JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 2009, p. 3630–3634
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 47, No. 11
Multicenter Evaluation of Bactec MGIT 960 System for Second-Line
Drug Susceptibility Testing of Mycobacterium tuberculosis Complex?
S.-Y. Grace Lin,1* Edward Desmond,1Donald Bonato,2Wendy Gross,2and Salman Siddiqi3
Microbial Diseases Laboratory, DCDC, CID, California Department of Public Health, Richmond, California1;
Tuberculosis Reference Laboratory, Veterans Affairs Medical Center, West Haven, Connecticut2; and
Becton Dickinson Diagnostic Systems, Sparks, Maryland3
Received 20 April 2009/Returned for modification 18 June 2009/Accepted 30 August 2009
The Bactec MGIT 960 system for testing susceptibility to second-line drugs was evaluated with 117 clinical
strains in a multicenter study. The four drugs studied were levofloxacin, amikacin, capreomycin, and ethio-
namide. The critical concentration established for levofloxacin and amikacin was 1.5 ?g/ml, that established
for capreomycin was 3.0 ?g/ml, and that established for ethionamide was 5.0 ?g/ml. The overall level of
agreement between the agar proportion method and the MGIT 960 system was 96.4%, and the levels of
agreement for the individuals drugs were 99.1% for levofloxacin, 100% for amikacin, 97.4% for capreomycin,
and 88.9% for ethionamide. The rate of reproducibility of the drug susceptibility testing results between the
participating laboratories was 99.5%.
The increase in the incidence of multidrug-resistant tuber-
culosis (MDR TB) and the emergence of extensively drug-
resistant tuberculosis present tremendous challenges to the
global efforts to combat tuberculosis (1, 5, 16, 21). Rapid meth-
ods enabling accurate susceptibility testing of first-line and
second-line drugs are critical for the early diagnosis of MDR
TB and extensively drug-resistant tuberculosis and the initia-
tion of effective regimens. Various drug susceptibility testing
(DST) methods that use solid media, including the agar pro-
portion method (AP) and other methods, have the drawback of
prolonged turnaround times (TATs). The World Health Or-
ganization and the U.S. Centers for Disease Control and Pre-
vention have recommended the use of liquid culture systems
for the diagnosis of tuberculosis and DST to improve TATs
(22, 25). The Bactec 460 (Becton Dickinson Diagnostic Sys-
tems, Sparks, MD), a radiometric liquid system, provided an
excellent alternative for testing of the susceptibilities of Myco-
bacterium tuberculosis complex (MTBC) isolates to streptomy-
cin, isoniazid, rifampin (rifampicin), and ethambutol (SIRE)
and to pyrazinamide (PZA) with improved TATs. The MGIT
960 liquid, nonradiometric SIRE DST (Becton Dickinson Di-
agnostic Systems), whose performance is comparable to that of
the Bactec 460 system, has been commercially available since
April 2002 (4, 20, 23). The Microbial Diseases Laboratory
(MDL) of the California Department of Public Health imple-
mented SIRE DST with the MGIT 960 system in 2004. With
confidence in the SIRE DST with the MGIT 960 system, a
study that used the same platform to test the susceptibilities of
MTBC isolates to four classes of second-line drugs, levofloxa-
cin (LVX), amikacin (AMK), capreomycin (CAP), and ethio-
namide (ETH), was initiated in November 2004. The study was
conducted at two laboratories: MDL and the TB Reference
Laboratory of the Veteran Affairs Medical Center (VA) in
West Haven, CT. Here we report the results of the multicenter
study, in which the critical concentrations of the test drugs
were established, the performance of the MGIT 960 system
was compared to that of AP, and the interlaboratory repro-
ducibility of the method was evaluated.
(Part of this work was presented at the 46th Interscience
Conference on Antimicrobial Agents and Chemotherapy,
2006, San Francisco, CA.)
