however, does not imply infection. It is only with invasion of organisms into viable tissue that
they gain access to the bloodstream and spread to other tissues where they release toxins and
induce the severe inflammatory response that characterizes burn wound sepsis. Surface swabs
and even quantitative cultures, therefore, do not reliably differentiate colonization from
invasion (31,32). Histologic examination of a biopsy specimen is the only means of accurately
identifying and staging invasive burn wound infection (33). Using a scalpel, a 500 mg
lenticular tissue sample is obtained from the area of the wound showing changes indicative of
invasive infection. The biopsy must include not only eschar, but also underlying, unburned
subcutaneous tissues as histologic diagnosis of invasive infection requires identification of
microorganisms that have crossed the viable–nonviable tissue interface to take residence and
proliferate in viable tissue. A local anesthetic agent if used should be injected at the periphery
of the biopsy site to avoid or minimize distortion of the tissue to be examined histologically.
One-half of the biopsy specimen is processed for histologic examination to determine the depth
of microbial penetration and identify microvascular invasion. The other half of the biopsy is
quantitatively cultured to determine the specific microorganisms causing the invasive infection.
The culture results are used to guide systemic antibiotic therapy. In the case of fungal invasion,
firm identification of the causative organism is problematic even with both histology and
culture, since histology results do not necessarily correlate with culture results (34). Therefore,
antifungal coverage should be such that all organisms identified are covered to maximize
outcomes.
The biopsy specimen is customarily prepared for histologic examination by a rapid
section technique that affords diagnosis in three to four hours. Burn wound infection, if
present, can then be staged on the basis of microbial density and depth of penetration to guide
treatment. Alternatively, the specimen can be processed by frozen section technique that yields
a diagnosis within 30 minutes, but is associated with a 0.6% falsely positive diagnosis rate and
a 3.6% falsely negative diagnosis rate (35). If the frozen section technique is utilized, permanent
sections must be subsequently examined to confirm the frozen section diagnosis and exclude
false negatives. The microbial status of the burn wound is classified according to the staging
schema detailed in Table 2. In stage I (colonization), the bacteria are limited to the surface and
nonviable tissue of the eschar. Stage I consists of three subdivisions (A, B, and C) defined by
depth of eschar penetration and proliferation of microorganisms. Stage II (invasion) also
consists of three subdivisions (A, B, and C) defined by extent of invasion of microorganisms
into nonviable tissue and involvement of lymphatics and microvasculature. Subsequent
Figure 4 (A) Gross appearance and histologic finding of invasive Aspergillus infection on the arm in a patient
who succumbed to infection. Note the discolored, dark, hemorrhagic appearance of the skin. Note organisms
present in viable tissue surrounding blood vessels. (B) The photomicrograph shows the presence of hyphae in
viable tissue (Stage II B).
366 Wolf et al.