WSLH Testing and Surveillance Updates

Transcription

1 WSLH Testing and Surveillance Updates Wisconsin Mycobacteriology Laboratory Network annual conference November 4, 2015, Madison, WI

2 Updates Outline Collection and Transport Smear and Culture Nucleic Acid Amplification Testing (NAAT) Identification of Mycobacteria at WSLH Molecular Detection of Drug Resistance Susceptibility testing at WSLH MTBC NTM 2014 WI laboratory surveillance

3 Specimen Collection and Transport

4 Transport Updates Recommend cold pack for transport of sputum to the laboratory Assessing State-wide courier service to assure optimal delivery of specimens Free courier service is available: Dunham Express WSLH no fee account is #7271

5 Submission of Patient Specimens to WSLH for Mycobacteriology Testing WSLH Respiratory Collection Kit #8 Order: Kits are free Insulated mailer with labels Absorbent pad Cold pack Sterile plastic conical tube with label Sealable biohazard specimen transport bag Instruction sheet 5

6 Smear and Culture Updates Sputum Recommended volume of sputum is 5-10 ml (no less than 3 ml). Specimen will be rejected if 1 ml of clear liquid is received Blood no longer performing smear on peripheral blood specimens due to very low bacterial concentrations in these specimens For other specimen requirements see the WSLH Clinical Testing reference manual hp

7 Nucleic Acid Amplification Testing (NAAT) Updates

8 WSLH Nucleic Acid Amplification Testing (NAAT) Updates Respiratory Non- Respiratory Smear Positive TB and MAC PCR Smear Negative* TB PCR TB and MAC PCR TB PCR (*) Smear Negative TB PCR testing is fee-exempt if patient is considered a TB suspect and reported to the State TB Program

9 WSLH Nucleic Acid Amplification Testing (NAAT) Updates Mycobacterium tuberculosis complex (MTBC) includes: M. tuberculosis, M. bovis, M. bovis BCG, M. caprae, M. microti, M. africanum, M. canettii, M. pinnipedii, and M. mungi M. avium complex includes: including M. avium, M. avium subsp. avium, M. avium subsp. avium M. avium subsp. paratuberculosis, M. intracellulare, M. chimaera, M. arosiense, M. colombiense, M. marseillense, M. bouchedurhonense, and M. timonense.

10 WSLH Nucleic Acid Amplification Testing (NAAT) Updates For diagnostic purposes, best practice to perform smear and culture even if PCR/NAAT is being performed Culture is more sensitive than PCR Culture needed for phenotypic (culture-based) drug susceptibility testing

11 WSLH Initial Smear Positive Primary Specimens 11

12 WSLH Nucleic Acid Amplification Testing (NAAT) Updates Year Number of TB PCR tests performed (49.5%) smr (56.4%) smr+ Number of patients tested by TB PCR Number of positive TB PCR results % of culturepositive pulmonary TB patients identified in 48 hours /37 = 67% (3 positive at ACL) /31= 77% (2 positive at DynaCare)

13 Sensitivity of TB PCR, initial specimens on culture-positive TB patients Smear Positive Smear Negative Total Respiratory 172/178 (96.6%) Non-respiratory 18/18 (100%) 17/27 (62.9%) 1/6 (16.6%) 189/205 (92.1%) 19/24 (79.1%) Total 190/196 (96.9%) 18/33 (54.5%) 208/229 = 90.8% WSLH data, 9/2010 to 6/2015

14 Sensitivity of MAC PCR, on culturepositive MAC patients 1+ Smear Positive 2+ Smear Positive 3+ Smear Positive 4+ Smear Positive Overall Respiratory* 7/22 (31.8%) 5/9 (55.5%) 5/6 (83.3%) 13/15 (86.6%) 30/52 (57.6%) Nonrespiratory 11/13 (84.6%) (*) WSLH data, Primary only 8/2013 to 6/2015

15 State-wide NAAT Availability WSLH: Laboratory-developed real-time PCR for MTBC and MAC Milwaukee City Health Department Laboratory: Cepheid Xpert MTB/RIF Wisconsin Diagnostic Laboratories (Dynacare), Milwaukee: Cepheid Xpert MTB/RIF ACL (Rosemont, IL) real-time PCR for detection of MTBC

16 Mycobacteria ID

17 WSLH Mycobacteria Identification WSLH uses a combination of MALDI-TOF, PCR and DNA sequencing for identification No longer performing HPLC No longer performing Accuprobe testing No longer performing biochemical reactions for ID Advantages and challenges to our new testing algorithm

18 WSLH Complicated ID Algorithm Specimen Triage Positive Culture containing AFB Amount of growth Patient history Time to positive in MGIT instrument TB/MAC PCR MALDI-TOF Inconclusive results 16S and rpob DNA sequencing Report Conclusive Results 18

19 Detection of Drug Resistance

20 Universal Molecular Detection of Drug Resistance for new TB patients MTBC-positive sediment or culture Patients with known risk factors for drug resistance Patients without known risk factors for drug resistance CDC MDDR Program Pyrosequencing and Sanger sequencing for detection of mutations that confer resistance Milwaukee City Health Department Laboratory Cepheid GeneXpert for detection of mutations that confer resistance 20

