PRINCIPLE

The tests used in the RapID STR System are based upon the microbial degradation of specific substrates detected by various indicator systems. The reactions employed are a combination of conventional tests and single-substrate chromogenic tests and are described below in Table 1.

RapID STR System

INTENDED USE
REMELs RapID STR System is a qualitative micromethod employing conventional and chromogenic substrates for the identification of medically important streptococci and related organisms which have been isolated from human clinical specimens. The RapID STR System is intended to aid in the identification of Lancefield groups A, B, C, D, and G streptococci, viridans streptococci and Streptococcus pneumoniae, Enterococcus spp., Aerococcus spp., Gemella spp., Leuconostoc spp., Pediococcus spp., 1-10 A complete listing Weisella confusus, and Listeria monocytogenes. of the organisms addressed by the RapID STR System is given in the RapID STR Differential Chart.

REAGENTS*
RapID STR Reagent (provided with kit) (15 ml/Btl.) Reactive Ingredient per Liter: o-Dianisidine ..........................................................................0.9 ml RapID Inoculation Fluid (REMEL # 8325102 supplied separately) (1 ml/Tube) KCl .........................................................................................6.0 g CaCl2......................................................................................0.5 g Demineralized Water .......................................................1000.0 ml
*Adjusted as required to meet performance standards.

SUMMARY AND EXPLANATION

The RapID STR System is comprised of (1) RapID STR Panels and (2) RapID STR Reagent. Each RapID STR Panel has several reaction cavities molded into the periphery of a plastic disposable tray. Reaction cavities contain dehydrated reactants and the tray allows the simultaneous inoculation of each cavity with a predetermined amount of inoculum. A suspension of the test organism in RapID Inoculation Fluid is used as the inoculum which rehydrates and initiates test reactions. After incubation of the panel, each test cavity is examined for reactivity by noting the development of a color. In some cases, reagents must be added to the test cavities to provide a color change. The resulting pattern of positive and negative test scores is used as the basis for identification of the test isolate by comparison of test results to reactivity patterns stored in a database or through the use of a computer-generated Code Compendium.

PRECAUTIONS
This product is For In Vitro Diagnostic Use and should be used by properly trained individuals. Precautions should be taken against the dangers of microbiological hazards by properly sterilizing specimens, containers, media, and test panels after their use. Directions should be read and followed carefully. Caution! 1. RapID STR Reagent is toxic and may cause harm to the environment. Harmful by inhalation, contact with skin or eyes, or if swallowed. May cause cancer, impair fertility, or cause harm to unborn child. Danger of serious irreversible effects. 2. Refer to Material Safety Data Sheet for detailed information on reagent chemicals.

Table 1. Principles and Components of the RapID STR System

Cavity # Test Code Reactive Ingredient Quantity Principle Hydrolysis of arginine releases basic products which raise the pH and change the indicator. Hydrolysis of the glucoside releases esculetin which reacts with ferric ion forming a black compound. Utilization of the carbohydrate substrate produces acidic products which lower the pH and change the indicator. Bibliography # Before Reagent Addition 1 2 3 4 5 6 7 8 9 10 7 8 9 10 ARG ESC MNL SBL RAF INU GAL GLU NAG PO4 TYR HPR LYS PYR L-arginine Esculin Mannitol Sorbitol Raffinose Inulin p-Nitrophenyl-,D-galactoside p-Nitrophenyl-,D-glucoside p-Nitrophenyl-n-acetyl,D-glucosaminide p-Nitrophenyl phosphate Tyrosine -naphthylamide Hydroxyproline -naphthylamide Lysine -naphthylamide Pyrrolidine -naphthylamide 2.0% 0.5% 1.5% 1.5% 1.2% 1.5% 0.1% 0.1% 0.1% 0.2% 0.05% 0.08% 0.08% 0.1% Hydrolysis of the arylamide releases free -naphthylamine which is detected with RapID STR Reagent. 15, 17-19 Hydrolysis of the colorless p-nitrophenyl-substituted glycoside or phosphoester releases yellow p-nitrophenol. 14-16 1, 2, 11 11-13 12

After Reagent Addition

STORAGE
The RapID STR System should be stored in its original container at 2-8C until used. Allow product to come to room temperature before use. DO NOT interchange reagents among different RapID systems. Remove only the number of panels necessary for testing. Reseal the plastic pouch and promptly return to 2-8C. Panels must be used the same day they are removed from storage. RapID Inoculation Fluid should be stored in its original container at room temperature (20-25C) until used.

