UNIVERSITY OF COLORADO SCHOOL OF MEDICINE DEPARTMENT OF PATHOLOGY RESIDENCY TRAINING PROGRAM

1. UNIVERSITY OF COLORADO SCHOOL OF MEDICINE DEPARTMENT OF PATHOLOGY RESIDENCY TRAINING PROGRAM MICROBIOLOGY Case Studies: Beta Hemolytic Streptococci

2. Endometritis In the left frame are illustrated the observation of a Gram stain prepared from A 25 year old woman is in her 37th week of gestation. A vaginal-rectal culture was sent it to the laboratory for culture. The right frame is a photograph of the colonies growing on the surface of a sheep blood agar plate. What is the most probable diagnosis? What follow-up tests should be performed? ANSWER

3. Endometritis Illustrated in the photographs are the rapid 4-hour and the overnight 18 – 24 hour CAMP reactions. Describe the principle and the procedure for each of these two tests. What is the most likely species identification? What other bacterial species also produce a positive CAMP reaction? How are they ruled out in this case? Why is it important to incubate the plates at ambient temperature? ANSWER

4. Endometritis ADDITIONAL QUESTIONS 1. What is the importance of detecting vaginal colonization of Group B streptococci during the last trimester of pregnancy? 2. What therapeutic measures are recommended? 3. In cases of the development of pre-term or parinatal infection, what rapid tests might be performed to confirm group B Strep? (Clue: Look up “Carrot Agar” (Hardy Diagnostics), and “Granada Agar” (J Clin Microbiol 1992. 30:1019-1021) 4. Molecular Techniques: Check which procedures are being used in each of the on-site microbiology laboratories ANSWER ANSWER ANSWER 1 ANSWER 2 ANSWER

5. Abbreviated Identification of Streptococcus agalactiae Zone of beta hemolysis on sheep blood agar Catalase Negative Gram Positive Cocci in Chains and Pairs Positive Rapid CAMP Factor Test Hydrolysis of sodium hippurate Carotene produced in serum starch broth STREPTOCOCCUS AGALACTIAE (Group B) QUIT

6. Answers for Page 2 The gram stain reveals gram-positive cocci in chains, most consistent with a Streptococcus species. The blood agar plate reveals relatively large colonies surrounded by a distinctive zone of beta hemolysis. The case history makes the likely presumptive identification as Streptococcus Group B (S. agalactiae). Although a full biochemical work-up might be indicated, a more direct first step would be to perform a CAMP test. In situations where an identification is urgent, the rapid 4-hour disk test could be performed. In this case, the overnight streak test would be adequate. RETURN

7. Answers for Page 3 • The positive CAMP reaction, both by the disk and the streak techniques, helps to confirm the presumptive identification of Group B streptococcus (S. agalactiae). • Several species of Gram-positive bacilli also produce positive CAMP reactions: • Listeria monocytogenes: The CAMP reaction is positive, but the area of inhanced hemolysis is rectangular rather than arrow-head in shape. • Corynebacterium auris • Corynebacterium coylae • Corynebacterium glucuronolyticum • Corynebacterium imitans • Some Group-A streptococci may be falsely CAMP positive if the test plate is incubated in a candle jar, in a CO2 atmosphere, or under anaerobic conditions. Therefore, ambient air incubation should be used for accurate results. RETURN

8. Answers for Page 4 Question #1 and #2 • 10-35% of women are asymptomatic carriers of Group B Strep (GBS). • gastrointestinal tract main reservoir with contamination of genital areas • Leading cause of neonatal morbidity and mortality in US in 1970’s • Case-fatality as high as 50% • 1980’s: administration of intra-partum antibiotics to women at risk during labor could reduce early onset disease (first 7 days of life; 60-70% of the GBS infections) • 1996: recommendation for intrapartum prophylaxis to prevent GBS perinatal disease • ACOG, CDC and AAP (1997) Abstracted from CACMLE Teleconference by Gerri S. Hall, Ph. D., March 23, 2005 RETURN

9. Answers for Page 4 Question #3 (1) • RECOMMENDED CULTURE MEDIA FOR RECOVERY OF GROUP B STREPTOCOCCI • BAP (Columbia or TSA) - With or without antibiotics • Broth enrichments • - LIM Broth (Todd Hewitt with colistin and nalidixic) with subculture to BAP - Carrot Broth (Hardy Diagnostics, Santa Maria, CA.) • Granada Agar (Hardy Diagnostics) Abstracted from CACMLE Teleconference by Gerri S. Hall, Ph. D., March 23, 2005 RETURN

10. Answers for Page 4 Question #3 (2) Abstracted from CACMLE Telecon-ference by Gerri S. Hall, Ph. D., March 23, 2005 • GRANADA AGAR • Orange pigmented colonies of GBS - Enhancement of pigment with methotrexate - Said to be equivalent to selective broth for recovery • No pigment for non-ß-heme colonies • May require anaerobic conditions; may require 48 hrs. • Short shelf-life • More expensive than BAP • No broth enhancement necessary CARROT BROTH Orange to red color change in 6 to 24 hrs. with ß-hemolytic GBS. Negatives should be subcultured to selective streptococcal agar for non-hemolytic GBS (~4%). RETURN

11. Answers for Page 4 Question #4 GBS Identification from LIM Broth • SUMMARY OF APPROACHES TO THE RECOVERY AND IDENTIFICATION OF GROUP b STREPTOCOCCI • Culture • Sub onto BAP or selective media • ß- hemolytic colonies; catalase positive gpc in chains; hippurate + and/or CAMP (Christie-Atkins-Munch-Peterson) + • AccuProbe (Gen-Probe, San Diego,CA) • > 8 hrs incubation; better when > 18 hrs • OIA • Amplification • Smart Cycler PCR Abstracted from CACMLE Teleconference by Gerri S. Hall, Ph. D., March 23, 2005 RETURN