MATERIALS AND METHODS
Study design. This study consisted of five phases: phase I, determination of
the MIC of each drug for the susceptible control strain, Mycobacterium
tuberculosis H37Rv (ATCC 27294); phase II, determination of the critical
concentration of each drug; phase III, testing of clinical strains at the critical
concentration of each drug determined in phase II; phase IV, study of inter-
laboratory reproducibility; and phase V, study of the reproducibility of the
medium and reagents.
Strains. A total of 117 clinical strains of MTBC, including 49 pansusceptible
strains and 68 resistant strains whose drug susceptibility results were previously
determined by AP, were tested. MDL contributed 89 strains (42 susceptible and
47 resistant strains), and VA contributed 28 strains (7 susceptible and 21 resis-
tant strains). At MDL, the pansusceptible strains were randomly chosen from
cultures in which MTBC was isolated between 2003 and 2006. As for the resistant
strains, we included as many strains from our archived collections as we could
find, excluding those found to be nonviable or contaminated. Strains from VA
were selected from cultures in which MTBC was isolated between 2002 and 2003
to include as many resistant strains as possible, and susceptible strains from the
same period were randomly chosen.
Antimicrobial agents. AMK, CAP, and ETH were purchased from Sigma-
Aldrich Corporation (St. Louis, MO); and LVX was provided by Johnson &
Johnson Pharmaceutical Research & Development, L.L.C. (Spring House, PA).
Each laboratory prepared drug solutions according to the protocol described
below. The stock solutions of AMK (120 ?g/ml), CAP (75 ?g/ml), and LVX (120
?g/ml) were prepared in sterile water. All drug concentrations, including the
concentrations in the stock solutions, the MICs, and the critical concentrations,
are expressed as the final concentrations used in tests with the MGIT 960 system.
These stock solutions were filtered through 0.22-?m-pore-size Millex-GS filter
units (Millipore, Bedford, MA) and were stored at ?20°C. The working solutions
of AMK, CAP, and LVX were diluted from the stock solution, aliquoted, and
frozen for future use. The stock solution of ETH (120 ?g/ml) was prepared in
ethylene glycol (Spectrum Quality Products, Inc., Gardena, CA) and was incu-
bated at 37°C overnight. It was then filtered through 0.22 ?m-pore-size Mil-
lex-GS filter units, aliquoted, and stored at ?20°C. The working solution was
* Corresponding author. Mailing address: Microbial Diseases Lab-
oratory, CDPH, 850 Marina Bay Parkway, Richmond, CA 94804.
Phone: (510) 412-3929. Fax: (510) 412-6099. E-mail: grace.lin@cdph
?Published ahead of print on 9 September 2009.
by on November 3, 2009
made fresh from the stock solution in sterile water for each test run. (After this
study was completed, some aliquots of the ETH stock solution were found to
contain ETH precipitates. Those precipitates did not readily dissolve at room
temperature. Storage of the ETH stock solutions at 2 to 8°C and periodic testing
of the MIC of ETH for the control strain is recommended. Our experience
indicated that the MIC of ETH for the H37Rv control strain remained un-
changed over a 2-year period.)
Preinoculation preparation. The LVX, AMK, CAP, and ETH drug panel
consisted of five MGIT tubes: one tube for the growth control and four tubes
for the drugs (one for each drug). Preinoculation preparation included ad-
dition of 0.8 ml SIRE supplement to each MGIT tube and addition of 0.1 ml
drug working solution to each designated MGIT 960 system tube. No drug
was added to the growth control tube. The SIRE supplement containing oleic
acid, albumin, dextrose, and catalase (OADC) is one of the components of
the SIRE drug kit (Becton Dickinson Diagnostic Systems). The Middlebrook
OADC enrichment from BBL (Becton Dickinson Diagnostic Systems) is an
alternative to the SIRE supplement. According to Becton Dickinson Diag-
nostic Systems (personal communication), the two products are identical.
