Another requirement of microbiologic specimens
is that they must be collected without undue micro-
bial contamination. The avoidance of microbial con-
tamination applies primarily to contamination by
organisms on the skin and the mucous membranes.
Contamination from the patient’s skin is controlled
by disinfecting the patient’s skin overlying the body
site to be sampled. Contamination from the physi-
cian’s skin is avoided by using aseptic technique.
Complete avoidance of contamination from the
mucous membranes is not possible if the specimen
must pass along the membrane. Expectorated spu-
tum, for instance, will always be contaminated with
organisms from the oral cavity. Contaminating
organisms from the mucous membranes may be
recognizably innocuous but their presence may make
the pathogenic species hard to detect. In that case,
processing and growth techniques can be used that
suppress contaminating organisms and enhance
pathogenic organisms. If the contaminating organ-
isms are potential pathogens, it may be possible to
determine when they are merely contaminants by
quantitative analysis. Contaminating organisms are
usually present in low density in a fresh specimen
while organisms causing an infection are usually
present in high density. In situations in which
microbial contamination by the mucous membranes
presents a serious impediment to clinical care, it can
be avoided altogether by bypassing the membrane
route and obtaining the specimen by percutaneous
aspiration or by biopsy. Transtracheal aspiration of
sputum is one example.
The amount of material contained in a microbio-
logic specimen must be large enough to contain a
detectable number of organisms. The actual amount
of material needed will, of course, vary according to
the density of the pathogen in the material. For
example, only a small volume of pus, just 1 to 2 ml,
is needed for the microbiologic study of an abscess
because the density of bacteria in pus is very high.
In contrast, the density of bacteria in the blood of a
patient with septicemia is very low, necessitating
that a large volume of blood be obtained, preferably
two 10 ml specimens. A particular concern relating
to the size of the specimen is the popularity of swabs
for specimen collection as swabs absorb only a tiny
volume of fluid. Their use is best limited to the
sampling of skin and mucous membranes where
there is no accumulated fluid to collect.
Finally, microbiologic specimens must be expe-
ditiously placed into the appropriate transport
containers and rapidly transferred to the laboratory.
Transport containers protect the specimen from
microbial contamination during handling and often
contain a transport medium that prevents the dehy-
dration of the specimen. Transport media are
usually non-nutritive in order to suppress microbial
growth but a nutritive medium may be used for the
transport of fastidious organisms. Special container
systems are available for the transport of specimens
to be cultured for anaerobic bacteria.
Specimens that manifestly fail to achieve
adequate standards of collection or transport are
usually rejected by the laboratory. Explicit criteria
for rejection should include: unlabeled or improperly
labeled specimens; specimens received in inappropri-
ate, leaking, or broken containers; specimens with
obvious contamination; and unpreserved specimens
received too long the time of collection (Wilson
1996).
Microbial substances
Microbial substances that are present at the site
of infection include proteins, nucleic acids, secreted
products, and metabolic products. Their detection in
material obtained from the site of infection offers a
means of rapidly demonstrating the presence of a
pathogen in settings in which microscopy may not
always be informative. Tests for these substances
can, of course, also be used in combination with
microscopy to maximize sensitivity for the rapid
demonstration of pathogens.
Microbial proteins, measured using immunologic
techniques, are the substances most often studied.
Their diagnostic performance as markers of infection
can be quite good. As an example, the latex aggluti-
nation test for cryptococcal antigen in CSF is both
highly sensitive and highly specific for the diagnosis
of
Cryptococcus neoformans
meningitis. Such
reliability is not a always attained, however. For
instance, it has been demonstrated that, using current
techniques, bacterial protein detection in CSF has an
unacceptably low specificity for the diagnosis of
bacterial meningitis (Perkins
et al.
1995). This
study also showed that it is very rare that a true
positive result is the sole finding indicative of infec-
tion. In almost all cases, routine microbiologic
studies also revealed the presence of infection, albeit
not always as rapidly. The detection of microbial
nucleic acids, measured using molecular diagnostic
techniques, is proving to be a consistently reliable
method of demonstrating pathogens. Because of the
Tissue Injury
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