Frances Jamieson, MD and Kevin May, BSc November 15 th,
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1 Frances Jamieson, MD and Kevin May, BSc November 15 th,
2 TB Elimination: Back To Basics Financial Interest Disclosure (over the past 24 months) Dr. Frances amieson Company Speaker Advisory Research None to declare
3 Financial Interest Disclosure (over the past 24 months) Dr. Frances Jamieson I have no conflict of interest.
4 TB Elimination: Back To Basics Financial Interest Disclosure (over the past 24 months) Kevin May Company Speaker Advisory Research None to declare
5 Financial Interest Disclosure (over the past 24 months) Kevin May I have no conflict of interest.
6 Objectives 1. Understand the basic microbiologic features and epidemiology in Ontario of Mycobacteria and M. tuberculosis and the implications for laboratory diagnosis of tuberculosis 2. Review the public health laboratory services available for the diagnosis of tuberculosis in Ontario and how to access these services 3. Understand the current laboratory assays and testing algorithms in use by the public health laboratory, including new molecular methods and testing limitations/caveats
7 Active Tuberculosis Disease 7
8 Active TB Testing Algorithm for TB Suspects* Chest radiography Sputum smear microscopy Microbiological culture and phenotypic drug susceptibility testing Nucleic acid amplification testing direct specimens Who should be tested? Everyone who has signs and symptoms compatible with disease or are at high risk know your patient s risk profile! *Canadian Tuberculosis Standards, 7 th Edition, Chapter 3 8
9 Consider: Demographics Country of origin; family setting; immigrant and date of landing Age Ethnicity (e.g. First Nations or Inuit) Host factors Immunocompromised or other co-morbidity (e.g. diabetes) HIV status Risk behaviours e.g. drug abuse Environment Contact with known or suspect TB case Work environment e.g. shelter worker, healthcare worker Living status e.g. homeless, incarceration 9
10 Tuberculosis: A Global Problem WHO Estimates (2015 data): 10.4 million New Incident Cases 1.4 million Deaths 10
11 TB in Canada 2013 WHO Global Tuberculosis Report
12 Public Health Agency of Canada,
13 TB in Ontario Source: (Ontario Data) Ontario Ministry of Health and Long-Term Care, integrated Public Health Information System (iphis) database, extracted by Public health Ontario [2016/09/13]. (Canadian data) Public Health Agency of Canada. Tuberculosis in Canada 2014 Pre-release. Ottawa (Canada): Minister of Public Works and Government Services Canada; 2016
14 Number Proportion of MtbC and NTM Isolated 2015 PHL Toronto Total positive NTM TB Year 14
15 By the Numbers Largest TB and Mycobacteriology laboratory in North America (by volume) - avg ,000 specimens processed annually Provide >95% of diagnostic testing and 100% of reference testing for Ontario Over 180 AFB smears read daily (one primary reader and one secondary) - Toronto Over 250 culture positive specimens and referred cultures identified weekly - Toronto
16 Laboratory Testing Algorithm - PHOL New smear positive 16
17 Smear Culture Incubate up to 7 wks TB/MAC NAAT Daily DST; report in 8-10 days New smear positive, smear negative on request Smear read and reported In 24 hours TB or NTM ID by line-probe/lat flow assay (TB) 17 and report ID
18 TB and Mycobacteriology Labs in Ontario Public Health Lab Network PHL-Toronto and 3 Regional PHLs Ottawa Hospital Dynacare (community lab) Timmins Ottawa TORONTO London
19 TB and Mycobacteriology: Specimen Collection Ensure specimens are appropriately labelled, transported to the lab asap do not pool specimens (24 hour) specimen will be diluted and potentially contaminated do not collect sputa or other specimens in waxed containers can produce false positive smear results indicate induced sputa specimens on requisition (can look like saliva as they appear watery) Obtain a post-bronchoscopy sputum wherever possible and submit with BAL and washings 19
20 TB and Mycobacteriology: Specimen Collection Gastric lavage specimens must be received neutralized (specimen collection container available from PHOL) frozen specimens may decrease yield of culture Swabs are sub-optimal specimens (e.g. collect abscess fluid in syringe remove needle prior to submission and cap end) Swabs in anaerobic transport media and dry swabs are unacceptable blood collected in EDTA, coagulated blood, serum or plasma are unacceptable for culture 20
21 Microbiologic characteristics of MtbC Aerobic, non-motile bacteria Facultative intracellular pathogens Rod-shaped, x um Acid-alcohol fast (ZN or Kinyoun stain) due to thick, lipid-rich, waxy cell wall Divide every hours (E. coli divides roughly every 20 minutes)