4.

Suspensions should be mixed thoroughly and vortexed if required. Suspensions should be used within 15 minutes of preparation.

An agar plate may inoculated for purity and any additional testing that may be required using a loopful of the test suspension from the inoculation fluid tube. Incubate the plate for 18-24 hours at 35-37C. Peel back the lid of the panel over the inoculation port by pulling the tab marked Peel to Inoculate up and to the left. Using a pipette, gently transfer the entire contents of the Inoculation Fluid tube into the upper right-hand corner of the panel. Reseal the inoculation port of the panel by pressing the peel-back tab back in place. After adding the test suspension, and while keeping the panel on a level surface, tilt the panel back away from the reaction cavities at approximately a 45-degree angle (see below).
Reaction Cavities Inoculating Trough (Back of Tray)

PRODUCT DETERIORATION
This product should not be used if (1) the color of the reagent has changed, (2) the expiration date has passed, (3) the plastic tray is broken or the lid is compromised, or (4) there are other signs of deterioration.

Inoculation of RapID STR Panels

1. 2.

SPECIMEN COLLECTION, STORAGE, AND TRANSPORTATION

Specimens should be collected and handled following recommended 20,21 guidelines.

3.

MATERIALS SUPPLIED
(1) 20 RapID STR Panels, (2) 20 report forms, (3) RapID STR Reagent (one plastic dropper-bottle containing reagent sufficient for 20 panels), (4) 2 chipboard incubation trays, (5) Instructions for use.

MATERIALS REQUIRED BUT NOT SUPPLIED

(1) Loop sterilization device, (2) Inoculating loop, swab, collection containers, (3) Incubators, alternative environmental systems, (4) Supplemental media, (5) Quality control organisms, (6) Gram stain reagents, (7) Microscope slides, (8) Cotton swabs, (9) RapID Inoculation Fluid - 1 ml (REMEL # 8325102), (10) McFarland #1 turbidity standard or equivalent (REMEL #20411), (11) Pipettes, (12) RapID STR Code Compendium (REMEL #8323003) or ERIC (Electronic RapID Compendium, REMEL #8323600). 4.

While tilted back, gently rock the panel from side to side to evenly distribute the inoculum along the rear baffles as illustrated below.

PROCEDURE Inoculum Preparation

1. Test organisms must be grown in pure culture, examined by Gram stain, and tested for hemolysis prior to use in the system. Note: Hemolysis is enhanced by incubation anaerobically or in 5-7% CO2. 2. Test organisms may be removed from nonselective agar growth media. The following types of media are recommended: Tryptic Soy Agar (TSA) with or without 5% Sheep Blood; Nutrient Agar; Chocolate Agar. Notes: Some media containing or supplemented with mono- or disaccharides are not recommended since they may suppress glycolytic activity and reduce test selectivity. Plates used for inoculum preparation should preferably be 18-24 hours old. Slow growing isolates may be tested using 48-hour plates. The use of media other than those recommended may compromise test performance. 3. Using a cotton swab or inoculating loop, suspend sufficient growth from the agar plate culture in RapID Inoculation Fluid (1 ml) to achieve a visual turbidity equal to a #1 McFarland turbidity standard or equivalent. Notes: Suspensions significantly less turbid than a #1 McFarland standard will result in aberrant reactions. Bacterial suspensions that are slightly more turbid than a #1 McFarland standard will not affect test performance and are recommended for stock cultures and quality control strains. However, suspensions prepared with a turbidity far greater than a #1 McFarland standard will compromise test performance. 5. While maintaining a level, horizontal position (best achieved by using the bench top against the reaction cavity bottoms), slowly tilt the panel forward toward the reaction cavities until the inoculum flows along the baffles into the reaction cavities (see below). This should evacuate all of the inoculum from the rear portion of the panel. Note: If the panel is tilted too quickly, air may be trapped at the test cavity junction, restricting fluid movement.