Inoculum preparation. Inocula were prepared from cultures of all 117 strains
grown in Lowenstein Jensen (LJ) slants. The cultures were 2 to 5 weeks old. The
manufacturer’s protocol for the preparation of inocula from solid media for use
in the SIRE DST was followed, with one exception: sterile normal saline instead
of Middlebrook 7H9 broth or MGIT broth was used to prepare the cell suspen-
sions and to adjust the turbidity of the suspension to a 0.5 McFarland standard.
In addition, the procedure was further validated for the testing of positive MGIT
tubes. A subset of 23 strains was subcultured to MGIT tubes and tested after
growth was detected by the MGIT 960 system. The inoculum prepared from
MGIT tubes was also standardized to a 0.5 McFarland standard. Briefly, the
growth at the bottom of a positive MGIT tube was transferred to a sterile tube
containing 2 ml sterile normal saline and the components were gently mixed.
Clumps were allowed to settle. The supernatant containing the homogeneous
cell suspension was transferred to another sterile tube, and its turbidity was
adjusted to a 0.5 McFarland standard. After the cell suspension was standard-
ized, it was diluted 1:5 with sterile normal saline for inoculation into drug-
containing MGIT tubes. The 1:5 cell suspension was further diluted 100-fold with
sterile normal saline for inoculation of the growth control tube. The volume of
each inoculum was 0.5 ml.
Determination of the MIC of each drug for H37Rv. On the basis of our
experiences with the Bactec 460 system, three concentrations of each drug were
chosen for testing. These were 0.25, 0.5, and 1.0 ?g/ml for AMK, LVX, and ETH
and 0.5, 1, and 1.5 ?g/ml for CAP. Three experiments were performed at each
laboratory, and the results obtained from the two laboratories were compared. This
was designed to confirm that the drug concentrations prepared at each laboratory
step in the establishment of a sound basis for a multicenter study.
Determination of critical concentration for each drug. Determination of the
critical concentration for each drug was done at MDL. A subset of 29 strains
from the 117-strain pool, including 8 pansusceptible strains and 21 resistant
strains with various drug susceptibility profiles (15 strains resistant to AMK and
CAP, 13 resistant to ETH, and 10 resistant to LVX), were studied. An additional
four quinolone-resistant strains with known gyrA mutations, obtained from B.
Kreiswirth at the Public Health Research Institute Tuberculosis Center (Newark,
NJ), were tested only for their susceptibilities to LVX. By testing both suscep-
tible and resistant strains, this phase was designed to determine the critical
concentration for each drug that could best distinguish the susceptible group
from the resistant group. Three concentrations were tested for each drug. AMK
and LVX were tested at 1, 1.5, and 2 ?g/ml; CAP was tested at 2, 3, and 4 ?g/ml;
and ETH was tested at 3, 4, and 5 ?g/ml.
Testing of additional clinical strains at the critical concentration of each drug.
After determination of the critical concentrations, an additional 88 clinical
strains (41 susceptible and 47 resistant strains) were tested to further establish
that the critical concentrations would correctly discriminate susceptible and
resistant strains on a larger scale. MDL tested 60 strains, and VA tested 28
Study for reproducibility between laboratories. A total of 48 strains from the
study pool (28 strains submitted by VA to MDL and 20 strains submitted by
MDL to VA) were tested at the critical concentrations at both laboratories. Of
the 48 strains, 17 were pansusceptible strains and 31 were resistant strains with
various drug susceptibility profiles. Two sets of 192 data points were compared to
determine the reproducibility between the two laboratories.
Study of reagent lot-to-lot reproducibility. A subset of six strains from the
study pool (three susceptible strains and three resistant strains) were tested at
the critical concentrations with three lots of MGIT medium and three lots of
SIRE supplement in three different test runs at MDL. Cell suspensions were
prepared fresh for each test run. Three sets of 24 data points were analyzed to
determine the reagent reproducibility.
QC. A strain of Mycobacterium tuberculosis, H37Rv (ATCC 27294), was used
as a quality control (QC) strain and was tested with each batch of DST at the
critical concentration of each drug. This QC strain is pansusceptible to the four
drugs tested in the present study. If the QC strain did not yield the expected
results, the test with that batch had to be repeated.
