Journal of Clinical Tuberculosis and Other Mycobacterial Diseases 27 (2022) 100303

Contents lists available at ScienceDirect

Journal of Clinical Tuberculosis and Other

Mycobacterial Diseases
journal homepage: www.elsevier.com/locate/jctube

Evaluation of TBMDR® and XDRA® for the detection of multidrug resistant

and pre-extensively drug resistant tuberculosis
Eunjin Cho a, Su jin Lee b, Jiyoung Lim b, Dong Sik Kim b, Namil Kim b, Han Oh Park b, Ji-im Lee a,
Eunsoon Son a, Sang Nae Cho a, Wah Wah Aung c, Jong Seok Lee a, *
a
ITRC (International Tuberculosis Research center): 236 Gaposunhwan-ro, Masanhappo-gu, changwon-si, Gyeongsangnam-do, Republic of Korea
b
BIONEER: 8-11, Munpyeongseo-ro, Daedeok-gu, Daejeon 34302, Republic of Korea
c
Advanced Molecular Research Centre, Department of Medical Research, Yangon, Republic of the Union of Myanmar

A R T I C L E I N F O A B S T R A C T

Keywords: This study evaluated the diagnostic performance of the AccuPower® TB&MDR Real-Time PCR (TBMDR®) and
TBMDR®, XDRA® AccuPower® XDR-TB Real-Time PCR Kit-A (XDRA®) to detect multidrug-resistant (MDR-TB) and pre-extensively
Diagnosis drug-resistant tuberculosis (pre-XDR-TB) in comparison with phenotypic drug susceptibility testing (DST) using
MDR-TB
MGIT 960 on 234 clinical Mycobacterium tuberculosis isolates. Discrepant results were confirmed by direct-
pre-XDR-TB
sequencing. Sensitivity and specificity of TBMDR and XDRA for cultured isolates were 81.2% and 95.8% for
isoniazid (INH) resistance, 95.7% and 95.7% for rifampicin (RIF) resistance, 84.1% and 99.1% for fluo­
roquinolone (FQ) resistance, and 67.4% and 100% for second-line injectables resistance. The sensitivities of each
drug were equivalent to other molecular DST methods. High concordance was observed when compared to
direct-sequencing. We also found that TBMDR and XDRA assays can detect INH, RIF and FQ resistance in isolates
with low level resistance-associated mutations which were missed by phenotypic DST. Our study showed TBMDR
and XDRA assays could be the useful tools to detect MDR-TB and pre-XDR-TB.

1. Introduction resistant to any FQ [4]. Thus second-line injectable drugs (SLID) were no
longer the part of XDR-TB nor widely recommended. However, it will
Drug-resistant tuberculosis (TB) is still remaining a worrisome public take times to change all previous injectable drug containing regimens to
health problem even though the overall TB incidence has been all oral drug regimens including newly introduced TB drugs. Thus, the
decreased. In 2019, an estimated 10 million people developed TB and use of currently available drug resistant detection methods must be
1.4 million people died. Among the newly developed TB cases, almost a continued and development of new improved methods is still needed.
half million were rifampicin-resistant TB (RR-TB), of which 78% were Culture-based phenotypic drug susceptibility testing (DST) remains
multidrug-resistant TB (MDR-TB). Considering the large gap between as a gold standard for drug resistance determination to detect MDR-TB
notified 7.1 million and estimated 10 million new cases in 2019, drug and XDR-TB although it is labor-intensive and time-consuming [5].
resistance can be more serious than reported. Another gap (38%) be­ Currently used molecular-based DSTs such as Xpert MTB/RIF assay and
tween estimated MDR/RR-TB and enrolled in treatment might support line probe genotypic assays (LPAs) which are designed for rapid detec­
such assumption [1]. tion of specific drug-resistance conferring mutations in Mycobacterium
To minimize such gaps, it is essential to expend TB diagnosis and tuberculosis (MTB), have some limitations in each test. Xpert MTB/RIF
drug resistance detection in various extents. Recently, the World Health assay is a cartridge based nucleic acid amplification test which detects
Organization (WHO) recommended to use oral drugs more extensively TB and RR-TB rapidly but it has limitation for ruling out rifampicin (RIF)
[2,3] and revised the definition of extensively drug-resistant TB (XDR- sensitive polyresistant TB. The World Health Organization (WHO)
TB). The updated XDR-TB is defined as infection with an MDR-TB strain endorsed LPAs for rapid molecular detection of MDR-TB, FQ and SLID
that is also resistant to any fluoroquinolone (FQ) and at least one resistance but there is requirement of laboratory infrastructure and
additional Group A drug and pre-XDR-TB is MDR/RR-TB that is also trained persons to perform the tests [6–9].

