1. WISCONSIN STATE LABORATORY OF HYGIENE 1 WSLH: Laboratory Update Tuberculosis Summit Verona, WI April 24, 2014 Julie Tans-Kersten, MS, BS-MT (ASCP) Tuberculosis Laboratory Program Coordinator Wisconsin State Laboratory of Hygiene tanskejl@mail.slh.wisc.edu (608) 263-5364

2. WISCONSIN STATE LABORATORY OF HYGIENE Laboratory Update Objectives Background Specimen Collection Specimen Preparation and Transport Testing performed at WSLH Reporting and Interpretation of Results, Expected turn-around times 2

3. WISCONSIN STATE LABORATORY OF HYGIENE 3 Mycobacteriology Testing at WSLH WSLH serves as a public health laboratory for the Wisconsin State Department of Public Health and Wisconsin local public health agencies. WSLH serves as a primary diagnostic facility and reference laboratory for clinicians and private mycobacteriology laboratories located throughout Wisconsin Full-service mycobacteriology laboratory Biosafety Level-3 facility

4. WISCONSIN STATE LABORATORY OF HYGIENE 4 Submission of Patient Specimens to WSLH for Mycobacteriology Testing

5. WISCONSIN STATE LABORATORY OF HYGIENE Specimen Quality is Important The results of tests, as they affect patient diagnosis and treatment, are directly related to the quality of the specimen collected and delivered to the laboratory. 5

6. WISCONSIN STATE LABORATORY OF HYGIENE Sputum Recently discharged material from the bronchial tree, with minimal amounts of oral or nasal material Expectorated: from deep productive cough Indications for sputum collection –To establish an initial diagnosis of TB –To monitor the infectiousness of the patient –To determine the effectiveness of treatment 6

7. WISCONSIN STATE LABORATORY OF HYGIENE Sputum Quality Specimens are thick and contain mucoid or mucopurulent material Ideally, 3–5 ml in volume, although smaller quantities are acceptable if the quality is satisfactory Poor quality specimens are thin and watery. Saliva and nasal secretions are unacceptable Laboratory requisition form should indicate when a specimen is induced to avoid the specimen being labeled as “unacceptable” quality Clinical and Laboratory Standards Institute. Laboratory detection and identification of mycobacteria; approved guideline. CLSI Document M48-A. Wayne, PA: CLSI; 2008. http://www.stoptb.org/wg/gli/assets/documents/29_specimen_condition_transport.doc 7

8. WISCONSIN STATE LABORATORY OF HYGIENE Sputum Quality Thick, Mucopurulent Hemoptysis Watery (induced?) Salivary 8

9. WISCONSIN STATE LABORATORY OF HYGIENE Sputum Collection Initial diagnosis of TB: Collect a series of three sputum specimens, 8-24 hours apart, at least one of which is an early morning specimen Optimally, sputum should be collected before the initiation of drug therapy Centers for Disease Control and Prevention. Guidelines for Preventing the Transmission of Mycobacterium tuberculosis in Health-Care Settings, MMWR 2005:54, RR-17 9

10. WISCONSIN STATE LABORATORY OF HYGIENE Sputum Collection Provide supervised sputum collection for at least the first sputum specimen, until the patient demonstrated the ability to properly collect the specimen. Use respiratory precautions when collecting sputum specimens All mycobacteria specimens are collected into a sealed leak proof container Label the specimen with patient name, collection date/time and specimen type. 10

11. WISCONSIN STATE LABORATORY OF HYGIENE Storage and Transport of Sputum Specimens Collection sites should refrigerate samples that cannot be transported immediately to reduce growth of contaminating organisms Specimens should be delivered to the laboratory as soon as possible, within 24 hours of collection is optimal (avoid batching) Recommended: include a cold pack with specimen transport materials 11

12. WISCONSIN STATE LABORATORY OF HYGIENE 12 Submission of Patient Specimens to WSLH for Mycobacteriology Testing  Insulated mailer with labels  Absorbent pad  Cold pack  Sterile plastic conical tube with label  Sealable biohazard specimen transport bag  Instruction sheet WSLH Respiratory Collection Kit #8 Order: 1-800-862-1088 Kits are free

13. WISCONSIN STATE LABORATORY OF HYGIENE 13 Submission of Patient Specimens to WSLH for Mycobacteriology Testing Requisition Form A Order: 1-800-862-1088 Preprinted with account number Submit one form with each specimen.

