OUTLINE Laboratory Detection and Reporting of Streptococcus agalactiae I. Importance of prenatal screening strategies II. Past approaches Erik Munson Clinical Microbiology Wheaton Franciscan Laboratory Milwaukee, Wisconsin The presenter states no conflict of interest and has no financial relationship to disclose relevant to the content of this presentation. 1 III. Current guidelines A. General indications for prophylaxis B. Laboratory methods and reporting C. Adaptations of molecular approaches D. Antimicrobial susceptibility testing 2 Streptococcus agalactiae Colonizes 15-4% of pregnant women Importance of Prenatal Screening J. Infect. Dis. 143: 761-766; 1981 Am. J. Obstet. Gynecol. 142: 617-6; 1982 J. Infect. Dis. 145: 794-799; 1982 J. Infect. Dis. 148: 82-89; 1983 Obstet. Gynecol. 88: 811-815; 1996 Obstet. Gynecol. 96: 498-53; Vertical transmission 3 4 Streptococcus agalactiae INTERVENTION Neonatal incidence rate per 1 live births: Infection 3. Septicemia 2. Case fatality 1. J. Pediatr. 82: 77-718; 1973 Group B streptococcal disease 83 women screened at 36 weeks gestation 173 (21.5%) positive for S. agalactiae Early onset Late onset -72 hours; pneumonia bacteremia 1-3 months; meningitis 8 received intrapartum ampicillin 93 did not receive antimicrobials 5 J. Clin. Microbiol. 23: 489-492; 1986 6
INTERVENTION INTERVENTION Intrapartum Ampicillin Treatment Number of Colonized Moms Number (%) of Colonized Babies Yes 8 (.) No 93 43 (46.2) Demographic Number of Moms Number of Births GBS Sepsis/ 1 Births Incidence Resultant Fatality GBS screen + and treated; GBS screen - 71 71.. GBS screen + and not treated; Not screened for GBS 1269 1274 5.49 2.35 Not treated; Not screened for GBS 395 311 2.25.32 J. Clin. Microbiol. 23: 489-492; 1986 7 J. Clin. Microbiol. 23: 489-492; 1986 8 DISEASE REDUCTION Second trimester assessment Screening- or risk-based 65% reduction in early-onset disease prevalence from 1993-1998 9 N. Engl. J. Med. 342: 15-; 1 SCREENING- VERSUS RISK-BASED Adjusted relative risk for early-onset GBS disease associated with screening approach was.48 N. Engl. J. Med. 347: 233-239; 2 11 12
SCREENING-BASED METHODS 35- to 37-week assessment Screening-based Blood agar plate Blood agar plate + Todd Hewitt (LIM) broth plus subculture Increases yield -35% 13 J. Matern. Fet. Med. 7: 172-176; 1998 Arch. Pathol. Lab. Med. 127: 718-7; 3 14 ADDITIONAL (RECTAL) SAMPLING Study Patients Carriage Rate (%) Recovery Only by Rectal Sampling (%) Badri et al. 1977 789.5 5. Dillon et al. 1982 254 35. 51.4 Philipson et al. 1995 383.4 31.1 Platt et al. 1995* 651 16.9 26.4 Quinlan et al. 222 24.3 18.5 Kovavisarach et al. 7 3 41.9 24.6 J. Infect. Dis. 135: 38-312; 1977 J. Infect. Dis. 145: 794-799; 1982 Obstet. Gynecol. 85: 437-439; 1995 *Diagn. Microbiol. Infect. Dis. 21: 65-68; 1995 J. Fam. Pract. 49: 447-448; J. Med. Assoc. Thai. 9: 171-1714; 7 15 Investigation WHO S SAMPLING?? Location S. agalactiae Culture Sensitivity (%) Patient Collection Provider Collection Mercer et al. 1995 Tennessee 91.7 7.8 Molnar et al. 1997 Ontario 97.4 82.1 Price et al. 6 Ontario 87.5* 96.9 Arya et al. 8 Ireland 84.3 94.3 P <.5 *P =.11 Am. J. Obstet. Gynecol. 173: 1325-1328; 1995 Fam. Pract. 14: 43-46; 1997 J. Obstet. Gynaecol. Can. 28: 183-188; 6 Eur. J. Obstet. Gynecol. Reprod. Biol. 139: 43-45; 8 16 SCREENING-BASED METHODS CARROT BROTH (observed at 24h) Blood agar plate + Todd Hewitt (LIM) broth plus subculture 87.% sensitivity Blood agar plate + plus subculture 96.3% sensitivity Negative for S. agalactiae Positive for S. agalactiae Wheaton Franciscan Laboratory in-house data 17 18
