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Laboratory Diagnosis of
Infection
Satisfying Koch’s Second Postulate
Lesson objectives
 Discuss general procedures for the collection and preservation of
bacterial specimens for the diagnosis of an infectious disease.
 Discuss the variables that can affect the quality of a specimen.
 Relate bacterial growth requirements and generation time to
specimen collection and processing.
 Discuss specific specimen collection procedures to assist in the
diagnosis of an infectious disease: wound, ear, eye, blood, urine,
feces, and sputum.
 Discuss the importance of the Gram stain and other staining
procedures as a tool in determining specimen quality, identification,
and empiric therapy.
 Briefly discuss laboratory procedures used to isolate and identify
infectious microorganisms.
 Explain non-culture based methods for diagnosing infectious disease.
Diagnosis of infectious disease starts at
the patient
Specimen collection: The following safeguards
must always be in place when a specimen is
collected for analysis in the microbiology lab:
1)
asepsis:
2) Correct sample: any part of the body can be
sampled for microbiological analysis. Each body
part has specific considerations.
• blood: collect 2 samples in special tubes or
bottles from one venipuncture. Collect 2 more
30 minutes later, preferably from a different
venipuncture site. Collect before antibiotic
therapy. One bottle is aerobe, the other is
anaerobe. If blood volume is limited, submit
aerobe only.
Disinfect skin with iodine or chlorhexidine (not
alcohol).
• Urine: may be collected through urinary catheter or
“clean catch mid-stream” (ccms).
Ccms: disinfect genital area. Collect specimen (at
least 10 ml) midstream. Why not collect immediate
void?
suprapubic catheter: catheter is surgically inserted
through the top of the bladder.
Advantages: a) bypasses the urethra
b) can collect anaerobic urine specimen
• Sputum: mucus obtained from lower respiratory
system. Not generally produced by healthy
individuals. May be produced during infection,
cancer, trauma, or by smokers.
Sputum is usually collected when the patient
awakes (more concentrated). Methods of
collection:
a) deep coughing (with/without chest
percussion)
b) using a suction catheter (Luken’s Trap)
can be used to collect anaerobic specimen.
Sputum must be distinguished from saliva. Saliva
contains a large variety of bacteria and fungi
(“normal oropharyngeal flora”). If a sputum
specimen contains abundant saliva, the microbes
that grow will be those found in the mouth, and
pathogens from the lungs may be missed.
How to distinguish sputum from saliva: the Gram
stain:
saliva: many squamous epithelial cells, very few
polymorphonucleocytes (neutrophils) or PMNLs,
large variety and numbers of microbes.
This specimen should not be
cultured. Lab will request recollect. Report “oropharyngeal
contamination”
A good sputum sample will have large numbers
of PMNLs and one or two bacterial types:
What to do?
Many PMNLs and many
epithelial cells?
No PMNLs nor epithelial cells?
3) Throat swab, wound culture: Use a
moistened transport swab. The medium is a
minimal medium—all needed nutrients, but
1/10 concentration. Keeps pathogens alive
without promoting growth.
Take care to culture only the site ordered.
Throat swabs must not touch
4) Body fluids:
•
•
•
•
Synovial fluid (joints—viscous)
Peritoneal, pericardial, pleural
Exudate (unexpected fluid, such as in a cyst, abscess)
Cerebrospinal (csf)
strict antisepsis of puncture area and aseptic technique
is followed.
Collected through needle aspiration, often by M.D.
with nurse assisting.
Note appearance (bloody, milky, cloudy, colored)
Fluids should be considered “STAT” specimens.
5) Stool: Main concern with stool specimens is
that the specimen does not contaminate the
environment. Over 90 % of the stool’s mass is
bacteria.
Note the appearance of the stool (formed,
loose, liquid). Also note any abnormalities
(unusual color, presence of frank blood, visible
parasites or undigested objects).
6) Reproductive specimens: (vaginal swab,
discharge) Organisms that cause STIs are often
fragile, and must be transported to lab quickly.
Specimens that cannot be transported to the lab
immediately (within 15 minutes) should be
refrigerated if delay is unavoidable.
Exceptions:
• Csf (meningitis organisms are killed by
refrigeration)
• Vaginal/penile specimens and discharges (agents
of STI are killed by refrigeration)
• Anaerobe specimens (refrigeration encourages
oxygenation.)
Getting the specimen ready for the lab
Lab slip: sample on page 411.
- accession number: corresponds to that specimen only. Other
specimens from the same patient will have different accession
numbers.
- patient name and ID number (always confirm identity of patient by
hospital bracelet)
- Exact tests ordered—check specimen type
- Date and time of specimen collection
- Tentative diagnosis (indicate if diagnosis is confirmed or tentative.
