Molecular Epidemiology of Mycobacterium tuberculosis-Barry Kreiswirth PhD

Molecular Epidemiology of Mycobacterium tuberculosis-Barry Kreiswirth PhD

• 1.
  Molecular Epidemiology of Mycobacteriumtuberculosis Barry N. Kreiswirth, PhD Director, PHRI TB Center Presented at the 41st Annual Symposium “Global Movement of Infectious Pathogens and Improved Laboratory Detection” Eastern PA Branch-American Society for Microbiology November 17, 2011 Thomas Jefferson University, Philadelphia
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  Slow grower; doubles24hrs; 3-4 weeks to culture 3-4 weeks for susceptibility testing Highly transmissible; requires BL3 facilities
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  TB Statistics 2 billion infected (1/3 world population)  8-9 million new cases each year  1.6 million deaths per year (25% of all preventable deaths)  This means 4, 000 deaths each day, a death every 20 seconds  85% of the mortality in developing countries 2008 WHO report on TB
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  HIV and MultidrugResistance HIV and Tuberculosis  Co-infection of M.tb and HIV a deadly-duet  11% co-infected (range from 1% to over 60%)  Reactivation of tuberculosis or rapid progression to disease are markers for HIV Multidrug Resistance (INH & RIF)  Multidrug resistance is emerging in virtually every country  425,000 new MDR cases annually  Estimated 50 million infected with MDR
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  M. tuberculosis Genome– H37Rv Genome size (bp): 4,411,532 3,959 predicted ORFs (90.8%) 2,441 attributed functions 912 conserved hypotheticals 606 unkonwns
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  Comparative Sequence Analysis  M. tuberculosis genome is highly conserved  M. tuberculosis is a monomorphic species  Synonymous base pair changes are rare  M. tuberculosis “young” human pathogen  M. tuberculosis evolved from M. bovis
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  A new evolutionaryscenario for the Mycobacterium tuberculosis complex. Brosch R, Gordon SV, Marmiesse M, Brodin P, Buchrieser C, Eiglmeier K, Garnier T, Gutierrez C, Hewinson G, Kremer K, Parsons LM, Pym AS, Samper S, van Soolingen D, Cole ST. Proc Natl Acad Sci U S A 2002 Mar 19;99(6):3684-9
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  Evolution of theMtb Complex Brochet et al, PNAS, 2002
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  Molecular Tools -Genotyping Methods Primary Genotyping Method  IS6110 Southern blot hybridization  Secondary Genotyping Methods  Spoligotyping Binary typing, DR region  PGRS Southern blot hybridization  VNTR, MIRU PCR, multiple targets  IS6110 mapping Southern blot hybridization  DNA sequencing Resistance targets, SNP  Array analysis Deletion mapping
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  Genotyping Targets toDiscriminate M. tuberculosis Strain 210 Strain HN5 Barnes et al. NEJM – 2003;349:1149
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  Biology of IS6110 Unique to M. tuberculosis complex  Copy number from 1 - 26 insertions  Insertions dispersed around genome  Chromosomal “hot-spots” identified  Insertions stable over time  Movement is a replicative process
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  Insertion Sequence IS6110
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  IS6110 DNA Fingerprinting Standardized Methodology  Southern blot hybridization  PvuII restriction digest  Common right-side hybridization probe  Common molecular weight standards  Digitized patterns  Pattern matching software
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  DNA Fingerprints ofM. tuberculosis Strains
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  Searching the Databasefor Strain W4
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  SRO Outbreak inSan Francisco
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  Genotyping Data –Public Health Issues   Evaluate nosocomial and community transmission  Evaluate suspected cases of laboratory contamination  Distinguish relapse vs re-infection  Genotype drug resistance genes to distinguish spread vs acquisition  Distinguish recent transmission and endemic strains
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  TREATING TUBERCULOSIS
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  The last TBdrug, ethambutol, was discovered in 1968 at “Wyeth” Pyrazinamide Streptomycin Ethionamide Capreomycin Isoniazid Kanamycin/amikacin PAS Thioacetazone Cycloserine Rifampin Ethambutol 1965 1966 1967 1968 1944 1946 1952 1955 1956 1957
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  Antimycobacterial Agents First Line Drugs Second Line Drugs  Streptomycin  Ethionamide  Isoniazid  Amikacin / Kanamycin  Rifampin  Capreomycin  Ethambutol  Fluoroquinolones  Pyrazinamide  PAS  Cycloserine
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  Current therapy fortuberculosis disease  Advantages: 2HRZE/4HR – 100% effective – Low relapse rate (3-4%) • Induction phase: 2 – Inexpensive months isoniazid, – Universally available – Can be given intermittently rifampin, pyrazinamide, ethambutol  Disadvantages – 6 months duration – High relapse rate in some • Continuation phase: 4 subgroups (10-15%) – Adverse effects common months isoniazid, – Interactions with HIV rifampin treatment – Not useful against MDR strains
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  Drug Resistant M.tuberculosis Multidrug Resistance (MDR) Resistance to at least isoniazid (INH) and rifampin (RIF) Multidrug Resistance (Plus) Resistance to at least isoniazid and rifampin plus resistance to fluoroquinolones Extensively Drug Resistance (XDR) Resistance to at least isoniazid and rifampin plus resistance to fluoroquinolones AND one of the second line injectable aminoglycoside drugs (amikacin, kanamycin or capreomycin)
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  Cure Rates forMDR-TB and XDR-TB MDR-TB cure rate 1993-8: 94% (fluoroquinolones & surgery were critical variables) MDR+-TB cure rate 1993-8: 60% XDR-TB cure rate 1993-8: 20% Case studies from National Jewish TB Center, Denver CO
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  DRUG SUSCEPTIBILITY TESTING
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  Mycobacteriology testing inthe clinical laboratory It is not the black sheep of the lab - - - It is the pig!
