Mycobacteria Diagnostic Testing in Manitoba Dr. Michelle Alfa Medical Director, DSM Clin Micro Discipline
Acknowlegements: Assunta Rendina: Charge Tech HSC Lab Joyce Wolf & Dr. Meenu Sharma: NML Dr. Kanchana Manickam: DSM Section Lead Mycobacteriology
Overview: Provincial TB services: - Sites offering services - Benchmarks for TATs Genetic resistance testing -new service Followup for TB patients: - Time to smear negative Future: Direct TB testing
TB. or Not TB?? That is the question Pathogen: M.tuberculosis Transmission: Airborne Disease: -Active TB disease - Latent TB Infection (LTBI) ~5% active TB Diagnosis of Active TB: Culture is the Gold standard Respiratory Non-Respiratory
TB Culture in Manitoba: DSM Cadham Provincial Lab: Rural site specimen referral point St. Boniface Hospital Site: AFB, Culture, Probe, ID & Sens HSC Health Sciences Centre Site: Full service: AFB, Culture, Probe, Sensitivity testing, ID NML National Microbiology Lab: M.tuberculosis ID Sensitivity testing
Processing & smear volumes to maintain competency [MMWR Nov 4, 2005;volume 54:RR-12] Competency Benchmarks: - Smears: 10-15 AFB smears/week (~500-750/yr) - Cultures: 20 AFB cultures/week (~1000/yr) Manitoba: ~ 10,000 specimens processed/year - HSC: ~8000/year (includes rural, WL & Wpg private labs) - SBGH: ~2000/year
Specimen delivery time-frame Manitoba Sites Site: WRHA Winnipeg (private) Rural North of Gimli Rural South of Gimli Average # days for Specimen transit (range): 1 (1 day) 3 (1-8 days) 4 (1-9 days) 2 (1-7 days) Longer transit times cause delays in reporting risk of TB transmission
MTB Benchmarks for Transport & Turn Around Time CDC/ATS MMWR Nov 4, 2005;volume 54:RR-12 AFB Smear: 24 hours from receipt of specimen Culture (detect growth & identify isolate by probe): 21 days from receipt of specimen Antimicrobial Susceptibility Testing (AST) results: 30 days from receipt of specimen
Specimen Collection/Processing: Suspected Respiratory TB Three early morning sputum samples (refridgerate if transit > 24 hrs) Induced sputum Bronchoalveolar Lavage (BAL) Gastric aspirate Liquid & Solid media Automated: liquid culture Decontaminated & concentrated Microscopy Solid media: slow Kinyoun AFB stain
Index Cases: TB ~34% are AFB smear positive from concentrated sample Smear TAT meets 1 day benchmark except for weekends (range 1 3 days) Average time to detect growth: 16.6 days, MTB complex probe same day (meets 21 day benchmark) MP bottles sent to NML (Level III containment) for AST and ID of M.tuberculosis
Testing at NML: Subculture MP bottle to MGIT bottle After 3-5 days growth in MGIT: confirm ID as M.tuberculosis detect resistance mutations (takes ~3 days to complete) set up AST for 1 st line agents (takes 14 days to finalize) April 16, 2012 Genetic resistance mutations report issued
Testing at NML: Genetic resistance test:~ 7-10 days If no problems and all agents are susceptible: ~ 3 week TAT in total If resistance detected:ast testing must be repeated: ~ 4 5 week TAT in total
Molecular resistance reporting Rifampin: will be reported Isoniazid: will be reported Pyrazinamide: will be reported Ethambutol: will NOT be reported The correlation between the detection of mutations in embb gene and ethambutol resistance is ~65%. Da Silva PEA, Palomina JC Molecular basis and mechanisms of drug resistance in M.tuberculosis: classical and new drugs. J Antimicrob Chemother 2011 66:1417-1430
Wording on reports: Rifampin This isolate is suspected to be resistant due to genetic mutations in the rpob gene. An rpob mutation is present in 95% of rifampin resistant isolates. Isoniazid This isolate is suspected to be resistant due to genetic mutations in the inha or katg genes. An inha and/or katg mutation is present in approximately 80% of isoniazid resistant isolates Pyrazinamide This isolate is suspected to be resistant due to genetic mutations in the pnca gene. A pnca mutation is present in >95% of pyrazinamide resistant isolates. Comment added to all reports: For advice regarding interpretation of this report: contact the Microbiologist on call. For advice regarding TB therapy: contact the Respirology Service or the Infectious Diseases Service.
Molecular resistance Report Isoniazid This isolate is suspected to be resistant due to genetic mutations in the inha or katg genes. An inha and/or katg mutation is present in approximately 80% of isoniazid resistant isolates. Rifampin and Pyrazinamide: No molecular mutations associated with resistance were found. For advice regarding interpretation of this report: contact the Microbiologist on call. For advice regarding TB therapy: contact the Respirology Service or the Infectious Diseases Service. Further report to follow
Final Report 0.1 and 0.4 ug/ml R Isoniazid This isolate is suspected to be resistant due to genetic mutations in the inha or katg genes. An inha and/or katg mutation is present in approximately 80% of isoniazid resistant isolates. Rifampin and Pyrazinamide: No molecular mutations associated with resistance were found. For advice regarding interpretation of this report: contact the Microbiologist on call. For advice regarding TB therapy: contact the Respirology Service or the Infectious Diseases Service.