Patient diagnosis may determine how testing is done)
- Medications (especially antibiotics)
- STAT if ordered. Where/how should results be reported?
Now the lab has the specimen. Now
what???
1) Direct examination of the specimen:
Gram stain: early indicator that microbes are present and
what they might be.
Acid fast stain (AFB) (p. 55): Similar to Gram stain, but
decolorizer is acidified alcohol. Detects the presence of
Mycobacterium tuberculosis.
India ink stain: Used on csf to detect budding yeast with
capsules (Cryptococcus neoformans).
Physical assessment of specimen (stool characteristics,
appearance of urine).
2) Culture: Different specimens have different
procedures for routine culture and sensitivity.
In general:
prepare stains first
culture onto selective and differential media. If
using one swab, go from least selective to most
selective.
Inoculate broth last (if using a swab, break swab
off into broth).
Reading culture results
• Initial reading of plates: Pathogen identity can be narrowed down
on the following basis:
compare culture results to Gram stain: if one bacterial type (Gram
positive cocci in chains) is noted, and only one colony type is seen,
there’s a good chance they are the same organism.
Specimen type: Certain pathogens are commonly found in certain
parts of the body
urine: E. coli, Proteus species
csf: Neisseria meningiditis, Streptococcus pneumoniae
sputum: Klebsiella pneumoniae, Hemophilis influenzae
Biochemical tests: Organisms are further identified through a series
of biochemical tests. Sometimes (though not often) these tests are
related to the organism’s pathogenic characteristics.
Common biochemical tests:
Oxidase: used to categorize Gram negative organisms
Fermentation: Many Gram negative rods ferment sugars (use sugars
without oxygen, producing acid). Which sugars are fermented give an
indication to the identity of the microbe. In particular lactose
fermentation separates many pathogenic Gram negative rods.
Enzyme production: some bacteria are identified by the enzymes they
excrete. Some of these are exotoxins, but not all:
coagulase: Staphylococcus aureus is positive (pathogen),
Staphylococcus epidermidis (non-pathogen) is negative.
Some bacteria excrete enzymes that can be identified even without
culture in rapid ID kits.
C. dificile is diagnosed by exposing suspected stool to human cells in
culture. The organism does not need to be grown.
What if the pathogen can’t be cultured?
• Immunologic typing: If the pathogen can’t be grown
outside the host, it is possible to detect antibodies to
the pathogen using direct or indirect methods.
Direct: latex agglutination for meningitis:
csf is boiled and concentrated. It is then exposed to
suspension of latex particles with antibodies to various
pathogens. Positive result = agglutination. Negative
result = no agglutination.
Direct immunologic test detects antigens of the
pathogen.
Indirect: ELISA for antibodies p. 423
1) Commercially prepared antigens are adsorbed
to test plate. Wash excess
2) Patient’s serum sample is applied. Wash excess.
3) Antibodies to human IgG is applied (raised in a
different animal—commercially prepared) This
antibody is chemically bound to an enzyme.
4) Substrate for the enzyme is added. The
enzyme/substrate complex is colored. A colored
result is positive, no color is negative.
Genetic tests: It is possible to purchase DNA
samples of a variety of pathogens. Only certain
DNA sequences are available (therefore, the DNA is
non-pathogenic).
PCR: Polymerase Chain Reaction: If the pathogen is
only present in small numbers (e.g. tuberculosis), it
is possible to selectively clone large numbers of
copies of the pathogen’s DNA.
PCR is also used to identify agents of bioterrorism
(anthrax) and to identify DNA samples in forensic
analysis.
Lab Considerations
• Quality Control:
Every procedure, media, and piece of equipment
in the laboratory must be routinely tested for its
abilities to detect positive results and to
determine negative results as negative.
Durable equipment (incubators, refrigerators,
autoclaves): Monitored daily for temperature, air
quality, and asepsis. Records are maintained and
audited.
• Media: All growth media must be identified by lot
number, and tested for sterility and performance.
Reference organisms are maintained, cultured on new
batches of media.
Example: EMB agar: new batch must include culture
of S. aureus (Gram positive), E. coli (Gram negative,
lactose fermenter), P. vulgaris (Gram negative, nonlactose fermenter).
Acceptable Quality: S. aureus does not grow
E. coli grows, green colonies
P. vulgaris grows, pink colonies
Test anomalies
• False negative: Pathogen is present, test fails
to detect it. If a false negative occurs, the test
has low sensitivity.
• False positive: Pathogen is not present, but
test identifies it as present. If a false positive
occurs, the test has low specificity.
Remedy: