Evaluation of BACTEC MGIT 960 System for Recovery of Mycobacterium tuberculosis Complex in Pakistan

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Abstract
We evaluated the performance of MGIT 960 system in terms of recover rate, detection time of mycobacteria and contamination rate from various human clinical specimens and compared it with already in use BACTEC 460 TB system and conventional LJ medium. This is the first reported study on MGIT 960 and its comparison with BACTEC 460 system in Pakistan. A total of 260 different clinical specimens received for the culture of mycobacteria were dealt during the six months study period. All the specimens were digested and decontaminated according to the standard N-acetyl-Lcysteine NaOH method. All the processed specimens were inoculated on both the liquid systems and solid medium and incubated for six weeks and eight weeks consecutively. A total of 44 mycobacterial isolates (Mycobacterium tuberculosis, n=43; Mycobacteria other than tuberculosis, n=1) were recovered from 260 clinical specimens. The recovery rate of M. tuberculosis complex was 97.6% on BACTEC MGIT 960 system and 93.0% on BACTEC 460 system and 83.7% on LJ medium. The mean detection time of mycobacteria on BACTEC MGIT 960 system was 11.2 days in smear positive cases, 14.2 days in smear negative cases and 14.8 days in smear positive cases on BACTEC 460 system. Contamination rates were 9.6% and 5.6% and 3.4% for BACTEC MGIT 960, BACTEC 460 system and LJ medium respectively. The non-radiometric, fully automated BACTEC MGIT 960 system has better diagnostic ability as compared with radiometric, semi-automated BACTEC 460 system and LJ medium, so it can be used as a reliable alternative in over burden laboratories.
Malaysian Journal of Microbiology, Vol 6(2) 2010, Uncorrected proof
Evaluation of BACTEC MGIT 960 system for recovery of Mycobacterium
tuberculosis complex in Pakistan
Luqman Satti,
*
Aamer Ikram, Shahid Abbasi, Tariq Butt, Nasarullah Malik and Irfan Ali Mirza
Department of Microbiology, Armed Forces Institute of Pathology (AFIP), Rawalpindi, Pakistan.
E-mail: luqmansatti@hotmail.com
Received 4 December 2009; received in revised form 26 February 2010; accepted 4 March 2010
_______________________________________________________________________________________________
ABSTRACT
We evaluated the performance of MGIT 960 system in terms of recover rate, detection time of mycobacteria and
contamination rate from various human clinical specimens and compared it with already in use BACTEC 460 TB system
and conventional LJ medium. This is the first reported study on MGIT 960 and its comparison with BACTEC 460 system
in Pakistan. A total of 260 different clinical specimens received for the culture of mycobacteria were dealt during the six
months study period. All the specimens were digested and decontaminated according to the standard N-acetyl-L-
cysteine NaOH method. All the processed specimens were inoculated on both the liquid systems and solid medium and
incubated for six weeks and eight weeks consecutively. A total of 44 mycobacterial isolates (Mycobacterium
tuberculosis, n=43; Mycobacteria other than tuberculosis, n=1) were recovered from 260 clinical specimens. The
recovery rate of M. tuberculosis complex was 97.6% on BACTEC MGIT 960 system and 93.0% on BACTEC 460 system
and 83.7% on LJ medium. The mean detection time of mycobacteria on BACTEC MGIT 960 system was 11.2 days in
smear positive cases, 14.2 days in smear negative cases and 14.8 days in smear positive cases on BACTEC 460
system. Contamination rates were 9.6% and 5.6% and 3.4% for BACTEC MGIT 960, BACTEC 460 system and LJ
medium respectively. The non-radiometric, fully automated BACTEC MGIT 960 system has better diagnostic ability as
compared with radiometric, semi-automated BACTEC 460 system and LJ medium, so it can be used as a reliable
alternative in over burden laboratories.
Keywords: BACTEC, tuberculosis, Mycobacterium tuberculosis complex.