Reaction Cavities (Front of Tray)

6.

Return the panel to a level position. If necessary, gently tap the panel on the bench top to remove any air trapped in the cavities. Notes: Examine the test cavities, which should appear bubble-free and uniformly filled. Slight irregularities in test cavity fills are acceptable and will not affect test performance. If the panel is grossly misfilled, a new panel should be inoculated and the misfilled panel discarded. Complete the inoculation of each panel receiving inoculation fluid before inoculating additional panels. Do not allow the inoculum to rest in the back portion of the panel for prolonged periods without completing the procedure.

Incubation of RapID STR Panels

Incubate inoculated panels at 35-37C in a non-CO2 incubator for 4 hours. For ease of handling, panels may be incubated in the chipboard incubation trays provided with the kit.

4.

5.

Scoring of RapID STR Panels

RapID STR panels contain 10 reaction cavities that, in addition to hemolysis, provide 15 test scores. Test cavities 7 through 10 are bifunctional, containing two separate tests in the same cavity. Bifunctional tests are first scored before the addition of reagent providing the first test result, and then the same cavity is scored again after the addition of reagent to provide the second test result. The bifunctional test cavities that require RapID STR Reagent are indicated with the first test above the bar and the second test below the bar. RapID STR Panel Test Location
Cavity # 1 2 3 4 5 6 INU 7 GAL TYR 8 GLU HPR 9 NAG LYS 10 PO4 PYR

6.

Allow at least 30 seconds but no more than 3 minutes for color development. Read and score cavities 7 through 10. Record the scores in the appropriate boxes of the report form using the test codes below the bar for bifunctional tests. Record the hemolysis reaction for the test isolate in the box provided on the report form. The hemolysis reaction serves as th the 15 test and should be scored as positive for beta-hemolytic isolates only. Alpha and gamma hemolysis should be scored as negative. Reference the microcode obtained on the report form in the RapID STR Code Compendium or ERIC (Electronic RapID Compendium) for the identification.

RESULTS AND RANGE OF EXPECTED VALUES

The RapID STR Differential Chart illustrates the expected results for the RapID STR System. Differential Chart results are expressed as a series of positive percentages for each system test. This information statistically supports the use of each test and provides the basis, through numerical coding of digital test results, for a probabilistic approach to the identification of the test isolate. Identifications are made using individual test scores from RapID STR panels in conjunction with other laboratory information (i.e. Gram stain, hemolysis, colonial morphology, growth on differential or selective media) to produce a pattern that statistically resembles known reactivity for taxa recorded in the RapID System database. These patterns are compared through the use of the RapID STR Differential Chart, or by derivation of a microcode and the use of the RapID STR Code Compendium or ERIC (Electronic RapID Compendium).

Test Code ARG ESC MNL SBL RAF

1. 2.

3.

While firmly holding the RapID STR panel on the benchtop, peel off the label lid over the reaction cavities by pulling the lower right hand tab up and to the left. Without the addition of the reagent, read and score cavities 1 (ARG) through 10 (PO4) from left to right using the interpretation guide presented in Table 2. Record the test scores in the appropriate boxes on the report form using the test code above the bar for bifunctional tests. Add 2 drops of RapID STR Reagent to cavities 7 (TYR) through 10 (PYR).

Table 2. Interpretation of RapID STR System Tests*

Cavity # Test Code Reagent Positive Before Reagent Addition 1 2 3 4 5 6 7 8 9 10 ARG ESC MNL SBL RAF INU GAL GLU NAG PO4 None Yellow Clear, tan, or very pale yellow Only the development of a significant yellow color should be scored as positive. Very pale or questionable colors should be scored as negative. None Yellow, yelloworange, or orange Red Any significant change from red should be scored as positive. None Yellow or yelloworange Red or orange Any significant yellow or yellow-orange color should be scored as positive. None None Red or dark orange Black Yellow or yelloworange Clear, tan, or light brown deposit Only a red or dark orange color should be scored as positive. Any development of a dark black color should be scored as positive. Reaction Negative Comments

After Reagent Addition 7 8 9 10 TYR HPR LYS PYR RapID STR Reagent RapID STR Reagent Light purple or purple Very dark purple Clear, tan, or yellow Clear, tan, or light to medium purple Any shade of purple should be scored as positive. Only the development of a distinct, very dark purple color should be scored as positive. Very pale or questionable colors should be scored as negative.