Reference methods and discrepancy resolution. AP was the primary refer-
ence method for this study. All strains were previously tested by AP with
AMK at 2 ?g/ml (strains from VA were tested at 6 ?g/ml), CAP at 10 ?g/ml,
ETH at 5 ?g/ml, and OFX at 2 ?g/ml. LVX is the L form of OFX. In addition
to using the results obtained by AP with OFX as a reference, we also tested
LVX at 2 ?g/ml with the Bactec 460 system (19). For the resolution of
discrepancies, DST was repeated at VA by AP and at MDL with the MGIT
960 and Bactec 460 systems. In tests with the Bactec 460 system, the test
concentrations were 1 ?g/ml for AMK, 1.25 ?g/ml for CAP, 2.5 ?g/ml for
ETH, and 2 ?g/ml for LVX.
Interpretation of results. The MGIT 960 system supports
the testing of various combinations of SIRE and PZA panels
configured by the manufacturer, but second-line drug panels
are not available. Testing of second-line drugs must be regis-
tered in the MGIT 960 system as one of the SIRE panels, and
the user manually enters the drug identification on the printout
of the results. The MGIT 960 system flags the completion of a
DST when the growth unit (GU) of the growth control reaches
400 and reports S for susceptible or R for resistant, as well as
a GU value for each drug-containing MGIT tube on the print-
out. An isolate is interpreted to be susceptible when the GU of
a drug-containing MGIT tube is equal to or less than 100 or as
resistant when the GU is greater than 100. If an isolate was
interpreted to be resistant, a smear was made and stained to
prove the presence of acid-fast bacilli (AFB) with morphology
compatible with that of MTBC and the absence of contami-
MIC of each drug for H37Rv (ATCC 27294). The results
obtained at both participating laboratories demonstrated
100% reproducibility in the three runs performed at each lab-
oratory. The MIC results for AMK, LVX, and ETH obtained
at VA and MDL were the same (0.5 ?g/ml for the three drugs),
but the MIC of CAP was slightly lower at VA (1.0 ?g/ml at VA
and 1.5 ?g/ml at MDL). To resolve this discrepancy, CAP was
tested with an additional concentration of 1.25 ?g/ml at MDL,
and the MIC of CAP was determined to be 1.25 ?g/ml three of
three times. Thus, the difference in the MIC for CAP at MDL
and VA was minimal and acceptable.
Critical concentration of each drug. The critical concentra-
tion of each drug established by this study is shown in Fig. 1. To
yield the best discrimination between susceptible and resistant
strains, the critical concentrations were determined to be 1.5
?g/ml for AMK and LVX, 3.0 ?g/ml for CAP, and 5.0 ?g/ml
for ETH. Determination of the critical concentrations for
AMK, LVX, and CAP was straightforward, since we were able
to decide on a concentration at which 100% of the resistant
strains grew in the presence of the drug and 100% of the
susceptible strains were inhibited. For ETH, the critical con-
centration was set at 5.0 ?g/ml to yield the best discrimination
between susceptible and resistant strains, yet 3 strains with
discordant results were found among the 29 strains tested,
which yielded a 90% (26/29) correlation between the results
VOL. 47, 2009TESTING SECOND-LINE DRUGS AGAINST MTBC WITH MGIT 960 3631
by on November 3, 2009
obtained with the MGIT 960 system and by AP. Twelve of 13
(92.3%) resistant strains yielded resistant results, and 14 of 16
(87.5%) susceptible strains yielded susceptible results. Among
the three discrepancies, one strain, which tested resistant by
AP but susceptible by MGIT 960, had a MIC of 4.0 ?g/ml by
MGIT 960, and two strains, which tested susceptible by AP but
resistant by MGIT 960, had MICs greater than 5.0 ?g/ml (the
MIC for one strain was 8.0 ?g/ml and the MIC for the other
one was greater than 10 ?g/ml) by MGIT 960.
