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

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.

 

Gibson Laboratories, Inc.
 

© Lakewood Biochemical Co., Inc. Dallas, TX 75214


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