STREP B CARROT BROTH™ KIT

Cat. no. Z140 Strep B Carrot Broth™ Kit 20 tests/kit
Provided Materials within Kit
Z140A - Strep B Carrot Broth™, 13x100mm Tube, 4ml
Z140B - Strep B Carrot Broth™ Tiles, 1/4" x 1/2"
20 tubes
20 tiles/jar
Cat. no. Z144BX







Strep B Carrot Broth™ Kit 100 tests/kit
Provided Materials within Kit
Z144A - Strep B Carrot Broth™, 12x80mm Tube, 4ml
Z144B - Strep B Carrot Broth™ Tiles, 1/4" x 1/2"

100 tubes
100 tiles/jar

Cat. no. Z146BX Strep B Carrot Broth™ Kit 100 tests/kit
Provided Materials within Kit
Z146A - Strep B Carrot Broth™, 16x100mm Tube, 6ml
Z146B - Strep B Carrot Broth™ Tiles, 1/4" x 1/2"

100 tubes
100 tiles/jar

Replacement item sold separately:
Cat. no. Z140T Strep B Carrot Broth™ Tiles (only), 1/4" x 1/2" 20 tiles/jar

INTENDED USE

Hardy Diagnostics Strep B Carrot Broth™ Kit is used for the selective enrichment and screening for beta-hemolytic group B Streptococci (Streptococcus agalactiae) from clinical specimens, especially for pregnant women.

SUMMARY

Approximately 10-35% of women are asymptomatic carriers of group B streptococci (GBS) in the genital and gastrointestinal tracts.(1) GBS remains a leading cause of serious illness and death in newborn populations and, therefore, the detection of GBS in the vaginal-anorectal area is critical to the prevention of neonatal GBS disease. Several surveys have been conducted that show the incidence of neonatal sepsis and meningitis due to GBS is currently 0.5 - 3 cases per 1,000 live births, although there are substantial geographical and racial differences.(2) The case-fatality ratios are now declining due to prompt recognition and proper treatment.(3)

The Centers for Disease Control and Prevention (CDC) recommends the screening of all pregnant women for vaginal and rectal GBS colonization between 35 and 37 weeks of gestation using an enrichment broth followed by subculture to a Blood Agar (Cat. no. A10) plate or other appropriate media. The use of a selective enrichment broth that incorporates chromogenic pigments, such as Strep B Carrot Broth™, has recently been included in the CDC's Prevention of Perinatal Group B Streptococcal Disease.(4) Strep B Carrot Broth™ demonstrates increased sensitivity and specificity, reduced incubation time, and reduced need for additional plated media.(5-9,23-26)

The production of orange, red, or brick red pigment is a unique characteristic of hemolytic GBS due to reaction with substrates such as starch, peptone, serum, and folate pathway inhibitors. These components serve as the basis for culture media used to detect and identify these organisms. Since the original description of starch serum agar by Islam in 1977, there have been many improvements to the original formula.(10) Presently, Granada Medium is a reliable plate method for the detection and identification of beta-hemolytic GBS.(8-10, 21) GBS detection with Granada media is only possible with beta-hemolytic colonies, thus providing evidence of a direct genetic linkage between pigment production on Granada media and hemolysin production. Beta-hemolytic, pigment producing GBS occurs with 95.3 to 99.5% of all GBS strains isolated from clinical specimens.(18-20) Non-hemolytic strains of GBS may be further tested on Hardy's GBS Detect™ Agar plates (A300).

