Rapid Detection of Multidrug Resistance Using the Bactec MGIT 960
System: a Multicenter Study
Salman Siddiqi,aAltaf Ahmed,bSunil Asif,bDigamber Behera,cMona Javaid,bJasmine Jani,dArora Jyoti,cRadhika Mahatre,e Dewanand Mahto,dElvira Richter,fCamilla Rodrigues,ePotharaju Visalakshi,cand Sabine Rüsch-Gerdesf
Becton Dickinson, Sparks, Maryland, USAa; Indus Hospital, Karachi, Pakistanb; LRS Institute of TB and Respiratory Diseases, New Delhi, Indiac; BD Diagnostic Systems, Gurgaon, Indiad; P.D. Hinduja National Hospital and Medical Research Center, Mumbai, Indiae; and National Reference Center for Mycobacteria, Borstel, Germanyf
Conventional indirect drug susceptibility testing of
Mycobacterium tuberculosis
with liquid medium is well established and
of-fers time-saving and reliable results. This multicenter study was carried out to evaluate if drug susceptibility testing (DST) can be
successfully carried out directly from processed smear-positive specimens (direct DST) and if this approach could offer
substan-tial time savings. Sputum specimens were digested, decontaminated, and concentrated by the laboratory routine procedure and
were inoculated in Bactec MGIT 960 as well as Lowenstein-Jensen (LJ) medium for primary isolation. All the processed
speci-mens which were acid-fast bacterium (AFB) smear positive were used for setting up direct DST for isoniazid (INH) and rifampin
(RIF). After the antimicrobial mixture of polymyxin B, amphotericin B, nalidixic acid, trimethoprim, and azlocillin (PANTA)
was added, the tubes were entered in the MGIT 960 instrument using the 21-day protocol (Bactec 960 pyrazinamide [PZA]
pro-tocol). Results obtained by direct DST were compared with those obtained by indirect DST to establish accuracy and time
sav-ings by this approach. Of a total of 360 AFB smear-positive sputum specimens set up for direct DST at four sites in three
differ-ent countries, 307 (85%) specimens yielded reportable results. Average reporting time for direct DST was 11 days (range, 10 to 12
days). The average time savings by direct DST compared to indirect DST, which included time to isolate a culture and perform
DST, was 8 days (range, 6 to 9 days). When results of direct DST were compared with those of indirect DST, there was 95.1%
con-cordance with INH and 96.1% with rifampin. These findings indicate that direct DST with the Bactec MGIT 960 system offers
further time savings and is a quick method to reliably detect multidrug resistance (MDR) cases.
A
ccording to the WHO, drug resistance in tuberculosis (TB) is
a global problem (30). Resistance against isoniazid (INH) and
rifampin (RIF), defined as multidrug resistance (MDR), is
in-creasing in many countries (3, 4, 31). If these cases are not treated
properly, they can develop resistance to other drugs as well, such
as fluoroquinolones and injectable aminoglycosides, defined as
extensive drug resistant (XDR), and may in turn infect others with
a drug-resistant strain (5, 10, 22). For a better management of
drug-resistant cases, early detection of resistance is extremely
im-portant so that effective treatment can be prescribed. Rapid drug
susceptibility testing plays an important role in the detection and
control of MDR/XDR TB (23, 31).
Drug susceptibility testing (DST) of
Mycobacterium
tuberculo-sis
is generally carried out after a culture is isolated from a clinical
specimen. This takes a long time, first to isolate a culture and then
to perform drug susceptibility testing (indirect DST). If DST
could be set up at the same time as when a processed specimen is
inoculated in solid and or liquid medium (direct DST), it could
save significant time for the detection of drug resistance.
Direct DST in the conventional solid medium has been well
established (9, 14, 15). The only disadvantage is that it takes a long
time to obtain results on solid medium, as the growth rate on such
media is lower. With the introduction of Bactec 12B liquid
me-dium (Becton Dickinson Diagnostic Systems, Sparks, MD), the
time to report results was significantly reduced (13, 19, 24, 25). In
1993, the CDC recommended to use liquid medium based on its
better performance and earlier results (27). Direct DST with the
Bactec 460 liquid system has been tried successfully (16).
How-ever, the use of the Bactec 460 radiometric method is phasing out
due to the concerns of the radioactive waste disposal. This system
is being replaced by the nonradiometric Bactec MGIT 960 system
(Becton Dickinson Diagnostic Systems, Sparks, MD). Indirect
DST is well established in this liquid system (1, 2, 6–8, 12, 20). In
2001, a small study using the manual BBL MGIT system was
re-ported on direct DST for INH and RIF with excellent results (11).
In 2007, WHO published a policy statement on the
recommenda-tion for the use of liquid medium for low- and middle-income
countries (32).
