the presence of the virulence factor (capsule and toxin) genes, and hence that an isolate is, or is not, virulent B. anthracis. For routine purposes, primers to one of the toxin genes (usually the Protective Antigen gene) and to one of the enzymes mediating capsule formation are adequate. In laboratories not equipped for PCR tests, if doubt remains to the definitive identity of a suspect B. anthracis isolate, inoculation into a mouse or guinea-pig may be the only way remaining to determine conclusively if it is virulent B. anthracis. However this should be a last resort procedure and confined to situations where a definitive identification is essential.