Hardy Diagnostics Strep B Carrot Broth™ Kit utilizes the Granada Medium reaction and contains the necessary components for pigment detection of beta-hemolytic GBS, including peptone, starch and buffers which are supplied in the Strep B Carrot Broth™ (Z140A, Z144A or Z146A). In addition, supplemental enrichment factors necessary for GBS growth and pigment production are provided on the Strep B Carrot Broth™ Tiles (Z140B, Z144B, Z146B or Z140T). This modification of Granada Broth (the separation of the supplements) increases the shelf life of the product and ensures well-defined and reliable pigment production in the presence of GBS. The advantage to this medium is that it is a single tube system that will produce positive results in as little as six hours and does not require subculturing to a blood agar plate, unless the results are negative. Enrichment broth procedures are known to be more sensitive than plate methods in their ability to detect GBS colonization. Strep B Carrot Broth™ is used to detect beta-hemolytic GBS without the need for further testing and, as an enrichment broth, provides greater sensitivity. A multi-center study conducted by Schreckenberger et al. in 2005 found the sensitivity of Strep B Carrot Broth™ to exceed that of the LIM Broth method (97% vs. 93%).(15) Other studies have shown that Strep B Carrot Broth™ can be successfully used in conjunction with PNA FISH™ or broth-enhanced PCR procedures.(21,22)

A companion product for Strep B Carrot Broth™ is also available that will assist in the isolation and identification of the non-hemolytic strains of GBS (which will comprise 0.5 to 4.7% of the total GBS isolates).(18-20) The GBS Detect™ (Cat. no. A300) plate is used when subculturing the negative Strep B Carrot Broth™ cultures. It contains selective agents and hemolysis enhancers to cause non-hemolytic strains to appear hemolytic, so they can be easily recognized and identified within 24 hours.

FORMULA

Ingredients per liter of deionized water:*

Strep B Carrot Broth™ (Z140A, Z144A, Z146A):
Peptone 25.0gm
Soluble Starch 20.0gm
Selective Agents 12.2gm
Morpholinepropanesulfonic Acid (MOPS) 11.0gm
Disodium Phosphate 8.5gm
Dextrose 2.5gm
Sodium Pyruvate 1.0gm
Magnesium Sulfate 20.0mg

Final pH 7.4 +/- 0.1 at 25ºC.

Strep B Carrot Broth™ Tiles (Z140B, Z140T, Z144B, or Z146B):
Growth and pigment promoting factors

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

STORAGE AND SHELF LIFE

Storage: Product is temperature sensitive. Upon receipt, store media at 2-8ºC. and protect from excessive heat, moisture, and freezing. For best performance, always store media in a refrigerated atmosphere.

Product is extremely light sensitive: protect against damage from excessive illumination and store away from any direct light source.

Do not use media after the expiration date. Sensitivity is not optimal after expiration date or if the product has been stored inadequately. The expiration date applies to the product in its intact packaging when stored as directed.

PRECAUTIONS

PROCEDURE

A white precipitate may be present in the tube. Prior to inoculation, invert the tube once to resuspend.

1. Collect a vaginal and/or rectal specimen (or other suspect specimen) following the appropriate guidelines for swab sample collection.

2. Submit the specimen to the laboratory in Amies, Stuart's or other appropriate transport media without delay. If transport time is to be more than 24 hours, then the specimen must be kept at 2 to 8ºC.(14)

3. Using sterile forceps, aseptically drop one Strep B Carrot Broth™ Tile (Z140B, Z144B, Z146B, or Z140T) into one tube of Strep B Carrot Broth™ (Z140A, Z144A, or Z146A). Ensure tile remains submerged within the broth. For optimum performance, tiles should be added to the tube at the time of inoculation. If inoculating a series of tubes, it is permissible to add the tiles to all of the tubes in the series up to 5 hours before inoculating the tubes with patient specimens. Please see "TILE INOCULATION STUDY" section.

4. Insert the specimen swab into the Strep B Carrot Broth™ tube. If using a gel-based transport medium, rotate the swab in the broth to emulsify the gel in the broth. Do not shake, agitate, or vortex. Carefully break the swab shaft, leaving the swab in the tube. Replace the tube cap and screw down tightly. It is important that the caps be tightly sealed in order to create the necessary anaerobic conditions at the bottom of the tube.

5. Incubate the inoculated Strep B Carrot Broth™ tube with swab and tile for 6 to 24 hours at 35ºC.

6. Examine tubes for an orange to red color change and/or spots typical of group B streptococci.
See "Interpretation of Results" section below.