*NOTE: Panels should be read by looking down through the reaction cavities against a white background.

RapID STR Differential Chart

Organism
Beta-hemolytic Streptococci

ARG 99 99 99 0 48 96 97 96 99 0 0 0 0 0 98 92 97 95 9 2 0 98 17 0 0 0 0 0 0 93 2 0 95

ESC 5 2 8 96 99 99 97 97 99 99 96 97 98 99 0 98 89 96 2 94 91 79 0 61 0 99 3 88 98 38 96 1 98

MNL 17 1 4 97 98 0 95 96 95 99 94 98 0 88 0 17 0 6 0 94 0 0 0 84 1 29 0 18 1 1 0 0 0

SBL 1 1 3 93 81 0 90 0 0 94 91 0 0 0 0 0 4 2 4 92 0 4 1 70 1 0 0 0 0 0 0 0 0

RAF 1 1 0 0 94 11 1 22 95 99 99 96 96 6 0 32 2 26 59 86 90 26 96 18 0 2 90 86 0 0 0 71 0

INU 0 0 0 0 66 0 0 0 94 0 0 77 0 0 0 0 0 0 0 82 40 66 0 0 0 0 0 0 0 0 0 69 0

GAL 9 2 1 33 95 69 1 76 98 7 90 99 98 0 92 80 2 18 38 94 0 23 86 17 7 2 99 96 0 0 0 93 0

GLU 96 96 98 1 4 4 99 1 5 0 0 99 99 9 0 22 88 99 96 92 96 16 99 98 9 99 16 80 95 0 0 93 0

NAG 72 0 12 1 96 30 95 92 90 1 3 5 1 85 0 0 0 99 92 0 1 74 39 0 1 0 0 4 98 11 0 84 2

PO4 99 94 99 0 9 0 8 1 8 1 6 1 1 55 12 95 93 95 79 1 10 79 96 18 64 0 0 0 21 0 0 0 0

TYR 90 1 96 12 2 2 17 1 9 1 0 1 48 0 15 93 84 92 90 12 11 94 89 7 19 0 0 2 48 4 0 79 1

HPR 1 2 1 56 2 86 9 36 12 14 26 1 1 12 12 3 1 5 1 1 1 0 1 19 74 0 0 0 0 0 0 5 0

LYS 99 92 99 9 90 33 90 90 89 6 2 95 98 81 98 96 98 99 96 92 98 95 96 87 82 29 3 4 56 80 18 96 0

PYR 99 0 0 99 99 99 99 99 99 99 99 1 0 0 0 0 0 1 0 0 0 0 0 90 42 0 0 0 0 0 0 0 0

HEM 97 92 99 0 0 6 7 0 0 0 0 0 0 0 0 28 46 3 0 4 0 0 0 0 0 0 0 0 58 0 0 0 0

Group A (S. pyogenes) Group B (S. agalactiae) Group C/G E. avium E. casseliflavus / mundtii

Enterococci
Group D Streptococci

E. durans / hirae E. faecalis E. faecium E. gallinarum E. malodoratus E. raffinosus S. bovis S. bovis var S. equins S. acidominimus S. anginosus

Viridans Streptococci Other

S. constelatus S. intermedius S. mitis S. mutans S. salivarius / vestibularis S. sanguis / gordonii S. sanguis II Aerococcus spp. Gemella morbillorum Leuconostoc citreum L. lactis L. mesenteroides group Listeria monocytogenes Pediococcus acidilactici P. pentosaceus Streptococcus pneumoniae Weisella confusus

QUALITY CONTROL
All lot numbers of the RapID STR System have been tested using the following quality control organisms and have been found to be acceptable. Testing of control organisms should be performed in accordance with established laboratory quality control procedures. If aberrant quality control results are noted, patient results should not be reported. Table 3 lists expected results for the selected battery of test organisms. Notes RapID STR Reagent quality control is accomplished by obtaining the expected reactions for tests requiring the addition of the reagent (cavities 7-10). Organisms which have been repeatedly transferred on agar media for prolonged periods may provide aberrant results.