This multicenter study was designed to establish direct DST
feasibility in four different clinical laboratories with different
pa-tient populations and test parameters. The primary objective of
this study was to establish a standard protocol for the direct DST
using the Bactec MGIT 960 automated system. Results were
com-pared with the indirect DST results to establish accuracy as well as
time savings with this approach.
MATERIALS AND METHODS
Study sites.This study was carried out at four different sites in three countries: (i) site 1, P.D. Hinduja National Hospital and Medical Research
Received29 September 2011Returned for modification26 October 2011
Accepted29 November 2011
Published ahead of print7 December 2011
Address correspondence to Sabine Rüsch-Gerdes, srueschg@fz-borstel.de.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
doi:10.1128/JCM.05188-11
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Center, a tertiary care hospital, Mumbai, India; (ii) site 2, LRS Institute of Tuberculosis and Lung Diseases, a dedicated TB hospital and national reference laboratory (NRL), New Delhi, India; (iii) site 3, Indus Hospital, a general charity hospital focused on TB, especially MDR management, Karachi, Pakistan; (iv) site 4, National Reference Center for Mycobacteria, national center (with access to clinical samples from areas of high MDR endemicity in other countries), Borstel, Germany.
Specimens.Only sputum specimens from patients strongly suspected of tuberculosis as well as those from old chronic cases, especially those expected to have MDR TB, were included in this study. Specimens which were found to be smear positive for acid-fast bacteria (AFB) irrespective of the degree of smear positivity were included in the study. Sputum speci-mens were transported to the laboratory with minimum delay and were refrigerated if the processing was not done immediately.
Specimen processing.All specimens were processed following the standard NALC-NaOH method for digestion, decontamination, and concentration (14, 26). The concentrated sediment was resuspended in about 2 to 3 ml phosphate buffer (pH 6.8) and mixed thoroughly. A smear was prepared for acid-fast staining, and culture media were inoculated according to the laboratory standard procedure for pri-mary isolation. It was ensured that a little more than 1 ml of suspen-sion was left for the direct DST.
AFB smears.All the smears were stained with the Ziehl-Neelsen and/or fluorochrome methods. Smears were graded following WHO guidelines (28) and based on the number of AFB found during
examina-tion. The smears were graded as scanty (1 to 9 AFB/100 fields), 1⫹(10 to
99/100 fields), 2⫹(1 to 10 AFB/field), or 3⫹(more than 10 AFB/field).
Two laboratories did not have the scanty category in their grading system
(these smears were included in the 1⫹category).
Inoculation of culture media for primary isolation.All four sites used the Bactec MGIT 960 system for liquid, and for the solid medium they used one Lowenstein-Jensen (LJ) slant. These media were inoculated following the established individual laboratory standard operating proce-dure (SOP). For MGIT medium, standard recommended proceproce-dures were followed (manufacturer’s recommendations and the MGIT manual by FIND [26]). After preparing smears and inoculations for culture, the remainder of specimen was refrigerated immediately at 2°C to 8°C and was used for setting up direct DST as soon as the smear results were available.
Direct DST procedure.There were three major differences in the di-rect DST procedure compared to the standard indidi-rect DST procedure for MGIT 960: (i) direct DST was a 4- to 21-day protocol, while indirect DST was a 4- to 13-day protocol; (ii) the control was diluted 1:10 in direct DST, while in indirect DST it was diluted 1:100; (iii) in the direct DST, an antimicrobial mixture of polymyxin B, amphotericin B, nalidixic acid, trimethoprim, and azlocillin (PANTA) (Becton Dickinson Diagnostic Systems, Sparks, MD) was added to the control as well as in the drug-containing MGIT tubes to suppress contamination.
All the media and other reagents were the same as those used in the routine indirect DST: MGIT medium (7-ml bar-coded MGIT tubes), SIRE supplement for DST, and the lyophilized Bactec MGIT INH and RIF drugs (Becton Dickinson Diagnostic Systems, Sparks, MD).
Prior to setting up the direct DST, lyophilized PANTA was reconsti-tuted using 15 ml of SIRE supplement (not growth supplement) (Becton Dickinson Diagnostic Systems, Sparks, MD) and mixed well until com-pletely dissolved. Drug vials (lyophilized drugs, same as those used in MGIT indirect DST) of INH and RIF were reconstituted with 4 ml of sterile deionized (DI) water and mixed well. Sets of four MGIT tubes were prepared per specimen for performing the direct DST. Two tubes were labeled “growth control” (GC), one for INH and the other for RIF. The third tube was labeled “INH” and the fourth was labeled “RIF.”
