ORIGINAL ARTICLE

Direct Susceptibility Testing on MGIT 960 TB System:

A Rapid Method for Detection of Drug Resistant Tuberculosis
Nadia Tayyab, Gohar Zaman, Luqman Satti, Aamer Ikram, Adeel Hussain Gardezi and Muhammad Tahir Khadim

ABSTRACT
Objective: To evaluate direct drug susceptibility testing on MGIT 960 system for detection of multidrug resistant
tuberculosis from smear positive pulmonary specimens.
Study Design: Cross-sectional analytical study.
Place and Duration of Study: Microbiology Department, Armed Forces Institute of Pathology, Rawalpindi, from July 2016
to September 2017.
Methodology: Smear positive specimens were pretreated according to guidelines and then tested on MGIT 960 TB system
for direct drug susceptibility testing (DST) of isoniazid and rifampin. Samples were also processed by gold standard
indirect method, which comprises culture and then DST from positive growth by MGIT 960 TB system.
Results: Out of 108 specimens, 95 (88%) DST results were reportable. Out of 95 reportable specimens, 17 isolates were
resistant to both isoniazid (INH) and rifampin (RIF) by direct DST. The sensitivity, specificity, positive predictive value,
negative predictive value and diagnostic accuracy for INH were 92%, 93%, 82%, 97% and 92.6%, respectively; and 95%,
96%, 86.3%, 98.6% and 95.7%, respectively for RIF. Average time to report DST by indirect method was 23.6 ±3.9 days,
while it was 11.4 ±2.7 days for the direct method.
Conclusion: Direct susceptibility testing on MGIT 960 system showed very good agreement when compared with indirect
method. Time saving is crucial factor in initiation of early effective therapy, especially in drug resistant cases. Further
studies on large scale are required for more accurate evaluation of this method.

Key Words: BACTEC MGIT 960. Culture. Direct drug susceptibility testing. Multidrug resistant tuberculosis.

INTRODUCTION the second line injectable drugs.4 Drug resistance TB is

Tuberculosis (TB) is one of the most important infectious
diseases caused by mycobacterium tuberculosis. It is
the biggest killer among communicable diseases in most
productive years of human life in developing countries
that are densely populated. Nearly half of the world's TB
burden lies in developing countries of South East Asia.
In year 2016, an estimated 1.3 million people died due
to TB and an additional 0.37 million deaths in HIV
positive individuals.1
Resistance to anti-tuberculosis drugs has posed a
serious threat in curtailing this disease. Multidrug
resistant tuberculosis (MDR-TB) is defined as the
resistance against two primary drugs Isoniazid (INH)
and Rifampicin (RIF).2 Incidence of MDR-TB is
continuously rising globally.3 Poor compliance,
substandard anti-TB drugs, and delay in treatment of
MDR cases can develop into extensively drug resistant
tuberculosis (XDR-TB). XDR-TB is defined as MDR with
additional resistance to any fluoroquinolones and one of
Department of Microbiology, Armed Forces Institute of Pathology
(AFIP), Rawalpindi.
Correspondence: Dr. Luqman Satti, Consultant
Microbiologist, Department of Microbiology, Armed Forces
Institute of Pathology (AFIP), Rawalpindi.
E-mail: luqmansatti@hotmail.com
Received: January 11, 2018; Accepted: May 17, 2018.
a continuous threat to community and in year 2016, 0.49
million new cases of MDR TB were diagnosed.1
Rapid, reliable and standardised drug susceptibility
testing (DST) methods are crucial for effective treatment
and prevention of transmission. The gold standard
indirect DST technique takes longer time for reporting
results as first the isolate is cultured from the processed
specimen and then DST is carried out from positive
cultured specimen.5
MGIT 960 TB system is an automated, non-radiometric,
continuously monitoring system for detection of MDR-TB.6
MGIT 960 is considered as gold standard culture system
in many parts of the world as it has reduced the
turnaround time by rapid growth of MTB as compared
to its growth in solid media.7 This system has been
evaluated against the other methods for detection of
bacterial growth and DST; and has shown sensitivity of
100% for RIF, and INH and specificity ranging between
89 to 100%.8
DST of MTB is based on growth of mycobacterium in a
drug containing tube and comparing it with a drug-free
growth control tube. Continuous analysis of
fluorescence emitted by growth of MTB is detected by
this system automatically. However, the main
disadvantage is that culture and DST is a two-step
process; first the isolate is cultured and then in second
step, processed for DST. Isolation of MTB takes about

