StrepQUICK™

Cat. no. Z122 StrepQuick™ 25 tests/kit

INTENDED USE

Hardy Diagnostics StrepQuick™ test kit is intended to aid in the identification of gram-positive, catalase-negative cocci based on pyroglutamate aminopeptidase (PYR), leucine aminopeptidase (LAP), and esculin hydrolysis (ESC) activity. This test kit simplifies identification of Enterococcusspp. and group A streptococci (Streptococcus pyogenes).

SUMMARY

Hardy Diagnostics StrepQuick™ is a rapid means of determining three key biochemical reactions for the differentiation and identification of gram-positive, catalase-negative cocci. StrepQuick™ detects activity of three enzymes; pyroglutamate aminopeptidase (PYR), leucine aminopeptidase (LAP), and esculinase (ESC). A positive PYR reaction is characteristic of group A streptococci and Enterococcus spp.(1) Aerococcus viridans and Leuconostoc spp. typically show negative reactions for LAP activity.(1) Enterococcus spp. and group D streptococci are positive for esculin hydrolysis.

PYR is a rapid colorimetric method for the detection of pyroglutamate aminopeptidase activity. L-pyroglutamic acid beta-naphthylamide impregnated into the test card serves as the substrate for the detection of pyroglutamate aminopeptidase. Hydrolysis of the substrate yields beta-naphthylamide which combines with the cinnamaldehyde reagent to form a bright pink to cherry red pigment.

L-leucine-beta-naphthylamide is impregnated in the filter paper in the LAP test circle. This substrate is hydrolyzed by the enzyme leucine aminopeptidase. Like the PYR reaction, released beta-naphthylamide combines with the cinnamaldehyde reagent to form a bright pink to cherry red pigment.

Esculin and ferric ammonium citrate are contained within the third reaction circle. Esculin is hydrolyzed by organisms capable of producing the enzyme esculinase. Esculetin, the end product of the hydrolysis reaction, complexes with ferric ions to produce a light gray to gray color.

FORMULA

Pyroglutamic aminopeptidase (PYR):
L-pyroglutamic acid beta-naphthylamide

Leucine aminopeptidase (LAP):
L-leucine-beta-naphthylamide

Esculin hydrolysis (ESC):
Esculin
Ferric Ammonium Citrate

StrepQuick™ Developer:
p-dimethylaminocinnamaldehyde

* Adjusted and/or supplemented as required to meet performance criteria.

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-8ºC. away from direct light. The test kit should not be used if there are any signs of deterioration, discoloration, contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.

PRECAUTIONS

PROCEDURE

Specimen Collection:

This product is not intended for primary isolation of patient specimens. It should be used only with cultures of isolated organism. This product is used in conjunction with other biochemical or serological tests to identify cultures of isolated organism.

Method of Use:

Note: Perform Gram stain and catalase test prior to inoculating StrepQuick™. Test organisms should be gram-positive cocci that are aerobic or facultatively anaerobic and catalase-negative.

1) Moisten each test circle slightly with a single drop of distilled or deionized water. Do not saturate.

2) Using a sterile plastic loop, pick 2-3 well isolated, 18-24 hour colonies and rub into a small area of the PYR reaction circle so that there is a visible paste.

3) Repeat step 2 for the LAP and ESC test circles.

Note: Take care not to carry over substrate from one circle to the next.

4) After the test organism has been inoculated onto the test circles, allow it to react for ten minutes.

5) After the 10 minute incubation period add one drop of chromogenic developer to the PYR and LAP circles.

6) Immediately after addition of the chromogenic developer, observe for the development of a bright pink or cherry red color in the PYR and LAP circles. The reaction should be read only in the area of the card where the organism was inoculated. A diffuse, light pink color covering the entire test area is not considered positive and should be disregarded. Any color development in the PYR and LAP circles appearing after 1 minute should be disregarded. Color development of orange, yellow or salmon in the PYR circle should be interpreted as a negative reaction.

7) Observe for a light gray to gray color to form in the ESC circle. Any darkening at all should be considered positive. Allow 10-15 minutes for full color development.

INTERPRETATION OF RESULTS

Test Positive Reaction Negative Reaction
PYR Cherry red or bright pink color develops Development of a orange, yellow or salmon color or no change

LAP Cherry red or bright pink color develops No color development*
ESC Light gray to gray color develops Any color other than gray

* Refer to Procedure step 6 above.