Once dissolved, 0.8 ml of PANTA-SIRE supplement mixture was added into each of the four labeled MGIT tubes. The next step was the addition of drugs. In the INH-labeled tube, 0.1 ml of reconstituted
lyoph-ilized INH drug was added (0.1g/ml final concentration). Similarly 0.1
ml of reconstituted RIF was added to the RIF-labeled tube (1.0g/ml).
These concentrations were the same as those in the indirect DST proce-dure (package insert [26]). After mixing the medium, 0.5 ml of the well-mixed reconstituted sediment was inoculated in each of the two drug-containing tubes. For the control, the resuspended sediment was diluted 1:10 by adding 0.2 ml of the well-mixed sediment into 1.8 ml of sterile saline or water. After being thoroughly mixed, 0.5 ml was inoculated into each of the two GC tubes. The tubes were mixed again by inverting several times.
For direct DST, an extended protocol which is used routinely for in-direct PZA DST setup was followed since in-direct DST requires a 21-day protocol to complete the test (18, 26). Two two-tube DST set carriers were used. Growth control and INH tubes were placed in one set carrier (GC and INH), and growth control and RIF tubes were placed in the other set carrier (GC and RIF). These set carriers were entered in the instrument as the PZA test. The first tube in the set carrier was always the control tube. At one site, another approach of handling the 21-day protocol was followed. In this second option for direct DST, the procedures were the same except that only one GC was used. The GC and INH tubes were placed in one two-tube carrier and entered as the PZA DST. The RIF tube was entered as a regular growth tube (42-day protocol) and was placed close to the INH set carrier.
Safety precautions.All four sites have well-equipped BSL III labora-tory facilities. Standard safety precautions were followed for specimen processing, inoculation, and DST (29).
Interpretation of direct drug susceptibility testing.When the GC reached the growth unit (GU) value of 400 or more, the instrument indi-cated that the test was complete, the susceptibility set was removed after scanning, and an inventory report was printed. Susceptibility results for both INH and RIF (in the first option) were interpreted by the instrument as “S” or “R.” At the time the GU value of the GC was 400 or more and if the GU value of the drug tube was less than 100, the test result was re-ported as “susceptible,” while if the GU value of the drug tube was 100 or more the result was interpreted as “resistant.” The GU values of both the DST sets were retrieved and recorded. In case the GU value of the control did not reach 400 within 21 days, the instrument indicated an X200 error, indicating insufficient growth. On the other hand, if the GU reached 400 earlier than day 4, the instrument gave an X400 error, indicating contam-ination or overinoculation.
In the second option, the GC and INH tubes were taken out once the instrument indicated that the test was complete and results were retrieved. The GU values of the INH set were retrieved. Since the RIF tube was incubated separately in the system as a regular growth tube, the instru-ment would not interpret the results. At this point, the GU value of the RIF tube was also retrieved by printing an instrument inventory report. The results were interpreted manually following the criteria given above.
For calculation of time to obtain direct DST results, the time it took for the instrument to complete the DST test was recorded. Since the instru-ment gives time in hours, any time equal to half a day or more was taken as a full day.
Reference method: indirect drug susceptibility testing.The primary isolation tubes of the specimens that were included in the study were followed. Once an inoculated specimen was culture positive in MGIT and
confirmed to have pure culture ofM. tuberculosis, the indirect DST was set
up following the manufacturer’s recommended procedures for the MGIT 960 system. Results of indirect DST and time to complete the test were retrieved from the instrument and recorded.
For calculation of time required for reportable indirect DST results for a specimen, time to get a positive culture plus time to achieve indirect DST results was added to get the total time. Since indirect DST is set up after 1 to 5 days after the instrument gives a positive signal, additionally an aver-age of 2 days was added to the total time. Occasionally, the DST was not set up within 5 days of culture positivity in MGIT and a subculturing was required. This time has not been documented. The time required for
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indirect DST was compared with that of direct DST of the same specimen. The difference was considered the time savings.
At site 4, routine indirect DST was also carried out on LJ medium, but results have not been included in this analysis.
Identification of the isolated mycobacteria.The MGIT 960 DST
method is recommended forM. tuberculosis, and therefore the isolated
mycobacteria from the culture-positive specimens were identified using
the routine method used in the laboratories to identifyM. tuberculosis.
Only those cultures confirmed asM. tuberculosiswere included in this
study. Direct DST results on those specimens which were contaminated or
where a mycobacterium was other thanM. tuberculosiswere excluded.
Discrepancy testing.The specimens that showed discrepant results between the direct and indirect methods were retested by repeating the indirect method. If the second testing results were the same as the first indirect results, the direct susceptibility results were considered discrep-ant. If the repeat testing results disagreed with the previous indirect results and were in agreement with the direct results, then the results were con-sidered the correct one.
