1. Journal Club Tiffany Ohta, PL-2 29 Aug 2006

2. Case Kari is a 32 y/o G3P2 female who comes in to L&D at 37+1 weeks gestation after ROM 1 hour ago and now with contractions every 5 minutes Kari is a 32 y/o G3P2 female who comes in to L&D at 37+1 weeks gestation after ROM 1 hour ago and now with contractions every 5 minutes Prenatal labs: O+, RPR NR, RI, HepBsAg neg, HIV neg, GBS unknown but negative last 2 pregnancies, negative UA and UCx Prenatal labs: O+, RPR NR, RI, HepBsAg neg, HIV neg, GBS unknown but negative last 2 pregnancies, negative UA and UCx Previous 2 pregnancies were uncomplicated SVD’s, has missed some prenatal appts with last appt 3 weeks ago Previous 2 pregnancies were uncomplicated SVD’s, has missed some prenatal appts with last appt 3 weeks ago

3. Case On exam, membranes found to be grossly ruptured and on cervical exam found to be 8/90%/+1 On exam, membranes found to be grossly ruptured and on cervical exam found to be 8/90%/+1 Within 5 minutes of being transferred to a labor room, Kari felt the urge to push, and after 5 minutes of pushing, a healthy infant male was born with APGAR’s 9 and 9 weighing 2.9 kg Within 5 minutes of being transferred to a labor room, Kari felt the urge to push, and after 5 minutes of pushing, a healthy infant male was born with APGAR’s 9 and 9 weighing 2.9 kg

4. Case Since the mother’s GBS status was unknown and did not receive any IAP, the infant was placed on q4 vitals and would be observed for 48 hours per hospital procedure Since the mother’s GBS status was unknown and did not receive any IAP, the infant was placed on q4 vitals and would be observed for 48 hours per hospital procedure After 24 hours, the mother became insistent that she wanted to go home as she has 2 other kids at home, and “my mom is an RN, and she will know if anything is wrong with Joey” After 24 hours, the mother became insistent that she wanted to go home as she has 2 other kids at home, and “my mom is an RN, and she will know if anything is wrong with Joey”

5. Question Is there a test other than a 48 hour GBS culture that would produce results sooner? YES! Is there a test other than a 48 hour GBS culture that would produce results sooner? YES! Could this test potentially prevent delayed discharge and reduce costs associated with it? YES! Could this test potentially prevent delayed discharge and reduce costs associated with it? YES! Let’s evaluate this test further….. Let’s evaluate this test further…..

6. Real Time Polymerase Chain Reaction for the Rapid Detection of Group B Streptococcal Colonization in Neonates Natarajan et al. Pediatrics. 2006; 118; 14-22

7. What is the big deal about GBS? A leading cause of morbidity and mortality in newborns, resulting in sepsis, pneumonia, and meningitis A leading cause of morbidity and mortality in newborns, resulting in sepsis, pneumonia, and meningitis GBS carried in the vaginal and anorectal flora of up to 30% of women – colonization can be intermittent or persistent GBS carried in the vaginal and anorectal flora of up to 30% of women – colonization can be intermittent or persistent –Those with GBS bacteriuria are known to be more frequently and more heavily colonized

8. What is the big deal about GBS? Colonization rate in newborns born to GBS positive mother is 40-70% Colonization rate in newborns born to GBS positive mother is 40-70% Risk factors for neonatal colonization: Risk factors for neonatal colonization: –Heavily colonized mother –Vaginal delivery –Prolonged ROM >18 hrs –Preterm delivery –Maternal temp >38°C –Lack of intrapartum antibiotic prophylaxis (IAP)

9. What is the big deal about GBS? “Early onset” infection defined as occurring within first 7 days “Early onset” infection defined as occurring within first 7 days –Incidence is 0.5-3/1000 live births with 4-10% mortality (preemies 20-30%) Goal of preventive strategies is to reduce or eliminate transmission by giving antibiotics to GBS-colonized women during delivery and selectively giving antibiotics to newborns after delivery Goal of preventive strategies is to reduce or eliminate transmission by giving antibiotics to GBS-colonized women during delivery and selectively giving antibiotics to newborns after delivery –Can reduce early onset GBS rates by 80-95%

10. GBS unknown… Occasions when GBS status will be unknown – missed appts, lost specimens, lab errors, or preterm delivery Occasions when GBS status will be unknown – missed appts, lost specimens, lab errors, or preterm delivery IAP indicated if GBS status unknown within 6 weeks of delivery and if risk factors develop IAP indicated if GBS status unknown within 6 weeks of delivery and if risk factors develop Infants are observed for 48 hrs and work- up initiated with antibiotics if s/s concerning for infection or GA <35 wks Infants are observed for 48 hrs and work- up initiated with antibiotics if s/s concerning for infection or GA <35 wks