14. WISCONSIN STATE LABORATORY OF HYGIENE 14 Submission of Patient Specimens to WSLH for Mycobacteriology Testing CodeTest Description MM00250Mycobacteria (AFB) Smear and Culture MM00253Mycobacteria Isolate Identification MM00202Mycobacterium avium complex (MAC) susceptibility MM00204Mycobacterium tuberculosis susceptibility 1 st line drugs MM00207Mycobacteria rapid grower susceptibility MM00256Mycobacterium tuberculosis PCR

15. WISCONSIN STATE LABORATORY OF HYGIENE 15 Submission of Patient Specimens to WSLH for Mycobacteriology Testing Wrap specimen in absorbent material Place in zipper portion of biohazard bag and zip closed Place requisition form in rear pouch of biohazard bag Place bags and cold pack in insulated mailing container and seal with tape Label mailer with WSLH address and UN3373 (“Biological Substance Category B”) label Arrange for pre-paid Dunham Express Pick- up: 1-800-236-7127 (WSLH account 7271)

16. WISCONSIN STATE LABORATORY OF HYGIENE Mycobacteriology Testing Performed at WSLH Smear Microscopy PCR for Direct Detection Culture Identification Drug Susceptibility Testing 16

17. WISCONSIN STATE LABORATORY OF HYGIENE AFB Smear Microscopy Small amount of processed and concentrated patient specimen is placed on a microscope slide and stained acid-fast organisms Rapid and inexpensive screening tool Positive AFB smear results provide a first indication of mycobacterial infection and potential TB disease Must be accompanied by additional testing including culture for confirmatory diagnosis 17

18. WISCONSIN STATE LABORATORY OF HYGIENE AFB Smear Microscopy

19. WISCONSIN STATE LABORATORY OF HYGIENE AFB Smear Microscopy: Interpreting Results WSLH ReportGraded ScaleQualitative Scale Interpretation Negative Potentially infectious 1-9 AFB per 100 fields 1+Positive (rare)Low-level infectious 1-9 AFB per 10 fields 2+Positive (few) Moderately infectious 1-9 AFB per field 3+Positive (moderate) >9 AFB per field 4+Positive (many) Highly infectious 19

20. WISCONSIN STATE LABORATORY OF HYGIENE Limitations of AFB Smear Microscopy Does not distinguish between viable and dead organisms –Follow-up specimens from patients on treatment may be smear positive yet culture negative Limited sensitivity –High bacterial load 5,000-10,000 AFB /mL is required for detection –Misses >45% of U.S. TB cases Limited specificity –All mycobacteria are acid fast –Does not provide species identification –Local prevalence of MTB and NTM determine the predictive values of a positive smear for MTB 20

21. WISCONSIN STATE LABORATORY OF HYGIENE AFB Smear Microscopy Results Guide Decisions Clinical management –Patient therapy may be initiated for TB based on smear result and clinical presentation –Changes in smear status important for monitoring response to therapy Public health interventions –Smear status and grade useful for identifying the most infectious cases –Contact investigations prioritized based on smear result –Decisions regarding respiratory isolation based on smear result 21

22. WISCONSIN STATE LABORATORY OF HYGIENE PCR for Direct Detection 22

23. WISCONSIN STATE LABORATORY OF HYGIENE WSLH TB/MAC PCR Detection of M. tuberculosis complex and M. avium complex (MAC) directly from patient specimens Healthy People 2020 Goal: Identify new TB patients within 48 hours –Respiratory isolation –Start therapy Identify smear positive MAC patients –Release from isolation –Alter therapy decisions Presumptive rapid results for 59% of smear positive patients 23

24. WISCONSIN STATE LABORATORY OF HYGIENE WSLH TB/MAC PCR Specimen Requirements TestSpecimen Type Smear Result TB PCRRespiratory and non-respiratory Smear positive and smear negative MAC PCR Respiratory onlySmear positive only 24

