Advances in 1st and 2nd Line Drug Susceptibility Testing in ...

Advances in 1 st and 2 nd Line Drug
Susceptibility Testing of
Mycobacterium tuberculosis
Alternative title: mix of interesting stuff about drug
susceptibility testing for TB
Edward Desmond, Ph.D., D (ABMM)
San Leandro, CA

Cepheid GeneXpert
What’s new?
Vaya con Dios, BACTEC 12B
Hello, 2 nd line drugs in MGIT
Moxifloxacin in MGIT
Trek broth MIC system under evaluation
Detecting drug resistance by sequencing
CDC’s MDDR service (molecular detection of drug
resistance)
Pyrosequencing
From culture
From sputum sediment
Ethambutol worries

The journey from radioactive
to non-
BD to discontinue production of BACTEC 12B after
Sept. 2011
November, 2009 CDC performance evaluation event
• 59 labs used MGIT as their 1 drug sus test system
• 5 labs used Versa Trek as their 1 drug sus test system
• 23 labs used BACTEC 12B as their 1 drug sus test
system

Can we transition 2 nd line testing from 12B
to MGIT?
Rusch-Gerdes, S., et al. 2006. JCM 44: 688
Van Ingen, J., et al. 2010. JCM 48: 2749
Lin, S.Y., et al. 2009. JCM 47: 3630
WHO 2008 Policy guidance on drug-
susceptibility testing (DST) of second-line
antituberculosis drugs

Drug Levofloxacin Amikacin Capreomycin
Concentration
(ug/mL)
MGIT test concentrations
ug/mL 6
Ethionamide
1.5 1.5 3 5
Drug Levo Amikacin Capreo Ethion
Conc
J. Clin. Microbiol. 2009 47(11)3630-4
WHO 2008 guidelines for MGIT 960
No
recomm.
1.0 2.5 5.0

Inoculating a MGIT drug sus
test from a primary MGIT tube
BD package insert calls for mixing the MGIT
tube and using a sample of this or a 1:5
dilution of it (day 3 to 5 after positivity) as
inoculum
Problem: variable amounts of M. tb cells likely have
been removed for smear and AccuProbe
Problem: anecdotes of inconsistent results
Problem: studies in which no M. tb cells were
removed from inoculum tube don’t really evaluate
accuracy of this method as performed using primary
cultures/clinical isolates
7

Calif. protocol for inoculum prep from 1 MGIT
Pipet 2 mL from bottom of primary MGIT tube
into a 1 dram vial & let settle for 20 min.
Pipet supernate to another 1 dram vial & let
settle for 15 min
Pipet supernate to another 1 dram vial and
dilute to McFarland 0.5 using normal saline;
dilute 1:5 (or 1:3)
Protocol compensates for variable amounts
removed from primary tube, and works with
single cells or small clumps
Full disclosure: BD provided free MGIT medium for
evaluation of this protocol (Lin 2009. JCM 47: 3630)
8

Fall 2010 CDC Model Performance Evaluation
Program (MPEP) results
• 2 cultures sent in duplicate
• MGIT showed significantly less ethambutol
resistance than agar or BACTEC 460 methods
• Is ethambutol concentration in MGIT too high?
• When proficiency testing reveals a flaw in a testing
method, is a laboratory required to repeat the testing
by another method?

Ethambutol in MGIT—what to do?
o If there was no consensus in testing by the reference method,
no action should be required
o If testing by the reference method did show consensus, and
your laboratory’s result using MGIT yielded a different result,
corrective action should be considered
o Wait for report for this event to see what conclusions are

Some musings about corrective actions
o May want to repeat testing if false ethambutol
susceptibility is suspected
o Madison, et al. 2002. JCM 40(11):3976-79 showed (table 2)
that 97% of TB isolates that are truly resistant to
ethambutol (by both agar and radiometric methods) were
also resistant to INH
o Maybe not necessary to re-test pan-susceptible strains.
These are very unlikely to be resistant to EMB
o Re-testing of cultures older than 6 months probably not
clinically relevant—treatment likely completed
o Maybe re-test strains

Repeat testing to confirm ethambutol
susceptibility
o Re-testing by same method (MGIT) seems
pointless
o Alternative methods would be BACTEC 12B,
agar proportion, or DNA sequencing,
depending on which you have in your
laboratory

Moxifloxacin results compared with LEV
by MGIT 960
MOX
by MGIT 960
LEV-R
By MGIT 960
LEV-S
By MGIT 960
>0.25 ug/ml 40 0
≤ 0.25 ug/ml 0 64
The abstract was presented at 2010 ASM General meeting.

