Att. 12-Final Aggregate Report

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Mycobacterium tuberculosis Complex
Drug Susceptibility Testing Program
Model Performance Evaluation Program
Report of Results
September 2020

CS 323576-A

Mycobacterium tuberculosis Complex
Drug Susceptibility Testing Report
for September 2020 Survey
Purpose
The purpose of this report is to present results of the U.S. Centers for Disease Control and Prevention (CDC) Model
Performance Evaluation Program (MPEP) for Mycobacterium tuberculosis complex (MTBC) drug susceptibility testing
survey sent to participants in September 2020.

Report Content
The material in this report was developed and prepared by:
Cortney Stafford, MPH, MT (ASCP), Health Scientist, Laboratory Capacity Team, NCHHSTP, DTBE, LB
Acknowledged NCHHSTP, DTBE, LB contributors:
Tracy Dalton, Lois Diem, Stephanie Johnston, Beverly Metchock, James Posey, Mitchell Yakrus,
and Angela Starks

Contact Information
Comments and inquiries regarding this report should be directed to:
TBMPEP@cdc.gov
404-639-4013
CDC TB MPEP Website
The findings and conclusions in this report are those of the author(s) and do not necessarily represent the views of
the Centers for Disease Control and Prevention.
Use of trade names and commercial sources is for identification only and does not imply endorsement by the U.S.
Department of Health and Human Services.

Note on Accessibility:
Find descriptions and explanations of figures in Appendix 1: Accessible Explanation of Figures on page 35.

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CDC MPEP MTBC DST Report for September 2020 Survey

Contents
Mycobacterium tuberculosis Complex Drug Susceptibility Testing Report for September 2020 Survey. . . . . . . . . . . .
Purpose. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Report Content. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Contact Information. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2
2
2
2

Abbreviations and Acronyms..

4

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Introduction: Overview of MPEP Final Report. .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5

Expected Drug Susceptibility Testing Results..

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

6

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

7

Technical Notes.

Descriptive Information about Participant Laboratories. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Primary Classification.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Annual Number of MTBC Drug Susceptibility Tests Performed.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
MTBC DST Methods Used by Participants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Antituberculosis Drugs Tested by Participants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Isolate 2020F. .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

12

Isolate 2020G.

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

17

Isolate 2020H.

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

20

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25

Isolate 2020I.

Isolate 2020J. .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

29

Equivalent Critical Concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Agar Proportion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Broth Based Media. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
References. .

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Appendix 1: Accessible Explanations of Figures. .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3
CDC MPEP MTBC DST Report for September 2020 Survey

34
35

Abbreviations and Acronyms
Acronym Definition
AMK amikacin
AP agar proportion—performed on Middlebrook 7H10 or 7H11
Bp base pair
CAP capreomycin
CDC U.S. Centers for Disease Control and Prevention
CIP ciprofloxacin
CLSI Clinical and Laboratory Standards Institute
CYS cycloserine
DNA deoxyribonucleic acid
DST drug susceptibility testing
EMB ethambutol
ETA ethionamide
FQ fluoroquinolones
INH isoniazid
KAN kanamycin
LEV levofloxacin
MDR multidrug resistant
MGIT BACTEC MGIT 960—Mycobacteria Growth Indicator Tube
MIC minimum inhibitory concentration
MOX moxifloxacin
MPEP Model Performance Evaluation Program
MTBC Mycobacterium tuberculosis complex
Nt nucleotide
PAS p-aminosalicylic acid
PZA pyrazinamide
OFL ofloxacin
R resistant
RBT rifabutin
RMP rifampin
RNA ribonucleic acid
S susceptible
Sensititre Thermo Scientific Sensititre MYCOTB AST or customized plate
STR streptomycin
TB tuberculosis
VersaTREK Thermo Scientific VersaTREK Myco susceptibility
XDR extensively drug resistant

4
CDC MPEP MTBC DST Report for February 2020 Survey

Introduction: Overview of MPEP Final Report
The Model Performance Evaluation Program (MPEP) is an educational self-assessment tool in which five isolates of M. tuberculosis
complex (MTBC) are sent to participating laboratories biannually for staff to monitor their ability to determine drug resistance
among the isolates. It is not a formal, graded proficiency testing program. The associated report includes results for a subset
of laboratories performing drug susceptibility tests (DST) for MTBC in the United States. MPEP is a voluntary program, and this
report reflects data received from participating laboratory personnel. This aggregate report is prepared in a format that will
allow laboratory personnel to compare their DST results with those obtained by other participants using the same methods and
drugs, for each isolate. We encourage circulation of this report to personnel who are either involved with DST or reporting and
interpreting results for MTBC isolates.
CDC is neither recommending nor endorsing testing practices reported by participants. For standards, participants should
refer to consensus documents published by the Clinical and Laboratory Standards Institute (CLSI), “M24: Susceptibility Testing
of Mycobacteria, Nocardiae spp., and Other Aerobic Actinomycetes” [1]. Recently, World Health Organization (WHO) published
two technical reports investigating critical concentrations, by method, for INH, RMP, EMB, PZA and twelve second-line antituberculosis drugs [2, 3]. Based on the systematic review data, recommendations were made for adjustments to critical
concentrations for RMP, MOX, LEV, AMK and KAN for some methods.

5
CDC MPEP MTBC DST Report for September 2020 Survey

Expected Drug Susceptibility Testing Results
Anticipated growth-based and molecular results for the panel of MTBC isolates sent to participants in September 2020 are
shown in the tables below. Although CDC recommends broth-based methods for routine first-line DST of MTBC isolates, the
results obtained by the reference agar proportion method (except for pyrazinamide, in which MGIT was performed) are shown
in Table 1. Molecular results obtained by DNA sequencing are listed in Table 2 [4].

Table 1. Expected Growth-based Results for September 2020 Survey
Note—S=susceptible, R=resistant , V=variable
Isolate

RMP

INH

EMB

PZA

Second-line Drugs Resistant to:

2020F*

S

R

†

S

S

ETA

2020G

S

R

S

S

ETA

2020H

S

R

R

S

ETA, STR¥

2020I*

S

R†

S

S

ETA

2020J

R

S

S

S

*Isolates 2020F and 2020I are the same isolate
†
Although INH resistance was expected, 80% consensus for a single categorical result of either susceptible or resistant was not achieved for this isolate among
participating laboratories. Variable resistance was observed depending on growth-based DST method.
¥
Although STR resistance was expected, 80% consensus for a single categorical result of either susceptible or resistant was not achieved for this isolate among
participating laboratories. Variable resistance was observed depending on growth-based DST method.

Table 2. Expected Molecular Results (Mutations Detected in Loci Associated with Resistance) for
September 2020 Survey
Note—Empty cell=No mutation detected
Isolate

rpoB¥

2020F

Arg447Arg*
(Arg528Arg)†

katG

inhA

fabG1

embB

ethA

Met306Val

(partial deletion)

Leu203Leu

2020G

C-15T

2020H

Ser315Thr

2020I

Arg447Arg*
(Arg528Arg)†

2020J

Ser450Leu*
(Ser531Leu)†

Leu203Leu

Mutation is listed using both the M. tuberculosis and E.coli numbering system [5, 6]
*M. tuberculosis numbering system used
†
E. coli numbering system used
¥

6
CDC MPEP MTBC DST Report for September 2020 Survey

Technical Notes
The following information pertains to all of the tables and figures
for the 2020 MTBC isolates F, G, H, I, and J included in this report.

■ The source of data in all tables and figures is the September 2020
MPEP MTBC DST survey.

■ First-line and second-line drugs have been separated into

individual tables for each isolate. Streptomycin is classified as a
second-line drug for this report.

■ Separate tables for molecular testing are included.
■ Laboratories that use more than one DST method are

encouraged to test isolates with each of those methods at either
CLSI-recommended or equivalent critical concentrations. Some
laboratories have provided results for multiple DST methods.
Consequently, the number of results for some drugs may be
greater than the number of participating laboratories. This report
contains all results reported by participating laboratories.