7. If no orange color (including pale peach) is present, subculture the specimen to a Blood Agar (Cat. no. A10), Selective Strep Agar (Cat. no. A70), or preferably a GBS Detect™ (Cat. no. A300) plate. Examine the Blood Agar and Selective Strep Agar plates for hemolytic colonies and/or non-hemolytic colonies typical of group B streptococci. Perform group specific latex testing on colonies suspicious of being GBS. Examine GBS Detect™ plates for colonies with large transparent zones of hemolysis with a soft edge. Non-hemolytic GBS will appear beta-hemolytic on this medium due to its hemolysis enhancing supplements.

For Copan WASP users, results show no adverse events if incubation of Strep B Carrot Broth™ tubes occurs without the addition of the transport swab, provided users swirl swabs in the broth for no less than three seconds to ensure appropriate inoculation from the specimen. Alternatively, 30µl of the transport medium from a flocked swab may be aliquoted to the Strep B Carrot Broth™ tube and incubated as appropriate without issue. Please see the “WASP COMPATIBILITY STUDY” below for more information.

INTERPRETATION OF RESULTS

Growth of beta-hemolytic group B Streptococci in Strep B Carrot Broth™ results in the development of an orange to red pigment within 6 to 24 hours. The color will be most intense in the lower portion of the tube. Visualization of any orange to red pigment in the Strep B Carrot Broth™ is indicative of the presence of beta-hemolytic group B Streptococci in the specimen.

In cases where the GBS count is low in the specimen, development of small orange to red spots on the swab or at the bottom of the tube can be observed rather than the entire tube turning orange-red. These should also be considered a positive result for GBS.

LIMITATIONS

Although rare, a small percentage of GBS may not produce beta-hemolysis. GBS detection with the Strep B Carrot Broth™ Kit is only possible with beta-hemolytic colonies. There is evidence of a direct genetic linkage between pigment production using the Strep B Carrot Broth™ and hemolysin production. Beta-hemolytic, pigment producing GBS occurs with 95.3 to 99.5% of all GBS strains isolated from clinical specimens.(18-20) For this reason, do not use S. agalactiae ATCC®13813 for quality control purposes because it will not produce the characteristic orange pigment.

It is recommended to subculture negative Strep B Carrot Broth™ tubes to a GBS Detect™ (Cat. no. A300) plate for simplified detection of rare cases of non-hemolytic GBS. This plate will cause non-hemolytic strains to appear hemolytic, thus making them more easily recognized. Further testing may be conducted by using the StrepPro™ B Grouping Latex (Cat. no. PL032HD).

Since positive reactions can appear on the swab tip, the use of swabs containing charcoal are not suitable.

When using gel-based transport media, emulsify by mixing the swab in the broth to ensure that organisms are not trapped in the gel. Do not vortex.

Failure to place the Strep B Carrot Broth™ tile into the Strep B Carrot Broth™ tube before incubation will prevent color development and will produce erroneous results.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as swabs, transport media, other culture media, forceps, and incubators, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Streptococcus agalactiae**
ATCC® 12386
A 6-24hr 35°C Aerobic Growth; bright orange to red color change
Streptococcus agalactiae
clinical strain
A 18-24hr 35°C Aerobic Growth; light orange to red color change
Streptococcus pyogenes
ATCC® 19615
A 18-24hr 35°C Aerobic Growth; no color change
Escherichia coli
ATCC® 25922
B 18-24hr 35°C Aerobic Partial to complete inhibition; no color change
Proteus mirabilis
ATCC® 12453
B 18-24hr 35°C Aerobic Partial to complete inhibition; no color change

*Prepare a cell suspension equivalent to a 0.5 McFarland Standard, then dilute to 1:100 for A or 1:10 for B method and inoculate media with 0.01mL of this suspension.