25. WISCONSIN STATE LABORATORY OF HYGIENE Patient Criteria for Fee-Exempt TB/MAC PCR Testing Patient must have signs and symptoms of pulmonary TB Patient must be reported to the local or state public health department as a suspect TB case Patient must be in respiratory isolation Patient must not have been diagnosed with TB or a non-tuberculous mycobacterial infection within the last 12 months Patient must have received ≤7 days of anti- mycobacterial therapy or no such treatment within the last 12 months 25

26. WISCONSIN STATE LABORATORY OF HYGIENE 26 Interpretation of PCR Results WSLH Lab ReportInterpretation “Mycobacterium tuberculosis complex DNA detected” Positive for TB “Mycobacterium avium complex DNA detected” Positive for MAC “No Mycobacterium tuberculosis complex DNA detected” Negative for TB “No Mycobacterium avium complex DNA detected” Negative for MAC “Inhibitory substances that prevent nucleic acid amplification were detected” Test is of no diagnostic help

27. WISCONSIN STATE LABORATORY OF HYGIENE Culture for Mycobacteria 27

28. WISCONSIN STATE LABORATORY OF HYGIENE 28 Culture for Mycobacteria Detects viable mycobacteria from patient specimens Most sensitive method for detecting mycobacteria (“Gold standard”) Slowest Method –Average time to detection for TB = 15 days –Range for detection of TB: 8-30 days Smear and Culture always performed together Broth and solid media used to grow mycobacteria

29. WISCONSIN STATE LABORATORY OF HYGIENE 29 Automated system that uses a fluorescent method for detection of oxygen consumption Mycobacteria Growth Indicator Tube (MGIT) Solid media plate

30. WISCONSIN STATE LABORATORY OF HYGIENE Identification of Mycobacteria 30

31. WISCONSIN STATE LABORATORY OF HYGIENE Identification of Mycobacteria at WSLH Multifaceted approach –Colony morphology and pigment –High performance liquid chromatography (HPLC) –Matrix-Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) –DNA Probes (M. tuberculosis complex, M. avium complex, M. gordonae, M. kansasii) –DNA sequencing 31

32. WISCONSIN STATE LABORATORY OF HYGIENE 32 Interpretation of Culture Results Test ResultInterpretation “Negative for mycobacteria” No mycobacteria detected during 6 weeks of incubation “Isolated: M. tuberculosis complex” Confirmation of TB by culture

33. WISCONSIN STATE LABORATORY OF HYGIENE The laboratory identification of MTBC is the most important finding in the clinical mycobacteriology laboratory. The finding of this species has vital epidemiologic and public health consequences. –MTBC is not found in the environment –Isolation almost always signifies disease MTBC culture is important for conventional drug susceptibility testing Significance of MTBC Culture Results 33

34. WISCONSIN STATE LABORATORY OF HYGIENE Progress of TB treatment is measured by culture conversion Recommend 2 negative cultures by the end of 2 months (intensive phase) to document culture conversion. If cultures are still positive after 4 months, the patient is deemed to have failed treatment (patient management must be re-assessed) Patients with MDR-TB may be kept under airborne precautions until culture conversion is documented Culture Results for Patient Management 34

35. WISCONSIN STATE LABORATORY OF HYGIENE Susceptibility Testing 35

36. WISCONSIN STATE LABORATORY OF HYGIENE Drug Susceptibility Testing of M. tuberculosis complex isolates Automatically performed for all new culture-confirmed TB patients (no need to order) For “conventional” culture-based susceptibility testing –Need culture growth –Need pure growth

37. WISCONSIN STATE LABORATORY OF HYGIENE WSLH TB First Line drugs MGIT 960 broth system –INH (0.2 ug/ml) –INH (1.0 ug/ml) –rifampin (1.0 ug/ml) –ethambutol (5.0 ug/ml) –PZA (100 ug/ml) Repeat testing if resistant