Resistance in ANY First Line Agent Set up Second Line agents Report all Second Line agents tested
Second Line Agents: MGIT evaluation of susceptibility Second Line agent: Critical Concentration (μg/ml) < CC = Sensitive > CC = Resistant Amikacin 1 Capreomycin 2.5 Ethionamide 5 Kanamycin 2.5 Linezolid 1 Moxifloxacin 0.25 Ofloxacin 2 PAS 4 Rifabutin 0.5 Sharma M et al. Canadian multi-centre lab study for standardized second-line antimicrobial susceptibility testing for M.tuberculosis, JCM 2011; DOI 10.1128/JCM.05195-11
M.tuberculosis: Manitoba 2011 New MTB (+) patient isolates INH resistant MTB 2011 5.2% (5/97 isolates) 2010; 9.7% (11/113 isolates) 2009; 4.7% ( 5/106 isolates) 2008; 3.4% ( 4/116 isolates) 2007; 10.6% ( 9/ 85 isolates) MDR-MTB: defined as resistance to at least INH & Rifampin 2011; 2.1% (2/97) 2010; 0.9% (also 1 XDR-TB) 2009; 0.0% 2008; 0.9% 2007; 0.0%
TB Cases: 2011:NEW TB Cases: 97 (34% smear Pos) Pulmonary TB Cases: 74 Non-pulmonary TB Cases: 23 2010:NEW TB Cases: 119 Pulmonary TB Cases: 83 Non-pulmonary TB Cases: 36
Followup of Smear (+) patients: Duration of therapy to become Smear (-) Newly diagnosed cases (2010) AFB Smear Positive 42.2% Smear Positive: 1+ or 2+ Average # days to Smear (-) Median # days to Smear (-) Smear Positive: 3+ or 4+ Average # days to Smear (-) Median # days to Smear (-) Smear (+) proven to become Culture Negative 24 days 16 days 43 days 37 days 14.3%
5 4+ 4 3+ 3 2+ 2 1+ 1 0 Smear Positive 3+: Days to Smear Negative 78 Days to Smear Negative 2 Culture Positive 1 Negative 0 AFB Score 26-Mar 03-Apr 05-Apr 10-Apr 12-Apr 22-Jan 23-Jan 24-Jan 05-Feb 08-Feb 10-Feb 11-Feb 20-Feb 21-Feb 22-Feb 27-Feb 28-Feb 01-Mar 06-Mar 07-Mar 08-Mar 12-Mar 13-Mar 14-Mar 19-Mar 21-Mar 22-Mar 14 Days to Follow-Up Culture Smear Culture
Follow up of Patients who are AFB smear (+) AFB POS: 1 2 (+): initiate follow-up samples on day 12 of therapy AFB POS: 3 4 (+): initiate follow-up samples on day 19 of therapy If ANY of the 3 followup samples are AFB(+) wait 7 days then initiate another series of 3 followup samples.
Epidemiology: MIRU testing Frequency of MIRU-VNTR Pattern in 2011 2% 2% 3% 5% MIRU report: ~ 30 days after ID of M.tuberculosis 46% 15% 224315143327234634423383 222425153322242424223352 224315143326234634423383 224425133533424244223384 227625173433324??4434364 223125153322242134223552 Patterns with one occurrence in 2011 27% Total culture submissions to NRCM/NML = 101
Manitoba Future: in the fight against TB???
Genetic Tests on Respiratory Specimens Test Method (# Specimens) RotorGene Concentrated (N = 220) GeneXpert Concentrated (N = 74) GeneXpert Direct (N = 74) Sensitivity 86% 100% for AFB smear (+) 63.2% for AFB smear (-) 88% 100% for AFB smear (+) 67% for AFB smear (-) 83% 100% for AFB smear (+) 56% for AFB smear (-) DSM Research funds for studies: Dr. Phillipe Lagace-Weins and Dr. Kanchana Manickam Specificity 98% 98%* 98%* * 2 PCR (+), Culture negative follow-up specimen; patient on TB therapy
GeneXpert MTB assay TARGET: High Risk patients in Rural sites 1 2 3 4
Direct PCR Testing: TB High Risk Index Patients: -WRHA - Rural hubs Add-on Test: Expedites diagnostic confirmation of M.tuberculosis -new service
Summary: Provincial TB services: - Sites offering services - Benchmarks for TATs Genetic resistance testing -new service Followup for TB patients: - Time to smear negative Future: Direct TB testing Diagnosis and follow-up testing for TB require integrated communication
Provincial services need to work together to provide optimal care for patients with TB