* Corresponding author.
E-mail address: cosmosljs@gmail.com (J. Seok Lee).

https://doi.org/10.1016/j.jctube.2022.100303

Available online 9 February 2022

2405-5794/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
E. Cho et al. Journal of Clinical Tuberculosis and Other Mycobacterial Diseases 27 (2022) 100303

Recently two new assays (TBMDR® and XDRA®), which are rapid Real-Time PCR, totaling 3 h. This workflow includes all processes from
and affordable in-vitro diagnostics solutions using Real-Time PCR plat­ sample preparation through the actual extraction process using the
form, were designed for rapid simultaneous detection of RIF/isoniazid ExiPrep™ 16Dx, which automatically deposit the extracted genomic
(INH) resistance and FQ/SLID resistance. These assays are based on fully DNA into the Elution buffer cartridge.
automated system which minimize all human handling processes such as Target gene region of AccuPower® TB & MDR Real-Time PCR
pipetting to reduce cross-contamination for better results. Total running (TBMDR) and AccuPower® XDR-TB Real-Time PCR Kit-A (XDRA) assays
time of the assays is short as it takes about three hours. In this study, were shown in Table 1.
these two new assays were evaluated to access their diagnostic perfor­
mance to detect MDR-TB and pre-XDR-TB. 2.4. Limit of detection (LoDs) of the assays

2. Methods The experiment followed the CLSI guideline EP17-A2. Six or more
dilutions were used, including the concentration values before and after
2.1. Clinical isolates the estimated minimum detection limit. The experiment was carried by
diluting step by step from a high concentration. More than 24 repetitions
Clinical Mycobacterium tuberculosis (MTB) isolates cultured from per concentration were tested, and Probit Analysis was performed.
sputum samples of active pulmonary TB patients who enrolled in a Through the analysis, the minimum concentration showing the 95%
prospective observational cohort study (ClinicalTrials.gov identification detection rate was set as the LoD value, and the value calculated as the
number, NCT00341601) at International Tuberculosis Research Center 95% Confidence interval (CI) was set as the confidence interval.
(ITRC) in South Korea during the study period 2005–2018 were used for
the present study. The study was reviewed and approved by the NMH 2.5. DNA sequencing
ethics review board. A total of 234 phenotypically well-characterized
MTB isolates comprising XDR (as previous definition), FQ/SLID resis­ Nucleotide sequence alterations in each target gene from test sam­
tant MDR, MDR, mono-resistant to any drugs, and pan-susceptible based ples were characterized by sequencing. Genes or genetic loci that were
on previous phenotypic DST were selected. These isolates were sub- known as involved in drug resistances according to the updated WHO
cultured on the Lowenstein-Jensen (LJ) egg slants and incubated at recommendation were characterized [12]. Primers to amplify targets for
37 ◦ C for 4 weeks [10]. sequencing and amplification conditions are described in Supplemen­
tary Table 1. Direct sequencing was carried out on the ABI3730 in
2.2. Phenotypic drug susceptibility test (DST) Bioneer (Daejeon, South Korea).