_______________________________________________________________________________________________
INTRODUCTION
Pakistan ranks sixth among the high TB burden countries
with incidence and prevalence rate of 181/100,000 and
359/100,000 population (Hasan et al., 2006). The growing
number of tuberculosis cases specially in underdeveloped
countries has made it necessary to develop new tools for
rapid detection and identification of mycobacteria from
various clinical samples (Hanna et al., 1999). In resource
poor countries, direct AFB microscopy and Lowenstein
Jensen (LJ) media are still main modalities used for the
diagnosis/screening of tuberculosis, but these methods
have low sensitivity which also depends upon the number
of tubercle bacilli present in the specimen (Farnia et al.,
2002). In some areas, patients are often treated on the
presumptive diagnosis due to lack of diagnostic facilities.
The broth based BACTEC 460 system, introduced in
1980, has considerably improved the detection time of
mycobacteria and it was a milestone in the advancement
in mycobacteriology (Morgan et al., 1983; Roberts et al.,
1983). However this system had many drawbacks like
radiation hazard, manual loading and unloading of vials,
no inbuilt incubation system and lack of computer
software. In low burden laboratories, fully automated
systems like MB/Bact (Organon Teknika, Turnhout,
Belgium) and ESP II (Difco Laboratories, Detroit, Mich)
serve the purpose but they have low capacity (Rohner et
al., 1997). The fully automated, high capacity BACTEC
MGIT 960 system is now being used as a rapid diagnostic
system for tuberculosis in many developed countries. This
system is easy to use, fully automated, non-invasive, non-
radiometric with high performance. It has an oxygen
sensitive fluorescent sensor embedded in silicon base
which serves as an indicator of mycobacterial growth
(Tortoli et al., 1999). Combination of a sold media with
one broth-based method such as BACTEC 460 system is
now widely accepted as "gold standard" (CDC, 1995). It is
recommended that all the new diagnostic systems for
tuberculosis must be evaluated fully (Chitra and Prasad,
2001). At present we are using BACTEC 460 as reference
system along with conventional LJ media for culture of
mycobacteria. The objective of this study is to evaluate
the performance of BACTEC MGIT 960 system in terms of
recovery rate, mean time to detection of mycobacteria and
contamination rate and comparing it with reference
BACTEC 460 system and LJ media.
*
Corresponding author
Mal. J. Microbiol. Vol 6(2) 2010, Uncorrected proof
MATERIALS AND METHODS
The study was carried out at the department of
microbiology, Armed Forces Institute of Pathology,
Rawalpindi, Pakistan from May 2008 through Oct 2008.
This laboratory receives various clinical samples from
tertiary care hospitals like Combined Military Hospital,
Military Hospital, Bone Marrow Transplant Centre and
from Northern areas of Pakistan. A total of 260
consecutive clinical specimens (sputum 90,
bronchoalveolar lavage 72, pus 20, tissue 36, pleural fluid
12, lymph node 10, peritoneal fluid 10, urine 6, synovial
fluid 3 and CSF 1) were dealt during the study period. For
patients already on anti-tuberculous treatment, blood and
bone marrow specimens and improper specimens like
saliva and pus swabs were excluded from the study.
Decontamination of specimens was carried out by
standard N-acetyl-L-cysteine NaOH digestion-
decontamination
method (Kent and Kubica, 1985). A final
concentration of 4% NaOH was used for decontamination.
A 2-3 drops of the processed specimens were placed on a
glass slide to make smear, stained with Ziehl-Neelsen
(ZN) acid fast staining and index is reported according to
the protocol described by Ellis (Ellis and Zabrowarny,
1993). The remaining suspension was used for inoculation
in both the liquid media (first in BACTEC 460) and then LJ
medium.
BACTEC 960 System
Before inoculation of specimen into 7mL MGIT tube, MGIT
PANTA vial was reconstituted with 15 mL of MGIT growth
supplement and mixed by gentle shaking. Each MGIT
tube was checked for any turbidity or contamination and
labelled properly. 0.8 mL of MGIT growth
supplement/PANTA was added aseptically into MGIT tube
with the help of insulin syringe. A 0.5 mL of the processed
specimen was added to the tube and the cap was
replaced immediately. The contents were mixed by
inverting the tube 3-4 times. All the inoculated tubes were
inserted in the MGIT 960 system after scanning the bar
code. All the tubes were incubated at the temperature of
37 °C. In case of a positive growth, the system
automatically detects the growth and signals positive.