QC testing.M. tuberculosisH37Rv strain (ATCC 27294) was used for quality control (QC) testing in DST. This strain was introduced at each time when a batch of DST was set up or as every 6th isolate in a run. If any resistance in the QC strain was observed, all the other results in that batch were considered invalid.
RESULTS
Of the total 360 specimens processed, 307 (85%) DST results were
reportable (Table 1). The majority of those which were not
report-able were those where the control did not reach the required
threshold (X200 error), while some were contaminated (X400
er-ror). Site 1 processed 126 sputum specimens and reported 113
results (90%), site 2 processed 122 specimens and reported 103
results (84%), site 3 processed 74 specimens and reported 58 results
(84%), while site 4 had 33 reportable results out of 38 specimens
processed (87%). Specimens which were negative for culture, were
contaminated, or had nontuberculous mycobacteria (NTM) were
excluded to calculate reportable results. There were a total of 15
spec-imens which had contamination, 8 were identified as NTM, 19 had
the X200 error, and 5 had the X400 error.
The time to report results of positive cultures was analyzed
according to the degree of AFB smear positivity (Table 2). The
majority of specimens were 2 to 3
⫹
smear positive at all the sites.
The time to detection of culture positive was not significantly
different between different smear grades. Overall, cultures were
positive at an average of 8 days, with a range of 8 to 10 days.
The time to complete direct DST from processed specimens
was calculated according to the smear-positive categories (Table
3). There was no significant difference between times to complete
direct DST results in different smear-positive categories. The
ma-jority of results were ready within 8 to 14 days, with an overall
average of 10 days at site 1, 11 days at sites 2 and 3, and 12 days at
site 4.
In Table 4, the average time to complete indirect DST from
isolated cultures is reported without taking into consideration the
time to culture positivity. The average time to report indirect DST
from isolated culture ranged from 6 days (site 4) to 10 days (site 2).
The total time required from the time the specimen was
pro-cessed to the time when indirect DST results were available is
included in Table 6. It ranged from 18 to 20 days.
Discrepant results were analyzed on all direct DST tests on
which confirmed indirect DST results were available (Table 5). Of
the 113 specimens reported by site 1, there were 5 specimens that
showed discrepant results between direct and indirect methods
for INH (4.4%): three results were reported as false resistant and
two results as false susceptible. For RIF, there were three
discrep-ant results (2.7%), two being false susceptible and one being false
resistant to RIF. At site 2, out of 103 specimens, three showed
discrepant results for INH (2.9%), all false susceptible. Also for
RIF, there were a total of three discrepant results (2.9%): two false
resistant and one false susceptible. Site 3 had six discrepant results
out of 58 tests for INH (10.3%), four being false resistant and two
false susceptible. For RIF, there were five discrepant results
(8.6%), two being false resistant and three false susceptible. At site
4, out of 33 tests there was only one (3.0%) false resistant to INH
and one (3.0%) false susceptible to RIF. Thus, there were overall
4.9% discrepant results for INH and 3.9% for RIF among all the
sites.
Findings of time savings by direct DST have been given in
Table 6. Overall time saving was 8 days, ranging from 6 to 9 days
among all the sites.
TABLE 1Overall summary of testing
Sitea No. of tests set up No. of reportable results
1 126 113
2 122 103
3 74 58
4 38 33
Total 360 307 (85%)
aSites: 1, PDH, India; 2, LRS, India; 3, Indus, Pakistan; 4, TB reference lab, Germany.
TABLE 2Time to detect MGIT-positive cultures
Smear grading by site
No. of specimens turned positive after days:
Total no. of specimens turned positive
Avg time to detect (no. of days)
3–7 8–14 15–21 ⬎21
Site 1
Scanty 4 6 1 0 11 9
1⫹ 14 7 3 0 24 9
2⫹ 14 14 1 0 29 8
3⫹ 45 3 1 0 49 6
Total 77 30 6 0 113 8
Site 2
Scanty 0 0 0 0 0 0
1⫹ 10 18 4 0 32 10
2⫹ 17 11 2 0 30 8
3⫹ 35 6 0 0 41 6
Total 62 35 6 0 103 8
Site 3
Scanty 0 0 0 0 0 0
1⫹ 8 15 3 0 26 10
2⫹ 4 1 1 0 6 8
3⫹ 21 3 1 1 26 6
Total 33 19 5 1 58 8
Site 4
Scanty 1 4 1 0 6 12
1⫹ 1 6 0 1 8 12
2⫹ 2 3 1 0 6 11
3⫹ 10 3 0 0 13 7
Total 14 16 2 1 33 10
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For quality control, H37Rv was set up along with direct and
indirect DST batches. There was not a single incidence at any site
where H37 Rv failed to give expected results.