The standard protocol for DST in MGIT 960 was strictly followed as 3. Results
recommended for isoniazid (INH) (0.1ug/ml), rifampicin (RIF) (1ug/
ml), ofloxacin (OFX) (2.0ug/ml), moxifloxacin (MOX) (0.25ug/ml), 3.1. Phenotypic DST profile
kanamycin (KM) (2.5ug/ml), and capreomycin (CAP) (2.5ug/ml). To
each 7 ml MGIT tube, 0.8 ml of MGIT 960 Growth Supplement and 0.1 Of 234 M. tuberculosis isolates included in the study, 37 (15.8 %)
ml of the drug stock solutions were aseptically added, and finally 0.5 ml were susceptible, 6 (2.6%) were mono-resistant, 20 (8.5%) were poly-
of the test inoculum was added. For each isolate, a growth control (GC) resistant other than MDR-TB, 41 (17.5%) were MDR-TB, 33 (14.1%)
tube with Growth Supplement and without drug was included. For this were MDR-TB plus FQ resistant, 13 (5.6%) were MDR-TB plus SLID
GC, the inoculum was prepared by pipetting 0.1 ml of the test inoculum resistant and 84 (35.9%) were XDR-TB. Total resistance to individual
with 10 ml of sterile water to make a 1:100 dilution; 0.5 ml of GC drug/drug group were 186 (79.5%), 188 (80.3%), 126 (53.8%) and 92
inoculum was added to a drug-free MGIT. All of the inoculated tubes (39.3%) for INH, RIF, FQ and SLID respectively (Table 2).
were placed into the BACTEC MGIT 960 instrument on the same day of
inoculation. The relative growth ratio between the drug-containing tube 3.2. Limit of detection (LoD)s of TBMDR and XDRA
and drug-free GC tube was determined by the system’s software algo­
rithm. If the relative growth in the drug-containing tube was equal to or LoDs of each gene conferring drug resistance were in an average
exceeded that of the GC tube, the isolate was considered drug resistant; range of 97.7 to 380.2 copies/test (Table 3).
if the relative growth was less than in the GC tube, the isolate was
considered drug susceptible. The instrument did the final interpretation
3.3. Diagnostic performance of MDRTB and XDRA
and reported the susceptibility results automatically [11].
Sensitivity and specificity of TBMDR and XDRA assays for INH, RIF,
2.3. AccuPower® TB & MDR Real-Time PCR (TBMDR) and
FQ and SLID were calculated compared to phenotypic DST results.
AccuPower® XDR-TB Real-Time PCR Kit-A (XDRA) assays
Diagnostic accuracy of TBMDR was 84.2 % and 95.7% for INH and RIF
respectively and that of XDRA was 91.0% and 87.2% for FQ and SLID
Preparation of kit materials and specimens, assay protocol, and data
respectively. Discordant samples (37 isolates for INH, 10 for RIF, 21 for
analysis were carried out according to the ExiStation™ system User
Guide (Bioneer, Daejeon, South Korea). The process included two steps:
Table 1
i) DNA extraction by Exiprep™ 16Dx ii) real-time PCR reaction, and data
Target regions of Accupower® TBMDR and XDRA Real-Time PCR Kits.
analysis by Exicycler™ 96. After preparing samples, DNA is extracted
using Exiprep™ 16DX (A-5050) instrument using the Exiprep™ Dx Kit Drug Gene Codon Nucleotide
target region
Mycobacteria genomic DNA Kit (K-4418). After the kit is installed in the
instrument, DNA extraction proceeds automatically, and DNA is Accupower® Rifampicin rpoB 501–531 –
TBMDR Isoniazid inhA − 15
dispensed into the PCR reaction tube. After that, the PCR tube is taken –
Real-Time promoter
out from the instrument, goes through the vortexing, spins down, and PCR Kit katG 315 –
then installed in the Exicycler™ 96 Real-Time Quantitative Thermal Accupower® Fluoroquinolones gyrA 90,91,94 –
Block (A-2060-1) perform Real-Time PCR. After PCR is finished, Exist­ XDRA Real- injectable second- rrs – 1401,1402,1484
ation S/W automatically analyzes the results. The running time is Time PCR line drugs
Kit
approximately 1 h and 15 min for DNA extraction and 1 h and 40 min for

