ID-AE TECHNICAL INFORMATION
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 overight 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
T he
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.
I t
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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