590 Journal of the College of Physicians and Surgeons Pakistan 2018, Vol. 28 (8): 590-593
Direct susceptibility testing on MGIT 960 TB system
2 - 4 weeks through MGIT system, followed by further
two weeks for DST.9 In the year 2012, a multicenter
study was carried out to get earlier results by direct DST
method on MGIT 960 system on smear positive
processed specimens omitting the first step of culture.
Results of this study were convincing with 95 to 96%
concordance with results obtained through conventional
two-step indirect DST method on same system.10
The rationale of this study was to investigate whether
direct susceptibility testing of INH and RIF on MGIT 960
system is feasible and expedites the detection of MDR TB
when compared with the standard indirect method.
The objective of this study was to evaluate direct drug
susceptibility testing on MGIT 960 system for detection
of multidrug resistant tuberculosis from smear positive
pulmonary specimens.
METHODOLOGY
This cross-sectional validation study was carried out at
Department of Microbiology, Armed Forces Institute of
Pathology, Rawalpindi, Pakistan. It is a tertiary care
reference laboratory with BSL-3 facilities for MTB culture
and sensitivity. Permission from Institutional Ethical
Committee was taken for the study.
All the consecutive smear positive pulmonary specimens
from fresh patients (patients who never took anti-TB
treatment) were pretreated with standard sodium
hydroxide-N-acetyl-L-cysteine method for digestion,
decontamination, homogenisation and concentration.11,12
A smear was made from the deposit and stained with
Ziehl-Neelsen stain. Smears were graded according to
WHO guidelines depending on the number of acid-fast
bacilli (AFB) observed under microscope.13 All the processed
specimens were inoculated in BACTEC MGIT 960
system both for direct DST as well as for conventional
indirect DST. M. tuberculosis H37Ra (ATCC 25177) was
used as a sensitive strain, and institutional MDR strain
was used as resistant strain for quality control (QC)
testing in DST. These strains were inoculated each time
when a new batch of DST was set up.
In direct DST, lyophilized antimicrobial mixture PANTA
(Becton Dickinson Diagnostic Systems, Sparks, MD)
containing polymyxin B, amphotericin B, nalidixic acid,
trimethoprim, and azlocillin was reconstituted with 15 ml
of SIRE supplement (not growth supplement) (Becton
Dickinson Diagnostic Systems, Sparks, MD) and mixed
thoroughly. A set of three MGIT tubes was prepared per
specimen. One tube was labelled as GC, one for INH
and the other for RIF. 800 ul of PANTA-SIRE supplement
mixture was added into each of the three-labelled MGIT
tubes. Then respective drugs were added in drug
labelled tubes. In the INH-labelled tube, 100 ul of
reconstituted lyophilized INH drug was added (0.1 g/ml
final concentration) and in RIF labelled tube, 100 l of
Journal of the College of Physicians and Surgeons Pakistan 2018, Vol. 28 (8): 590-593
reconstituted lyophilized RIF was added (1.0 g/ml).
Then 500 l of the well-mixed smear positive processed
specimen was inoculated in each of the two drug-
containing tubes. For GC tube, 500 l of 1:10 dilution of
processed specimen was added. For the direct DST, an
extended protocol, which is used routinely for indirect
PZA DST setup, was followed since direct DST requires
a 21-day protocol to complete the test.12 Tubes were
then entered in the instrument. The first tube in the set
carrier was always the growth control tube. When the
GC reached the required growth unit (GU) value of 400
or more, the susceptibility set was removed after
scanning, and an inventory report was printed. If GU
value of the drug tube was less than 100, the test result
was reported as susceptible (S); while if the GU value of
the drug tube was 100 or more, the result was interpreted
as resistant (R). In case the GU value of the control did
not reach 400 within 21 days, the instrument indicated
insufficient growth or contamination. The time it took for
the instrument to complete the DST test was recorded.
In the indirect DST procedure, the processed smear
positive specimens were inoculated in MGIT system for
culture of bacteria, according to manufacturer's
recommendations. Once an inoculated specimen
yielded positive growth indicated by signal on MGIT
system, positive growth was subsequently processed for
DST. Results of the indirect DST and time to complete
the test were retrieved from the instrument and
recorded. The time required for indirect DST was
compared with that of direct DST for every specimen.
The specimens which showed discrepant results
between the direct and indirect methods were retested
by repeating the indirect method.
Performance of direct DST by MGIT 960 TB system was
analysed by using SPSS (Statistical Package for the
Social Sciences), version 21 to calculate sensitivity,
specificity, positive predictive value, negative predictive
value and diagnostic accuracy. Chi-square test was used
to find association between qualitative variables and
p-value of <0.05 was considered as significant. In this
study, sensitivity represents the ability of a test to detect
true resistance, and specificity indicates the ability of a
test to detect true sensitivity of an isolate.
RESULTS
Out of 108 smear positive specimens, 47 (43.5%) were
endobronchial washings, 43 (39.8%) sputum samples,
15 (13.8%) bronchoalveolar lavage, and 3 (2.7%) pleural
fluid. From processed specimens, 95 (88%) DST results
were reportable by both the methods. Thirteen results
were not included in the final analysis. Out of invalid
results, 5 specimens had contamination (X400 error),
4 did not reach the required threshold for GC (X200
error), 3 were identified as mycobacteria other than
tuberculosis (MOTT) and 1 did not yield positive culture
591
Nadia Tayyab, Gohar Zaman, Luqman Satti, Aamer Ikram, Adeel Hussain Gardezi and Muhammad Tahir Khadim