Overall agreement. The overall agreement between the re-
sults obtained with MGIT 960 and by AP was 96.4% (451/468),
and a comparison of the results between the two methods after
discrepancy resolution is shown in Table 1. For AMK, there
was 100% agreement. For CAP and LVX, a total of four
discrepancies were detected, but the results obtained with
Bactec 460 were in concordance with those obtained with the
MGIT 960 system. For ETH, there were 13 discrepancies. Two
strains (designated group A) tested resistant by AP but sus-
ceptible by MGIT 960, and 11 strains (designated group B)
tested susceptible by AP but resistant by MGIT 960. Both
strains in group A demonstrated pinpoint colonies on the
ETH-containing quadrant on Middlebrook 7H10 plates by AP.
It indicated some degree of inhibition by the drug, although
the test result was interpreted to be resistant. One of these two
strains tested susceptible by Bactec 460; the other strain tested
equivocal (in one test it was susceptible and in the other test it
was resistant and had a low increase in the growth index). Ten
of the 11 strains in group B tested resistant and 1 tested
susceptible by Bactec 460. The overall rate of agreement was
88.9% (104/117) between AP and MGIT 960; however, the
results for 11 of the 13 strains with discrepancies were in
concordance between the Bactec 460 and MGIT 960 systems.
Interlaboratory reproducibility. A subset of 48 strains from
the study pool was tested at both laboratories. The interlabo-
ratory reproducibility study was analyzed on the basis of the
results of first-shot testing at each laboratory. One discrepancy
was found between the two sets of 192 test results, which
yielded an overall reproducibility of 99.5% (191/192) (Table 2).
No discrepancies were found for LVX, AMK, and ETH, which
yielded 100% reproducibility for each of the three drugs. The
only discrepancy occurred with CAP. The particular strain with
the discrepancy tested susceptible at VA but resistant at MDL
and had a GU of 177. Upon retesting at MDL, this strain
tested susceptible and had a GU of 6, but substantial growth in
the MGIT tube was observed, which indicated the presence of
Reproducibility of reagents. Three lots of MGIT medium
and three lots of SIRE supplement were tested with three
susceptible strains and three resistant strains. A total of 72 data
points were generated, and 100% reproducibility was realized.
The duration for the completion of a DST in the MGIT 960
system is called “time in protocol” (TIP) by the manufacturer.
The average TIPs for the three sets of tests were 8 days and
9.2 h, 8 days and 15.4 h, and 8 days and 8.3 h. The MGIT 960
system demonstrated excellent reproducibility with different
lots of MGIT medium and growth supplement.
FIG. 1. MIC of each drug determined by use of the MGIT 960 system with strains that tested susceptible (S) or resistant (R) by AP.
TABLE 1. Comparison of results obtained by use of MGIT 960
No. of strains with indicated
results by AP/MGIT 960b
aA total of 117 strains were tested.
bR, resistant; S, susceptible.
cThe data represent percent agreement (number of strains with the correct
result/total number of strains tested).
dTested resistant with the Bactec 460 system.
eOne of two strains tested susceptible with the Bactec 460 system.
fTen of 11 strains tested resistant with the Bactec 460 system.
gThe results obtained with the MGIT 960 and Bactec 460 systems were
concordant for 11 of 13 discrepancies.
3632 LIN ET AL.J. CLIN. MICROBIOL.
by on November 3, 2009
Inoculum source: growth from LJ slants versus that from
MGIT medium. Inocula were prepared from 23 seeded MGIT
tubes (which included 8 pansusceptible strains and 15 resistant
strains with various drug susceptibility profiles), and the results
were compared with those prepared from the LJ slants. There
was a 100% reproducibility in the results between the inocula
prepared from MGIT tubes and LJ slants. The reproducibility
was excellent, which may be due to the use of homogeneous
cell suspensions and standardization of the inocula, regardless
of the source of growth.