■ The Sensititre system allows determination of a minimum

inhibitory concentration (MIC) for each drug in the panel.
Laboratories using this method may establish breakpoints to
provide a categorical interpretation of S or R.

■ For participant result tables for first- and second-line DST that

have drug-method totals equal to 0, results were not received or
the test was not performed.

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CDC MPEP MTBC DST Report for September 2020 Survey

Descriptive Information about Participant Laboratories
Primary Classification
This report contains DST results submitted to CDC by survey participants at 69 laboratories in 34 states.
The participants were asked to indicate the primary classification of their laboratory (Figure 1). MPEP participants self-classified as:

■ 50 (73%): Public health laboratory (e.g., local, county, state)
■ 9 (13%): Hospital laboratory
■ 7 (10%): Independent/Reference laboratory (non-hospital based)
■ 2 (3%): Federal government laboratory
1 (1%):1Other (Medical Manufacturing Company)
■ FIG
Figure 1. Primary Classification of Participating Laboratories, September 2020

Other
(Medical Manufacturing
Company)
1%

Federal government
laboratory
3%
Independent/
Reference
laboratory
10%

Hospital
laboratory
13 %

Public health
laboratory
73%

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CDC MPEP MTBC DST Report for September 2020 Survey

The number of MTBC isolates tested for drug susceptibility by the 69 participants in 2019 (excluding isolates used for quality
control) is shown in Figure 2. In 2019, the counts ranged from 0 to 1,039 tests. Participants at 28 (41%) laboratories reported
testing 50 or fewer DST isolates per year. Laboratories with low MTBC DST volumes are encouraged to consider referral of testing
because of concerns about maintaining proficiency [7].

Figure 2. Distribution of the Annual Volume of MTBC Isolates Tested for Drug Susceptibility by
Participants in Previous Calendar Year (n=69)

30
Number of Laboratories Responding

2

Annual Number of MTBC Drug Susceptibility Tests Performed

28

25
20
15

13

10
6

5
0

7

5

6

2
51-100

101-150 151-200 201-300
Number of Isolates Tested

2
301-500

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CDC MPEP MTBC DST Report for September 2020 Survey

501-1000

G3
MTBC DST Methods Used by Participants
The DST methods that were used by participating laboratories for this panel of MTBC isolates are displayed in Figure 3. Of
participating laboratories, 46 (66%) reported results for only one method, 20 (29%) reported two methods, and 4 (5%) noted
three susceptibility methods.

Figure 3. MTBC Drug Susceptibility Test Method Used by Participants (n=97)

Number of Laboratories
Responding

70

64

60
50
40
30
20
10
0

17
4
MGIT

10
2

Agar Proportion
Sensititre
VersaTREK
Drug Susceptibility Test Method

Molecular
Methods

Ten molecular methods reported by participants are shown in Figure 4. The method used most frequently by laboratories (5) was
targeted DNA sequencing (50%), including pyrosequencing and Sanger sequencing. Three (30%) laboratories reported use of the
Cepheid Xpert MTB/RIF assay, one (10%) reported results for line probe assays, Genotype MTBDRplus and MTBDRsl by Bruker, and
one (10%) reported results from whole genome sequencing.

Figure 4. Molecular Method Reported (n=10)
Whole Genome
Sequencing,
1
Bruker
Line
Probe,
1

Cepheid
Xpert,
3

Targeted
DNA
Sequencing,
5

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CDC MPEP MTBC DST Report for September 2020 Survey

Antituberculosis Drugs Tested by Participants
The number of participating laboratories that reported testing each antituberculosis drug in the September 2020 survey is
presented in Figure 5. CLSI recommends testing a full panel of first-line drugs (rifampin [RMP], isoniazid [INH], ethambutol
[EMB] and pyrazinamide [PZA])[1] because it represents a combination of tests that provides the clinician with comprehensive
information related to the four-drug antituberculosis therapy currently recommended for most patients. All participants reported
results for three of the first-line drugs (RMP, INH and EMB) and 64 (93%) also reported results for PZA by growth-based DST
methods. One laboratory performs molecular testing for PZA via sequencing of pncA, in place of growth-based DST.
For 21 laboratories reporting second-line drug results (with the exception of streptomycin), four (19%) tested all three secondline injectable drugs and at least one fluoroquinolone needed to confidently define XDR TB. The second-line injectable drugs are
amikacin, kanamycin and capreomycin. Fluoroquinolones include ofloxacin, ciprofloxacin, levofloxacin and moxifloxacin

Figure 5. Antituberculosis Drugs Tested by Participants

Rifampin
Isoniazid
Ethambutol
Pyrazinamide

69
69
69
64

Antituberculosis Drugs Tested

Streptomycin

44

Ofloxacin
Moxifloxacin
Ciprofloxacin
Levofloxacin

13
6
5
5

Capreomycin
Kanamycin
Amikacin

14
13
12

Ethionamide
Para-aminosalicylic Acid
Rifabutin
Cycloserine

18
13
11
0

7
10

20
30
40
50
60
70
Number of Participating Laboratories Responding

11
CDC MPEP MTBC DST Report for September 2020 Survey

80

Isolate 2020F
Expected Result: Resistant to INH at 0.2 µg/ml and ETA at 5.0 µg/ml by agar proportion
Isoniazid
Isoniazid (INH) is the most widely used first-line antituberculosis drug and is a cornerstone of regimens used to treat TB disease
and latent TB infection. INH is a prodrug and is activated by the catalase-peroxidase enzyme encoded by the katG gene [4, 8].
The target of activated INH is enoyl-acyl-carrier protein reductase (encoded by the inhA gene); this binding inhibits cell wall
mycolic acid biosynthesis. There are two mechanisms that account for the majority of INH resistance [4, 8, 9]. The most common
mechanism, mutations in katG, is generally associated with high-level resistance to INH. Resistance to INH can also occur by
mutations in the promoter region of the inhA gene, which are generally associated with low-level resistance to INH and are
less frequent than katG mutations. Approximately 10–15% of isolates found to be INH-resistant have no mutations detected in
either of these loci. Numerous loci have been investigated to identify additional genes correlated with INH resistance. The fabG1
(also known as mabA) gene, like inhA, is involved in mycolic acid biosynthesis and at least one mutation in this region has been
associated with low-level INH resistance [10, 11]. In MTBC, ahpC codes for an alkyl hydroperoxide reductase that is associated
with resistance to reactive oxygen and reactive nitrogen intermediates; consequently, it is believed that mutations in the
promoter region could be surrogate markers for INH resistance [8].
DNA sequence analysis of inhA, katG, fabG1, and ahpC for Isolate 2020F revealed a G>A point mutation in fabG1 at codon 203
resulting in the synonymous/silent mutation Leu203Leu; inhA, katG, and ahpC were wild-type (i.e., no mutations were detected).
Within fabG1, the silent/synonymous mutation (i.e., nucleotide change but no corresponding change in amino acid) Leu203Leu
has been found to confer INH resistance through the formation of an alternative promoter, thereby increasing the transcriptional
levels of inhA [11]. Although silent mutations were previously believed to not play a role in drug resistance, the Leu203Leu
mutation demonstrates that silent mutations could be associated with resistance depending on the specific gene and the
location of the mutation.
The recommended critical concentration and additional higher concentrations for testing INH using the AP method are 0.2 µg/
ml and 1.0 µg/ml, respectively. The equivalent concentrations for MGIT and VersaTREK are 0.1 µg/ml and 0.4 µg/ml [1].
For Isolate 2020F, 78 INH results were reported for the critical concentration. This isolate was reported resistant to INH by
method, as follows:

■ 94% (15/16) of the results when using AP
■ 49% (28/57) of the results when using MGIT
■ 0% (0/3) of the results when using Sensititre
■ 100% (2/2) of the results when using VersaTREK
No results were reported as resistant at the higher concentrations of INH. Only 28 (48%) laboratories performing MGIT DST
reported a result for the higher concentration of INH, although some may have tested the higher concentration by a different
method.
Of the 6 molecular results reported for INH, 3 (50%) laboratories reported detection of a mutation in fabG1, all specifically noting
the Leu203Leu mutation.
Three of the laboratories performing Sensititre reported INH MIC values as 0.12 µg/ml (n=2) and 0.25 µg/ml (n=1).