11. GBS rapid testing? Recent investigation into rapid methods of GBS detection, especially in the laboring women with unknown GBS status Recent investigation into rapid methods of GBS detection, especially in the laboring women with unknown GBS status Fluorescent real-time PCR is reported to be highly sensitive and specific in laboring women and gives results in 30-45 min Fluorescent real-time PCR is reported to be highly sensitive and specific in laboring women and gives results in 30-45 min –Not yet used at NNMC

12. GBS rapid testing? Currently limited data on the validity of rapid methods of GBS detection in neonates Currently limited data on the validity of rapid methods of GBS detection in neonates Could more accurately recognize colonized infants that would need further evaluation and treatment Could more accurately recognize colonized infants that would need further evaluation and treatment Could prevent delayed discharge, increased nursing time for frequent vitals, and unnecessary evaluation of those not at risk Could prevent delayed discharge, increased nursing time for frequent vitals, and unnecessary evaluation of those not at risk

13. Methods – Subject Selection Study done at Hutzel Women’s Hospital in Detroit from July 2002 to March 2004 Study done at Hutzel Women’s Hospital in Detroit from July 2002 to March 2004 Subjects included neonates >32 weeks GA whose maternal GBS status was unknown Subjects included neonates >32 weeks GA whose maternal GBS status was unknown –Unknown due to lack of prenatal care, unavailability of results, or PTL <35 wks Excluded critically ill infants Excluded critically ill infants

14. Methods – Clinical Data Recorded maternal data: age, race, previous hx of infants with GBS, +/- IAP, presence of GBS bacteriuria, chorio, and duration of ROM Recorded maternal data: age, race, previous hx of infants with GBS, +/- IAP, presence of GBS bacteriuria, chorio, and duration of ROM Recorded neonatal data: gestational age, gender, birth wt, results of diagnostic eval if done, +/- antibiotics, course in hospital, duration of hospitalization Recorded neonatal data: gestational age, gender, birth wt, results of diagnostic eval if done, +/- antibiotics, course in hospital, duration of hospitalization

15. Methods - Procedures Neonates had collection of specimens from 4 locations for both standard cx and PCR (before 1 st bath within 4 hrs of delivery) – nares, ear canals, rectum, and gastric aspirate Neonates had collection of specimens from 4 locations for both standard cx and PCR (before 1 st bath within 4 hrs of delivery) – nares, ear canals, rectum, and gastric aspirate On culture, GBS identity confirmed by ß- hemolysis on blood agar and serologic grouping On culture, GBS identity confirmed by ß- hemolysis on blood agar and serologic grouping

16. Methods - PCR LightCycler technology – combines PCR reaction with real-time detection of fluorescently tagged amplified products after each cycle LightCycler technology – combines PCR reaction with real-time detection of fluorescently tagged amplified products after each cycle –Technique used to simultaneously amplify and quantify a specific part of a DNA molecule Positive controls - genomic DNA from 5 commercially available strains of GBS Positive controls - genomic DNA from 5 commercially available strains of GBS

17. Methods - PCR Melting curve was generated for each specimen to confirm the amplified product (All PCR products should have same melting point unless contaminated or other lab error) Melting curve was generated for each specimen to confirm the amplified product (All PCR products should have same melting point unless contaminated or other lab error) –All 5 positive controls had identical melting curves, so only one was used to compare to all specimen runs Gel electrophoresis was also used to confirm the real-time PCR products Gel electrophoresis was also used to confirm the real-time PCR products

18. Melting Curve

19. Results – patient characteristics Results – patient characteristics 94 infants 94 infants Mean birth wt 3002g and GA 38 wks Mean birth wt 3002g and GA 38 wks Males 53.2% Males 53.2% CBC/cx done in 47.9% CBC/cx done in 47.9% 26.6% received empiric antibiotics – clinical s/s sepsis or suggestive findings on CBC 26.6% received empiric antibiotics – clinical s/s sepsis or suggestive findings on CBC No cases of early onset GBS in this cohort No cases of early onset GBS in this cohort Median duration of stay – 3 days Median duration of stay – 3 days

20. Results – maternal characteristics Mean age 25.7, 81.9% black Mean age 25.7, 81.9% black 24 with chorio 24 with chorio 11 with PROM 11 with PROM 4 with GBS bacteriuria 4 with GBS bacteriuria 3 with h/o previous child with GBS 3 with h/o previous child with GBS 22 received IAP for risk factors 22 received IAP for risk factors

21. Results – rates of colonization 16 infants (17%) found to be colonized at 1+ sites based on culture 16 infants (17%) found to be colonized at 1+ sites based on culture 48 infants (51%) found to be colonized at 1+ sites based on PCR 48 infants (51%) found to be colonized at 1+ sites based on PCR