User Quality Control

Physical Appearance

Strep B Carrot Broth™

Showing positive (left tube) and negative (right tube) Strep B Carrot Broth™ Reactions (Cat. no. Z140)
A Strep B Carrot Broth™ Tile was aseptically added to each tube with sterile forceps. The left and right tubes were inoculated with Streptococcus agalactiae ATCC® 12386) and Streptococcus pyogenes (ATCC® 19615), respectively. The tubes were incubated aerobically for 24 hours at 35ºC. The orange to red color change was indicative as positive for a beta-hemolytic group B Streptococci.

PERFORMANCE CHARACTERISTICS

Three independent studies have been performed to evaluate the Strep B Carrot Broth™.

Study 1: Forty-six hemolytic GBS isolates of several serotypes (Ia, Ib, II, III, IV, V, VI, and VII) were tested and the Strep B Carrot Broth™ successfully detected all of the strains in concentrations as low as 200 CFU/tube.(17)

Study 2: Eighty-one clinical isolates were diluted in several concentrations and inoculated into Strep B Carrot Broth™ and LIM broth. In concentrations of 102 CFU/mL, Strep B Carrot Broth™ showed sensitivity of 98.8% in detecting 80 of 81 samples, while the LIM broth method detected 76 of 81 samples (93.8%).(12)

Study 3: Strep B Carrot Broth™ was compared to the recommended LIM broth method in a multi-center clinical trial by eight hospitals across the United States. Strep B Carrot Broth™ sensitivity/specificity was 96.8% and 100%, and LIM broth was 93% and 99.4%.(15)

ANTIMICROBIAL SUSCEPTIBILITY TESTING

A positive Strep B Carrot Broth™ can be subcultured for susceptibility testing; however, all GBS are universally susceptible to penicillin, since resistance to this antibiotic has not been reported. Penicillin remains the primary therapeutic option. The CDC does not recommend testing susceptibility of penicillin.(13)

Susceptibility testing of erythromycin and clindamycin is recommended only when the patient is allergic to penicillin.(13) If the isolate is resistant to these antimicrobials, the last therapeutic option is vancomycin.

POSITIVE Strep B Carrot Broth™ VIABILITY STUDY

Strep B Carrot Broth™ has been proven to be reliable for the preservation of GBS should the physician request susceptibility testing at a later date. In a study performed by DiPersio, 21 positive specimens were evaluated and all remained viable for at least 15 days at room temperature. The viability period ranged from 15 to 81 days with an average of 45 days.(16) It is presumed that viability would be increased if tubes had been refrigerated.

TILE INOCULATION STUDY(27)

Due to increasing usage of Strep B Carrot Broth™ by large volume clinical laboratories, the possibility of inoculating Strep B Carrot Broth™ tiles in advance to streamline and batch the inoculation process was assessed. An in-house inoculation study, where tiles were pre-inoculated at different time intervals (0.5 hour, 1 hour, 2 hours, 3 hours and 5 hours) prior to inoculation of ten clinical GBS isolates at concentration of 100 CFUs, was completed.

Based on this brief in-house study involving pure and diluted GBS, Strep B Carrot Broth™ may be setup with the reagent tile and left at room temperature for up to five hours with no measurable effect on performance. Further extensive testing and validation should be done with clinical specimens, at discretion of each labortory.

WASP COMPATIBILITY STUDY

A procedure was developed for the use of Strep B Carrot Broth™ with the COPAN WASP system. A study was conducted to validate the use of commonly used traditional transport swabs where the swabs were used to inoculate Strep B Carrot Broth™ (in a red-capped plastic tube, Cat. no. Z144BX) by swirling or by vortexing in Strep B Carrot Broth™ before swabs were removed and discarded. Nylon Tipped Flocked Transport Swabs were vortexed, followed by inoculation of 30µl of the suspension into Strep B Carrot Broth™.

Overall, the results were consistent and all 100% strains tested were successfully recovered by Strep B Carrot Broth™/GBS Detect™ system, indicating that alternative methods may be successfully employed to process samples with the Copan WASP. Full study results are on file, and can be provided upon request.

