1. Management of Streptococcal Pharyngitis: Role of the Laboratory and POC Testing Arthur E. Crist, Jr., Ph.D. Director, Clinical Microbiology York Hospital

2. Outline n Organism n Nomenclature n Pathogenesis & Spectrum of Disease n Clinical Diagnosis n Laboratory Diagnosis –Culture –Rapid Antigen Testing –Molecular Methods

3. Gram Stain – Blood Culture

4. Structure

5. Extracellular products: Pyrogenic exotoxin = erythrogenic toxin scarlet fever, toxic shock lysogenic bacteriophage, Spe-A,B,C,F, Ssa. Hemolysins streptolysin Ostreptolysin S O 2 sensitiveO 2 resistant antigenic (AST) non antigenic Enzymes DNases A-DHyaluronidase Streptokinase NADase

6. Classification

7. Beta hemolysis on SBA

8. Ignaz P. Semmelweiss

9. Joseph Lister

10. Why Diagnose Streptococcal Pharyngitis? Treatment Prevents Sequelae Treatment Prevents Sequelae - Rheumatic Fever - Rheumatic Fever - Scarlet Fever - Glomerulonephritis - Invasive Streptococcal Disease - (Flesh-eating Strep)

11. Local Infections n Pharyngitis - Winter-Spring months - School aged child, esp. 3-10 years - Abrupt onset n Pyoderma (Impetigo) n Otitis Media n Sinusitis

12. Streptococcal Sore Throat

13. Impetigo

14. Invasive Infections n Erysipelas n Cellulitis n Necrotizing Fasciitis n Puerperal Fever n Sepsis n Endocarditis n Scarlet Fever n Toxic Shock Syndrome

15. Erysipelas

16. Cellulitis

17. Necrotizing Fasciitis

18. Nonsuppurative Sequelae n Acute Rheumatic Fever –1 to 5 weeks following acute pharyngitis –6 to 15 years of age –Attack rate 0.4% (civilian) to 3% (military) –M types 1,3,5,6,12,18,19,24 –Inflammation of the heart (heart valves), joints, blood vessels, and subcutaneous tissues –Probably immunological (autoimmune?) –Recurrences are common

19. Nonsuppurative Sequelae n Acute Glomerulonephritis –Approx. 10 days following acute pharyngitis; (3 weeks following pyoderma) –Winter/Spring (Summer/Fall-pyoderma) –6 to 15 years of age (preschool-pyoderma) –Attack rate 10-15% with known neph. strain –Limited M types involved –Probably immunological (immune complex) –Recurrences are rare

20. Which Streptococci Cause Sore Throat? n Group A streptococcus (S. pyogenes) n Group C streptococcus (S. dysgalactiae subsp. equisimilis) n Group G streptococcus (S. dysgalactiae subsp. equisimilis)

21. Signs of Streptococcal Tonsillopharyngitis n Characteristic signs -Tonsillopharyngeal erythema -Tonsillopharyngeal exudate -Soft-palate petechiae (doughnut lesions) -Beefy red, swollen uvula -Anterior cervical lymphadenitis -Scarlatiniform rash n Uncharacteristic signs -Conjunctivitis -Anterior stomatitis -Discrete ulcerative lesions

22. Symptoms of Streptococcal Tonsillopharyngitis n Characteristic symptoms -Sudden onset of sore throat -Headache -Pain on swallowing -Abdominal pain -Fever -Nausea and vomiting n Uncharacteristic symptoms -Coryza -Cough -Hoarseness -Diarrhea (Bisno, NEJM)

23. Clinical Diagnosis of Streptococcal Pharyngitis n Based on signs and symptoms n Not very accurate n 50% of patients with pharyngitis will be treated but will not be infected with Group A Streptococci n 30% of patients with pharyngitis will not be treated but will be infected with Group A Streptococci

24. Microbial Causes of Acute Pharyngitis (Bisno, NEJM) Pathogen Estimated Percentage of Cases Viral Rhinovirus (100 types and 1 subtype)20 Coronavirus (3 or more types) 5 Adenovirus (types 3, 4, 7, 14, and 21)5 Herpes simplex virus (types 1 and 2)4 Parainfluenza virus (types 1-4)2 Influenzavirus (types A and B)2 Coxsackievirus A (types 2, 4-6, 8, and 10)<1 Epstein-Barr virus<1 Cytomegalovirus<1 Human immunodeficiency virus type 1<1

25. Microbial Causes of Acute Pharyngitis (Bisno, NEJM) Pathogen Estimated Percentage of Cases Bacterial Streptococcus pyogenes (Group A -hemolytic streptococci) 15-30 Group C -hemolytic streptococci 5 Neisseria gonnorrhoeae <1 Corynebacterium diphtheriae <1 Arcanobacterium haemolyticum 1 Chlamydial Chlamydia pneumoniae Unknown Mycoplasmal Mycoplasma pneumoniae <1

26. Laboratory Diagnosis n Culture –Specimen Collection (10% FN) –Gold Standard ?? –Selective vs. non-selective medium –Aerobic, 5-10% CO 2, anaerobic atmosphere –1 or 2 day incubation –Broth enhancement

