WSLH: Laboratory Update Tuberculosis Summit Verona, WI April 24, 2014

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. Laboratory UpdateObjectives • Background • Specimen Collection • Specimen Preparation and Transport • Testing performed at WSLH • Reporting and Interpretation of Results, Expected turn-around times

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. Submission of Patient Specimens to WSLH for Mycobacteriology Testing

5. 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.

6. 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

7. 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

8. Sputum Quality Thick, Mucopurulent Watery (induced?) Hemoptysis Salivary

9. 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

10. 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.

11. 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

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

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. Submission of Patient Specimens to WSLH for Mycobacteriology Testing

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. Mycobacteriology Testing Performed at WSLH • Smear Microscopy • PCR for Direct Detection • Culture • Identification • Drug Susceptibility Testing

17. 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

18. AFB Smear Microscopy

19. AFB Smear Microscopy: Interpreting Results

20. 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

21. 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

22. PCR for Direct Detection

23. 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

24. WSLH TB/MAC PCR Specimen Requirements

25. 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

26. Interpretation of PCR Results

27. Culture for Mycobacteria

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. Mycobacteria Growth Indicator Tube (MGIT) Automated system that uses a fluorescent method for detection of oxygen consumption Solid media plate

30. Identification of Mycobacteria

31. 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

32. Interpretation of Culture Results

33. Significance of MTBC Culture Results • 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

34. Culture Results for Patient Management • 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

35. Susceptibility Testing

36. 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. 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. CDC TB Drug Susceptibility Testing

39. Interpretation of Drug Susceptibility Test Results

40. 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

41. CDC MDDR Results

42. Expected Turn-around TimesReporting Results

43. Summary of WSLH Turn-Around-Times

44. Turn-Around Times for Drug Susceptibility Testing

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

46. 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).

47. WSLH Reporting of Critical Laboratory Results

48. 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

49. 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. WSLH Laboratory Team Nate Youngmi and Ana Dave Don Julie B. Julie TK