DISCUSSION
Results obtained with the conventional indirect susceptibility
test-ing methods, especially with solid media, become available too
late to influence a timely decision on patient management. Hence,
more rapid TB susceptibility tests directly applied on clinical
spec-imens are needed. Some of the noncommercially available direct
tests include the nitrate reductase assay (NRA) and microscopic
observation drug susceptibility (MODS) assay. These tests have
been developed as “in house” assays with the aim to overcome the
high costs of the commercially available techniques. In NRA, the
addition of the NRA reagent requires tubes to be regularly opened,
which poses significant risk of aerosol generation. Reading of the
MODS plates has to be performed on a daily basis and is laborious
and time-consuming (34). Commercially available molecular
as-says, such as Genotype MTBDR Plus (Hain Lifescience, Nehren,
Germany), can be applied directly to smear-positive specimens
and have less turnaround time, thus saving several weeks.
How-ever, none of the established molecular tests target all possible
genes involved in resistance, and thus a variable proportion of
resistant strains may not be detected (17, 33). Liquid culture has
been established as a gold standard and is most rapid for
pheno-typic DST. This was a research study designed to establish time
savings with the direct DST approach compared to the routine
indirect approach in liquid medium, and thus molecular testing
was not included in the study.
Liquid culture offers a more sensitive and rapid method for
isolation of
M. tuberculosis
and performing susceptibility testing
against a variety of first-line and second-line antituberculosis
drugs (13, 21). However, most of the DST studies have been
car-ried out by the routine indirect method using isolated cultures.
This is the first large-scale multicenter study to evaluate direct
susceptibility testing of
M. tuberculosis
from clinical specimens
using the MGIT 960 automated liquid culture system.
Since MDR tuberculosis is one of the main concerns in a TB
control program and DST results play an important role in the
control of TB, we focused on direct DST on only two drugs, INH
TABLE 3 Time to report direct drug susceptibility results from processed specimens
Smear (no. of specimens)
No. of tests after days: Avg time to
detect (no. of days)
3–7 8–14 15–21
Scanty (11) 1 7 3 12
1⫹(24) 0 20 4 12
2⫹(29) 2 21 6 11
3⫹(49) 18 31 0 8
Avg time to detect of total
10
Site 2
Scanty 0 0 0 0
1⫹(32) 3 21 8 13
2⫹(30) 3 23 4 11
3⫹(41) 17 21 3 9
Avg time to detect of total
11
Site 3
Scanty 0 0 0 0
1⫹(26) 3 13 10 13
2⫹(6) 2 4 0 9
3⫹(26) 8 16 2 10
Avg time to detect of total
11
Site 4
Scanty (6) 0 4 2 15
1⫹(8) 0 3 5 14
2⫹(6) 0 6 0 11
3⫹(13) 1 11 1 10
Avg time to detect of total
12
TABLE 4Time to report results by indirect susceptibility testing from
isolated culturesa
Site
No. of tests with reportable results after days:
Avg time to report (no. of days)
4–7 8–13
1 27 86 8
2 16 87 10
3 18 40 9
4 30 3 6
aTime to report indirect DST is the total time required from the day of setting up DST
to the day results were ready. It does not include the time to get a positive culture from processed specimen, 1 to 5 additional days for setting up DST from the day the instrument gives a positive signal, plus in some cases additional time required for subculturing if needed.
TABLE 5Discrepant results between direct and indirect DST methods
Site (total no. of tests)
No. (%) of specimens
INH RIF
False S False R Total False S False R Total
1 (113) 2 (1.8) 3 (2.7) 2 (1.8) 1 (0.9)
2 (103) 3 (2.9) 0 1 (1) 2 (1.9)
3 (58) 2 (3.4) 4 (7) 3 (5.2) 2 (3.4)
4 (33) 0 1 (3) 1 (3) 0
Total (307) 7 (2.3) 8 (2.6) 15 (4.9) 7 (2.5) 5 (1.6) 12 (3.9)
TABLE 6Time savings by direct susceptibility testing from processed specimens
Site
Avg time to report (days)
Time savings (no. of days)
Indirecta Direct
1 18 10 8
2 20 11 9
3 19 11 8
4 18 12 6
All sites 19 11 8
aTime to report indirect DST is the total time from inoculation of a processed
specimen into MGIT and the time when indirect DST results were available. This includes additional time, averaging 2 days for setting up DST from the day an MGIT culture was instrument positive. In some cases, additional time was required for subculturing if needed, which has not been documented.