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  METHODS  Middlebrook 7H10or 7H11 agar – Incorporate drug  Middlebrook 7H10 or 7H11 agar - E-test  Lowenstein-Jensen slants  BACTEC 460 – radioactive CO2  BACTEC MGIT 960 – oxygen consumption LIMITATIONS  Susceptibility requires a growing culture – 3 weeks  First-line drugs tested first – 3 weeks  Second-line drugs tested for MDR – 3 weeks  MDR treatment delayed
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  Genotyping Drug Resistance Nearly all drug resistance target genes identified  With two exceptions, non-synonymous mutations in drug resistance target genes predicts resistance  Molecular approaches are able to genotype resistance in less than 24 hrs – too costly even for developed countries
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  Drug Resistance TargetGenes
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  Rifampin Resistance &rpoB Mutations WT: CTG AGC CAA TTC ATG GAC CAG AAC CCG CTG TCG GGG TTG ACC CAC AAG CGC CGA CTG TCG GCG CTG RIF: CTG AGC CAA TTC ATG GAC CAG AAC CCG CTG TCG GGG TTG ACC TAC AAG CGC CGA CTG TCG GCG CTG
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  Fluoroquinolone Resistance andgyrA Mutations Wild Type: CAC GGC CAC GCG TCG ATC TAC GAC AGC CTG Resistance: CAC GGC CAC GCG TCG ATC TAC GGC AGC CTG
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  Genotyping Drug Resistance Advantages Challenges  Speed  Bypass culturing  Resistance genes identified  Infectious & Contaminated  No synonymous mutations  Cost  Mutation predicts resistance  Sensitivity / Specificity  Simplicity  Flexibility
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  Genotyping Drug Resistance Hainreverse hybridization line probe assay (Hain Lifescience, Nehren, Germany)  Multiplex PCR and reverse hybridization  Identifies major mutations in rpoB, katG and inhA – Detects MDR  Demonstrated with specimens and culture Cepheid (Sunnyvale, CA)  PCR and molecular beacon detection  Detection of rpoB – surrogate for MDR  Closed system with primary specimens Abbott’s Ibis, PLEX ID (Abbott Park, IL)  Multiplex PCR and mass spectrometry  Detection platform able to detect XDR  Not evaluated with primary specimens
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  Tuberculosis in NewYork City
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  AIDS
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  168th Street Men’sShelter – March, 1992
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  W Strain MDROutbreak in NYC  January 1990 - August 1993  43 Months - 8,021 Cases  357 Patients with W strain tuberculosis  Spread in NYC hospitals and state prisons  All resistant to first line drugs  86% HIV infected; >90% Mortality  160 Patients identified since study  22 Patients identified outside of NYC Bifani et al., JAMA 1996:275;452. Munsiff et al., JID 2003:188;356.
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  Outbreak of theMultidrug Resistant “W” Tuberculosis Clone
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  W MDR Outbreak:1990-1993  Isoniazid (100%) katG - 315:AGC>ACA; Ser>Thr  Rifampin (100%) rpoB - 526:CAC>TAC; His>Tyr  Streptomycin (100%) rpsL - 43:AAG>AGG: Lys>Arg  Ethambutol (100%) embB - 306:ATG>GTG; Met>Val  Pyrazinamide (55%) pncA - 139:ACC>GCC; Thr>Ala  Kanamycin (92%) rrs - nucleotide 1400  Fluoroquinolones (0%)
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  W MDR OUTBREAK:1993-1999 W34 W31 W40 W12 W25 W1 W1 W W
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  Creating Extremely DrugResistant W Strains Munsiff et al., JID 2003:188;356
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  XDR-TB OUTBREAK IN AN HIV-POSITIVE POPULATION IN SOUTH AFRICA 15 YEARS LATER
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  Jan 2005 –March 2006 1,539 TB Diagnosed 542 Culture Positive Cases 168 MDR Cases 53 XDR Cases 52 / 53 Died 44 Patients tested: All HIV+ MEDIAN SURVIVAL OF 16 DAYS FROM THE TIME OF DIAGNOSIS
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  XDR-TB OUTBREAK INAN HIV-POSITIVE POPULATION IN SOUTH AFRICA IS6110 Fingerprint
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  WHO ESTIMATES: 25,000CASES OF XDR-TB EMERGING EVERY YEAR
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   As welearned in NYC during our HIV – MDR outbreak, it will take strong political will, dedicated medical and public health teams and a great deal of money to deal with this emerging epidemic –  If not, XDR and all its baggage will appear at your doorsteps
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  Case Studies
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  Identity Crisis 8/5/05: patientKD admitted to hospital “A” with diagnosis of suspected pulmonary tuberculosis  X-ray abnormal; sputum smear positive  Culture positive; resistant to INH 8/10/05: patient threatens to leave hospital AMA  No health officer restraining order requested  1:1 monitoring in place 8/11/05: patient leaves hospital AMA  Numerous attempts to locate patient prove futile  Fictitious identity and locating information provided to hospital by patient  Case filed as lost to follow-up
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  DNA Fingerprint ofthe M. tuberculosis from patient KD
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  Identity Crisis  4/5/06 Patient CB admitted to hospital “B” with diagnosis of suspected pulmonary tuberculosis  X-ray abnormal; sputum smear positive  Culture positive; resistant to INH, EMB, and PZA  4/6/06 patient threatens to leave hospital AMA  Patient uncooperative; Legal intervention requested  Health Officer hospital restraining order served  Despite completely different demographic information subtle physical similarities existed between patients KD and CB  CB denies any knowledge of KD
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  1 2 JI strains isolated from patient “CB/KD” 1. 2005 2. 2006