Table I: Comparison of results of direct DST with indirect DST.

Drugs Direct DST Indirect DST Direct DST %
R S Sensitivity Specificity PPV NPV DA
INH R 23 5 92 93 82 97 92.6
S 2 65
RIF R 19 3 95 96 86.3 98.6 95.7
S 1 72
PPV = Positive predictive value; NPV = Negative predictive value; DA = Diagnostic accuracy.

Table II: Average time for detection by both methods and average time
saved (days).
Smear No. of Average TT-DST
index specimens (Mean ± SD)
Indirect Direct
Method Method
1+ 14 23.8 ±3.4 11.57 ±2.9
2+ 39 23.4 ±3.8 11.33 ±2.9
3+ 30 23.1 ±3.7 11.1 ±2.2
4+ 12 24.3 ±5.2 11.9 ±3.2
TT-DST: Total time to drug susceptibility testing results, SD: Standard deviation.
by direct method. The sensitivity, specificity, positive
predictive value, negative predictive value, and diagnostic
accuracy for INH and RIF are shown in Table I.
Out of 95 reportable results, 62 (65%) isolates were
sensitive to both INH and RIF, while 17 (18%) isolates
were MDR by direct DST. A total of 16 (16.8%) results
showed mono resistance, 11 (11.5%) isolates showed
resistance to INH, whereas 5 (5.2%) were resistant to
RIF only by direct DST. By indirect DST, 66 isolates were
sensitive to both drugs, 16 were MDR, 9 were resistant
to INH only, and 4 were resistant to RIF. All the
inconsistent results were retested by indirect method for
confirmation.
The time to report results of positive cultures was
analysed according to the degree of AFB smear
positivity. Smear grades were 2+ 39 (41.1%) and 3+ 30
(31.6%) for the majority of specimens. The time interval
taken to yield positive result by indirect DST was not
significantly different between different smear grades
(p=0.90). Overall, cultures were positive at an average
of 12 days, with a range of 10 to 20 days, whereas
average time to report indirect DST from isolated
cultures was 9 days ranging from 6 days to 14 days. The
total time required from the time the specimen was
processed to the time when indirect DST results were
available ranged from 15 to 34 days. Maximum results
were available within 24 days. The time to complete
direct DST from processed specimens was calculated
according to the smear-positive grades, but it did not
reveal any significant association (p = 0.94), similar to
that noted in indirect DST. Results were ready within 7 to
19 days, out of which maximum results were ready
within 12 days. (Table II).
DISCUSSION
Early detection of MDR strains and timely management
of patients are very crucial to control spread of these
resistant strains in population. Basically, there were three
main differences in the direct DST procedure compared
Average time to the conventional indirect DST procedure on MGIT 960
saved system.9 (i) Direct DST was a 4- to 21-day protocol,
while indirect DST was a 4- to 13-day protocol; (ii) Growth
control was diluted 1:100 in indirect DST, while in direct
12.23
DST it was diluted 1:10; (iii) PANTA was added to the
12.1
12.0
control as well as in the drug-containing MGIT tubes in
12.4
direct DST; whereas in indirect DST, PANTA was not
added. To the best of our knowledge, this is the third
reported study on direct DST by MGIT 960 system for
the detection of MDR TB from smear positive clinical
specimens. The first study was carried out by Siddiqi
et al. in 2012, which was a multicenter study and second
study by Zhang et al. in 2015.10,14 However, in both the
studies, authors only included smear positive sputum
specimens; while in this study, the authors included all
the smear positive pulmonary specimens from new
patients. In another study, Demers et al. tested the
susceptibility of pyrazinamide by direct method on