After a maximum of six weeks (42 days), the instrument
flags a tube negative if there is no growth. In case of a
positive growth, the tube is taken out of the instrument,
stained for AFB presence and subcultured on to Columbia
agar (Oxoid, Basingstoke, UK) to look for any
contamination. The day in which the tubes were kept
inside the system for incubation was taken as day zero
while the time to detection of mycobacteria was taken on
the day of instrument positivity alone with AFB smear
positivity. After confirmation of a positive growth in solid or
broth based media, the parallel media for that particular
specimen was read daily. When AFB were seen in a
smear prepared from a positive MGIT tube, the growth
was further confirmed as Mycobacterium tuberculosis
complex (MTBC) by para-nitrobenzoic acid (PNB) test
(Giampaglia et al., 2005). At the end of protocol
incubation, all the instrument negative tubes were again
subjected to ZN staining to look for false negative tubes.
BACTEC 460 TB System
All the 12B vials containing 4 mL of modified Middlebrook
7H12 broth were supplemented with 0.1 mL of the
antimicrobial supplement PANTA prior to inoculation of
the specimens to minimize the chances of contamination.
0.5 mL of the specimen concentrate was inoculated in the
BACTEC 460 12B vial and incubated aerobically at 37
0
C
for up to 6 weeks. Growth of mycobacteria was monitored
twice weekly in first two weeks and weekly thereafter for
the next 4 weeks. A Growth index (GI) of 10 or more was
considered tentatively positive and was read daily
thereafter. An AFB smear was made when a GI of 100 or
more was reached. Contamination of the medium was
checked by Gram stain smears and by inoculation on to
Columbia agar (Oxoid, Basingstoke, UK) containing 5%
horse blood (blood agar), incubated aerobically at 37
0
C
overnight and examined for any growth. The positive
growth showing presence of AFB was further confirmed
as MTBC by radiometric p-nitro-
α
-acetylamino-
β
-hydroxy
propiophenone (NAP) test (Becton Dickinson Diagnostic
Systems). At the end of protocol period, all the negative
vials were stained with ZN stain to rule out false negative
results.
LJ Medium
A 0.2 mL of the processed specimen was inoculated on
two LJ slants that is one with PNB (500 µg/mL of medium)
and other without PNB (Giampaglia et al., 2005) and
incubated at 37 °C in 5-10% CO
2
incubator. All the LJ
slants were checked twice weekly for the first two weeks
and then weekly for 8 weeks for the appearance of visible
colonies on agar. Colonies of MTBC will not grow on LJ
medium with PNB.
Statistics
Independent sample T-test was used to compare mean
time to detection of mycobacteria and recovery rate in
different culture systems/medium. P-value < 0.05 was
considered significant.
Controls
H37Ra ATCC 25177 of Mycobacterium tuberculosis was
used as positive control. An uninoculated MGIT tube was
used as negative control. Institutional control strain of
Mycobacterium chelonei was used to check MGIT PNB
and BACTEC NAP.
RESULTS
Out of total 260 specimens, 44 mycobacterial isolates
(Mycobacterium tuberculosis complex, n=43;
nontuberculous mycobacteria, n=1) were isolated from all
the culture media. Pulmonary samples were among the
Mal. J. Microbiol. Vol 6(2) 2010, Uncorrected proof
dominant (62.3%) followed by tissue (13.8%) and pus
(7.6%). Maximum number of positive isolates 41 (93.2%)
were recovered from pulmonary specimens as shown in
Figure 1, highlighting the heavy burden of pulmonary
tuberculosis in our setup. Percentage of recovery of
mycobacteria on BACTEC MGIT 960, BACTEC 460 and
LJ medium was 97.6%, 93.0% and 83.7% respectively as
shown in Table 1. P-value was less than 0.05 for recovery
of mycobacteria between MGIT 960 and LJ medium which
is a significant difference. No isolate was recovered from
urine, pleural fluid, peritoneal fluid, synovial fluid and CSF.
When combined together, BACTEC MGIT 960 and LJ
medium detected 42 mycobacterial isolates (97.6%) while
BACTEC 460 plus LJ medium recovered 40 mycobacterial
isolates (93.0%). In combination with solid media, the
recovery rates of both the liquid system were same as
when considered individually. This is because of the fact
that LJ medium gave positive growth only in smear
positive specimens and all the smear positive specimens
were also culture positive on both the liquid systems.