Transport Time and Temperature Study

Due to increasing usage of Strep B Carrot Broth™ by reference laboratories with many locations, the possibility of transporting samples at room temperature for up to 72 hours was assessed. In an in-house study, mixed samples (Lactobacillus, E. coli, C. albicans, E. faecalis and GBS) were made to mimic clinical vaginal samples and held for up to 72 hours prior to inoculating the Strep B Carrot Broth™.

The overall recovery rates of GBS from all transport media held at room temperature was 87.5% at 24hrs, 81.3% at 48hrs and 78.1% at 72hrs. All negative Strep B Carrot Broth™ tubes (100%) containing GBS were detected as positives upon subculture on GBS Detect™ media and confirmed by latex agglutination.

Based on this study, GBS is optimally transported under refrigerated conditions. However, when refrigeration during transit is unavailable, Strep B Carrot Broth™ was able to detect the presence of GBS in 78% of specimens left at room temperature for 72 hours without the need for subculture. Most significantly, the combination of Strep B Carrot Broth™/GBS Detect™ media system ensures 100% recovery even if the specimens are left un-refrigerated for up to 72 hours. Full study results are on file, and can be provided upon request.

REFERENCES

1. Regan, J.A., Klebanoff, M.A., Nugent, R.P. 1991. The epidemiology of group B streptococcal colonization in pregnancy. Vaginal Infections and Pregnancy Study Group. Obstet. Gynecol.; 77:604-10.

2. Schrag, S.J., E.R. Zell, R. Lynfield, et al. 2002. A population-based comparison of strategies to prevent early-onset group B streptococcal disease in neonates. N. Engl. J. Med. 25;347(4):233-9.

3. Schuchat, A. 2001. Group B streptococcal disease: from trials and tribulations to triumph and trepidation. Clin. Infect. Dis. 5;33(6):751-6.

4. The Centers for Disease Control and Prevention. 2010. Prevention of Perinatal Group B Streptococcal Disease. Revised Guidelines. MMWR 59 (RR-10). Internet: http://www.cdc.gov/mmwr/pdf/rr/rr5910.pdf

5. Overman, S.B., D.D. Eley, B.E. Jacobs, J.A. Ribes. 2002. Evaluation of methods to increase the sensitivity and timeliness of detection of Streptococcus agalactiae in pregnant women. J. Clin. Microbiol.; 40(11):4329-31.

6. de la Rosa, M., M. Perez, C. Carazo, L. Pareja, J.I. Peis and F. Hernandez. 1992. New Granada Medium for detection and identification of group B streptococci. J. Clin. Microbiol.; 30:1019-1021.

7. Garcia Gil, E., M.C. Rodriguez, R. Bartolome, B. Berjano, L. Cabero and A. Andreu. 1999. Evaluation of the Granada Agar plate for detection of vaginal and rectal group B streptococci in pregnant women. J. Clin. Microbiol.; 37:2648-2651.

8. Rosa-Fraile, Manuel, J. Rodriguez-Granger, M. Cueto-Lopez, A. Sampedro, E. Biel Gaye, J.M. Haro and A. Andreu. 1999. Use of Granada Medium to detect group B streptococcal colonization in pregnant women. J. Clin. Microbiol.; 37:2674-2677.

9. Rosa-Fraile, Manuel, A. Sampedro, J. Varela, M. Garcia-Pena, and G. Gimenez-Gallego. 1999. Identification of a peptide pigment from mammal albumins responsible for enhanced pigment production by group B streptococci. Clin. Diag. Lab. Imm.; 6:425-426.

10. Islam, AKMS. 1977. Rapid recognition of group B streptococci. Lancet 309(8005):256-257.

11. B. Spellerberg, B. Pohl, G. Haase, S. Martin, J. Weber-Heynemann and R. Lütticken. 1999. Identification of Genetic Determinants for the Hemolytic Activity of Streptococcus agalactiae by ISS1 Transposition. J. Bacteriol.; 181: 3212-3219.

12. Hardy Diagnostics and Fleury Medical Diagnostic Center. 2004. Evaluation of Three Methods for Recovery of Group B Streptococci. A poster presentation at American Society for Microbiology 104th General Meeting, New Orleans, LA.

