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Any tissue or body fluid can be subjected to microbiological investigation
with the aim of identifying the infecting pathogen and
predicting response to therapy.
To optimize the diagnostic benefit it is necessary to:
- understand in which tissues/specimens the organism is to be
found and when in the natural history of the infection;
- take samples carefully (e.g. poor aseptic technique may lead to
contamination of sterile samples causing false-positive results);
- transport samples rapidly to the laboratory in a suitable medium.
It is important to remember that some organisms survive poorly
outside the body (e.g. numbers of strict anaerobes are reduced by
atmospheric oxygen) and in some cases suitable media needs to be
inoculated directly in the clinic (e.g. for isolation of Neisseria
Specimens may be examined grossly, for example to detect adult
worms in faeces. Although microscopy is rapid, it is insensitive and
requires considerable expertise; specificity may also be a problem
if commensal organisms can be mistaken for pathogens. Microscopy
can also be used to define specimen quality, for example
identifying salivary contamination of sputum by the presence of
Special stains can be used to help identify organisms, such as
Giemsa staining of blood films and tissues, which is used to identify
malaria and Leishmania (Malaria, leishmaniasis and trypanosomiasis
can provide precise identification of a pathogen by using antibodies
that are directed against a specific organism.
- Antibiotic therapy administered presampling can falsely render
samples culture negative.
- Most human pathogens are fastidious, requiring media supplemented
with nutrients to support growth and increase their
numbers to detectable levels.
- Growth on solid media allows organisms to be separated into
individual colonies; a pure (clonal) population permits subsequent
identification and susceptibility testing.
- Selective agents such as antibiotics or dyes may be used to suppress
unwanted organisms in specimens with a normal flora.
- An appropriate atmosphere must be provided: fastidious anaerobes
require an oxygen-free atmosphere.
- Most pathogenic bacteria are incubated at 37 °C, but some fungi
are incubated at 30 °C.
- Identification predicts pathogenicity: Vibrio cholerae causes
severe watery diarrhoea, whereas Shigella sonnei infection is
- Identification of some organisms should prompt public health
action, for example contact tracing for a patient found to have
Bacterial identification depends on colonial morphology on
agar, microscopic morphology, biochemical tests and, increasingly,
nucleic acid amplification tests (NAATs) and gene sequencing.
This is especially important for organisms that are slow
growing (e.g. Mycobacterium tuberculosis) or impossible to grow
(e.g. Trophyrema whippelii) to grow.
Susceptibility testing aims to determine whether treatment with a
given antibiotic will be successful. A susceptible organism should
respond to a standard dose of an antimicrobial, a moderately
resistant strain should respond to a larger dose, whereas a resistant
organism is likely to fail therapy with the given antibiotic. Clinical
response depends on host factors, and in vitro tests only provide
an approximate guide to therapy.
Several bodies including the British Society of Antimicrobial
Chemotherapy (BSAC) and the Clinical Laboratory Standards
Institute (CLSI) define methods and standardized conditions to
ensure testing is reproducible. Both are based on measurement of
the diameter of the zone of inhibition of confluent growth for the
test organism that is caused by an antimicrobial incorporated into
a paper disc. The minimum inhibitory concentration, which is the
lowest dose that completely inhibits growth, is a more objective
method and enables resistance levels to be related to the concentration
of antibiotic that is achievable in the tissues.
Susceptibility can be assessed rapidly by hybridization or
sequence-based methods that detect specific antibiotic-resistance
An infection can be diagnosed by detecting the immune response
to the pathogen: for example by detection of rising or falling antibody
concentrations more than a week apart, or by the presence
of a specific IgM or specific antigen. These techniques are used for
organisms that are difficult to grow such as viruses (e.g. HIV or
Southern blotting and nucleic acid hybridization
A labelled DNA probe will bind to the specimen if it contains the
specific sequence that is being sought. The captured probe is
detected by the activity of it attached label. This technique is specific
and rapid, but less sensitive than other methods that involve
Nucleic acid amplification tests
Nucleic acid amplification tests (NAATs) make the diagnosis by
amplifying specific regions of the genome from the pathogen.
Although different methods are used to amplify pathogen-specific
DNA or RNA the aim is the same, to produce sufficient copies for
detection. For example, nucleic acid from the pathogen is separated
into single strands and primers are designed to bind to target
sequences. A polymerase then catalyses synthesis of new nucleic
acid and this process is repeated for multiple cycles. Automated
systems and commercial kits have made these tests available in
many laboratories. Real-time PCR machines measure rising concentrations
of target DNA and determine positivity when the concentration
passes a set threshold. NAATs have the advantage that
they can detect slow-growing organisms or those that are difficult
to grow (e.g. M. tuberculosis) or make a diagnosis when samples
are rendered falsely negative by antibiotic therapy. Methods to
detect antibiotic-resistance genes can also be used to provide surrogate
susceptibility results (e.g. detection of the rpoB gene mutation
for rifampicin resistance in M. tuberculosis).