33
8
1
1
1
Sputum
BAL
Lymph node
Pus
Tissue
Figure 1: Number of mycobacterial isolates from various
specimens (n=44)
Table 1: Relationship of concentrated specimen microscopy with mycobacterial culture on individual system/medium
Culture
system/media
Smear positive
culture positive
Smear negative
culture positive
Smear negative
culture negative
Total
MGIT 960 36 6 218 260
BACTEC 460 36 4 220 260
LJ medium 36 0 224 260
Mean time to detection of mycobacteria for smear
positive specimens on BACTEC MGIT 960 system was
11.2 days and 14.2 days for smear negative specimens. It
was 14.8 days on BACTEC 460 system for smear positive
cases and 15.6 days for smear negative cases as shown
in Table 2. Individual sample T-test was applied to check
the significance level between mean time to detection of
mycobacteria on both the liquid systems and P-value was
found to be 0.037 which is a significant difference.
Similarly a statistically significant difference was found
between the individual liquid systems and LJ medium (P <
0.05). Mean time to detection of mycobacteria was directly
related to mycobacterial slide index as shown in Table 4.
Contamination rates on MGIT 960, BACTEC 460 and
LJ medium were 9.6%, 5.6% and 3.4% respectively.
Contamination rates were high in the initial two months
but then decreased gradually in the subsequent months
as shown in Table 3.
Table 2: Relationship of mean detection time of
mycobacteria with smear positivity on
individual systems
BACTEC
system
Mean detection time in days of
mycobacteria (range)
AFB (+) smear
AFB (
-
MGIT 960
11.2 (1-30)
14.2 (6-30)
BACTEC 460
14.8 (2-31)
15.6 (3-36)
LJ medium
28.3 (16-52)
-
Table 3: Contamination rates (%) on individual system
Culture
System
May-
June
July-
Aug
Sept-
Oct
6 Months
Average
MGIT 960 14.5 9.8 4.7 9.6
BACTEC 460 7.8 4.1 5.1 5.6
LJ medium 3.8 3.4 2.9 3.4
Mal. J. Microbiol. Vol 6(2) 2010, Uncorrected proof
Table 4: Relationship of mycobacterial index with detection time of mycobacteria on individual system/medium
DISCUSSION
Due to increase in the spread of tuberculosis worldwide,
early diagnosis and prompt treatment has become
important for the control of this disease (ATS, 2000). The
advancement in development of new diagnostic methods
is an important element of the “Global Plan to Stop TB”
and the WHO new global “Stop TB Strategy” (Pai et al.,
2008). According to WHO study in 2006, it has been found
that the world’s expenditure per year on diagnosis of TB is
about US$ 1 billion (WHO, 2006). The methods presently
used for the diagnosis and sensitivity of mycobacteria are
either costly or time consuming. Therefore, a rapid, cost
effective and simple method is required for under
developed countries. Although BACTEC 460 system is
being withdrawn worldwide, its use in recent years have
significantly improved the recovery rate of mycobacteria
but the culture procedure on this system is time
consuming and labor-intensive. MGIT 960 system has
been developed recently for the rapid diagnosis and
sensitivity testing of mycobacteria (Somoskovi et al.,
2004). It has also overcome the drawbacks of previous
BACTEC 460 system which was and still being used as a
gold standard for the culture of mycobacteria
in various
countries (Ganeswire
et al., 2004).
A number of studies have been conducted worldwide
to evaluate the diagnostic efficacy of MGIT 960 system
from various clinical specimens. We evaluated the
performance of the new MGIT 960 system in terms of
recovery rate of MTBC, mean time to detection of
mycobacteria and contamination rates from various
clinical specimens in our settings and compared it with the
already in use gold standard BACTEC 460 system and
conventional LJ medium. In our study, out of total 260
clinical specimens, 44(16.9%) yielded growth of
mycobacteria. 15.9% were MTBC and only one isolate
was nontuberculous mycobacteria (NTM). Four isolates of
MTBC were recovered only on MGIT 960 system and not
on BACTEC 460 system while only one isolate was
recovered on BACTEC 460 system but not on MGIT 960
system and that was identified as NTM. The possible
reason for recovery of four isolates only on MGIT 960 and
not on BACTEC 460 could be that the mycobacteria
present in the 12B vial did not metabolize the C
14
palmitic
acid completely. Another reason could be that the MGIT
960 tube contains 7 mL broth while 12B vial contains 4 mL
broth. The high volume of broth in MGIT 960 tube might
have diluted the potential growth inhibitors thus providing
a better chance for mycobacteria to grow. A number of
other studies have revealed that all the isolates of MTBC
or NTM were not recovered on both the systems
(Somoskovi et al., 2004). The possible reason why the
only NTM in our study was not recovered on MGIT 960
system might be that concentrated specimen was first
inoculated into BACTEC 460 vial followed by MGIT 960
tube. The number of organisms might be low in the
sample and uneven distribution had resulted in negative
culture in MGIT 960 system.