SCREENING-BASED METHODS + Blood agar Todd Hewitt plate (LIM) broth plus subculture 38.3% resulted on day 1 Blood agar plate + plus subculture 8.8% resulted on day 1 (P <.2) Is This Working? Wheaton Franciscan Laboratory in-house data 19 SUCCESS IN SCREENING 21 N. Engl. J. Med. 36: 2626-2636; 9 22 SUCCESS IN PROPHYLAXIS Early-onset GBS disease per 1 births 2.4 2.2 2 1.8 1.6 1.4 1.2 1.8.6.4.2 CDC Guidelines Revised CDC Guidelines Estimate * Ten-state surveillance 1973 1993 1998 1999-1 3-5 3-4* Interval N. Engl. J. Med. 36: 2626-2636; 9 23 J. Pediatr. 82: 77-718; 1973 N. Engl. J. Med. 342: 15-; JAMA 299: 56-65; 8 N. Engl. J. Med. 36: 2626-2636; 9 24
DISAPPOINTMENT??? DISAPPOINTMENT??? N. Engl. J. Med. 36: 2626-2636; 9 25 N. Engl. J. Med. 36: 2626-2636; 9 26 DISAPPOINTMENT??? BIG DISAPPOINTMENT??? Expected 44 to 86 cases of group B streptococcal disease among term infants Observed 116 cases N. Engl. J. Med. 36: 2626-2636; 9 27 N. Engl. J. Med. 36: 2626-2636; 9 28 BENEFIT FROM A RAPID RESULT Need improved diagnostics Increased attack rates and mortality in low birth weight neonates At same time, demographics may benefit from rapid & accurate diagnostics 29 N. Engl. J. Med. 342: 15-; MMWR. 59 (RR-1): 1-32; 1 3
BENEFIT FROM A RAPID RESULT BENEFIT FROM A RAPID RESULT Increased attack rates and mortality in low birth weight neonates Increased attack rates and mortality in low birth weight neonates Inadequate/no prenatal care Inadequate/no prenatal care Higher probability in African Americans Increased disease in those with inadequate care Increased disease in African American neonates Moms who screen negative at 35-37 weeks, but are colonized at parturition (estimated 4-9%) Obstet. Gynecol. 87: 575-58; 1996 Obstet. Gynecol. 89: 28-32; 1997 31 Pediatrics 115: 124-1246; 5 J. Infect. Dis. 148: 82-89; 5 32 COMMERCIAL PCR PERFORMANCE INDICES Rapid detection of S. agalactiae DNA in vaginal/ rectal specimens from prepartum or intrapartum women (direct swab) -enhanced PCR 33.% detection LIM broth-enhanced PCR 3.5% detection culture 29.6% detection 86-94% sensitivity (LIM broth reference) Parameter Carrot Broth PCR LIM Broth PCR Clin. Infect. Dis. 39: 1129-1135; 4 Sensitivity (%) 1. 92.5 Negative predictive value (%) 1. 96.4 Unresolved rate (%)..5 Processing time/specimen (min) 5.1 5.1 33 J. Clin. Microbiol. 46: 3615-36; 8 34 PCR-POSITIVE SPECIMENS; n = 67 COMMERCIAL PCR Parameter Timepoint of Carrot Broth Culture Observation Overnight Incubation Final Subculture Result Positive culture 34 6 Sensitivity (%) 5.7 89.6 Negative predictive value (%) 8.5 95.1 Rapid detection of S. agalactiae DNA in vaginal/ rectal specimens from prepartum or intrapartum women (direct swab) 86-94% sensitivity (LIM broth reference) Clin. Infect. Dis. 39: 1129-1135; 4 56-59% sensitivity (carrot broth reference) J. Clin. Microbiol. 46: 3615-36; 8 35 J. Clin. Microbiol. 46: 3615-36; 8 J. Clin. Microbiol. 48: 4495-45; 1 36