Ethionamide
Resistance to INH and ethionamide (ETA) can occur by mutations in the fabG1–inhA regulatory region, which are generally
associated with low-level resistance to INH. Mutations in ethA also confer resistance to ETA, without concomitant resistance to
INH [12].

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CDC MPEP MTBC DST Report for September 2020 Survey

Sequencing analysis of ethA was not performed and as previously noted, sequencing of the inhA gene revealed wild-type (i.e., no
mutations were detected). The synonymous/silent mutation Leu203Leu was detected in the fabG1 locus for Isolate 2020F.
For Isolate 2020F, 17 ETA results were reported. This isolate was reported resistant to ETA by method, as follows:

■ 84% (11/13) of the results when using AP
■ 67% (2/3) of the results when using MGIT
■ 0% (0/1) of the results when using Sensititre
One laboratory performing Sensititre reported an ETA MIC value as 2.5 µg/ml (n=1) with a categorical result of susceptible. One
additional laboratory reported an ETA MIC value as 10 µg/ml (n=1) but as no interpretation was indicated by this laboratory, the
result was excluded from Table 9.

Rifampin
Rifampin (RMP) is a bactericidal drug used as part of a standard first-line regimen for the treatment of TB. RMP’s mechanism
of action is to inhibit mycobacterial transcription by targeting DNA-dependent RNA polymerase [8]. The primary mechanism
of resistance is a mutation within the 81-bp central region of the rpoB gene that encodes the β-subunit of the bacterial DNAdependent RNA polymerase [9]. Mutations in codons 450, 445 and 435 (E. coli numbering system corresponding to 531, 526
and 516) are among the most frequent mutations in RMP-resistant isolates and serve as predictors of RMP resistance [8, 9]. The
activity of RMP on isolates with rpoB mutations depends on both the mutation position and the type of amino acid change.
CDC has recommended that RMP resistance detected by the Xpert MTB/RIF assay be confirmed by DNA sequencing of rpoB
[13]. The Xpert MTB/RIF assay could generate results that falsely indicate resistance when compared to growth-based methods
because of the presence of silent/synonymous mutations [14]. Sequencing of rpoB will allow for clarification of the result and
understanding of possible discordance between rapid molecular and growth-based testing results.
DNA sequence analysis of rpoB in Isolate 2020F revealed a C>T point mutation in codon 447 (E. coli numbering 528) of the
rpoB locus. However, this mutation does not result in an amino acid change; arginine remains arginine (Arg447Arg). Unlike the
fabG1 silent mutation in this isolate that was associated with INH resistance, the Arg447Arg synonymous (i.e., silent) mutation
in rpoB is not considered clinically significant and isolates with this mutation reliably test as RMP-susceptible in growth-based
systems. However, as noted above, the Xpert MTB/RIF assay could indicate RMP resistance for this isolate and sequencing of
rpoB should be performed.
For Isolate 2020F, 80 results for RMP were reported. This isolate was reported as susceptible to RMP by method, as follows:

■ 100% (16/16) of the results when using AP
■ 100% (59/59) of the results when using MGIT
■ 100% (3/3) of the results when using Sensititre
■ 100% (2/2) of the results when using VersaTREK
Of the 8 molecular results reported for RMP, 5 (63%) laboratories reported mutation detected with 4 laboratories specifically
noting the Arg447Arg silent mutation. Three laboratories reported mutation not detected, however this may be due to the
detection of a silent mutation not associated with resistance.
Three of the laboratories performing Sensititre reported RMP MIC values as ≤0.12 µg/ml (n=1), 0.12 µg/ml (n=1) and 0.25 µg/ml (n=1).
Complete first-line DST, second-line DST and molecular results submitted by all participants for Isolate 2020F are listed in Tables 3–10.
Three laboratories noted no growth for at least one antituberculosis drug tested for Isolate 2020F

13
CDC MPEP MTBC DST Report for September 2020 Survey

Table 3. Isolate 2020F—Participant Results for First-Line DST by AP
Drug

Susceptible

Resistant

Total

Rifampin

16

0

16

Isoniazid—Low

1

15

16

Isoniazid—High

16

0

16

Ethambutol

16

0

16

Table 4. Isolate 2020F—Participant Results for First-Line DST by MGIT
Drug

Susceptible

Resistant

Total

Rifampin

59

0

59

Isoniazid—Low

29

28

57*

Isoniazid—High

28

0

28

Ethambutol

58

1

59

Pyrazinamide

61

1

62

* One additional laboratory reported ‘Borderline’ for INH by MGIT.

Table 5. Isolate 2020F—Participant Results for First-Line DST by Sensititre
Drug

Susceptible

Resistant

Total

Rifampin

3

0

3

Isoniazid—Low

3

0

3

Isoniazid—High

1

0

1

Ethambutol

3

0

3

Table 6. Isolate 2020F—Participant Results for First-Line DST by VersaTREK
Drug

Susceptible

Resistant

Total

Rifampin

2

0

2

Isoniazid—Low

0

2

2

Isoniazid—High

2

0

2

Ethambutol

2

0

2

Pyrazinamide

1

0

1

Table 7. Isolate 2020F—Participant Results for Second-Line DST by AP
Drug

Susceptible

Resistant

Total

Streptomycin

14

0

14

Ofloxacin

10

0

10

Ciprofloxacin

4

0

4

Levofloxacin

2

0

2

Moxifloxacin

2

0

2

Amikacin

7

0

7

Kanamycin

10

0

10

Capreomycin

9

0

9

Ethionamide

2

11

13

Rifabutin

7

0

7

Cycloserine

4

0

4

p-Aminosalicylic acid

9

0

9

14
CDC MPEP MTBC DST Report for September 2020 Survey

Table 8. Isolate 2020F—Participant Results for Second-Line DST by MGIT
Drug

Susceptible

Resistant

Total

Streptomycin

30

0

30

Ofloxacin

3

0

3

Ciprofloxacin

0

0

0

Levofloxacin

2

0

2

Moxifloxacin

2

0

2

Amikacin

3

0

3

Kanamycin

1

0

1

Capreomycin

3

0

3

Ethionamide

1

2

3

Rifabutin

2

0

2

Cycloserine

0

0

0

p-Aminosalicylic acid

1

0

1

Table 9. Isolate 2020F—Participant Results for Second-Line DST by Sensititre
Drug

Susceptible

Resistant

Total

Streptomycin

2

0

2*

Ofloxacin

1

0

1*

Ciprofloxacin

0

0

0

Levofloxacin

1

0

1

Moxifloxacin

1

0

1*

Amikacin

2

0

2*

Kanamycin

1

0

1*

Capreomycin

1

0

1

Ethionamide

1

0

1*

Rifabutin

2

0

2*

Cycloserine

1

0

1*

p-Aminosalicylic acid

2

0

2*

* One additional laboratory reported ‘No Interpretation’ for STR, OFL, MOX, AMK, KAN, ETA, RBT, CYC, and PAS by Sensititre.

