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Detection of Group B streptococcus by SmartCycler

Detection of Group B streptococcus by SmartCycler. By: Josephine Soyemi. Objectives. State the type of organism Group B streptococcus (GBS) is State the two guidelines used for identifying candidates for treatment of GBS State the type of PCR used for detection of GBS

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Detection of Group B streptococcus by SmartCycler

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  1. Detection of Group B streptococcus by SmartCycler By: Josephine Soyemi

  2. Objectives • State the type of organism Group B streptococcus (GBS) is • State the two guidelines used for identifying candidates for treatment of GBS • State the type of PCR used for detection of GBS • State the genetic target of the BD GeneOhm™ strep B assay • State the type of probe used for detecting GBS

  3. Group B streptococcus (GBS) also known as streptococcus agalactiae is a catalase negative, gram-positive cocci arranged in pairs and chains Background health.uiowa.edu/CEID/Pathogen%20pages/GBStrep.html http://www.innermostsecrets.com/Left-Menu-Items/Pregnancy/Special-Investigations/Group-B-Streptococcus.aspx emedicine.medscape.com/article/228936-diagnosis

  4. Group B Streptococcus • Appears on blood plate as a 1- to 2-mm, gray-white colonies surrounded by a zone of β-hemolysis • Distinction of GBS from other streptococci • Resistance to bacitracin • Hydrolysis of sodium hippurate • CAMP test http://www.mullhaven.co.uk/gbs.htm

  5. Group B Streptococcus • Normal flora • Female genital tract • Lower gastrointestinal tract • May occasionally colonize the upper respiratory tract • 10% to 40% of pregnant women carry GBS asymptomatically in vagina or rectum http://www.suite101.com/view_image.cfm/767703

  6. Neonatal colonization • Early-onset infection • Occurs at less than 7 days of age • Usually vertically transmitted to neonate due to amniotic fluid infection or from the birth canal of a colonized mother • Late-onset GBS infection • Occurs at or beyond 7 days of age • Horizontal transmission through nosocomial spread in nursery by colonized nursery personnel or other colonized neonates www.glowm.com/?p=glowm.cml/section_view&articleid=179

  7. In 1996 the Center for Disease Control (CDC), in conjunction with American College of Obstetricians and Gynecologists (ACOG) and American Academy of Pediatrics (AAP) recommends Screening – based strategy GBS carriers at 35-37 weeks gestation, premature onset of labor or preterm membrane rupture, should be offered Intrapartum antimicrobial prophylaxis (IAP) Risk – based strategy Women who develop one or more risk conditions at the time of labor or membrane rupture should receive IAP Guidelines for treatment

  8. PCR Amplification

  9. Real-time PCR Systems Improved Sensitivity & Specificity Permits rapid target identification; 15- 30 min Permits quantification Develop multiplex assays Diagnostic tool Eliminate other technologies; Northerns, Southerns, RPAs, etc Real-Time PCR

  10. BD GeneOhm™ strepB assay Genetic target is cfb gene which is present in virtually all GBS isolates More sensitive than the standard selective culture method Less than 1 hour protocol Contains the sample buffer, master mix, positive control, negative control, internal control (IC) and diluents Positive control Monitors substantial reagent failure Negative control Detect reagent or environmental contamination (carry over) by either S. agalacitae or GBS amplicons Internal control Detects PCR inhibitory specimens and confirms the integrity of assay reagents Real-Time PCR Methods

  11. Specimen requirement for Assay • Recommended swab • BBL cultureswab with liquid stuart medium (LSM) • Copan Transystem with LSM • Copan Venturi Transystem with LSM • HealthLink TransPorter with (LSM) www.biosearchtech.com

  12. BD GeneOhm StrepB Test Procedure • Specimen Preparation • Place swab in sample buffer tube (blue cap) • Break swab • Let stand, then vortex at high speed briefly • Lysis – DNA Extraction • Transfer sample to the lysis tube (yellow cap) • Vortex, centrifuge briefly, then heat to inactivate potential inhibitors • Place lysis tube on a cooling block http://www.bd.com/geneohm/english/products/pdfs/StrepB_Test_Procedure.pdf

  13. Test procedure continue • Reconstitution of Master Mix • Reconstitute 1 master mix + 1 positive control + 1 negative control per assay run: • Add diluent (A) to the lyophilized master mix (C) and controls • Transfer lysate solution (B) to master mix reaction tube (C) • Centrifuge and vortex briefly • Place reaction tubes on specially adapted cooling block until ready to load http://www.bd.com/geneohm/english/products/pdfs/StrepB_Test_Procedure.pdf

  14. Real time PCR Analysis • Real-Time PCR Analysis • Insert each reaction tube into the instrument • Start run and obtain results in less than 45 minutes • Assay Performance • Sensitivity: 94% • Specificity: 96% • Negative Predictive Value: 99% • Positive Predictive Value: 84% http://www.bd.com/geneohm/english/products/pdfs/StrepB_Test_Procedure.pdf

  15. Real-Time PCR Analyzer Smartcycler System Smartcycler reaction tube and I-core http://www.cepheid.com/product-catalog/systems/smartcycler/

  16. Molecular Beacon Method • Amplified DNA targets are detected with molecular beacons • hairpin-forming single-stranded oligonucleotides labeled at one end with a quencher and at the other end with a fluorescent reported (fluorophore) • In the absence of target • the fluorescence is quenched • In the presence of the target • the hairpin structure opens upon beacon/target hybridization, resulting in emission of fluorescence www.molecular-beacons.org

  17. Molecular Beacon Method • Detection of GBS amplicons • molecular beacon contains the fluorophore FAM at the 5’ end and the non-fluorescent quencher moiety DABCYL at the opposite end of the oligonucleotide • Detection of the IC amplicons • molecular beacon contains the flourophore TET at the 5’ end and the quencher DABCYL 3’ end • Signal intensity is directly proportional to the amount of amplified DNA www.molecular-beacons.org

  18. Quantification Plateau Log-Linear Exponential Fluorescentsignal Baseline

  19. Light emitted from dye in excited state is received by a computer and shown on a graph display PCR cycles on the X-axis and a logarithmic indication of intensity on the Y-axis Quantification www.biosearchtech.com www.biosearchtech.com

  20. Recommended swabs with transport system must be used Improper specimen collection Presence of inhibitor Technical error Sample mix-up Number of organisms in the specimen organisms present at less than 105 organisms Mutation in primer or probe binding regions may affect detection Limitations

  21. Interfering substances • Includes • Amniotic fluid • Blood • Feces • Lubricant • Urine • Vaginal mucosa • Excessive vaginal secretions may inhibit PCR result • Presence of excessive blood • Concurrent antibiotics therapy

  22. Conclusion • GBS has emerged as the leading cause of neonatal morbidity and mortality • septicemia, pneumonia, meningitis and visual and auditory problems • Sensitivity of cultures in detecting GBS colonization varies from 54% to 87% • slow turnaround time requiring up to 36-72 hours • The BD GeneOhm™ Streptococcus group B real time PCR is a very sensitive and specific test • yield results in less than one hour

  23. Thank you Questions?

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