Use of the Bactec 460 system to test for susceptibility to the
primary drugs SIRE and PZA has been cleared by the U.S.
Food and Drug Administration (FDA). It has been standard-
ized and widely used, and its performance has been considered
a reference standard. Although use of the Bactec 460 system
for DST of second-line drugs has been studied and used by
some laboratories (15), FDA has not approved it for use with
those drugs. Many laboratories still use AP to perform testing
for susceptibility to second-line drugs. Therefore, we used AP
as the primary reference method for validation of the DST
with second-line drugs with the MGIT 960 system.
The basis for this study was, first, the establishment of com-
parable MICs of each drug for the control strain, H37Rv
(ATCC 27294), at both laboratories. The MIC study, which
demonstrated excellent interlaboratory agreement, provided a
sound basis and confidence for the remainder of the study.
Second, in the process of determining the critical concentra-
tion for each drug, we did not take the traditional twofold
dilution approach, which was used in two previous studies (9,
18). Instead, we tested additional drug concentrations within
twofold dilutions. This fine-tuning approach enabled us to se-
lect a drug concentration that could better discriminate sus-
ceptible and resistant strains.
The overall levels of agreement between the results obtained
by AP and with MGIT 960 were excellent for AMK, CAP, and
LVX, which were 100%, 97.4%, and 99.1%, respectively. How-
ever, the agreement for ETH was only 88.9%. This level of
correlation between the results obtained by use of MGIT 960
and AP for ETH was also demonstrated in a study conducted
by Martin et al. (12). The results obtained for ETH revealed a
problematic aspect of the DST with ETH. In our opinion, to
validate a new method, when the correlation between the new
method and the reference method is greater than 95%, it
provides a relatively high degree of confidence in the applica-
bility of the new method. However, we were unable to find a
critical concentration that could yield greater than 95% dis-
crimination between susceptible strains and resistant strains
when AP was used as the “gold standard” for DST with ETH.
Lefford and Mitchison (10) conducted a study and found that
the MIC distributions of ETH between probable susceptible
strains (specimens obtained from patients before treatment)
and probable resistant strains (specimens obtained after treat-
ment with ETH) were not well separated. In the process of
resolving the discrepancies for ETH, we tested the MICs for
the 11 strains which tested resistant by MGIT 960 but suscep-
tible by AP. We found that 7 of the 11 strains had MICs greater
than 10 ?g/ml, 3 strains had MICs of 10 ?g/ml, and 1 strain had
an MIC of 8 ?g/ml. These MICs were much higher than the
critical concentration (5 ?g/ml) established in this study, and
we inferred that the discrepancies were unlikely to be resolved
by choosing a higher drug concentration as the critical concen-
tration. To prove this, we retested all ETH-resistant strains at
6, 8, and 10 ?g/ml, and we found that 6 of the 43 resistant
strains became susceptible at 6 ?g/ml and that 15 strains be-
came susceptible at 10 ?g/ml. Thus, none of the higher con-
centrations would generate a better correlation. Furthermore,
we also used the Bactec 460 system to evaluate the strains with
discrepancies, and we found that the results between the
MGIT 960 and Bactec 460 systems were in concordance for 11
of 13 strains. The better correlation between the MGIT 960
and the Bactec 460 systems shown in our study was also dem-
onstrated in studies conducted by Rodrigues et al. (17) and
Rusch-Gerdes et al. (18). These results may indicate that the
causes of the discrepancies for ETH were perhaps attributable
more to methodology differences than to the selection of the
The overall reproducibility studied in phase IV was excel-
lent. We found only one discrepant result with CAP. For the
particular strain with that result, MDL not only could not
reproduce the result from VA but also could not reproduce its
own result when the strain was retested. Poor reproducibility
may be expected when a strain has an MIC that is at or very
close to the critical concentration (8, 24). As shown in this
study, this strain initially tested resistant at MDL and had a
GU of 177, but it was susceptible on retesting and had a GU of
6. The cutoff for resistance is a GU of 100. The first result was
interpreted to be resistant, and the second result was inter-
preted to be susceptible. How to interpret such results presents
a challenge. Although the majority of drug tests had GU values
of 0 for susceptibility and 400 for resistance, 2.7% (13/468) of
the tests in this study yielded GU values between 1 and 399. It
may be advisable to designate a borderline category for the
interpretation of results that fall in this range. We believe that
it is more reliable to report a borderline result as “borderline”
than “susceptible” or “resistant” by chance. However, estab-
lishment of the precise definition of “borderline” is beyond the
scope of this study.