27. CP1069709-1 Total positive cultures (%) Days SXT plus blood agars Blood agar plate only Aggregate antigen detection tests n=261 n=189 n=106 n=151 n=187 Cockerill. 2002. Mayo Clinic Data. AACC Internet Presentation

28. Culture Methods Roddey, et al. 1995. JAMA 274: 1863-1865

29. Presumptive Identification

30. Definitive Identification (Biochemical or Serological) Definitive Identification (Biochemical or Serological)

31. Positive PYR Test

32. Rapid Antigen Tests n Extract (acid) cell wall antigen from organism on throat swab n Detect presence of extracted antigen by –Latex agglutination (LA) –Enzyme immunoassay (EIA) –Optical immunoassay (OIA)

33. Rapid Antigen Test

34. FDA Approved Kits n 40 CLIA High Complexity (1993-1999) Effective Date –Abbott TestPack Plus Strep A 01-11-1999 –BD Directogen 1-2-3 Group A Strep 10-27-1995 –J & J CDI SureCell 10-06-1995 –Gen-Probe Group A Strep Direct 06-10-1994 n Almost all are used to type organisms from culture n Gen-Probe most frequently used direct specimen test in this group

35. FDA Approved Kits n 77 CLIA Moderate Com. (1993-2002) Effective Date –Abbott Signify Strep A03-08-2002 –Quidel Quickvue Strep A Test03-05-2002 –Abbott TestPack Plus Strep A w/OBC II01-11-1999 –Binax Strep A Test04-19-1996 –J & J CDI SureCell10-06-1995 –Abbott TestPack Plus Strep A 07-26-1993 –Baxter MicroScan Cards O.S.07-26-1993 –BD Directogen 1-2-3 Grp A Strep07-26-1993 –Binax Equate Strep A 07-26-1993 –BioStar Strep A OIA07-26-1993 –Hybritech ICON Strep A 07-26-1993 –Meridian Diagnostics ImmunoCard07-26-1993 –Quidel Group A Strep07-26-1993 –Wampole Bactigen Group A Strep07-26-1993

36. FDA Approved Kits n 24 CLIA Waived (1996-2002) Effective Date –Genzyme OSOM Ultra Strep A Test07-19-2002 –Quidel Quickvue In-Line Strep A07-08-2002 –Acon Strep A Rapid Test Strip12-18-2001 –Beckman Coulter ICON DS Strep A12-18-2001 –Quidel Quickvue Dipstick Strep A 07-26-2001 –ICON DS Strep A07-17-2001 –Beckman Coulter ICON FX Strep A03-16-2001 –Wyntek Diagnostics OSOM Ultra Strep A07-06-2000 –Fisher Sure -Vue Strep A09-22-1999 –Meridian ImmunoCard STAT Strep A05-19-1998 –Abbott Signify Strep A Test09-22-1997 –Biostar Acceava Strep A Test07-09-1997 –Binax NOW Strep A Test01-10-1997

37. Factors Affecting ADT Sensitivity n Culture methods selected in a study influence the calculated ADT sensitivities n Studies are likely to report higher ADT sensitivities when cultures are performed in physician offices n When culture methods result in identification of very small numbers of GABHS, the calculated sensitivity of an ADT will be lower than that calculated when a less rigorous culture method is used because ADTs are likely to fail to detect low numbers of GABHS

38. Comparison of a CLIA Waived & Moderately Complex Test Mod. Complex ADT Mod. Complex ADT Sens. (%) Spec. (%) Sens. (%) Spec. (%) Waived ADT91 93 Gold Standard Gold Standard Sens. (%) Spec. (%) Sens. (%) Spec. (%) Waived ADT80 95 Gold Standard Gold Standard Sens. (%) Spec. (%) Sens. (%) Spec. (%) Mod. Com ADT82 100

39. Comparison of the Abbott Signify Group A Test to Culture – York Hospital Dec. 1999 to Mar. 2000 (n=397) n Study Sens. (%) Spec. (%) PVP (%) PVN (%) __________________________________________________________________ n YH* 82.9 97.8 90.0 96.0 n PI** 96.0 97.8 ND ND *York Hospital *York Hospital ** Package Insert ** Package Insert

40. Sensitivity and Specificity of the OIA Study Sensitivity (%) Specificity (%) Harbeck 1993 97.495.6 Harbeck 1993 98.998.4 Dale1994 8197.5 Roe1995 8389 Heiter1995 92.395.4 Fries1995 9498 Harris 1995 9694 Baker1995 7890 Gerber 1997 8493 Hart1997 7762 Pitetti 1998 79.596.5 Kuhn1999 8996.5 Chapin2002 86.197.1

41. Comparison of Test Sensitivity Cockerill. 2002. Mayo Clinic Data. AACC Internet Presentation

42. Conclusions: Rapid Antigen Kit Analytical Sensitivity Comparison Detection of different isolates by different tests showed no differences Analytical Sensitivities were best for the OIA (3.3 x 10 4 CFU) and worst for Directigen (5 x 10 5 CFU) Cockerill. 2002. Mayo Clinic Data. AACC Internet Presentation