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and RIF. This does not mean that this approach is applicable to
only these two antimicrobials. It is anticipated that this study
could provide a guideline for a rapid broth-based direct DST for
other first-line and second-line anti-TB drugs, which will help
especially if a patient is suspected to have resistance to those drugs.
One of the main concerns was how to carry out direct DST. It
was established earlier that for the direct DST using liquid
me-dium, the length of the test protocol should be extended from 14
days to 21 days, since
M. tuberculosis
present in the clinical
speci-men does not grow as rapidly as it does from an isolated culture
(16). In the proposed protocol, the direct DST is set up from an
AFB smear-positive specimen with any degree of smear positivity
by any staining procedure; thus, in some cases, the bacterial count
present in the inoculum is low and may take longer than 14 days to
reach the required growth level or GU. The MGIT 960 indirect
DST procedure is designed with a 4- to 13-day protocol, except for
PZA DST, which is a 4- to 21-day test. However, the PZA test is
designed for only a two-tube system. We tried several different
workflow approaches to achieve results with the 21-day protocol.
The first approach was to set up each drug with its own control
and then enter in the instrument in a two-tube set carrier as a PZA
test. This is a simple, straightforward, and preferred procedure,
and the instrument interprets results automatically. However,
some investigators thought that this approach was costly, because
a control is needed for each drug, meaning requirement of more
MGIT tubes. To cut down the cost, we came up with another
approach, where the first drug, in this case growth control and
INH, is entered into the instrument with the PZA protocol. The
other drug tube, namely, RIF, is entered in the instrument as a
growth and not a DST tube. The tube is placed in the same drawer
close to the INH DST tubes. Once the INH set is ready (GU of
control reaches 400 or more), the instrument flags it as complete
and interprets results as “S” or “R.” At that time, the GU values of
the RIF tube are retrieved by asking the inventory GU values of the
incubated tubes in the instrument, and then the GU value of the
RIF tube is recorded. This is done by printing the inventory report
without scanning the tube out of the instrument prior to printing,
as the GU values may be lost. In case it is difficult to locate a single
tube in a drawer, there is another possibility of putting this RIF
tube in a two-tube DST set carrier, placing first a blank
uninocu-lated MGIT medium tube as a growth control and then an RIF
tube. This set carrier is entered in the instrument as the PZA DST
and is placed close to the INH set. Since the control is not
inocu-lated, this set carrier should be taken out at the same time as the
INH set carrier. The GU values are retrieved and recorded before
scanning out this second carrier set; otherwise, the GU values will
be lost. Interpretation of RIF DST is done manually based on the
formula given in the earlier section. The uninoculated growth
control tube may be used again and again. The above-described
procedure may be followed for any and as many drugs as needed.
This study was carried out in well-established laboratories. The
culture positivity rate of the smear-positive specimen was very
high (above 95%), with acceptable contamination rates (4 to 8%)
and very low prevalence of NTM. The overall success rate of DST
from smear-positive specimens was 85%. That means that only
about 10 to 15% of total DST setups were uninterpretable, due to
several reasons, such as contamination or presence of NTM (X400
errors) or no growth or not enough growth in the control (X200
errors). A few, though AFB smear positive (even 3
⫹
), either failed
to grow in the primary isolation tube and were culture negative or
the growth was not enough to interpret direct DST results (X200
error). The information on the success rate of direct DST is
im-portant to evaluate the cost-effectiveness of the direct DST
ap-proach.
The most important aspect of our findings is the time savings
by direct DST. The time to report Bactec MGIT indirect DST from
positive cultures varied from 6 to 10 days, which is concordant
with many earlier reports (1, 2, 7, 20). This time to report varies
depending on many factors, including variability in the standard
procedure followed in a laboratory, patient population, and
prev-alence of drug resistance. It is known that drug-resistant isolates
tend to take a longer time to grow than the drug-susceptible ones.
The total time for report of indirect DST was calculated as the time
to isolate a culture, the time required to set up DST, and the time
to get results of the susceptibility test from positive culture. On
the other hand, the time to report direct DST was the time to
achieve DST results after inoculation of a processed specimen. The
time savings by direct DST was overall 8 days and did not vary too
much from site to site. This time savings is significantly important,
as every day counts in an MDR case. The results indicate that
direct DST further reduces the time to report susceptibility results
significantly. Following direct DST, on average, sites 1 and 4
re-ported direct DST results after 2 days of culture-positive results,
and sites 2 and 3 reported after three days of cultupositive
re-sults. These sites handle a large number of MDR cases, and thus
there was a good representation of drug-resistant cases among the
total cases that we studied.