Our study showed that mean time to detection of
mycobacteria on MGIT 960 was shorter than BACTEC
460 and LJ medium . The minimum time to detection was
one day on MGIT 960 and 2 days on BACTEC 460.
Maximum time to detection was 30 days on MGIT 960 and
36 days on BACTEC 460. Findings of our study were also
consistent with another study (Hanna et al., 1999). They
found that the mean time to detection of mycobacteria
was 14.4 days for MGIT 960 and 15.2 days for BACTEC
460. The detection time of mycobacteria has a vital role in
the performance of a system. Early detection of
mycobacteria will limit the spread of infection by timely
treatment.
During the course of study, it was observed that
mycobacterial growth is seen as small clumps, grains or
cords settling down at the bottom of tube after shaking like
snow fall when visualized in good light while
contamination appeared as uniform turbidity in the entire
tube. Contamination rate was higher on MGIT 960 system
(9.6%) as compared to BACTEC 460 system (5.6%).
Higher contamination rate is the major draw back of the
fully automated system. However in a study (Somoskovi
et
al., 2004), they found that the contamination rates on
MGIT 960 and BACTEC 460 were 3.7% and 2.9%
respectively. In another study (Rishi et al., 2007),
contamination rate on MGIT 960 was 13.4% and 27.2%
on LJ medium which is very high. The higher
contamination rate on MGIT 960 may be due to the fact
that the cap of MGIT 960 tube is opened for specimen
inoculation while insulin syringes are used in BACTEC
460 vial for inoculation without opening them. Another
reason could be that MGIT 960 system uses enriched
broth whereas the 12B broth medium in BACTEC 460
system is low in nutrients as it uses C
14
palmitic acid.
Thus the enriched high volume broth is like a double-edge
sword, at one side it dilutes the inhibitors and improves
the recovery of mycobacteria while on the other side,
enrichment favors the contaminants.
Another interesting observation was that the
contamination rate for MGIT 960 system was high in the
Mycobacterial
Index
Number
of
mycobacterial
isolates
Mean detection time in days
MGIT 960
BACTEC 460
LJ
4+ 7 8.2(1-15) 9.7(3-16) 20.2(16-22)
3+ 12 9.9(3-20) 14(3-26) 24.8(18-28)
2+ 12 11.8(3-22) 16.3(5-30) 29.0(25-38)
1+ 5 12.4(5-25) 16.8(6-30) 39.1(34-52)
Mal. J. Microbiol. Vol 6(2) 2010, Uncorrected proof
first two months of the study but it declined significantly in
next four months. This could be due to the fact that it was
a new system introduced in our laboratory, and laboratory
staff were not well familiar with the system initially but their
handling of the system improved significantly in
subsequent months once they gained experience. Another
possibility could be that climatic conditions in our country
are hot in the month of May, June and July and
transportation was also delayed in some specimens thus
allowing the contaminants to overgrow.
In our study we observed that maximum number
(77.5%) of mycobacterial isolates were cultured from
sputum samples indicating that sputum is the most
frequent specimen submitted by the patients. This also
signifies the importance of early isolation of these cases
and prevention of spread of bacilli from open or infectious
cases.
There were no false negative results in our study that
is all the MGIT 960 instrument- negative tubes (at the end
of 42-day protocol) were examined by direct AFB
microscopy and subcultured to solid media. We observed
three false positive result in our study that is instrument
positive but smear negative and subculture negative but
after incubation for 3 to 4 more days, AFB was detected
under direct microscopy.