2
E. Cho et al. Journal of Clinical Tuberculosis and Other Mycobacterial Diseases 27 (2022) 100303

Table 2 genes or genetic loci which suspected to involve in INH resistance

Phenotypic DST profiles of 234 clinical M. tuberculosis isolates. (suspects around 10–15% of INH resistance) [14]. Two phenotypic DST
Drug resistance profile No. of isolates (%) susceptible/TBMDR resistant isolates showed katG mutation (S315N)
which is associated with low level resistance [15]. Most of the INH
Susceptible to all drugs 37 (15.8)
MDR-TB 41 (17.5) resistance discrepancies, phenotypic DST resistant/TBMDR sensitive
MDR-TB plus FQ resistant 33 (14.1) samples showed katG and inhA genes mutation points that TBMDR does
MDR-TB plus SLID 13 (5.6) not target and one sample was due to kit error as it failed to detect C-15 T
XDR-TB 84 (35.9) inhA mutation.
Mono-resistant 6 (2.6)
Poly-resistant other than MDR-TB 20 (8.5)
Much higher sensitivity and specificity were shown for RIF. Sensi­
Individual drug/drug group resistance tivity was 95.7% (95% CI 91.79–96.15%) and specificity was 95.7%
INH 186 (79.5) (95% CI 85.16–99.47%) which were similar to the 95% pooled sensi­
RIF 188 (80.3) tivity and 98% pooled specificity values of Xpert MTB/RIF assay [6] and
FQ 126 (53.8)
96–98.7% sensitivity and 88.9 to 99% specificity values of MTBDRplus
SLID 92 (39.3)
assays reported by the previous studies [7,8,9]. In comparison with
DST = Drug susceptibility testing, MDR-TB = Multidrug-resistant TB, XDR-TB = sequencing results of RRDR it showed 98.7% concordance. Of eight
Extensively drug-resistant TB, INH = Isoniazid, RIF = Rifampicin, FQ = Fluo­ phenotypic DST resistant/TB-MDR kit RIF susceptible samples, five were
roquinolones, SLID = Second-line injectable drugs.
confirmed as susceptible by sequencing, thus those five samples can be
phenotypic DST error or due to other resistance determinants. Two
samples had V176F and S522W mutations which kit does not target. Kit
Table 3
Limit of detection (LoD)s of TBMDR and XDRA. error was found in one sample which had S531L mutation. The results
also showed that TB-MDR kit can detect two RIF low level resistant
Name of assay Target Limit of detection
isolate with L511P and D516T mutations which were missed by
Average (Range) copies
numbers/test phenotypic DST [16,17].
gyrA and gyrB are two major genes that explain about 90% of FQ
Accupower TBMDR Mycobacterium 63.10 (40.74 ~ 95.50).
tuberculosis resistances. Similarly, rrs gene and eis promoter region are known to be
RIF 144.54 (95.50 ~ 213.80), major resistance determinants which can explain about 75–90% of SLIDs
INH 112.20 (69.18 ~ 181.97) resistance [18]. However, current Bioneer XDRA targets only gyrA for
Accupower XDRA FQ gyrA1W 309 (186.2 ~ 512) FQ and rrs for SLID. One phenotypic DST susceptible/XDRA resistant
assay FQ gyrA1M 104.7 (64.6 ~ 166.0) discordant result was found to be caused by isolate with A90V mutation
FQ gyrA2W 380.2 (213.8 ~ 660.7) which is associated with FQ low level resistance and it was missed by
FQ gyrA2M 92.72 (60.26–158.5)
phenotypic DST. However most of FQ and SLID resistance discordant
SLID rrs1W 281.8 (154.88 ~ 524.8)
SLID rrs1M 199.5 (123.03 ~ 323.6)
results were due to presence of gyrB and eis promotor mutations that
SLID rrs2W 288.4 (186.2 ~ 446.7) XDRA do not target. Regarding phenotypic DST error for FQ, some MOX
SLID rrs2M 158.5 (97.72 ~ 251.2) resistant isolates were suggested involved in low level resistance. In such
INH = Isoniazid, RIF = Rifampicin, FQ = Fluoroquinolones, SLID = Second-line a case, current drug concentrations might not good enough to detect FQ
injectable drugs. resistance [19–21].
Although XDRA targets only gyrA and rrs, sensitivities for FQs and
FQ and 30 for SLID) between phenotypic DST and TBMDR /XDRA assays SLIDs were comparable to other reports on XDR detection or even higher
were detected. High concordance rates (greater than95%) were [8,12,17]. Sequencing results of gyrA/gyrB for FQs and rrs/eis promoter
observed when compared to direct-sequencing for each drug resistant region for SLIDs showed significant concordance with XDRA assay;
conferring genes. Diagnostic performance of TBMDR and XDRA 95.3% for FQs and 96.6% for SLIDs, respectively.
compared to phenotypic DST, drug resistant gene mutations pattern and In the present study, we found that TBMDR and XDRA assays can
concordance rates between sequencing and TDMDR and XDRA assays detect INH, RIF and FQ resistance in isolates with low level resistance
were described in Table 4. Discordant results for each drug resistance which were missed by phenotypic DST. The sensitivities, specificities
and target gene sequence variation were shown in Supplementary and diagnostic accuracies of TBMDR and XDRA assays can also be
Table 2. improved if we take consider phenotypic DST error and low level
resistance.
4. Discussion We also observed two TBMDR kit error cases which failed to detect
RIF and INH common resistance mutations. This can be due to mutation
Bioneers’s TBMDR and XDRA are in vitro diagnostic Real-Time PCR- ratio of these isolates were below the limit of detection. “ExiStation™ is
based assays, designed for rapid detection of MDR-TB and XDR-TB. an automated molecular diagnostic system consisting of an automatic
These assays can be used in human samples such as sputum and bron­ nucleic acid extraction instrument (ExiPrep™16 Dx) and a nucleic acid
choalveolar lavage and culture isolates. The present study evaluated the amplification instrument (Exicycler™ 96). We minimized user errors by
diagnostic performance of these assays on culture isolates compared to automating the contamination prevention system and PCR reaction set
phenotypic DST. LoDs of each gene conferring drug resistance in TBMDR up in the instrument. In addition, errors were minimized by reducing the
and XDRA assays showed an average range of 97.7 to 380.2 copies/test. hands-on step using a pre-filled nucleic acid extraction cartridge, and
Overall diagnostic accuracy was satisfactory as it ranged from 84.2% to vacuum dried premix type PCR reagent. Minimizing TB -MDR kit error is
95.7%. This result is comparable to that of WHO recommended important because this could lead to wrong selection of the treatment
molecular-based GenoType MTBDRplus and MTBDRsl assays reported by regimen for patients.
other studies [7,8,13]. There were some limitations in our study. There was lack of sputum
For INH resistance detection, TBMDR showed a sensitivity of 81.2% smear data and we can not perform the testing directly on the sputum
(95%CI 74.81–86.53%) and specificity of 95.8% (95%CI samples. The present study mainly focused on diagnostic performance of
85.75–99.49%). Relatively low sensitivity might be explained from the the kits and we did not correlate the genotypic resistance profiles of the
fact that, i) several genes or genetic loci has been known to involve INH assays to the clinical treatment data. We can not use WHO endorsed
resistance (three major targets; katG, inhA, ahpC), ii) there are additional LPAs in our study for direct comparison with the tested assays. Further
study was suggested to evaluate the performance of these assays for