15
CDC MPEP MTBC DST Report for September 2020 Survey

Table 10. Isolate 2020F—Participant Results for Molecular Testing
Drug

Mutation Detected

Mutation Not Detected

Total

Rifampin

5

3

8

Isoniazid

3

3

6

Ethambutol

0

4

4

Pyrazinamide

0

2

2

Ofloxacin

0

3

3

Ciprofloxacin

0

3

3

Levofloxacin

0

4

4

Moxifloxacin

0

4

4

Amikacin

0

3

3

Kanamycin

0

3

3

Capreomycin

0

3

3

Ethionamide

2

0

2

Rifabutin

1

3

4

16
CDC MPEP MTBC DST Report for September 2020 Survey

Isolate 2020G
Expected Result: Resistant to INH at 0.2 µg/ml and ETA at 5.0 µg/ml by agar proportion
Isoniazid
DNA sequence analysis of inhA, katG, fabG1, and ahpC of Isolate 2020G revealed a C>T point mutation at nucleotide position
-15 of the promoter region of the inhA gene (C-15T); katG, fabG1 and ahpC were wild-type (i.e., no mutations were detected).
Mutations in the promoter region of the inhA gene are generally associated with low-level resistance to INH.
For Isolate 2020G, 82 INH results were reported. This isolate was reported resistant to INH by method, as follows:

■ 100% (17/17) of the results when using AP
■ 100% (61/61) of the results when using MGIT
■ 50% (1/2) of the results when using Sensititre
■ 100% (2/2) of the results when using VersaTREK
Five (9%) results were reported as resistant at the higher concentrations of INH. Only 34 (56%) laboratories performing MGIT DST
reported a result for the higher concentration of INH, although some may have tested the higher concentration by a second DST method.
Of the 6 molecular results reported for INH, all (100%) laboratories reported detection of a mutation with 5 laboratories
specifically noting the C-15T mutation.
Two of the laboratories performing Sensititre reported INH MIC values as 0.25 µg/ml (n=1) and 0.5 µg/ml (n=1). A third
laboratory reported an INH MIC value as 0.25 µg/ml (n=1) and indicated a result of borderline.
Complete first-line DST, second-line DST, and molecular results submitted by all participants for Isolate 2020G are listed in Tables 11–18
.Table 11. Isolate 2020G—Participant Results for First-Line DST by AP
Drug

Susceptible

Resistant

Total

Rifampin

17

0

17

Isoniazid—Low

0

17

17

Isoniazid—High

17

0

17

Ethambutol

17

0

17

Table 12. Isolate 2020G—Participant Results for First-Line DST by MGIT
Drug

Susceptible

Resistant

Total

Rifampin

62

0

62

Isoniazid—Low

0

61

61

Isoniazid—High

29

5

34

Ethambutol

62

0

62

Pyrazinamide

60

3

63

17
CDC MPEP MTBC DST Report for September 2020 Survey

Table 13. Isolate 2020G—Participant Results for First-Line DST by Sensititre
Drug

Susceptible

Resistant

Total

Rifampin

3

0

3

Isoniazid—Low

1

1

2*

Isoniazid—High

0

0

0*

Ethambutol

3

0

3

* One additional laboratory reported borderline for INH by Sensititre.

Table 14. Isolate 2020G—Participant Results for First-Line DST by VersaTREK
Drug

Susceptible

Resistant

Total

Rifampin

2

0

2

Isoniazid—Low

0

2

2

Isoniazid—High

2

0

2

Ethambutol

2

0

2

Pyrazinamide

1

0

1

Table 15. Isolate 2020G—Participant Results for Second-Line DST by AP
Drug

Susceptible

Resistant

Total

Streptomycin

15

0

15

Ofloxacin

10

0

10

Ciprofloxacin

5

0

5

Levofloxacin

2

0

2

Moxifloxacin

2

0

2

Amikacin

7

0

7

Kanamycin

11

0

11

Capreomycin

10

0

10

Ethionamide

1

13

14

Rifabutin

7

0

7

Cycloserine

5

0

5

p-Aminosalicylic acid

10

0

10

Table 16. Isolate 2020G—Participant Results for Second-Line DST by MGIT
Drug

Susceptible

Resistant

Total

Streptomycin

32

0

32

Ofloxacin

3

0

3

Ciprofloxacin

0

0

0

Levofloxacin

2

0

2

Moxifloxacin

2

0

2

Amikacin

3

0

3

Kanamycin

1

0

1

Capreomycin

3

0

3

Ethionamide

0

3

3

Rifabutin

2

0

2

Cycloserine

0

0

0

p-Aminosalicylic acid

1

0

1

18
CDC MPEP MTBC DST Report for September 2020 Survey

Table 17. Isolate 2020G—Participant Results for Second-Line DST by Sensititre
Drug

Susceptible

Resistant

Total

Streptomycin

2

0

2*

Ofloxacin

1

0

1*

Ciprofloxacin

0

0

0

Levofloxacin

1

0

1

Moxifloxacin

1

0

1*

Amikacin

2

0

2*

Kanamycin

1

0

1*

Capreomycin

1

0

1

Ethionamide

1

0

1*

Rifabutin

2

0

2*

Cycloserine

0

0

0*

p-Aminosalicylic acid

2

0

2*

* One additional laboratory reported ‘No Interpretation’ for STR, OFL, MOX, AMK, KAN, ETA, RBT, CYC, and PAS by Sensititre.

Table 18. Isolate 2020G—Participant Results for Molecular Testing
Drug

Mutation Detected

Mutation Not Detected

Total

Rifampin

0

8

8

Isoniazid

6

0

6

Ethambutol

0

4

4

Pyrazinamide

1*

1

2

Ofloxacin

0

3

3

Ciprofloxacin

0

3

3

Levofloxacin

0

4

4

Moxifloxacin

0

4

4

Amikacin

0

3

3

Kanamycin

0

3

3

Capreomycin

0

2

2

Ethionamide

2

0

2

Rifabutin

0

4

4

* One laboratory noted the detection of a mutation not associated with PZA resistance..

19
CDC MPEP MTBC DST Report for September 2020 Survey

Isolate 2020H
Expected Result: Resistant to INH at 0.2 µg/ml and 1.0 µg/ml, EMB at 5.0 µg/ml, ETA at 5.0 µg/ml and
STR at 2.0 µg/ml by agar proportion
Isoniazid
As previously noted, resistance to INH most commonly occurs due to mutations in the katG gene or the promoter region of the
inhA gene, however, mutations in fabG1 and ahpC can also cause resistance. DNA sequence analysis of Isolate 2020H revealed a
G>C point mutation at codon 315 in the katG locus resulting in wild-type serine being replaced by threonine (Ser315Thr); inhA,
fabG1, and ahpC were wild-type (i.e., no mutations were detected).
For Isolate 2020H, 82 INH results were reported. This isolate was reported resistant to INH by method, as follows:

■ 100% (17/17) of the results when using AP
■ 100% (61/61) of the results when using MGIT
■ 100% (2/2) of the results when using Sensititre
■ 100% (2/2) of the results when using VersaTREK
Fifty-five or 100% of results at the higher concentrations of INH were reported as resistant. Only 34 (56%) laboratories performing
MGIT DST reported a result for the higher concentration of INH, although some may have tested the higher concentration by a
second DST method.
Of the 6 molecular results reported for INH, all (100%) laboratories reported detection of a mutation with 5 laboratories
specifically noting the Ser315Thr mutation.
Two of the laboratories performing Sensititre reported INH MIC values as 4 µg/ml (n=2). One additional laboratory reported an
INH MIC value as 4 µg/ml (n=1).

Ethambutol
Ethambutol (EMB) is an important first-line drug for the treatment of TB and is used in combination with INH, RMP and PZA to
prevent emergence of drug resistance. EMB is a bacteriostatic agent that is active against growing bacilli and has no effect on
non-replicating bacilli [8, 9]. EMB targets the arabinosyl transferases (embCAB operon), thereby inhibiting the biosynthesis of the
cell wall components arabinogalactan and lipoarabinomannan [15].
Issues with false-susceptibility with some growth-based methods for EMB, particularly in broth-based media, have been reported
and remain a potential concern. Probable causes include the bacteriostatic nature of the drug, reduced drug activity in culture,
and an organism’s MIC for EMB falling too close to the critical concentration tested [16-18].
Sequence analysis of EMB-resistant clinical isolates has shown that EMB resistance is associated primarily with missense (nonsynonymous) mutations within the EMB resistance determining region of the gene embB at codons 306, 406 and 497 [4, 15].
DNA sequence analysis of embB of Isolate 2020H revealed a A>G point mutation at codon 306 in the embB gene resulting in
wild-type methionine being replaced by valine (Met306Val). Certain embB mutations at the 306 codon, such as Met306Val and
Met306Leu, are associated with EMB resistance [4].
For Isolate 2020H, 83 EMB results were reported. This isolate was reported resistant to EMB by method, as follows:

■ 88% (15/17) of the results when using AP
■ 16% (10/61) of the results when using MGIT
■ 100% (3/3) of the results when using Sensititre
■ 50% (1/2) of the results when using VersaTREK

20
CDC MPEP MTBC DST Report for September 2020 Survey

Of the 4 molecular results reported for EMB, all laboratories reported detection of a mutation and specifically noted the
Met306Val mutation.
Three of the laboratories performing Sensititre reported EMB MIC values as 8 µg/ml (n=3).