MGIT 960 system.15
Most of the available methods for DST of MTB are very
slow and cumbersome other than commercial molecular
methods.16 Although commercially available molecular
assays yield earlier results with good sensitivity and
specificity, yet these molecular tests are costly and
expertise is required.17 Couple of other DST methods
such as nitrate reductase assay (NRA), thin layer agar
(TLA), and microscopic observation drug susceptibility
(MODS) on smear positive clinical specimens have also
been studied in last two decades.18-20 Although these
methods are inexpensive but are laborious, and take
longer time to yield results with increased risk of aerosol
generation and contamination.
In this study, out of 108 specimens, 5 (4.6%) were
contaminated (X400 error), still in the acceptable range
for a laboratory. These results are consistent with the
study by Zhang et al. but in disagreement with the study
by Siddiqi et al. in which majority of the specimens,
which were not reportable, had X200 error. In this study,
out of total 95 specimens, individually INH had 88
(92.6%) while RIF had 91 (95.8%) concordance. The
results of this study are similar to study by Siddiqi et al.
in which there was 95.1% concordance for INH and
96.1% for RIF.
In this study, majority of the isolates had 2+ and 3+ smear
grades; a finding similar to studies by Siddiqi et al. and
Zhang et al. However, time to report DST had no

592 Journal of the College of Physicians and Surgeons Pakistan 2018, Vol. 28 (8): 590-593
Direct susceptibility testing on MGIT 960 TB system
significant association with grades of smear in both
direct and indirect methods. Siddiqi et al. also observed
this finding in their study, but Zhang et al. reported a
significant relation of smear grades with time to report
DST. Average time to report DST for indirect method was
23.6 days while it was 11.4 days for the direct method.
These results were similar to the studies done by Siddiqi
et al. and Zhang et al.
The main focus of this study was time-saving by direct
method when compared with indirect method. Overall
time-saving by direct method in our study was 12 days.
This study showed slightly more time-saving as
compared to studies by Siddiqi et al. and Zhang et al. in
which time savings were 8 days and 10.5 days,
respectively. The most likely reason could be that it was
a single centre study and also the number of specimens
was not too large. This time saving is very important as
it can help the clinicians in initiation of early therapy and
controlling the spread of MDR isolates.
Direct DST has two main disadvantages: Firstly, there
may be no or poor growth of mycobacteria, in which
case the indirect DST becomes mandatory thus further
delay in diagnosis and therapy. Secondly, the presence
of NTM can give invalid results, especially in areas
where NTM has high prevalence. This study has two
main limitations: Firstly it was carried out at one setup
and number of specimens was not too large as
compared to previous study by Siddiqi et al., which was
a multicenter study with 360 clinical specimens.
Secondly, the representation of MDR isolates was small
(18%) of the total isolates.
CONCLUSION
Direct DST is a reliable method with very good agreement
when compared with indirect method. The time saving is
significant, which can help in initiation of early effective
therapy, especially in MDR cases. Further studies on
large scale and on other drugs are needed to know its exact
accuracy and reliability with INH and RIF and other drugs.
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