22. Results – rates of colonization Real-time PCR had a higher detection rate when compared to cultures, with ears and nares being best sites for detection Real-time PCR had a higher detection rate when compared to cultures, with ears and nares being best sites for detection Authors believe the increased detection represents true colonization rather than false positives since “the assay was performed under stringent conditions for specificity, both inherent and imposed” Authors believe the increased detection represents true colonization rather than false positives since “the assay was performed under stringent conditions for specificity, both inherent and imposed”

23. Results – rates of colonization Interestingly, among the 22 infants whose mothers received antibiotics, GBS colonization observed in 4 (18.2%) by culture, and 11 (50%) by PCR Interestingly, among the 22 infants whose mothers received antibiotics, GBS colonization observed in 4 (18.2%) by culture, and 11 (50%) by PCR Among the infants without exposure to antibiotics, 12 (16.7%) had GBS by culture, and 37 (52.1%) had GBS by PCR Among the infants without exposure to antibiotics, 12 (16.7%) had GBS by culture, and 37 (52.1%) had GBS by PCR Not statistically significant differences, but authors do comment that the study was NOT powered to evaluate these differences Not statistically significant differences, but authors do comment that the study was NOT powered to evaluate these differences

24. Are the results valid? Was there an independent, blind comparison with a reference standard? Was there an independent, blind comparison with a reference standard? –YES! –Reference standard is the culture, which was independently evaluated from the PCR results Did the patient sample include an appropriate spectrum of patients to whom the diagnostic test will be applied in clinical practice? Did the patient sample include an appropriate spectrum of patients to whom the diagnostic test will be applied in clinical practice? –YES! –Neonates with GBS status unknown mothers

25. Are the results valid? Did the results of the test being evaluated influence the decision to perform the reference standard? Did the results of the test being evaluated influence the decision to perform the reference standard? –NO! –Reference standard and experimental test performed in all patients Were the methods for performing the test described in sufficient detail to permit replication? Were the methods for performing the test described in sufficient detail to permit replication? –YES! Procedures for cx and PCR very detailed

26. What are the results? Are likelihood ratios for the test results presented or data necessary for their calculation included? Are likelihood ratios for the test results presented or data necessary for their calculation included? –Sort of… –Authors state that pt and maternal characteristics/outcomes were compared in groups who had and did not have colonization, by culture and PCR, using t test, x 2 test and Fisher’s exact test to determine significance – results of these tests not presented, however data for calculation is included

27. What are the results? “Likelihood ratio” is similar to a chi square test, which is what was apparently used in this study to determine significance “Likelihood ratio” is similar to a chi square test, which is what was apparently used in this study to determine significance Authors do report specificity, sensitivity, PPV, and NPV Authors do report specificity, sensitivity, PPV, and NPV Having a high sensitivity and NPV are crucial to a good screening test Having a high sensitivity and NPV are crucial to a good screening test

28. What are the results? ▪ Negative predictive value is consistently high (with narrow CI’s) for all sites, which is especially useful when evaluating a screening test

29. What are the results? Sensitivity is another important measure for a screening test – a very sensitive test produces few false negatives Sensitivity is another important measure for a screening test – a very sensitive test produces few false negatives –PCR had high sensitivity for the ear and nose sites, however the CI’s were wide Sample size in this study is low (94) – increasing the sample size could decrease the standard error, which would narrow the CI and also increase the power Sample size in this study is low (94) – increasing the sample size could decrease the standard error, which would narrow the CI and also increase the power

30. Will the results help me in caring for my patients? Will the reproducibility of the test result and its interpretation be satisfactory in my setting? Will the reproducibility of the test result and its interpretation be satisfactory in my setting? –YES – results easily interpreted and reproducible Are the results applicable to my patient? Are the results applicable to my patient? –YES – common occurrence to have an infant with a GBS status unknown mother

31. Will the results help me in caring for my patients? Will the results change my management? Will the results change my management? –YES – advantages are that the test is noninvasive, easy to perform, and quick –Based on this study, ~50% of infants born to GBS unknown mothers are negative for GBS – this group could potentially need no additional testing or observation –However, preemies 32-35 weeks likely still do full eval/observation in most circumstances

32. Will the results help me in caring for my patients? Cost-benefit? Cost-benefit? –PCR test $26 (16-54) –Maternal prenatal culture $21 –Maternal antibiotic prophylaxis $63 (30-115) –In hospital care of well infant $1100 (500-1500) –NICU care of GBS infant $30100 (12000-128000) Primary goal would be to decrease hospital stay and nursing care Primary goal would be to decrease hospital stay and nursing care

33. Will the results help me in caring for my patients? Will patients be better off as a result of the test? Will patients be better off as a result of the test? –YES – prevent delayed discharge, decreased interventions in the immediate neonatal period, decrease costs

34. Questions? Questions?