Ethionamide
As previously noted, resistance to ETA is commonly due to mutations in the ethA gene or mutations in fabG1 or inhA resulting in
cross-resistance with INH.
DNA sequencing analysis revealed a partial deletion of ethA; inhA and fabG1 were wild-type (i.e., no mutations were detected).
For Isolate 2020H, 18 ETA results were reported. This isolate was reported resistant to ETA by method, as follows:

■ 64% (9/14) of the results when using AP
■ 100% (3/3) of the results when using MGIT
■ 0% (0/1) of the results when using Sensititre
Of the 2 molecular results reported for ETA, 1 (50%) laboratory reported detection of a mutation and specifically noted
an ethA deletion.
One of the laboratories performing Sensititre reported an ETA MIC value as 2.5 µg/ml (n=1). One laboratory reported an ETA MIC
value as 5 µg/ml (n=1) but as no interpretation was indicated by this laboratory, the result was excluded from Table 25.

Streptoymycin
Streptomycin (STR) belongs to the aminoglycoside class of drugs and its primary mechanism of action is to inhibit protein
synthesis by preventing the initiation of translation by binding to the 16s rRNA [8, 9]. In MTBC, the genetic basis of the majority of
resistance to STR is usually due to mutations in rrs or rpsL [9, 19]. CLSI recommended testing STR as a second-line drug based on
American Thoracic Society’s categorization of STR as a second-line drug for treatment due to increased resistance in many parts
of the world [1, 20].
DNA sequencing analysis did not reveal a mutation in rrs or rpsL; other mechanisms of resistance may exist.
Among three methods, 48 results for STR were reported for Isolate 2020H. This isolate was reported as resistant to STR by
method, as follows:

■ 76% (11/14) of the results when using AP
■ 48% (16/33) of the results when using MGIT
■ 100% (1/1) of the results when using Sensititre
One of the laboratories performing Sensititre reported an STR MIC value as 4.0 µg/ml (n=1). A second laboratory reported a STR
MIC value as 2 µg/ml (n=1) and indicated borderline resistance. A third laboratory reported STR MIC value as 4 µg/ml (n=1) but
as no interpretation was indicated, the result was excluded from Table 25.
Complete first-line DST, second-line DST and molecular results submitted by all participant for Isolate 2020H are listed in Tables 19–26.

Table 19. Isolate 2020H—Participant Results for First-Line DST by AP
Drug

Susceptible

Resistant

Total

Rifampin

16

1

17

Isoniazid—Low

0

17

17

Isoniazid—High

0

17

17

Ethambutol

2

15

17

21
CDC MPEP MTBC DST Report for September 2020 Survey

Table 20. Isolate 2020H—Participant Results for First-Line DST by MGIT
Drug

Susceptible

Resistant

Total

Rifampin

62

0

62

Isoniazid—Low

0

61

61

Isoniazid—High

0

34

34

Ethambutol

51

10

61*

Pyrazinamide

63

0

63

* One additional laboratory reported borderline for EMB by MGIT.

Table 21. Isolate 2020H—Participant Results for First-Line DST by Sensititre
Drug

Susceptible

Resistant

Total

Rifampin

3

0

3

Isoniazid—Low

0

2

2

Isoniazid—High

0

2

2

Ethambutol

0

3

3

Table 22. Isolate 2020H—Participant Results for First-Line DST by VersaTREK
Drug

Susceptible

Resistant

Total

Rifampin

2

0

2

Isoniazid—Low

0

2

2

Isoniazid—High

0

2

2

Ethambutol

1

1

2

Pyrazinamide

1

0

1

Table 23. Isolate 2020H—Participant Results for Second-Line DST by AP
Drug

Susceptible

Resistant

Total

Streptomycin

3

11

14*

Ofloxacin

10

0

10

Ciprofloxacin

5

0

5

Levofloxacin

2

0

2

Moxifloxacin

2

0

2

Amikacin

7

0

7

Kanamycin

11

0

11

Capreomycin

10

0

10

Ethionamide

5

9

14

Rifabutin

7

0

7

Cycloserine

5

0

5

p-Aminosalicylic acid

10

0

10

* One additional laboratory reported ‘Borderline’ for STR by AP.

22
CDC MPEP MTBC DST Report for September 2020 Survey

Table 24. Isolate 2020H—Participant Results for Second-Line DST by MGIT
Drug

Susceptible

Resistant

Total

Streptomycin

17

16

33

Ofloxacin

3

0

3

Ciprofloxacin

0

0

0

Levofloxacin

2

0

2

Moxifloxacin

2

0

2

Amikacin

3

0

3

Kanamycin

1

0

1

Capreomycin

3

0

3

Ethionamide

0

3

3

Rifabutin

2

0

2

Cycloserine

0

0

0

p-Aminosalicylic acid

1

0

1

Table 25. Isolate 2020H—Participant Results for Second-Line DST by Sensititre
Drug

Susceptible

Resistant

Total

Streptomycin

0

1

1*†

Ofloxacin

1

0

1*

Ciprofloxacin

0

0

0

Levofloxacin

1

0

1

Moxifloxacin

2

0

2*

Amikacin

2

0

2*

Kanamycin

1

0

1*

Capreomycin

1

0

1

Ethionamide

1

0

1*

Rifabutin

2

0

2*

Cycloserine

0

0

0*

p-Aminosalicylic acid

2

0

2*

* One additional laboratory reported ‘No Interpretation’ for STR, OFL, MOX, AMK, KAN, ETA, RBT, CYC, and PAS by Sensititre.
†
One additional laboratory reported ‘Borderline’ for STR by Sensititre.

23
CDC MPEP MTBC DST Report for September 2020 Survey

Table 26. Isolate 2020H—Participant Results for Molecular Testing
Drug

Mutation Detected

Mutation Not Detected

Total

Rifampin

0

8

8

Isoniazid

6

0

6

Ethambutol

4

0

4

Pyrazinamide

1*

1

2

Ofloxacin

0

3

3

Ciprofloxacin

0

3

3

Levofloxacin

0

4

4

Moxifloxacin

0

4

4

Amikacin

0

3

3

Kanamycin

0

3

3

Capreomycin

0

2

2

Ethionamide

1

1

2

Rifabutin

0

4

4

* This laboratory noted the detection of a mutation not associated with PZA resistance.

24
CDC MPEP MTBC DST Report for September 2020 Survey

Isolate 2020I
Expected Result: Resistant to INH at 0.2 µg/ml and ETA at 5.0 µg/ml by agar proportion
Isolate 2020I is a duplicate of Isolate 2020F. Therefore, laboratories should have the same results for both isolates. Overall,
laboratories reported similar results for INH, ETA, and RMP. Laboratories should consider performing an internal comparison of
results between these two isolates.