43. Molecular Tests for Detection of Group A Streptococcus from Throat Swabs - Not Point-of-Care Group A Streptococcus Direct Test (Gen-Probe) PCR – Conventional or Real Time

44. Gen-Probe Group A Streptococcus Direct Test Test detects rRNA sequences using a chemiluminescent single-stranded DNA probe Enhanced sensitivity vs. Antigen detection methods

45. Percent TestSensitivitySpecificity Positive predictive value Negative predictive value Heiter 199393.5 99.799.0 97.8 Pakorski 1994 85.7 97.883.9 98.1 Chapin 200294.8 10010096.9 Comparison Of The Gen-Probe Test and Culture

46. Polymerase Chain Reaction Unamplified DNA Denature and anneal primers Primer extension Denature and anneal primers Cycle 0 Cycle 1 Targeted sequence Cycle 2 Cycle 3 Denature and anneal primers Primer extension Cycles 4-25 At least 10 5 -fold increase in DNA

47. Comparison of PCR to Other Methods Test Sensitivity (%) Specificity (%) PVP (%) PVN (%) ____________________________________________________________________ OIA 76 92 85 87 Agar culture 79100 100 90 Both-enhanced cult. 86 100 100 93 Combined cult. 89100 100 94 OIA & agar cult. 89 92 86 94 PCR 96100 100 98 Kaltwasser, et al. 1997. Pediatr. Infect. Dis. J. 16: 748-753

48. Management of Streptococcal Pharyngitis Management of Streptococcal Pharyngitis Hofer, et al. 1997. Arch. Pediatr. Adolesc. Med. 151: 824-829.

49. Management of Streptococcal Pharyngitis Management of Streptococcal Pharyngitis % Physicians Ordering Test _____________________________________________________________________ Private HMO Residents Total (n=50) (n=50) (n=50) (n=150) ____________________________________________________________ % Physicians Ordering Test _____________________________________________________________________ Private HMO Residents Total (n=50) (n=50) (n=50) (n=150) ____________________________________________________________ Case A Throat culture 100 100 100 100 20-minute ADT 74 90 88 84 4-hour ADT 32 46 40 39 Case B Throat culture 80 64 66 70 Throat culture 80 64 66 70 20-minute ADT 54 54 54 54 4-hour ADT 18 30 20 23 20-minute ADT 54 54 54 54 4-hour ADT 18 30 20 23 Berwick, et al. 1987. Pediatr. Infect. Dis. J. 6: 1095-1102 Berwick, et al. 1987. Pediatr. Infect. Dis. J. 6: 1095-1102

50. Management of Streptococcal Pharyngitis Management of Streptococcal Pharyngitis % of Physicians Who Believe ADT _____________________________________________________________________ Private HMO Residents Total (n=40) (n=43) (n=36) (n=119) ____________________________________________________________ % of Physicians Who Believe ADT _____________________________________________________________________ Private HMO Residents Total (n=40) (n=43) (n=36) (n=119) ____________________________________________________________ Case A Positive 98 93 94 95 Negative 42 42 50 45 (n=44)(n=45) (n=43) (n=132) (n=44)(n=45) (n=43) (n=132) Case B Positive 89 93 72 85 Positive 89 93 72 85 Negative 73 82 79 78 Negative 73 82 79 78 Berwick, et al. 1987. Pediatr. Infect. Dis. J. 6: 1095-1102 Berwick, et al. 1987. Pediatr. Infect. Dis. J. 6: 1095-1102

51. Management of Streptococcal Pharyngitis Effect of ADT Availability on Treatment Management of Streptococcal Pharyngitis Effect of ADT Availability on Treatment Patients Treated (per100) _____________________________________________________________________ Private HMO Residents Total (n=50) (n=50) (n=50) (n=150) ____________________________________________________________ Patients Treated (per100) _____________________________________________________________________ Private HMO Residents Total (n=50) (n=50) (n=50) (n=150) ____________________________________________________________ Case A None 92 84 82 86 20-minute ADT 70 59 67 65 4-hour ADT 82 68 75 75 20-minute ADT 70 59 67 65 4-hour ADT 82 68 75 75 Case B None 19 6 8 11 None 19 6 8 11 20-minute ADT 17 16 16 16 4-hour ADT 19 10 11 13 20-minute ADT 17 16 16 16 4-hour ADT 19 10 11 13 Berwick, et al. 1987. Pediatr. Infect. Dis. J. 6: 1095-1102 Berwick, et al. 1987. Pediatr. Infect. Dis. J. 6: 1095-1102

52. Conclusions - Rapid Testing for Streptococcal Infection Rapid Antigen Detection (LA, EIA, or OIA-based, point-of-care) - Culture if Rapid Antigen Result Negative Molecular methods, e.g. Real-time PCR, may equal or exceed sensitivity provided by culture (American Academy of Pediatrics) (Infectious Disease Society of America)

53. Any Questions?