Expected reactions of select organisms:(1,5-7)

Organism Expected Reactions
PYR LAP ESC Vancomycin (30ug)** 6.5% NaCl* Hemolysis* Gram Morphology*
Aerococcus viridans + - V S + Alpha Cl
Aerococcus urinae - + V S + Alpha Cl
Enterococcus spp. + + + S/R + Alpha/Gamma Ch
Gemella haemolysans + + - S - Alpha/Gamma D,Cl,Ch,T
Gemella morbillorum + + - S - Alpha/Gamma D,Cl,Ch,T
Leuconostoc spp.*** - - V(+) R V(+) Gamma Ch
Pediococcus spp. - + + R V(-) Alpha Cl,T
Streptococcus pyogenes
(group A)
+ + V(-) S + Beta Ch
Streptococcus agalactiae
(group B)
- + - S V(+) Beta Ch
Streptococcus group D (non-Enterococci) - + + S - Alpha/Beta/Gamma Ch
Streptococcus pneumoniae - + V(-) S - Alpha D
Streptococcus viridans (non-group D) - + V(-) S - Alpha/Gamma Ch

V = Variable results
V(+) = Variable reactions where the majority of isolates are positive (>80%)
V(-) = Variable reactions where the majority of isolates are negative (>80%)(9)
S = Sensitive
R = Resistant
Cl = Cluster
Ch = Chain
D = Diplococci
T = Tetrad

* These tests are not included in the StrepQuick™ card, but are included to aid in identification.

** Results are included to help aid in identification, as this test separates Leuconostoc and Pediococcus spp. from the majority of listed species.

*** Weissella paramesenteroides ATCC® 33313 (previously known as Leuconostoc paramesenteroides) is consistently positive for LAP.(10)

LIMITATIONS

A Gram stain and catalase test should be performed to confirm the presence of gram-positive, catalase-negative cocci.

Only pure cultures or isolated colonies of gram-positive, catalase-negative cocci should be tested; mixed cultures may give false-positive reactions.

Staphylococcus, Aerococcus, most Corynebacterium haemolyticum, as well as some Enterobacteriaceae and other gram-negative bacilli, are PYR positive.(1,9)

For PYR and LAP tests, after addition of the developer, color development after 1 minute should be disregarded.(5)

For PYR and LAP test, after the addition of the developer, the development of a diffuse, light pink color covering the entire test area is not considered positive and should be disregarded.

False-negative results can occur for any of the reactions if inadequate inoculum is used.(5)

Bile tolerance is not included in the esculin hydrolysis test, so results obtained with StrepQuick™ may not correlate with results obtained from Bile Esculin media.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, other culture media, swabs, applicator sticks, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms
PYR Reaction
LAP Reaction
ESC Reaction
Streptococcus agalactiae
ATCC® 12386
Negative; no color change Positive; cherry red or bright pink color change Negative; no color change
Enterococcus faecalis
ATCC® 29212
Positive; cherry red or bright pink color change Positive; cherry red or bright pink color change Positive; light gray to gray color change
Aerococcus viridans
ATCC® 11563
Positive; cherry red or bright pink color change Negative; no color change directly on or in the adjacent area of the sample inoculum Negative; no color change

User Quality Control

E. faecalis Reactions

Showing StrepQuick™ (Cat. no. Z122) reactions for Enterococcus faecalis (ATCC® 12386). Growth from 18-24 hour colonies were rubbed onto each reaction circle leaving a visible paste. After a 10 minute room temperature incubation, one drop of chromogenic developer was added to each circle. Showing: PYR Positive (cherry red or bright pink color change); LAP Positive (cherry red or bright pink color change); ESC Positive (light gray to gray color change).

REFERENCES

1. Versalovic, J., et al. Manual of Clinical Microbiology. American Society for Microbiology, Washington, D.C.

2. Tille, P., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.

3. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.

4. Koneman, E.W., et al. Color Atlas and Textbook of Diagnostic Microbiology, J.B. Lippincott Company, Philadelphia, PA.

5. MacFaddin, J.F. Biochemical Tests for Identification of Medical Bacteria,, Lipincott Williams & Wilkins, Philadelphia, PA.

6. Facklam, R., et al. 1995. Evaluation of three disk tests for identification of enterococci, leuconostocs, and pediococci. J.Clin. Microbiol.; 33:885-887.

7. Bosley, G.S., et al. 1983. Rapid identification of enterococci. J. Clin. Microbiol.; 18:1275-1277.

8. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.

9. Holt, J.G., et al. Bergey's Manual of Determinative Bacteriology, Williams & Wilkins, Baltimore, MD.

10. Ruoff, K.L., D.R. Kuritzkes, J.S. Wolfson and M.J. Ferraro. 1988. J. Clin. Microbiol.; 26:2064-2068.


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031016gr