% Difference in Sensitivity 35 3 25 15 1 5 Direct swab PCR WHY?? LIM broth culture % Difference in Sensitivity 35 3 25 15 1 5 Direct swab PCR WHY?? LIM broth culture culture Clin. Infect. Dis. 39: 1129-1135; 4 37 Wheaton Franciscan Laboratory in-house data 38 % Difference in Sensitivity 35 3 25 15 1 5 Direct swab PCR WHY?? LIM broth culture culture - Enhanced PCR produces orange pigment Clinical specimen (vaginal/rectal swab) Inoculate carrot broth No pigment production Report as positive for group B Streptococcus Perform PCR on broth aliquot J. Clin. Microbiol. 46: 3615-36; 8 39 4 IN VITRO EXPERIMENTATION CAN THIS BECOME MORE RAPID?? Inoculate carrot broth tubes with 1 3, 1 2, 1 1 S. agalactiae Mock inoculation with 1 9 flora; simulating Anaerobic flora Urogenital flora Gastrointestinal flora Pathogenic flora Collect 5- L aliquots at specified intervals for carrot broth-enhanced PCR 41 42
CARROT BROTH-ENHANCED PCR CLINICAL EXPERIMENTATION inoculation Hold remaining swab from original patient collection Percentage Positive S. agalactiae Inoculum Time of aliquot collection (hours) 2 4 6 12 24 1 1. 33.3 41.7 25. 33.3 1 2 58.3 66.7 91.7 ND ND 1 3 1. 1. 1. ND ND Orange carrot broth pigment after overnight incubation YES x hours Aliquot removed and frozen (-7 C) ND; not determined J. Clin. Microbiol. 46: 4495-45; 1 43 1. Recover remaining swab from original patient collection for direct swab StrepB PCR 2. Retrieve frozen carrot broth aliquot for StrepB PCR 44 CLINICAL EXPERIMENTATION CLINICAL EXPERIMENTATION Orange carrot broth pigment after overnight incubation NO positive 1. Recover remaining swab from original patient collection for direct swab StrepB PCR 2. Retrieve frozen carrot broth aliquot for StrepB PCR enhanced StrepB PCR negative 3 of the prospective aliquots retrieved for early-aliquot StrepB PCR specificity validation 45 Number of Specimens Early-aliquot Carrot Broth-enhanced PCR Collection Interval (h) from Remnant Direct Swab PCR P value 33 < 3. 54.5 66.7.31 35 3.-3.99 4. 54.3.23 35 4.-4.99 51.4 48.6.81 41 5.-5.99 73.2 65.9.47 39 6.-6.99 82.1 46.2.9 44 > 7. 86.3 56.8.2 Total (227) 66.1 56.4.3 J. Clin. Microbiol. 46: 4495-45; 1 46 POSITIVE CARROT BROTH CULTURE Number of Specimens Early-aliquot Carrot Broth-enhanced PCR Collection Interval (h) from Remnant Direct Swab PCR P value 12 < 3. 83.3 91.7 ND 12 3.-3.99 5. 75. ND 1 4.-4.99 8. 8. ND 19 5.-5.99 94.7 89.5 ND 13 6.-6.99 1. 69.2 ND 1 > 7. 1. 7. ND Total (76) 85.5 8.3.39 NEGATIVE CARROT BROTH CULTURE Number of Specimens Early-aliquot Carrot Broth-enhanced PCR Collection Interval (h) from Remnant Direct Swab PCR P value 21 < 3. 38.1 52.4.35 23 3.-3.99 34.7 43.5.55 25 4.-4.99 4. 36..77 22 5.-5.99 54.5 45.5.55 26 6.-6.99 73.1 34.6.5 34 > 7. 82.4 52.9.1 Total (151) 56.2 44.4.4 J. Clin. Microbiol. 46: 4495-45; 1 47 J. Clin. Microbiol. 46: 4495-45; 1 48
35- to 37-week assessment Screening-based 49 INDICATIONS FOR PROPHYLAXIS Previous infant with invasive early-onset disease S. agalactiae bacteriuria during any trimester of current pregnancy Positive S. agalactiae vaginal/rectal screening culture in late gestation during current pregnancy Unknown S. agalactiae status at labor PLUS one: Delivery at < 37 weeks gestation Amniotic membrane rupture 18 hours Intrapartum temperature 1.4 F Positive intrapartum nucleic acid amplification test 5 SPECIMEN COLLECTION/TRANSIT Lower vaginal, then rectal collection 35-37 weeks gestation; can be self-collected Cervical, perianal, perirectal not acceptable Swabs placed into non-nutritive transport medium Recovery decreases over 1-4 days (room temp) Refrigerate swabs, if feasible Clinicians indicate if patient possesses