22 Recommendations for specimen submission CTS 7 th Ed Major New Recommendations At least three sputum specimens should be collected and tested with microscopy as well as culture. (Conditional recommendation, based on moderate evidence) Where feasible, three sputum specimens (either spontaneous or induced) can be collected on the same day, at least 1 hour apart. (Conditional recommendation, based on moderate evidence)
23 Sputum Smear Microscopy Still the mainstay of diagnosis; simple, quick, cheap Sensitivity limited by threshold of detection: 5 10,000 bacilli/ml of specimen (20 80%) Concentration and fluorescence microscopy improves sensitivity un-concentrated smears are at least 10-fold less sensitive Specimen quality extremely important (no saliva!) and at least 5 10 ml Incremental yield in sensitivity from 3 rd specimen is 2 5%(but culture yield is 5 10%)
24 Sputum Smear Microscopy More sensitive in respiratory than non-respiratory specimens Obtain post-bronchoscopy sputum Consider obtaining induced sputum if cannot produce Has decreased specificity in low-incidence settings (could be an NTM) Less sensitive for paediatric cases and HIV infection persons (more likely to be smear-negative) Cannot be used to determine drug resistance 24
25 PHOL - Improved AFB Staining Protocol Traditional A/R Staining Smear + / NAAT + / Culture + Bacilli/ml of concentrate 5000 PHO A/R Staining Smear + / NAAT + / Culture + Smear - / NAAT + / 1000 Culture + Smear - / NAAT + / 700 Culture + Smear - / NAAT - / Culture + 10 Smear - / NAAT - / Culture + 25
26 2015 PHL Toronto Total Specimens: 48, 417 (2.4% TB +, 14.8% NTM +) Smear Positives (5.8%) Mtb Complex NTM No growth 16% TB 25% 30.1% of sm +, culture % of sm +, culture + NTM 59%
27 Culture Gold standard for diagnosis of tuberculosis Requires only organisms Liquid culture systems improve recovery and time to detection of organism (10% more sensitive than solid) Automated detection system (e.g. MGIT) Average time to growth days Identification by TB Ag MPT64 RAPID lateral flow assay for Mtb (SD Bioline) or line-probe assay (GenoType, HAIN Lifesciences)
28 Number of Specimens PHOL Distribution of Turnaround Times for Central PHL Specimens with MTBC Isolated - Positive and Negative Smears Smear +'ve Smear -'ve Turnaround Time (days) 28
29 Suboptimal specimen example: Pleural Fluid Specimens (N) Smear Positive 6 (0.2%) 1 (0.03%) 1 (0.04%) TB Culture Positive 24 (0.9%) 10 (0.3%) 11 (0.4%) NTM Culture Positive 8 (0.3%) 4 (0.1%) 13 (0.5%) PCR positive/tested 0/22 0/8 0/11 29
30 Phenotypic Drug Susceptibility Testing (DST) 30
31 DST: Definitions Critical Concentration: The lowest concentration of a drug that inhibits 95% of wild-strains of Mtb that have never been exposed to the drug, and at the same time does not inhibit strains of Mtb considered resistant that were isolated from patients not responding to therapy Proportion method Resistance is defined as growth of greater than 1% (10% for PZA) of an inoculum of bacterial cells in the presence of the critical concentration of the drug; gold standard method is agar proportion Liquid culture systems (eg MGIT 960) equivalencies have been established 31
32 First-Line DST algorithm: First isolate, new patient DRUG Critical Concentration Result Reflex Testing INH 0.1 mg/l Resistant INH 0.4 mg/l, Ofloxacin, Moxifloxacin RMP 1.0 mg/l Resistant Full 2 nd Line Panel EMB 5.0 mg/l Resistant EMB 7.5 mg/l PZA 100 mg/l Resistant; repeat Test if M. bovis or M. bovis BCG by HAIN; pnca analysis Two or more drugs Resistant Full 2 nd Line Panel MDRTB = INH R and RMP R XDRTB = MDRTB and any FQ R and R to at least one of AK, CAP, KAN 32
33 Low-level INH R (critical concentration) Low-level INH R due to mutations in genes encoding for cell wall mycolic acid synthesis (inha) High-level INH R due to mutations in genes encoding for catalase/peroxidase enzymes needed to convert INH to its active form therefore no active drug available Low-level INH R Mtb will still convert the drug to its active form, and therefore active drug is available for susceptible organisms in combination with other drugs Higher doses of INH may be effective, however studies haven t shown differences in clinical outcome of high vs low-level INH R 33
34 Drug Name Second-line DST Amikacin 1.0 Capreomycin 2.5 Ethionamide 5.0 Kanamycin 2.5 Linezolid 1.0 Moxifloxacin 0.25 Ofloxacin (for Ciprofloxacin) 2.0 PAS 4.0 Rifabutin 0.5 Streptomycin 1.0 Critical Concentration mg/l *Using MGIT/960 as of October 1 st, 2010
35 Ontario MDRTB TB Drug Resistance in Canada
36 Direct Molecular Detection of M. tuberculosis in Specimens
37 APHL Molecular Detection and Identification of Mycobacteria
38 Direct Molecular Detection Caveats Does not replace conventional culture and DST Limited sensitivity for smear-negative specimens (but excellent in smear positive specimens) Positive result indicates presence of MtbC does NOT distinguish live and dead organisms Negative result does NOT mean the absence of MtbC- not rule-out Inhibition of amplification Target below the limit of detection (paucibacillary) CANNOT replace clinical judgement nor used as guide for therapy or to determine patient removal from isolation (not 100% sensitive or specific!) Assay performance improves with increased clinical suspicion for TB 38