Isoniazid
DNA sequence analysis for Isolate 2020I revealed a fabG1 G>A point mutation at codon 203 resulting in the synonymous/silent
mutation Leu203Leu; inhA, katG, and ahpC were wild-type (i.e., no mutations were detected).
The recommended critical concentration and additional higher concentrations for testing INH using the AP method are 0.2 µg/
ml and 1.0 µg/ml, respectively. The equivalent concentrations for MGIT and VersaTREK are 0.1 µg/ml and 0.4 µg/ml [1].
For Isolate 2020I, 80 INH results were reported. This isolate was reported resistant to INH by method, as follows:

■ 82% (14/17) of the results when using AP
■ 55% (32/58) of the results when using MGIT
■ 0% (0/3) of the results when using Sensititre
■ 100% (2/2) of the results when using VersaTREK
No results were reported as resistant at the higher concentrations of INH. Only 29 (49%) laboratories performing
MGIT DST reported a result for the higher concentration of INH, although some may have tested the higher concentration
by a different method.
Of the 6 molecular results reported for INH, 3 (50%) laboratories reported detection of a mutation, specifically noting the
Leu203Leu mutation.
Three of the laboratories performing Sensititre reported INH MIC values as 0.25 µg/ml (n=1), 0.50 µg/ml (n=1) and 1.2 µg/ml (n=1).

Ethionamide
DNA sequencing analysis of ethA was not performed and as previously noted, sequencing of the inhA gene revealed wild-type
(i.e., no mutations were detected). The synonymous/silent mutation Leu203Leu was detected in the fabG1 locus for Isolate 2020I.
For Isolate 2020I, 18 ETA results were reported. This isolate was reported resistant to ETA by method, as follows:

■ 71% (10/14) of the results when using AP
■ 67% (2/3) of the results when using MGIT
■ 0% (0/1) of the results when using Sensititre
One of the laboratories performing Sensititre reported an ETA MIC value as 5 µg/ml (n=1). Another laboratory reported an ETA
MIC value as 10 µg/ml (n=1) but as no categorical interpretation was provided, the data were not included in Table 33.

Rifampin
DNA sequence analysis of rpoB in Isolate 2020I revealed a C>T point mutation in codon 447 (E. coli numbering 528) of the
rpoB locus. However, this mutation does not result in an amino acid change; arginine remains arginine (Arg447Arg). Unlike the
fabG1 silent mutation in this isolate that was associated with INH resistance, the Arg447Arg synonymous (i.e., silent) mutation
in rpoB is not considered clinically significant and isolates with this mutation reliably test as RMP-susceptible in growth-based
systems. However, as noted above, the Xpert MTB/RIF assay could indicate RMP resistance for this isolate and sequencing of
rpoB should be performed.

25
CDC MPEP MTBC DST Report for September 2020 Survey

For Isolate 2020I, 82 results for RMP were reported. This isolate was reported as susceptible to RMP by method, as follows:

■ 100% (17/17) of the results when using AP
■ 100% (60/60) of the results when using MGIT
■ 100% (3/3) of the results when using Sensititre
■ 100% (2/2) of the results when using VersaTREK
Of the 8 molecular results reported for RMP, 5 (63%) laboratories reported mutation detected with 4 laboratories specifically
noting the Arg447Arg silent mutation. Three laboratories reported mutation not detected, however this may be due to the
detection of a silent mutation not associated with resistance.
Three of the laboratories performing Sensititre reported RMP MIC values as ≤0.12 µg/ml (n=1), 0.12 µg/ml (n=1) and 0.25 µg/ml (n=1).
Complete first-line DST, second-line DST and molecular results submitted by all participants for Isolate 2020I are listed in Tables 27–34.
Two laboratories noted no growth for at least one antituberculosis drug tested for Isolate 2020I.

Table 27. Isolate 2020I—Participant Results for First-Line DST by AP
Drug

Susceptible

Resistant

Total

Rifampin

17

0

17

Isoniazid—Low

3

14

17

Isoniazid—High

17

0

17

Ethambutol

17

0

17

Table 28. Isolate 2020I—Participant Results for First-Line DST by MGIT
Drug

Susceptible

Resistant

Total

Rifampin

60

0

60

Isoniazid—Low

26

32

58*

Isoniazid—High

29

0

29

Ethambutol

60

0

60

Pyrazinamide

60

1

61

* One additional laboratory reported ‘Borderline’ for INH by MGIT.

Table 29. Isolate 2020I—Participant Results for First-Line DST by Sensititre
Drug

Susceptible

Resistant

Total

Rifampin

3

0

3

Isoniazid—Low

3

0

3

Isoniazid—High

1

0

1

Ethambutol

3

0

3

26
CDC MPEP MTBC DST Report for September 2020 Survey

Table 30. Isolate 2020I—Participant Results for First-Line DST by VersaTREK
Drug

Susceptible

Resistant

Total

Rifampin

2

0

2

Isoniazid—Low

0

2

2

Isoniazid—High

2

0

2

Ethambutol

2

0

2

Pyrazinamide

1

0

1

Table 31. Isolate 2020I—Participant Results for Second-Line DST by AP
Drug

Susceptible

Resistant

Total

Streptomycin

15

0

15

Ofloxacin

10

0

10

Ciprofloxacin

5

0

5

Levofloxacin

2

0

2

Moxifloxacin

2

0

2

Amikacin

7

0

7

Kanamycin

11

0

11

Capreomycin

10

0

10

Ethionamide

4

10

14

Rifabutin

7

0

7

Cycloserine

5

0

5

p-Aminosalicylic acid

10

0

10

Table 32. Isolate 2020I—Participant Results for Second-Line DST by MGIT
Drug

Susceptible

Resistant

Total

Streptomycin

31

0

31

Ofloxacin

3

0

3

Ciprofloxacin

0

0

0

Levofloxacin

2

0

2

Moxifloxacin

2

0

2

Amikacin

3

0

3

Kanamycin

1

0

1

Capreomycin

3

0

3

Ethionamide

1

2

3

Rifabutin

2

0

2

Cycloserine

0

0

0

p-Aminosalicylic acid

1

0

1

27
CDC MPEP MTBC DST Report for September 2020 Survey

Table 33. Isolate 2020I—Participant Results for Second-Line DST by Sensititre
Drug

Susceptible

Resistant

Total

Streptomycin

2

0

2*

Ofloxacin

1

0

1*

Ciprofloxacin

0

0

0

Levofloxacin

1

0

1

Moxifloxacin

1

0

1*

Amikacin

2

0

2*

Kanamycin

1

0

1*

Capreomycin

1

0

1

Ethionamide

1

0

1*

Rifabutin

2

0

2*

Cycloserine

0

0

0*

p-Aminosalicylic acid

2

0

2*

* One additional laboratory reported ‘No Interpretation’ for STR, OFL, MOX, AMK, KAN, ETA, RBT, CYC, and PAS by Sensititre.

Table 34. Isolate 2020I—Participant Results for Molecular Testing
Drug

Mutation Detected

Mutation Not Detected

Total

Rifampin

5

3

8

Isoniazid

3

3

6

Ethambutol

0

4

4

Pyrazinamide

0

2

2

Ofloxacin

0

3

3

Ciprofloxacin

0

3

3

Levofloxacin

0

4

4

Moxifloxacin

0

4

4

Amikacin

0

3

3

Kanamycin

0

3

3

Capreomycin

0

2

2

Ethionamide

2

0

2

Rifabutin

1

3

4

28
CDC MPEP MTBC DST Report for September 2020 Survey

Isolate 2020J
Expected Result: Resistant to RMP at 1.0 µg/ml by agar proportion
Rifampin
DNA sequence analysis of rpoB in Isolate 2020J revealed a C>T point mutation in codon 450 (E. coli numbering 531) resulting
in wild-type serine being replaced by leucine (Ser450Leu). Isolates with Ser450Leu (Ser531Leu in E. coli numbering system)
mutations consistently test resistant to RMP in growth-based assays.
For Isolate 2020J, 81 results for RMP were reported. This isolate was reported as resistant to RMP by method, as follows:

■ 88% (15/17) of the results when using AP
■ 98% (58/59) of the results when using MGIT
■ 100% (3/3) of the results when using Sensititre
■ 100% (2/2) of the results when using VersaTREK
Of the 9 molecular results reported for RMP, all (100%) laboratories reported detection of a mutation. Five laboratories specifically
noted the Ser450Leu mutation and two laboratories reporting results for Xpert MTB/RIF noted no signal for Probe E.
Three of the laboratories performing Sensititre reported RMP MIC values as 16 µg/ml (n=1) and >16 µg/ml (n=2).