Quality control strains should be stored frozen or lyophilized. Prior to use, quality control strains should be transferred 2-3 times from storage on an agar medium that is recommended for use with the RapID STR System. Formulations, additives, and ingredients of culture media vary from manufacturer to manufacturer and may vary from batch to batch. As a result, culture media may influence constitutive enzymatic activity of designated quality control strains. If quality control strain results differ from the patterns indicated, a subculture onto medium from a different batch or from another manufacturer will often resolve quality control discrepancies.

Table 3. Quality Control Chart for RapID STR Panels

Organism Enterococcus faecalis ATCC 29212 Enterococcus durans ATCC 11576 or 49479 Streptococcus bovis ATCC 9809 Streptococcus pyogenes ATCC 19615 ARG + + + ESC + + + MNL + + SBL + RAF + INU + GAL + + GLU + + (+) NAG + + V PO4 () + TYR V V + HPR +* LYS + () + + PYR + + +

+, positive; , negative; V, variable; (), usually negative; (+), usually positive

*Note: Enterococcus durans may yield a very weak positive reaction in the HPR cavity. Gemella morbillorum ATCC 27824 can also be used as a quality control strain for the HPR reaction. However, E. durans should still be used to quality control the GLU and LYS cavities.

LIMITATIONS
1. The use of the RapID STR System and the interpretation of results require a competent laboratorian who is trained in general microbiological methods and who should judiciously make use of knowledge, experience, specimen information, and other pertinent procedures before reporting the isolate identity obtained using the RapID STR System. Characteristics such as Gram stain reaction, hemolysis, and cellular morphology must be considered when using the RapID STR System. The RapID STR System must be used with pure cultures of test organisms. The use of mixed microbial populations or direct testing of clinical material without culture will result in aberrant results. The RapID STR System is designed for use with the taxa listed in the RapID STR Differential Chart. The use of organisms not specifically listed may lead to misidentifications. Expected values listed for RapID STR System tests may differ from conventional test results or previously reported information. The accuracy of the RapID STR System is based upon the statistical use of a multiplicity of specially designed tests and an exclusive, proprietary database. The use of any single test found in the RapID STR System to establish the identification of a test isolate is subject to the error inherent in that test alone.

13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35.

2. 3.

4. 5. 6.

PERFORMANCE CHARACTERISTICS
The RapID STR System performance characteristics have been established by laboratory testing of reference and stock cultures at REMEL and by clinical evaluations using fresh clinical and stock 22-25 isolates.

BIBLIOGRAPHY
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. Lennette, E.H., A. Balows, W.J. Hausler, Jr., and J.P. Truant. 1980. Manual of Clinical Microbiology. 3rd ed., p. 88-110. ASM, Washington, D.C. Balows, A., W.J. Hausler, Jr., K.L. Herrmann, H.D. Isenberg, and H.J. Shadomy. 1991. Manual of Clinical Microbiology. 5th ed., p. 238-257. ASM, Washington, D.C. Poole, P.M. and G. Wilson. 1976. J. Clin. Microbiol. 29:740-745. Facklam, R.R. 1977. J. Clin. Microbiol. 5:184-201. Ruoff, K.L. and L.J. Kunz. 1982. J. Clin. Microbiol. 15:920-925. Berlutti, F., M.C. Thaller, S. Schippa, F. Pantanella, and R. Pompei. 1993. Int. J. Syst. Bacteriol. 43:63-68. Collins, M.D., D. Jones, J.A.E. Farrow, R. Klipper-Balz, and K.H. Schleifer. 1984. Int. J. Syst. Bacteriol. 34:220-223. Facklam, R.R. and M.D. Moody. 1970. Appl. Microbiol. 20:245-250. Holt J.G., N.R. Krieg, P.H.A. Sneath, J.T. Staley, and S.T. Williams. 1994. Bergeys Manual of Determinative Bacteriology, 9th ed. Williams and Wilkins, Baltimore, MD. Isenberg, H.D., E.M. Veilozzi, J. Shapiro, and L.G. Rubin. 1988. J. Clin. Microbiol 26:479-483. Blazevic, D.J. and G.M. Ederer. 1975. Principles of Biochemical Tests in Diagnostic Microbiology. John Wiley & Sons, New York, NY. Facklam, R.R. 1976. Crit. Rev. Clin. Lab. Sci. 6:287-316.

