1.
Molecular Epidemiology of
Mycobacterium tuberculosis
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

2.
Slow grower; doubles 24hrs; 3-4 weeks to culture
3-4 weeks for susceptibility testing
Highly transmissible; requires BL3 facilities

3.
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

4.
HIV and Multidrug Resistance
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

5.
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

6.
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

7.
A new evolutionary scenario 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

8.
Evolution of the Mtb Complex
Brochet et al, PNAS, 2002

9.
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

10.
Genotyping Targets to Discriminate M. tuberculosis
Strain 210
Strain HN5
Barnes et al. NEJM – 2003;349:1149

11.
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

12.
Insertion Sequence IS6110

13.
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

14.
DNA Fingerprints of M. tuberculosis Strains

15.
Searching the Database for Strain W4

16.
SRO Outbreak in San Francisco

17.
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

18.
TREATING TUBERCULOSIS

19.
The last TB drug, 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

20.
Antimycobacterial Agents
First Line Drugs Second Line Drugs
 Streptomycin  Ethionamide
 Isoniazid  Amikacin / Kanamycin
 Rifampin  Capreomycin
 Ethambutol  Fluoroquinolones
 Pyrazinamide  PAS
 Cycloserine

21.
Current therapy for tuberculosis 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

22.
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)

23.
Cure Rates for MDR-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

24.
DRUG SUSCEPTIBILITY TESTING

25.
Mycobacteriology testing in the clinical laboratory
It is not the black sheep of the lab - - - It is the
pig!

26.
METHODS
 Middlebrook 7H10 or 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

27.
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

28.
Drug Resistance Target Genes

29.
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

30.
Fluoroquinolone Resistance and gyrA Mutations
Wild Type: CAC GGC CAC GCG TCG ATC TAC GAC AGC CTG
Resistance: CAC GGC CAC GCG TCG ATC TAC GGC AGC CTG

31.
Genotyping Drug Resistance
Advantages Challenges
 Speed  Bypass culturing
 Resistance genes identified  Infectious & Contaminated
 No synonymous mutations  Cost
 Mutation predicts resistance  Sensitivity / Specificity
 Simplicity
 Flexibility

32.
Genotyping Drug Resistance
Hain reverse 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

33.
Tuberculosis in New York City

34.
AIDS

35.
168th Street Men’s Shelter – March, 1992

36.
W Strain MDR Outbreak 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.

37.
Outbreak of the Multidrug Resistant “W” Tuberculosis Clone

38.
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%)

39.
W MDR OUTBREAK: 1993-1999
W34
W31
W40
W12
W25
W1
W1
W
W

40.
Creating Extremely Drug Resistant W Strains
Munsiff et al., JID 2003:188;356

41.
XDR-TB OUTBREAK IN
AN HIV-POSITIVE
POPULATION IN SOUTH
AFRICA
15 YEARS LATER

42.
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

43.
XDR-TB OUTBREAK IN AN HIV-POSITIVE
POPULATION IN SOUTH AFRICA
IS6110 Fingerprint

44.
WHO ESTIMATES: 25,000 CASES OF XDR-TB EMERGING EVERY YEAR

45.
 As we learned 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

46.
Case Studies

47.
Identity Crisis
8/5/05: patient KD 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

48.
DNA Fingerprint of the M. tuberculosis from patient KD

49.
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

50.
1 2
JI strains isolated
from patient “CB/KD”
1. 2005
2. 2006