The non-radiometric, fully automated, 7 mL BACTEC
MGIT 960 system is rapid in terms of recovery of
mycobacteria and time to detection of mycobacteria as
compared with radiometric, semi-automated BACTEC 460
system and conventional LJ medium. It has eliminated the
use of needles for inoculation or testing of vials. It has
high capacity and less labor-intensive with minimum staff
required. As this system uses plastic tubes so there are
less chances of breakage and laboratory accidents. The
average cost per test that is AFB culture/sensitivity on LJ
medium is 4.7$ (400 PKRs) and 9.4$ (800 PKRs) on
BACTEC MGIT 960 system. Although expensive, but
BACTEC MGIT 960 system can be used as a reliable
alternative for culture of mycobacteria in high burden
laboratories keeping in view the financial resources of the
laboratory.
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    • "This direct inoculation sensitivity loss cannot be ignored and should be evaluated considering the signifycantly increased contamination rate. All these reports focused on broth cultures and to our best knowledge, there is little data available on alternative culturing methods involving Lowenstein Jensen medium, which is the main culture media available to resource limited settings (Satti et al., 2010). Our data show that time to culture positivity is not significantly increased in low bacterial load samples (scant, +1 vs. +2, +3 for microscopy examination) suggesting a minimum sensitivity loss while using the modified culture protocol (Figure 2). "
    [Show abstract] [Hide abstract] ABSTRACT: Mycobacteria culture remains the cornerstone of tuberculosis diagnosis. Naturally contaminated samples need pre-inoculation processing but some economically challenged medical facilities may benefit from a simpler and cheaper sputum decontamination procedure. The aim of this study was to test a simple decontamination method lacking a centrifugation step to be used in conjunction with the culture on Löwenstein-Jensen medium. A total of 7446 sputum samples collected from 3229 patients were microscopically examined and then cultured on Löwestein-Jensen medium using a simplified Petroff method. All positive cultures were confirmed by direct microscopic examination and biochemical identification. Culture and microscopic status and time to positivity were recorded. Mean and median times to culture and contamination rate were similar as compared to classical Löwenstein-Jensen culture method. Overall results suggest that the described modified of Petroff method may be used with adequate results in resource poor settings as the method does not require an aerosol safe centrifuge and relies on cheap, stable and readily available reagents.
    Full-text · Article · May 2015
    • "Considering these, the diagnosis efficacy of BACTEC MGIT 960 was evaluated in the present study. To date, a number of studies have been conducted worldwide on the diagnostic efficacy of the MGIT 960 system for the recovery of mycobacteria from clinical samples2627282930. The present study demonstrated that the MGIT 960 system provided 60% and 66.7% better isolation rate of mycobacteria than the LJ culture and LED fluorescence microscopy, respectively . "
    [Show abstract] [Hide abstract] ABSTRACT: Objective: Tuberculosis (TB) caused by Mycobacterium tuberculosis has been identified as a reemerging infectious disease with public health importance globally. Exploitation of new laboratory techniques for precise identification of mycobacteria in clinical specimens is of great importance to improve the diagnosis as part of the global TB control efforts. Methods: The current study was conducted for the evaluation of BACTEC MGIT 960 method in comparison with Lowenstein–Jensen (LJ) culture and light emitting diode (LED) fluorescence microscopy for isolation of mycobacteria among TB suspects from Bangladesh. A total of 421 specimens were tested with these methods. Results: Among the tested samples, 3.6% (n = 15) were LED fluorescence microscopy positive; while 18 (4.2%) and 45 (10.6%) were recovered from LJ and MGIT 960 culture. The relative positivity found through MGIT 960 system were 60% and 66.7% higher than that of LJ culture and LED fluorescence microscopy, respectively. Recovery rate of Mycobacterium tuberculosis complex ([MTC], 21 by MGITand 16 by LJ culture) and non-tubercular mycobacteria ([NTM], 24 by MGITand 2 by LJ culture) by MGIT 960 was 24% and 96% greater, respectively than LJ culture. Moreover, MGIT 960 was found to be highly sensitive (100%), specific (93.3%), accurate (93.6%) and a more rapid method in detecting mycobacteria when compared with LJ culture. Conclusion: Extended recovery of NTM and MTC through MGIT 960 urged frequent application of this method to detect mycobacteria more effectively and rapidly.
    Full-text · Article · Sep 2013
  • Full-text · Article · Jan 2011 · International Journal of Mycobacteriology
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