Gross, K.D, N.P. Houghton, and L.B. Senterfit. 1975. J. Clin. Microbiol. 1:54-60. Bridge, P.D. and P.H.A. Sneath. 1983. J. Gen. Microbiol. 129:565-597. Guilbault, G.G. 1970. Enzymatic Methods of Analysis. p. 43-51. Pergamon Press, New York, NY. Nord, C.E., A.A. Lindberg, and A. Dahlback. 1975. Med. Microbiol. Immunol. 161:231-238. Facklam, R.R., L.G. Thacker, B. Fox, and L.A. Eriquez. 1982. J. Clin. Microbiol 15:987-990. Norris, J.R. and D.W. Ribbons. 1976. Methods in Microbiology. Vol. 9, p. 1-14. Academic Press, New York, NY. Westley, J.R., P.J. Anderson, V.A. Close, B. Halpern, and E.M. Lederberg. 1967. Appl. Microbiol. 15-822-825. Murray, P.R., E.J. Baron, J.H. Jorgensen, M.A. Pfaller, and R.H. Yolken. 2003. Manual of Clinical Microbiology. 8th ed., Vol. 1. ASM, Washington, D.C. Forbes, B.A., D.F. Sahm, and A.S. Weissfeld. 2002. Bailey and Scotts Diagnostic Microbiology. 11th ed. Mosby. St. Louis, MO. Appelbaum, P.C., M.R. Jacobs, W.M. Palko, E.E. Frauenhoffer, and A. Duffett. 1986. J. Clin. Microbiol. 23:843-846. Arduino, M.J., S.K. McAllister, S.M. Aguero, and L.A. Bland. 1994. Abstract C-138. Abstracts of the 94th General Meeting of the American Society for Microbiology. ASM, Washington, D.C. Hinnenbusch, C.J., D.M. Nikolai, and D.A. Bruckner. 1991. Am. J. Clin. Pathol. 96:459-463. You. M.S. and R.R. Facklam. 1986. J. Clin. Microbiol. 24:607-611. Ball, L.C. and M.T Parker. 1979. J. Hyg. 82:63-78. Facklam, R.R. 1972. Appl. Microbiol. 23:1131-1139. Facklam, R.R., D. Hollis, and M.D. Collins. 1989. J. Clin. Microbiol. 27:724-730. Farrow, J.A.E., D. Jones, B.A. Phillips, and M.D. Collins. 1983. J. Gen. Microbiol. 129:1425-1432. Hardy, M.A., H.P. Dalton, and M.J. Allison. 1978. J. Clin. Microbiol. 8:534-544. Janda, W.M. 1994. Clin. Microbiol. Newsl. 16:21. Lee, M.R. and G.M Ederer. 1977. J. Clin. Microbiol. 5:290-292. Ruoff, K.L. 1994. Clin. Microbiol. Newsl. 16:20. Schleifer, K.H. and R. Klipper-Balz. 1984. Int. J. Syst. Bacteriol. 34:31-34. Welbourn, P.P., W. Keith Hadley, E. Newbraun, and D.M. Yajko. 1983. Int. J. Syst. Bacteriol. 33:293-299.

PACKAGING
REF 8311003, RapID STR System................................ 20 Tests/Kit Symbol Legend REF IVD
Catalog Number In Vitro Diagnostic Medical Device Consult Instructions for Use (IFU) Temperature Limitation (Storage Temp.)

LOT

Batch Code (Lot Number) Use By (Expiration Date)

RapID and ERIC are trademarks of REMEL Inc. ATCC is a registered trademark of American Type Culture Collection. IFU 8311003, Revised April 30, 2003 Printed in U.S.A.

12076 Santa Fe Drive, Lenexa, KS 66215, USA General Information: (800) 255-6730 Technical Service: (800) 447-3641 Order Entry: (800) 447-3635 Local/International Phone: (913) 888-0939 International Fax: (913) 895-4128 Website: www.remel.com Email: remel@remel.com