MOX
MIC
(ug/ml)
Mutations ( # of strains)
90gtg 94gcc 95acc 94ggc 91ccg 94cac 94tac
Total #
strains
0.5 1 1 1 6
0.75 6 3 1 10
1 1 1 1 1 1 5
1.5 1 2 5 1 2 11
2 1 3 2 1 8
3 2 2
4 1 1
6 1 1
Total #
strains 8 6 6 13 2 4 1 40

Recommendations
o Test MOX at 0.25 ug/ml.
o If susceptible, it will predict susceptibility to LEV.
o When resistant to MOX at 0.25, test MOX at 0.5, 1,
2 and 4 ug/ml.
o The quantitative determination of MOX resistance may
be used as a guide for MOX dosage modification.
o When MOX MIC is > 1? 2? 4? ug/ml, it indicates high
level resistance.

Detection of Drug Resistance
• Drugs of interest:
• For XDR screening
Mutations
• INH, RIF, KAN, AMK, CAP, fQs
• Targeted Genes
• katG, inhA promoter for INH
• rpoB for RIF
• rrs for KAN, AMK & CAP
• gyrA for Quinolones
16
G Lin PSQ 11-2-10

Sequencing other genes related to
drug resistance
o pncA: to detect resistance to pyrazinamide
o CDC study: 55/65 (85%) of PZA-resistant cultures
had a pncA mutation (Campbell et al AAC 2011 55:2032)
o 109/124 (86%) of PZA-susceptible cultures had no
mutation in pncA gene
o embB: to detect resistance to ethambutol
o CDC study: 121/154 (79%) of EMB-resistant cultures
had embB mutation
o 149/160 (93%) of EMB susceptible cultures had no
mutation in embB gene
o More data are needed to show which mutations
are or are not associated with drug resistance

6111138
Pyrosequencing
Primer DNA template
dATP
dTTP
dGTP
dCTP
dATP
chemiluminescence
chemiluminescence
degrades
degrades
degrades
G
GA
Copyright © motifolio.com

Instruments needed for
pyrosequencing
o Thermocycler for PCR.
o Plate shaker to keep streptavidin beads in
suspension and allow binding of biotin-DNA
onto beads.
o Vacuum work station to capture beads.
o Heating block to heat DNA. When cooling,
SQ primer anneals to DNA template.
o Pyrosequencer to do sequencing work.

20
G Lin PSQ 11-2-10

Detection of Mutations with a Molecular
Beacon
(Loop portion containing wildtype SQ)
Mutant Sequence
Wildtype Sequence
Fluorophor
e
+
Heat
Molecular
Beacon (off)
Fluorophore
Light
Loop
Amplicon
Quencher
Hybrid (Molecular Beacon - On)

MB Performance
(% Agreement between MB and phenotypic drug
results)
INH RIF
Cultures 98.4% 99%
Sediments 93.6% 94.9%
Overall 96.1% 97.2%

Limitations of molecular beacons
o Molecular beacons good for detecting absence of wild
type sequence in a small (about 15 bases) region of a
gene with a highly conserved sequence
o Fine for rpoB, katG, inhA promoter region
o Not good for detecting mutations associated with
resistance if the normal gene sequence is variable (e.g.
gyrA 95)
o Not good for detecting mutations that are spread out
over a wide region (e.g. pncA mutations associated with
PZA resistance)

Comparison of MB & PSQ
Interpretation of silent
mutations or mutations
not conferring R
Objectivity on
interpreting results
MB PSQ
Mutation present
or not
Misinterpret
as R
More
subjective
Exact SQ
Does not
misinterpret
More
objective
Hand-on time 1.5 hr, simple 2.5 hr, more steps
Total 24
test time 3.5 hr 5 hr

Trek Sensititre(TB) MIC plate
o MICs for OFL, MFX, RIF, AMI, STR, RFB, PAS, ETH, CYC,
INH, KAN, and EMB
o Plate is sealed with tape and wiped with disinfectant before
incubation
o Results quicker than agar proportion method (7-10 days vs 14-
21 days)
Personal observations (E.D.)
o Clinicians may want susceptible vs. resistant results rather than
numerical MICs
o Interpretation of MIC not straightforward for TB due to
intracellular growth and metabolic dormancy
o Still some unknown factors—cost, safety, & whether trailing
endpoints might occur

Thank you