13. National Center for Infectious Diseases, et al. 2002. Revised Guidelines from CDC: Prevention of Perinatal Group B Streptococcal Disease. MMWR Recommendations and Reports/51(RR11);1-22. Internet: http://www.cdc.gov/mmwr/preview/mmwrhtml/rr5111a1.htm.

14. Rosa-Fraile, M. et al. 2005. Specimen Storage in Transport Medium and Detection of Group B Streptococci by Culture. J. Clin. Microbiol.; 43:928-930.

15. Schreckenberger et al. 2005. Evaluation of Strep B Carrot Broth™ and LIM Broth Methods for Recovery of Group B Streptococci (GBS): Results of a Multi-Center Trial. A poster presentation at American Society for Microbiology, 105th General Meeting, Atlanta, GA.

16. DiPersio, J. 2005. GBS Preservation by Strep B Carrot Broth™ Inoculated with Patient Specimens. Unpublished data.

17. Facklam, R. et al. 2006. Evaluation of Accuracy of Strep B Carrot Broth™ in the Detection of Different Seryotypes of Group B Streptococci (GBS). A poster presentation at American Society for Microbiology, 106th General Meeting, Orlando, FL.

18. Young, Uh et al. 1998. Serotypes and Biochemical Reaction Patterns of Group B Streptococci. Korean J. Clin. Path.; 18:386-390.

19. Merrit, K. and Jacobs, N. 1978. Characterization and Incidence of Pigment Production by Human Clinical Group B Streptococci. J. Clin. Microbiol.; Vol. 8, No. 1, p. 105-107.

20. Noble, M., Bent, J., West, A. 1983. Detection and identification of group B streptococci by use of pigment production. J. Clin. Path.; 36:350-352.

21. de la Rosa, M., R. Villareal, D. Vega, C. Miranda, and A. Martinezbrocal. 1983. Granada Medium for Detection and Identification of Group B Streptococci. J. Clin. Microbiol.; Vol. 18, No. 4, p.779-785.

22. Rosa-Fraile, Manuel, J. Rodriguez-Granger, A. Haidour-Benamin, J.M. Cuerva, and A. Sampedro. 2006. Granadaene: Proposed Structure of the Group B Streptococcus Polyenic Pigment. J. Clin. Microbiol.; Vol. 72, No. 9, p.6367-6370.

23. da Gloria Carvalho, M., R. Facklam, D. Jackson, B. Beall, and L. McGee. 2009. Evaluation of Three Commercial Broth Media for Pigment Detection and Identification of a Group B Streptococcus (Streptococcus agalactiae). J. Clin. Microbiol.; Vol. 47, No. 12, p.4161-4163.

24. Block, T., E. Munson, A. Culver, K. Vaughan, and J. Hryciuk. 2008. Comparison of Carrot Broth- and Selective Todd-Hewitt Broth-Enhanced PCR Protocols for Real-Time Detection of Streptococcus agalactiae in Prenatal Vaginal/Anorectal Specimens. J. Clin. Microbiol.; Vol. 46, No. 11, p.3615-3620.

25. Church, D.L., H. Baxter, T. Lloyd, B. Miller, and S. Elsayed. 2008. Evaluation of Strep B Carrot Broth™ versus Lim Broth for Detection of Group B Streptococcus Colonization Status of Near-Term Pregnant Women. J. Clin. Microbiol.; Vol. 46, No. 8, p.2780-2782.

26. Czerepuszko, D.J., and M.J. Lewis. 2010. Comparison of LIM Broth with PNA FISH to Carrot Broth with PNA FISH for Identification of Group B Streptococcus in Prenatal Vaginal/Rectal Specimens. A poster presentation at American Society for Microbiology, San Diego, CA.

27. Massey C. and A. Hsiung 2013. Research Summary Report: Varying the Time of Insertion of Carrot Broth Tiles. In-house report.

ATCC is a registered trademark of the American Type Culture Collection.
PNA FISH is a trademark of AdvanDx, Inc., Woburn, MA.

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