allergy to penicillin or cephem agent 51 SPECIMEN PROCESSING Selective broth medium (Todd-Hewitt base) LIM broth Transvag broth Alternative selective media can be chromogenic Granada biphasic broth 18-24 hours in 35-37C ambient air or 5% CO 2 Direct plating may be included Lower sensitivity than broth enrichment Should not be used as sole means of recovery 52 RESULTS AND INTERPRETATION Selective broths subcultured to appropriate agar(s) Non-pigmented chromogenic broths subcultured to appropriate agar(s) Positive identification may be derived from: Biochemical or probe testing of isolated growth Pigmented broth ( -hemolytic S. agalactiae) Probe testing of selective broth Nucleic acid amplification testing of selective broth Latex agglutination of selective broth MMWR. 59(RR-1): 1-32; 1 53 54
DIRECT MOLECULAR DETECTION? Accurate results are more important than rapid turnaround time for antenatal screening. College of American Pathologists MIC.64817 A pre-enrichment step using a selective broth enrichment culture is performed for antepartum (35-37 weeks gestation) vaginal/rectal swab screening for Group B streptococci (GBS) colonization by nucleic acid amplification testing (NAAT). 55 ANTIMICROBIAL SUSCEPTIBILITY Disk diffusion or broth microdilution performed on antenatal S. agalactiae isolates from women at risk for anaphylaxis (related to penicillin or cephem) Anaphylaxis Respiratory distress Angiodedema Urticaria Inducible clindamycin testing on erythromycinresistant S. agalactiae CLSI M1 document recommends suppression of erythromycin susceptibility testing data 56 INDUCIBLE CLINDAMYCIN RESISTANCE CDC recommends D-test on erythromycin-r/ clindamycin-s isolates of S. agalactiae; allows for performance on other validated AST systems 2 g clindamycin disk Mueller-Hinton w/blood 15 g erythromycin disk 35C; 5% CO 2 12 millimeters apart -24 hours Percentage Susceptible 9 8 7 6 5 4 3 S. agalactiae versus erythromycin 1 57 1 2 3 4 5 6 7 8 9 1 11 12 1358 4 9 8 S. agalactiae versus erythromycin 9 8 S. agalactiae versus erythromycin S. agalactiae versus clindamycin 7 7 Percentage Susceptible 6 5 4 3 Percentage Susceptible 6 5 4 3 1 1 1 2 3 4 5 6 7 8 9 1 11 12 1359 4 1 2 3 4 5 6 7 8 9 1 11 12 136
9 8 S. agalactiae versus erythromycin S. agalactiae versus clindamycin INTRAPARTUM PROPHYLAXIS Percentage Susceptible 7 6 5 4 3 1 1 2 3 4 5 6 7 8 9 1 11 12 1361 62 THE TIMES THEY ARE A CHANGIN Fourteen non-invasive S. agalactiae isolates between 1995-5 had alterations in PBP2X Clinical significance unclear J. Antimicrob. Chemother. 65: 594-595; 1 Antimicrobial. Agents Chemother. 52: 289-2897; 8 63 64 S. agalactiae BACTERIURIA Marker for heavy genital tract colonization; risk factor for early-onset GBS disease Scand. J. Infect. Dis. 17: 195-199; 1985 1996 guidelines no threshold specification 2 guidelines report any concentration 1 guidelines 1 4 colony forming units/ml Few data available on risk for early-onset GBS in context of low-count bacteriuria MMWR. 45 (RR-7): 1-24; 1996 MMWR. 51 (RR-11): 1-24; 2 65 THE END Identification of candidates for intrapartum chemoprophylaxis is essential for prevention of earlyonset group B streptococcal disease Much of this has fallen into the hands of laboratory Situation has improved since the 197s; more work to be done Molecular diagnostics and antimicrobial susceptibility testing, when applied appropriately, play major role 66