Another objective of this study was to evaluate the accuracy of
results obtained by the direct method. In this calculation, indirect
DST was considered the gold standard, and in case of discordant
results indirect DST was repeated. Overall, among the four sites,
only 4.9% of results were discordant in case of INH and 3.9% in
case of RIF, with 2.3% false susceptible (very major error) for INH
and 2.5% for rifampin. False resistance (major error) was 2.6% for
INH and 1.6% for rifampin. One site had higher discordance than
the other three sites. There was no clear pattern of discordant
results, as both false resistant and false susceptible were observed
with INH and RIF.
In summary, this multicenter study established a standard
pro-tocol for performing direct susceptibility with the Bactec MGIT
automated system. DST results may be reported 2 to 3 days after
the culture positivity results are available. This significant time
savings could offer a great help in prescribing effective treatment,
especially in MDR cases. If cost is a concern and the prevalence of
monoresistance to RIF is not common, only RIF may be tested and
could be considered a surrogate marker for MDR. Direct DST is a
reliable test, as results obtained by direct DST had 95 to 96%
concordance with those obtained by the indirect method.
ACKNOWLEDGMENTS
We thank Kirsten Ott (National Reference Center for Mycobacteria, Bor-stel, Germany) for valuable contribution to this study. We thank Becton Dickinson for supplying the reagents.
Salman Siddiqi has a consulting agreement with BD along with several other organizations working in TB diagnostics. The remaining authors report no conflicts of interest.
REFERENCES
1.Adjers-Koskela K, Katila ML.2003. Susceptibility testing with the man-ual Mycobacteria Growth Indicator Tube (MGIT) and the MGIT 960
on May 16, 2020 by guest
http://jcm.asm.org/
system provides rapid and reliable verification of multiresistant
tubercu-losis. J. Clin. Microbiol.41:1235–1239.
2.Ardito F, et al.2001. Evaluation of BACTEC Mycobacteria Growth In-dicator Tube (MGIT 960) automated system for drug susceptibility testing ofMycobacterium tuberculosis.J. Clin. Microbiol.39:4440 – 4444. 3.Aziz MA, et al.2006. Epidemiology of anti-tuberculosis drug resistance
(the global project on anti-tuberculosis drug resistance surveillance): an
updated analysis. Lancet368:2142–2154.
4.Balabanova Y, et al.2005. Multidrug-resistant tuberculosis in Russia: clinical characteristics, analysis of second-line drug resistance and
devel-opment of standardized therapy. Eur. J. Clin. Microbiol. Infect. Dis.24:
136 –139.
5.Banerjee R, Schecter GF, Flood J, Porco TC.2008. Extensively drug-resistant tuberculosis: new strains, new challenges. Expert Rev. Anti Infect.
Ther.6:713–724.
6.Bastian I, Rigouts L, Palomino JC, Portaels F.2001. Kanamycin
suscep-tibility testing ofMycobacterium tuberculosis using Mycobacterium
Growth Indicator Tube and a colorimetric method. Antimicrob. Agents
Chemother.45:1934 –1936.
7.Bemer P, Palicova F, Rüsch-Gerdes S, Drugeon HB, Pfyffer GE.2002. Multicenter evaluation of fully automated BACTEC Mycobacteria
Growth Indicator Tube 960 system for susceptibility testing of
Mycobac-terium tuberculosis.J. Clin. Microbiol.40:150 –154.
8.Cambau E, et al.2000. Mycobacterial Growth Indicator Tube versus the proportion method on Lowenstein-Jensen medium for antibiotic
suscep-tibility testing ofMycobacterium tuberculosis.Eur. J. Clin. Microbiol.
In-fect. Dis.19:938 –942.
9.CLSI.2007. Susceptibility testing of mycobacteria, nocardiae, and aer-obic actinomycetes: approved standards, second edition, vol 26, no 23. CLSI document M24-2. Clinical and Laboratory Standards Institute, Wayne, PA.
10. Gandhi NR, et al.2006. Extensive drug-resistant tuberculosis as a cause of death in patients co-infected with tuberculosis and HIV in a rural area of
South Africa. Lancet368:1575–1580.
11. Goloubeva V, et al.2001. Evaluation of Mycobacteria Growth Indicator
Tube for direct and indirect drug susceptibility testing ofMycobacterium
tuberculosisfrom respiratory specimens in a Siberian prison hospital. J.
Clin. Microbiol.39:1501–1505.
12. Idigoras P, et al.2000. Comparison of the automated nonradiometric BACTEC MGIT 960 system with Lowenstein-Jensen, Coletsos, and Middlebrook 7H11 solid media for recovery of mycobacteria. Eur. J. Clin.
Microbiol. Infect. Dis.19:350 –354.
13. Kam KM, et al.2010. Determination of critical concentrations of second-line anti-tuberculosis drugs with clinical and microbiological relevance.