39 Direct Molecular Detection at PHOL AMTD Assay Performance 2015: Routinely utilized for respiratory specimens Sensitivity Smear Positive=98.7%, Smear Negative 50%; Specificity 99.6% PCR Assay (Seegene) Performance (fresh tissue) For non-respiratory specimens (eg lymph node) Sensitivity Smear Positive=100%, Smear Negative 57% Specificity 99% Direct detection of MtbC by real-time PCR assay (lab developed test) Replaced AMTD in June, 2016
40 Real-time PCR (qpcr) Allows detection and/or quantification of amplified target sequences in real time. Same reagents as traditional PCR, with addition of a fluorescent reporter. either a fluorescent dye that binds to double stranded DNA or a fluorescently labeled oligonucleotide probe specific for the target. Real-time PCR is performed in a specialized thermal cycler that can visualize the fluorescence during each cycle Fluorescence levels are directly proportional to the quantity of target present. APHL Molecular Biology
41
42 Mtb Complex and M avium Complex detection from specimens by real-time PCR Replaces the AMTD and the TB PCR assays, and detects MtbC and MAC directly in specimens Original assay developed by Wadsworth Laboratory, NY AFB Positive (%) AFB Negative (%) All Specimens (%) MtbC: Sensitivity Specificity PPV NPV MAC: Sensitivity Specificity PPV NPV
43 PHOL NAAT for MtB and MAC June 6 th October 14 th 2016 Sensitivity Specificity Indeterminate Inhibition Smear Positive TB 115/117 (98.3) 306/314 (97.4) 7 23 Smear Negative TB 5/6 (83.3) 16/16 (100) 0 1 Smear Positive MAC 114/196 (58.2) 203/206 (98.5) Smear Positive MAC (no mixed cultures included) 114/184 (62.0) N/A 35 N/A Smear Negative MAC 0 22/22 (100) 0 1 Respiratory TB 85/87 (97.7) 295/300 (98.3) 6 22 Non-Respiratory TB 35/36 (97.2) 27/29 (93.1) 1 2
44 Molecular Testing: Drug Resistance 44
45 Molecular DST (MDDR) Look for mutations in DNA known to be associated with phenotypic resistance Mutations in particular genes e.g. katg (INH) Mutations in areas of DNA hot spots or resistance determining regions, e.g. rpob 81 bp region (RRDR) for rifampin Different mutations within a gene can cause different levels of resistance Some mutations do not appear to cause phenotypic resistance -?no clinical significance or issue with DST? 45
46 Advantages and Caveats of MDDR Rapid results (24-48 hours) Do not need level 3 laboratory to perform assays (once organism inactivated or DNA extracted) Not all mechanisms of resistance known, therefore lack of mutation does not mean susceptible Not all mutations associated with phenotypic resistance, e.g. silent (synonymous) mutations and neutral polymorphisms Limited genes and sites usually targeted Emerging resistance in mixed populations may not be detected (limit of detection) 46
47 MDDR at PHOL: Current HAIN MTBDRplus v1 Detection of Mtb complex, and common mutations for INH (in katg and inha) and Rifampin (rpob) smear-positive cases where high suspicion of possible drug resistance, for cases failing therapy or had a known exposure to a drug-resistant case Can be performed on isolates Useful for mixed culture (ie MAC and MTB) where cannot perform phenotypic DST 47
48 MDDR: Future Status Leveraging Whole Genome Sequencing DNA sequencing Bioinformatics DRagon pipeline TB DNA extraction and preparation for WGS Compares output to reference genome with prediction of antibiotic resistance Clinical Report Modifed from: Musser K, Wadsworth Laboratory, NYDOH
49 Other Services PCR for M. leprae NTM DST (done at National Microbiology Laboratory, Winnipeg) Identification of NTM directly in specimens (research only) 49
50 TB CSI : Tracking Tuberculosis In Ontario 50
51 TB Genotyping: Current status Genetic analysis of Mycobacterium tuberculosis DNA using molecular techniques to produce a digital signature or DNA fingerprint: RFLP (no longer available) Spoligotype MIRU-VNTR 24 These signatures for all genotyped isolates are compared and analysed to identify clusters of identical matches 51
52 OUT-TB WEB ON-LINE SERVICE DELIVERY Ontario Universal Typing of Tuberculosis: Web-based Surveillance and Communication System To request access, contact
53 Genotyping: Future Status WGS DNA sequencing Bioinformatics pipeline TB DNA extraction and preparation for WGS Compares output to reference genome and build consensus for phylogeny and lineage (spoligotype) Clinical Report Modifed from: Musser K, Wadsworth Laboratory, NYDOH
54 Thank-you! Kevin May Shirley Brown PHOL TB Laboratory staff Alex Marchand-Austin Kirby Cronin Kelsie Jagt Carla Duncan Jennifer Ma Jennifer Guthrie Carolina Mehaffy David Alexander Nahuel Fittipaldi Sarah Teatero Taryn Athey Daniel Rosen 54
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