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INTENDED USE
ID-AE kit is intended for use as a
rapid colorimetric test for the presumptive identification of group A streptococcal and
enterococcal bacteria from clinical isolates.
SUMMARY AND EXPLANATION
PYR is a substrate which is
hydrolyzed by 100% of the enterococci and group A streptococci, but not by any other
streptococcal strains.1 ~
S. pyogenes are isolated from throat
cultures, blood, skin, wounds, vagina, and rectum. Typical colonies of S. pyogenes on
blood agar are approximately 0.5 mm in diameter surrounded by a zone of beta-hemolysis.
Colonies appear translucent, domed, and smcoth or semi-mat surface.
Enterococci are normal inhabitants
of the human gastrointestinal tract and may spread from this site to cause urinary tract
infections, and wound infections. Enterococci are typically larger in size than S.
pyogenes on blood agar medium.
PRINCIPLES
The Gibson ID- AETM test
utilizes filter paper strips impregnated with the substrate specific for PYRase. The
usefulness of pyroglutamyl aminopeptidase (PYRase) activity as an aid in the detection of
group A streptococci and enterococci is well documented.1~5 This test is based
on the ability of group A streptococci and enterococci to hydrolyze the chromogenic
substance PYR (L-pyrrolidonyl-beta-naphthylamide). The hydrolysis of PYR is detectable by
the presence of the color red when a Color Developer reagent is added.
REAGENTS
Each Gibson ID- AETM kit
contains the following reagents sufficient for 50 tests:
1-20 ml boffle Buffer Reagent
1-20 ml baffle Color Developer A
50 Test Cards
PRECAUTIONS: This test is intended
for use by those trained in appropriate laboratory and bacteriological procedures. This
test is for IN VITRO DIAGNOSTIC USE only. Precautions should be taken against the dangers
of microbiological hazards. Specimens, containers, media, and test cards should be
sterilized after use. Reagents should not come into contact with skin, eyes, or clothing.
Do not inhale or ingest reagents. In case of an accident, seek medical attention
immediately. The sodium azide buffer reagent may react with copper and lead in plumbing to
form highly explosive metal azides. Upon disposal, flush with large volumes of water to
prevent buildup of azides.
STORAGE INSTRUCTIONS: This kit must
be stored at 2-8C to prevent breakdown of the substrates and reagents. The kit must be at
room temperature before use, and may be left at rcom temperature for up to one hour.
EVIDENCE OF DETERIORATION:
Substrates and Reagents provided with this kit should not be used if the expiration date
has passed. Discard product if the white filter paper within the test cirde shows signs of
discoloration. If any deficiencies are noted with this product notify the manufacturer.
SPECIMEN COLLECTION
Clinical specimens should be
protected from excessive heat and cold and should be delivered to the laboratory without
delay. The specimen should be collected prior to the initiation of therapy. If the
specimen is collected after the initiation of therapy, the microbiologist should be
notified on the data form. Additional information on collection of clinical specimens may
be found in standard reference texts. Fresh cultures grown ovemight on nonselective media
give the best results. Use Gram positive and catalase negative colonies which
morphologically resemble group A streptococci and/or enterococci. Inoculate the test
circle with approximately eight, 0.5 mm or larger colonies.
PROCEDURES
OTHER MATERIAL REQUIRED BUT NOT
SUPPLIED: The standard clinical microbiological equipment such as Icop, burner, and
incubator are needed for procedures involving the use of this product. Other materials
required include the following: Gram Stain Reagents, Catalase Reagent, Microscope Slides,
and Culture media.
Use one card for each spedmen
tested. Before performing the test make sure the colonies are gram positive and
catalase negative.
1. Apply 34 drops of the buffer
reagent to the circle.
2. Using a swab, wcoden applicator
or an inoculating Icop, apply approximately four to five suspect colonies 0.5 mm or
larger to the center of the filter
paper within the circle. The smear should be visible to the naked eye.
3. Incubate the inoculated card at
room temperature for 10 minutes.
4. Apply 2 drops of Color Developer
to the filter paper with the test circle. The appearance of a red color indicates positive
PYRase activity.
INTERPRETATION OF RESULTS
The presence of PYRase activity
in Gram positive and catalase negative colonies presumptively identifies an organism as
either group A streptococci or enterococci. The confirmation of group A streptococci and
enterococci must be performed using additional biocnemical and/or serological procedures.
Interpretation Chart:
USER QUALITY CONTROL
Daily quality control should be
performed in accordance with proper laboratory procedures using organisms that will
produce known positive and negative reactions. The following American Type Culture
Collection strains are recommended:
Stneptococcus pyogenes (Group
A)
ATCC 19615
Enterococcus faecalis (Group
D)
ATCC 29212
Streptococcus
agalactiae (Group B)
ATCC 13813
Red Circle Red (PYRase +)
Red (PYRase +)
No ColorIYel low (PYRas~ -)
LIMITATIONS OF PROCEDURE
The ID- AETM test is intended
only for the presumptive identification of Gram positive, catalase negative cocci which
are morphologically similar to streptococci isolated from primary and secondary plated
media. Some species of staphylococci may produce a positive PYR test. Streptococci may be
differentiated from staphylococci by the catalase test and the benzidine test.1 Streptococci
are catalase-negative and, lacking cytochromes, are benzidine negative. Staphylococci are
catalase-positive and yield a positive benzidine test.
It should be noted that recentiy
described gmm-positive cocci with negative or weak catalase reactions (Lactococcus,
Gemella, Helcococcus, Globicatella, and Stomatococcus) may produce results similar to
group A streptococci.6
Kiebsiella (gram negative rod) may
also produce a positive PYRase reaction, a gram-stained smear should be examined to
differentiate this organism. Further biochemcial and/or serological procedures is required
to identify the colonies.
PERFORMANCE CHARACTERISITICS
The Gibson ID- AETM test was tested
on 180 dinical isolates at two different laboratories. The isolates consisted of 50 group
A streptococci, 50 Enterococci, and 80 non group A streptococci. The Gibson ID A.E.
tested correctly 178 out of the 180 isolates to show a 98.8% sensitivity and 100%
spedficity.
REFERENCES
1. Bosley, G.S~, R.R. Facklam, and
D. Grossman. 1983. Rapid identification of Enterococci. J. Clin. Microbiol.
18:1275-1277.
2. Elmer, P.D., D.A. Williams, M.E.
Hosmer and M. Cohenford. 1985. Preliminary evaluation of a rapid colorimetric method for
the presumptive identification of Group A Streptococci and Enterococci. J. Cl in.
Microbiol. 22:880-881.
3. Facklam, R.R. 1972. Recognition
of Group D Streptococcal Spedes of human origin by biochemical and physiological tests.
AppI. Microbiol. 23:11311-1139.
4. Facklam, R.R., L.G. Thacker, B.
Fox, and L. Eriquez. 1982. Presumptive identification of Streptococci with a new test
system. J. Clin. Microbiol. 15:987-990.
5. Facklam, R.R., and R.B. Carey.
1985. Streptococci and Aerococci. Pages 154-175 [[1E.H. Lennete, A. Balows, W.J. Hausler,
and Shadomy, eds. Manual of Clinical Microbiology. 4th ed. Amer. Soc~ for Microbiol~,
Washington, D.C.
6. Murray, B.E. 1990. The life and
times of the Enterococcus. Clin. Microbiol. Rev. 3:4~65.
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