38. WISCONSIN STATE LABORATORY OF HYGIENE CDC TB Drug Susceptibility Testing TB First-Line DrugsTB Second-line Drugs INH Rifampin Ethambutol PZA Streptomycin Rifabutin Ciprofloxacin Kanamycin Ethionamide Capreomycin PAS Ofloxacin Amikacin 38

39. WISCONSIN STATE LABORATORY OF HYGIENE 39 Interpretation of Drug Susceptibility Test Results ResultInterpretation SusceptibleStrain is likely to show responsiveness to the drug ResistantStrain is unlikely to show responsiveness to the drug IndeterminateTest is of no help in prediction of responsiveness to the drug

40. WISCONSIN STATE LABORATORY OF HYGIENE CDC Molecular Detection of Drug Resistance (MDDR) Program For rapid detection of drug resistance from cultures or smear positive patient specimens CDC performs sequencing to detect mutations that confer resistance –First-line drugs, fluoroquinolones, injectables Turn-around time is 2-3 days Requires CDC approval for submission –Patient must have risk factors for drug resistance Since 2010, we have rapidly detected all of our MDR-TB patients using this program 40

41. WISCONSIN STATE LABORATORY OF HYGIENE CDC MDDR Results Gene (region) examined ResultInterpretation rpoB (RRDR)No mutationRifampin susceptible inhA (promoter) No mutation INH resistant katG (ser315 codon) Mutation: Ser315Thr 41

42. WISCONSIN STATE LABORATORY OF HYGIENE Expected Turn-around Times Reporting Results 42

43. WISCONSIN STATE LABORATORY OF HYGIENE 43 Summary of WSLH Turn-Around- Times TestExpect Report Smear24 hours PCR24-48 hours CultureM. tuberculosis complex usually grows within 15 days. 6 weeks for final report

44. WISCONSIN STATE LABORATORY OF HYGIENE 44 Turn-Around Times for Drug Susceptibility Testing TestExpect Report Molecular Detection (CDC) 2-3 days from date of receipt at CDC TB first line conventional (WSLH) Average 30 days (range 16-98 days) TB second line conventional (CDC) 4-5 weeks from date of receipt at CDC

45. WISCONSIN STATE LABORATORY OF HYGIENE Testing Turn-around Times Primary specimen PCR 24-48 hours Culture 7-21 days Smear positive respiratory Identification 0-2 days TB first line drugs 4-20 days TB second-line drugs 3-4 weeks MDDR 2-3 days Conventional (Culture Based): 4-10 weeks Molecular: 4-6 days

46. WISCONSIN STATE LABORATORY OF HYGIENE WSLH Reporting of Laboratory Results All requested test results are reported to the submitter All reportable results are electronically transferred into the Wisconsin Electronic Disease Surveillance System (WEDSS). 46

47. WISCONSIN STATE LABORATORY OF HYGIENE 47 WSLH Reporting of Critical Laboratory Results Critical ValueNotification by telephone and fax Positive smear resultSubmitter and TB Program Positive PCR resultSubmitter and TB Program New positive cultures with M. tuberculosis complex isolated Submitter and TB Program Resistant TB first-line drug results (conventional or molecular) Submitter and TB Program

48. WISCONSIN STATE LABORATORY OF HYGIENE Summary Quality specimens are important for quality results Transport specimens ASAP using Dunham Express with cold pack Expect smear results in 24 hours Fee-exempt PCR testing is available for detection of MTBC and MAC directly from patient specimens Request CDC molecular detection of drug resistance for TB patients with risk factors for drug resistance Culture-based “conventional” testing for identification and susceptibility testing requires more time but is still considered the gold standard 48

49. WISCONSIN STATE LABORATORY OF HYGIENE For More Information Julie Tans-Kersten Wisconsin State Lab of Hygiene (608) 263-5364 Fax: (608) 890-2548 julie.tanskersten@slh.wisc.edu TB (Mycobacteriology) Lab: (608) 262-1618 Lorna Will, Philip Wegner, Pa Vang WI State TB Program 608-261-6319

50. WISCONSIN STATE LABORATORY OF HYGIENE 50 Dave Julie TK Julie B. Nate Youngmi and Ana WSLH Laboratory Team Don