Rifabutin
Participant results are consistent with rifabutin (RBT) results based on the presence of the rpoB Ser450Leu mutation [21].
Among three methods, 11 results for RBT were reported for Isolate 2020J. This isolate was reported as resistant to RBT by
method, as follows:

■ 100% (7/7) of the results when using AP
■ 100% (2/2) of the results when using MGIT
■ 100% (2/2) of the results when using Sensititre
Of the 4 molecular results reported specifically for RBT, 3 (75%) laboratories reported detection of a mutation.
Two of the laboratories performing Sensititre reported an RBT MIC value as 2.0 µg/ml (n=2). Another laboratory reported an RBT
MIC value as 0.5 µg/ml (n=1) but as no categorical interpretation was provided, the data were excluded from Table 41.
Complete first-line DST, second-line DST and molecular results submitted by all participants for Isolate 2020J are listed in Tables 35–42.
One laboratory noted no growth for at least one antituberculosis drug tested for Isolate 2020J.

Table 35. Isolate 2020J—Participant Results for First-Line DST by AP
Drug

Susceptible

Resistant

Total

Rifampin

2

15

17

Isoniazid—Low

17

0

17

Isoniazid—High

17

0

17

Ethambutol

17

0

17

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CDC MPEP MTBC DST Report for September 2020 Survey

Table 36. Isolate 2020J—Participant Results for First-Line DST by MGIT
Drug

Susceptible

Resistant

Total

Rifampin

1

58

59

Isoniazid—Low

59

0

59

Isoniazid—High

23

0

23

Ethambutol

59

0

59

Pyrazinamide

63

0

63

Table 37. Isolate 2020J—Participant Results for First-Line DST by Sensititre
Drug

Susceptible

Resistant

Total

Rifampin

0

3

3

Isoniazid—Low

3

0

3

Isoniazid—High

2

0

2

Ethambutol

3

0

3

Table 38. Isolate 2020J—Participant Results for First-Line DST by VersaTREK
Drug

Susceptible

Resistant

Total

Rifampin

0

2

2

Isoniazid—Low

2

0

2

Isoniazid—High

2

0

2

Ethambutol

2

0

2

Pyrazinamide

1

0

1

Table 39. Isolate 2020J—Participant Results for Second-Line DST by AP
Drug

Susceptible

Resistant

Total

Streptomycin

15

0

15

Ofloxacin

10

0

10

Ciprofloxacin

5

0

5

Levofloxacin

1

0

1

Moxifloxacin

1

0

1

Amikacin

7

0

7

Kanamycin

11

0

11

Capreomycin

10

0

10

Ethionamide

14

0

14

Rifabutin

0

7

7

Cycloserine

5

0

5

p-Aminosalicylic acid

10

0

10

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CDC MPEP MTBC DST Report for September 2020 Survey

Table 40. Isolate 2020J—Participant Results for Second-Line DST by MGIT
Drug

Susceptible

Resistant

Total

Streptomycin

32

0

32

Ofloxacin

3

0

3

Ciprofloxacin

0

0

0

Levofloxacin

2

0

2

Moxifloxacin

2

0

2

Amikacin

3

0

3

Kanamycin

1

0

1

Capreomycin

3

0

3

Ethionamide

3

0

3

Rifabutin

0

2

2

Cycloserine

1

0

1

p-Aminosalicylic acid

1

0

1

Table 41. Isolate 2020J—Participant Results for Second-Line DST by Sensititre
Drug

Susceptible

Resistant

Total

Streptomycin

2

0

2*

Ofloxacin

1

0

1*

Ciprofloxacin

0

0

0

Levofloxacin

1

0

1

Moxifloxacin

1

0

1*

Amikacin

2

0

2*

Kanamycin

1

0

1*

Capreomycin

1

0

1

Ethionamide

1

0

1*

Rifabutin

0

2

2*

Cycloserine

1

0

1*

p-Aminosalicylic acid

2

0

2*

* One additional laboratory reported ‘No Interpretation’ for STR, OFL, MOX, AMK, KAN, ETA, RBT, CYC, and PAS by Sensititre.

31
CDC MPEP MTBC DST Report for September 2020 Survey

Table 42. Isolate 2020J—Participant Results for Molecular Testing
Drug

Mutation Detected

Mutation Not Detected

Total

Rifampin

9

0

9

Isoniazid

0

6

6

Ethambutol

0

4

4

Pyrazinamide

0

2

2

Ofloxacin

0

3

3

Ciprofloxacin

0

3

3

Levofloxacin

0

4

4

Moxifloxacin

0

4

4

Amikacin

0

3

3

Kanamycin

0

3

3

Capreomycin

0

2

2

Ethionamide

0

2

2

Rifabutin

3

1

4

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CDC MPEP MTBC DST Report for September 2020 Survey

Equivalent Critical Concentrations
(Concentrations listed as µg/ml)

Agar Proportion
First-line Drugs

7H10 agar

7H11 agar

Isoniazid

0.2 and 1.0*

0.2 and 1.0*

Rifampin

1.0

†

1.0

Ethambutol

5.0

7.5

Not recommended

Not recommended

Pyrazinamide

NOTE—Critical concentrations as indicated in CLSI M24 document [1]
*The higher concentration of INH should be tested as second-line drugs after resistance at the critical concentration is detected.
†
CLSI critical concentrations for RMP differ from revised WHO recommendation of 0.5 µg/ml published in 2021 [2].

Second-line Drugs

7H10 agar

7H11 agar

Streptomycin

2.0

2.0

Amikacin

4.0

Not determined*

Capreomycin

10.0

10.0

Kanamycin

5.0¥

6.0¥

Levofloxacin

1.0

Not determined*

Moxifloxacin

0.5

0.5

Ethionamide

5.0

10.0

Rifabutin

0.5

0.5

p-Aminosalicylic acid

2.0

8.0

†

NOTE—Critical concentrations as indicated in CLSI M24-A2 document [1]
*Breakpoints for establishing susceptibility have not be determined.
†CLSI critical concentration for AMK differ from revised WHO recommendation of 2.0 µg/ml published in 2018 [3].
¥CLSI critical concentration for KAN differ from revised WHO recommendation of 4.0 µg/ml for 7H10. WHO recommended the withdrawal of the current KAN
critical concentration for 7H11 published in 2018 [3].

Broth Based Media
First-line Drugs

MGIT

VersaTREK

Isoniazid

0.1 (and 0.4*)

0.1 (and 0.4*)

Rifampin

1.0

Ethambutol

5.0

5.0 (and 8.0*)

100.0

300.0

Pyrazinamide

1.0

†

NOTE—Critical concentrations as indicated in applicable manufacturer package inserts
*The higher concentration of INH and EMB should be tested after resistance at the critical concentration is detected.
†CLSI critical concentrations for RMP differ from revised WHO recommendation of 0.5 µg/ml published in 2021 [2].

Second-line Drug
Streptomycin

MGIT†

VersaTREK

1.0 (and 4.0*)

Not available

NOTE—Critical concentrations as indicated in applicable manufacturer package inserts
*The higher concentration of STR should be tested after resistance at the critical concentration is detected.
†
Revised WHO recommendations provide LEV and MOX critical concentrations for MGIT published in 2018 [3].