Int. J. Tuberc. Lung Dis.14:282–288.
14. Kent PT, Kubica GP.1985. Public health microbiology, a guide for the level III laboratory. Centers for Disease Control, Division of Laboratory Training and Consultation, Atlanta, GA.
15. Kim SJ.2005. Drug-susceptibility testing in tuberculosis: methods and
reliability of results. Eur. Respir. J.25:564 –569.
16. Libonati JP, Stager CE, Davis JR, Siddiqi SH.1988. Direct antimicrobial
susceptibility testing ofMycobacterium tuberculosisby the radiometric
method. Diagn. Microbiol. Infect. Dis.10:41– 48.
17. Ling DI, Zwerling AA, Pai M.2008. Genotype MTBDR assays for the diagnosis of multi-drug resistant tuberculosis; a meta-analysis. Eur.
Re-spir.32:1165–1174.
18. Pfyffer GE, Palicova F, Rüsch-Gerdes S.2002. Testing of susceptibility of
Mycobacterium tuberculosisto pyrazinamide with the non-radiometric
BACTEC MGIT 960 system. J. Clin. Microbiol.40:1670 –1674.
19. Roberts GD, et al.1983. Evaluation of the BACTEC radiometric method
for recovery of mycobacteria and drug susceptibility testing of
Mycobac-terium tuberculosisfrom acid-fast smear-positive specimens. J. Clin.
Mi-crobiol.18:689 – 696.
20. Rüsch-Gerdes S, et al.1999. Multicenter evaluation of the mycobacterial
growth indicator tube for testing susceptibility ofMycobacterium
tubercu-losisto first-line drugs. J. Clin. Microbiol.37:45– 48.
21. Rüsch-Gerdes S, Pfyffer GE, Casal M, Chadwick M, Siddiqi S.2006. Multicenter laboratory validation of the BACTEC MGIT 960 technique
for testing susceptibilities of Mycobacterium tuberculosisto classical
second-line drugs and newer antimicrobials. J. Clin. Microbiol.44:688 –
692.
22. Shah NS, et al.2007. Worldwide emergence of extensively drug-resistant
tuberculosis. Emerg. Infect. Dis.13:380 –387.
23. Shinnick TM, Lademarco MF, Ridderhof JC.2005. National plan for reliable tuberculosis laboratory services using a systems approach. Recom-mendations from CDC and the Association of Public Health Laboratories Task Force on Tuberculosis Laboratory Services. MMWR Recomm. Rep.
54:1–12.
24. Siddiqi SH, Hawkins JE, Laszlo A.1985. Interlaboratory drug
suscepti-bility testing ofMycobacterium tuberculosisby radiometric procedure and
two conventional methods. J. Clin. Microbiol.22:919 –923.
25. Siddiqi SH, Libonati JP, Middlebrook G.1981. Evaluation of a rapid
radiometric method for drug susceptibility testing ofMycobacterium
tu-berculosis.J. Clin. Microbiol.13:908 –912.
26. Siddiqi SH, Rüsch-Gerdes S.2006. MGIT procedure manual. Founda-tion for Innovative New Diagnostics (FIND), Geneva, Switzerland. 27. Tenover FC, et al.1993. The resurgence of tuberculosis: is your laboratory
ready? J. Clin. Microbiol.31:767–770.
28. World Health Organization.1998. Laboratory series in tuberculosis con-trol. Part II, microscopy. WHO/TB/98.258. World Heath Organization, Geneva, Switzerland.
29. World Health Organization.2004. Laboratory biosafety manual, 3rd ed. WHO/CDS/CSR/LYO/2004.11. World Heath Organization, Geneva, Switzerland.
30. World Health Organization.2004. Anti-tuberculosis drug resistance in the world. Report no. 3. The WHO/IUATLD global project on anti-tuberculosis drug resistance surveillance. WHO/HTM/TB/2004.343. World Heath Organization, Geneva, Switzerland.
31. World Health Organization. 2006. Guidelines for the programmatic management of drug resistant tuberculosis. WHO/HTM/TB/2006.361. World Heath Organization, Geneva, Switzerland.
32. World Health Organization.2007. Use of liquid TB culture and drug susceptibility testing (DST) in low and middle income settings. Summary report of the expert group meeting on the use of liquid culture media. World Heath Organization, Geneva, Switzerland.
33.World Health Organization. 2008. Policy statement. Molecular line probe assays for rapid screening of patients at risk of multidrug-resistant tuberculosis (MDR-TB). World Heath Organization, Geneva, Switzer-land.
34. World Health Organization. 2010. Policy statement. Noncommercial culture and drug susceptibility testing for rapid screening of patients at risk of MDR-TB. World Heath Organization, Geneva, Switzerland.