The MGIT 960 system DST report contains a TIP with two
figures (days and hours) for each DST panel tested. The aver-
age TIP was 8 days and 6 h at MDL and 9 days and 8 h at VA.
We were not aware of the TIP difference when the MIC study
was conducted in phase I. Although it did not seem to affect
the results, it would have been better if the TIP issue had been
TABLE 2. Results of interlaboratory reproducibilitya
No. of strains with indicated result for
drug at MDLb
LVX AMKCAP ETH
330 290 291 250
0 150 190 180 23
aS, susceptible; R, resistant.
bThe overall agreement was 99.5% (191/192 strains); and the levels of agree-
ment for the individuals drugs were 100% (48/48 strains) for LVX, 100% (48/48
strains) for AMK, 98% (47/48 strains) for CAP, and 100% (48/48 strains)
VOL. 47, 2009TESTING SECOND-LINE DRUGS AGAINST MTBC WITH MGIT 9603633
by on November 3, 2009
addressed at the beginning of the study. An ideal DST protocol
would be one that can generate accurate results with the short-
est TIP. This study used a 1:5 dilution of the cell suspension
equivalent to a 0.5 McFarland standard as an inoculum. A
future study is required to find the optimal cell concentration,
which could shorten the average TIP yet still produce accurate
As a routine practice, only primary drugs are tested initially.
When strains test resistant to rifampin or any two of the pri-
mary drugs, second-line drugs are then tested. When resistance
to primary drugs is detected, the growth on LJ slants is usually
adequate for the testing of second-line drugs. In this study, we
used the growth from LJ slants as the main source for the
inoculum preparation. With advances in molecular technolo-
gies, isoniazid and/or rifampin resistance can be detected by
rapid methods, such as those that use molecular beacons, line
probes, or microchips (2, 3, 6, 7, 11, 13). Once rifampin resis-
tance or MDR TB is detected, testing for susceptibility to
second-line drugs should be performed along with testing for
susceptibility to first-line drugs as early as possible, and testing
should not wait for growth from LJ slants. Therefore, we in-
cluded a subset of 23 specimens for validation of this assay
using inocula prepared from positive MGIT tubes. Our proto-
col prepares inocula from MGIT tubes by standardization of
the growth to a 0.5 McFarland standard, as is done for LJ
slants. DST can be performed from a primary MGIT tube and
not subject to the manufacturer’s time schedule, as stated in
the package insert for SIRE DST. This has two advantages: the
inoculum does not contain coarse clumps, which helps to pro-
duce more consistent results, and it does not require seeding of
a MGIT tube and waiting for its growth for DST, thus improv-
ing the TAT and reducing the cost for medium.
This study presents a DST protocol that uses a homoge-
neous, standardized cell suspension to prepare the inoculum
regardless of the source of growth: solid or liquid medium. The
verification data were good, and the reproducibilities for in-
terlaboratory test results, different lots of reagents, and differ-
ent sources of inocula were excellent. This study and previous
studies (9, 17, 18), regardless of the reference methods used,
provided data that may be used to demonstrate that DST with
the MGIT 960 system is a reliable method for the testing of
We thank Becton Dickinson for providing the MGIT tubes, SIRE
supplements, and Bactec 12B vials and Johnson & Johnson Pharma-
ceutical Research & Development, L.L.C., for providing LVX. We
also thank Beverly Metchock at CDC for assistance with performing
DST by AP for several strains with discrepancies.
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