21 Molecular Detection of Drug Resistance for MTBC CDC MDDR Program City of Milwaukee Laboratory Method Used Initial molecular analysis Additional molecular analysis Pyrosequencing and Sanger sequencing INH and rifampin ethambutol, PZA, fluoroquinolones, injectables Cepheid GeneXpert rifampin

22 Still Need Culture-Based DST If we have molecular methods to detect resistance, why do we need to perform culture-based testing? Presence of a mutation does not always result in phenotypic resistance A wild-type sequence does not always confirm that a strain is susceptible We do not know every possible locus leading to resistance for any antibiotic 22

23 Culture-Based Susceptibility Testing for MTBC Drugs Tested Method Turn-Around Time (after positive culture) TB First-Line Drugs (WSLH and CDC) isoniazid (INH) 0.2 ug/ml isoniazid (INH) 1.0 ug/ml rifampin 1.0 ug/ml ethambutol 5.0 ug/ml pyrazinamide 100 ug/ml MGIT broth method (WSLH) Agar proportion method (CDC) 7-20 days 3 weeks TB Second-Line Drugs (CDC only) capreomycin (10ug/ml) ethambutol (10 ug/ml) ethionamide (5 ug/ml) kanamycin (6 ug/ml) ofloxacin 2 ug/ml) PAS (2 ug/ml) rifabutin (0.5 ug/ml) streptomycin 2 ug/ml) streptomycin (10 ug/ml) Bedaquiline (MIC) Agar proportion method 23

24 Second/third-line TB drugs Tested at National Jewish, Denver, CO Quinolones: Moxifloxacin Levofloxacin Ciprofloxacin Clofazimine Cycloserine Linezolid

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

26 NTM Drug Susceptibility Testing

27 Organism NTM DST at WSLH Test Code Drugs Tested M. avium complex MM00202 clarithromycin Rapidly-growing mycobacteria (e.g. M. fortuitum group, M. chelonae/abscessus group) MM drug panel amikacin, cefoxitin, ciprofloxacin, clarithromycin, doxycycline, imipenem, linezolid, moxifloxacin, trimethoprimsulfamethoxazole, tobramycin.

28 NTM DST at National Jewish, Denver CO Organism Test Code Drugs Tested M. avium complex slow-grower NTM NTM10 10 drug panel clofazimine, ciprofloxacin, moxifloxacin, amikacin, streptomycin, rifabutin, linezolid, clarithromycin, rifampin, ethambutol, rifampin-ethambutol synergy

29 NTM DST at National Jewish, Denver CO Organism Test Code Drugs Tested Rapidlygrowing mycobacteria Rapidlygrowing mycobacteria NTM4 15 drug panel NTM6 20 drug panel amikacin, kanamycin, tobramycin, cefoxitin, imipenem, ciprofloxacin, doxycycline, moxifloxacin, tigecycline, clarithromycin, azithromycin, augmentin, trimethoprim-sulfamthoxazole, linezolid, clofazimine, amikacinclofazimine synergy 15 drug panel plus: gentamicin, ceftriaxone, cefepime, cefotaxime, minocycline.

30 Mycobacteria Surveillance

31 State-wide Laboratory-based Mycobacteria Surveillance, 2014 ACL, West Allis Mayo (Franciscan Skemp), La Crosse Mayo (Luther), Eau Claire Gundersen, La Crosse Marshfield Laboratories Milwaukee City Health Department Lab Theda Care Laboratories, Appleton Waukesha ProHealth Wisconsin Diagnostic Laboratories, Milwaukee Zablocki VA, Milwaukee

32 (12.5%) (14%) (2.9%) (61%) State-wide Mycobacteria Surveillance, isolates

33 State-wide Mycobacteria Surveillance, 2014 Species Number of Isolates (percentage of total) Includes tuberculosis complex 44 (2.9) tuberculosis, bovis, bovis BCG avium complex 921 (61) avium, chimaera, intracellulare Rapid growers 210 (14) abscessus, chelonae, fortuitum, mucogenicum,, peregrinum, phocaicum gordonae 188 (12.5) Slow growers 68 (4.5) kansasii, marinum, terrae complex, xenopi Other mycobacteria 30 (2) Aerobic actinomycetes/afb 37 (2.4) Actinomadura, Gordonia, Nocardia, Streptomyces, Tsukamurella TOTAL 1498

34 State-wide Mycobacteria Surveillance, Culture-confirmed TB First Line Drug Results MTBC DST, 2014 Susceptible to all drugs 33 INH resistant 2 PZA resistant MDR TB (resistant to at least INH and RIF) TOTAL 39 Number of patients 1 (bovis) 2

35 Essentials for the Mycobacteriology Laboratory: Promoting Quality Practices Free interactive modules developed by APHL and CDC Safety Specimen collection, transport, handling, processing AFB smear microscopy Culture, ID DST Molecular 101 Molecular detection Molecular DST 35

36 New Building?? UW-Madison Campus Southeast side of Madison

37 WSLH TB Lab Team Ali Lopez Dave Warshauer Youngmi Kim Ana Guaracao Julie Brockman Don Busalacchi Julie Tans-Kersten

38 For More Information Julie Tans-Kersten Wisconsin State Lab of Hygiene (608) Fax: (608) TB (Mycobacteriology) Lab: (608)