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CDC MPEP MTBC DST Report for September 2020 Survey

References
1. CLSI, Susceptibility Testing of Mycobacteria, Nocardiae spp., and Other Aerobic Actinomycetes, in 3rd Ed. CLSI Standard M24. 2018,
Clinical and Laboratory Standards Institute: Wayne, PA.
2. World Health Organization, Technical Report on critical concentrations for drug susceptibility testing of isoniazid and the
rifamycins (rifampicin, rifabutin and rifapentine). 2021: Geneva.
3. World Health Organization, Technical Report on critical concentrations for drug susceptibility testing of medicines used in the
treatment of drug-resistant tuberculosis. 2018: Geneva.
4. Campbell, P.J., et al., Molecular detection of mutations associated with first- and second-line drug resistance compared with
conventional drug susceptibility testing of Mycobacterium tuberculosis. Antimicrob Agents Chemother, 2011. 55(5): p. 2032-41.
5. Andre, E., et al., Consensus numbering system for the rifampicin resistance-associated rpoB gene mutations in pathogenic
mycobacteria. Clin Microbiol Infect, 2017. 23(3): p. 167-172.
6. APHL, Issues in Mycobacterium tuberculosis complex (MTBC) Drug Susceptibility Testing: Rifampin (RIF), in APHL Issues in Brief:
Infectious Diseases. 2019, Association of Public Health Laboratories: Washington, D.C.
7. APHL, TB Drug Susceptibility Testing Expert Panel Meeting Summary Report. 2007, Association of Public Health Laboratories:
Washington, D.C.
8. Almeida Da Silva, P.E. and J.C. Palomino, Molecular basis and mechanisms of drug resistance in Mycobacterium tuberculosis:
classical and new drugs. J Antimicrob Chemother, 2011. 66(7): p. 1417-30.
9. Zhang, Y. and W.W. Yew, Mechanisms of drug resistance in Mycobacterium tuberculosis. Int J Tuberc Lung Dis, 2009. 13(11):
p. 1320-30.
10. Ramaswamy, S.V., et al., Single nucleotide polymorphisms in genes associated with isoniazid resistance in Mycobacterium
tuberculosis. Antimicrob Agents Chemother, 2003. 47(4): p. 1241-50.
11. Ando, H., et al., A silent mutation in mabA confers isoniazid resistance on Mycobacterium tuberculosis. Mol Microbiol, 2014. 91(3):
p. 538-47.
12. Morlock, G.P., et al., ethA, inhA, and katG loci of ethionamide-resistant clinical Mycobacterium tuberculosis isolates. Antimicrob
Agents Chemother, 2003. 47(12): p. 3799-805.
13. Centers for Disease Control and Prevention, Availability of an assay for detecting Mycobacterium tuberculosis, including rifampinresistant strains, and considerations for its use—United States, 2013. MMWR Morb Mortal Wkly Rep, 2013. 62(41): p. 821-7.
14. Van Deun, A., et al., Rifampin drug resistance tests for tuberculosis: challenging the gold standard. J Clin Microbiol, 2013. 51(8):
p. 2633-40.
15. Starks, A.M., et al., Mutations at embB codon 306 are an important molecular indicator of ethAmbutol resistance in Mycobacterium
tuberculosis. Antimicrob Agents Chemother, 2009. 53(3): p. 1061-6.
16. Angra, P.K., et al., Performance of tuberculosis drug susceptibility testing in U.S. laboratories from 1994 to 2008. J Clin Microbiol,
2012. 50(4): p. 1233-9.
17. APHL, Issues in Mycobacterium tuberculosis Complex Drug Susceptibility Testing: EthAmbutol, in APHL Issues in Brief: Infectious
Diseases. 2016, Association of Public Health Laboratories: Washington, D.C.
18. Madison, B., et al., Multicenter evaluation of ethAmbutol susceptibility testing of mycobacterium tuberculosis by agar proportion
and radiometric methods. J Clin Microbiol, 2002. 40(11): p. 3976-9.
19. Zhang, Y. and W.W. Yew, Mechanisms of drug resistance in Mycobacterium tuberculosis: update 2015. Int J Tuberc Lung Dis, 2015.
19(11): p. 1276-89.
20. Centers for Disease Control and Prevention, Treatment of Tuberculosis, American Thoracic Society, CDC, and Infectious Diseases
Society of America. 2003, MMWR. p. 4,11,19-20.
21. Whitfield, M.G., et al., The potential use of rifabutin for treatment of patients diagnosed with rifampicin-resistant tuberculosis.
J Antimicrob Chemother, 2018. 73(10): p. 2667-2674.

34
CDC MPEP MTBC DST Report for September 2020 Survey

Appendix 1: Accessible Explanations of Figures
Figure 1. The primary classification of the 69 laboratories participating in the September 2020 MPEP survey is shown in this pie
chart. The largest slice, at 73%, represents 50 laboratories that have self-classified as a health department laboratory. The next
major slice signifies 9 hospital laboratories. The remaining three slices of the pie chart represent 7 independent laboratories, 2
federal government laboratories, and 1 laboratory self-identified as a medical manufacturer. (page 8)
Figure 2. The annual volume of MTBC isolates tested for drug susceptibility by participating laboratories (N=69) in 2019 is
displayed in this vertical bar graph. The vertical y–axis is the number of laboratories responding and ranges from 0 to 30 using
increments of 5. Along the horizontal x-axis are eight vertical bars representing the number of isolates tested per year. From left
to right, 28 laboratories tested less than or equal to 50 isolates per year; 13 laboratories tested between 51 to 100 isolates per
year; 6 laboratories tested between 101 to 150 isolates per year; 7 laboratories tested between 151 to 200 isolates per year; 2
laboratories tested between 201 to 300 isolates per year; 5 laboratories tested between 301 to 500 isolates per year; 6 laboratories
tested between 501 to 1000 isolates per year, and 2 laboratories tested greater than or equal to 1001 isolates per year. (page 9)
Figure 3. The drug susceptibility testing methods used by MPEP participants (N=97) is displayed in this vertical bar graph. The
vertical y-axis is the number of laboratories reporting with ranges from 0 to 70, by increments of 10, and the horizontal x- axis
lists the susceptibility testing methods. Each bar represents the number of reporting laboratories performing a particular drug
susceptibility test method. From left to right: 64 used MGIT, 17 used agar proportion, 4 used Sensititre, 2 used VersaTREK, and 10
used molecular methods. (page 10)
Figure 4. The molecular methods used by MPEP participants (N=10) are displayed in this pie chart. The largest slice
represents the 5 laboratories that perform targeted DNA sequencing. The next three slices represent 3 laboratories that use
the Cepheid Xpert MTB/RIF assay, 1 laboratory that uses Bruker line probe assays and 1 laboratory that uses whole genome
sequencing. (page 10)
Figure 5. The antituberculosis drugs tested by MPEP participants is displayed in a horizontal bar graph. The vertical y -axis
contains a list of each drug tested and the horizontal x-axis contains the number of laboratories with ranges from 0 to 80,
by increments of 10. There are 16 horizontal bars with each bar representing the number of laboratories reporting a result
for a particular drug for susceptibility testing. 69 laboratories tested rifampin; 69 laboratories tested isoniazid; 69 laboratories
tested ethAmbutol; 64 laboratories tested pyrazinamide; 44 laboratories tested streptomycin; 13 laboratories tested ofloxacin;
6 laboratories tested moxifloxacin; 5 laboratories tested ciprofloxacin; 5 laboratories tested levofloxacin; 14 laboratories tested
capreomycin; 13 laboratories tested kanamycin; 12 laboratories tested amikacin; 18 laboratories tested ethionamide; 13
laboratories tested PAS; 11 laboratories tested rifabutin; and 7 laboratories tested cycloserine. (page 11)

35
CDC MPEP MTBC DST Report for September 2020 Survey
CS 323576-A

For more information please contact
Centers for Disease Control and Prevention
1600 Clifton Road NE, Atlanta, GA 33029-4027
Telephone: 1-800-CDC-INFO (232-4636)
MPEP Telephone: 404-639-4013
MPEP Email: TBMPEP@cdc.gov
MPEP Web: www.cdc.gov/tb/topic/laboratory/mpep/default.htm
Publication date: April 2021

CS 323576-A