3
E. Cho et al. Journal of Clinical Tuberculosis and Other Mycobacterial Diseases 27 (2022) 100303

Table 4
Diagnostic performance of TBMDR and XDRA assays compared to phenotypic DST and gene sequencing.
Test kit Ant- pDST (MGIT) % Sensitivity % Specificity Diagnostic Concordance
TB (95% CI) (95% CI) accuracy sequencing vs
Susceptible Resistant
drugs (%) AccuPower® kits
No. of Mutation No. of Mutation (%)
Isolates types Isolates types
(sequencing) (sequencing)

AccuPower® INH Susceptible 46 No mutation 35 katG 312GCG- 81.2 95.8 84.2 95.3
TBMDR GGG# (74.81–86.53) (85.75–99.49)
katG 390TAT-
>TGT#
inhA -34C-T#
inhA -15T§
Resistant 2 katG 315AGC- 151 Not shown
AACψ
RIF Susceptible 44 No mutation 8 522TCG-TGG# 95.7 95.7 95.7 98.7
176GTC-TTC# (91.79–96.15) (85.16–99.47)
531TCG-TTG §
Resistant 2 511CTG-CCGψ 180 Not shown
516GAC-TACψ

AccuPower® FQs Susceptible 107 No mutation 20 gyrB 540GAA- 84.1 99.1 91.0 95.3
XDRA GAC# (76.56–90.03) (94.95–99.98)
gyrB 538AAC-
GAC#
gyrB 539ACC-
GCC#
gyrB 551GGG-
AGG#
gyrB 486TCC-
TTC#
Resistant 1 gyrA 90GCG- 106 Not shown
GTGψ*
SLIDs Susceptible 142 No mutation 30 rrs A1401G§ 67.4 100.0 87.2 96.6
rrs C1402T§ (56.82–76.80) (97.44–100.0)
eis -8C-A#
eis -10G-A#
eis -12C-T#
eis -14C-T#
Resistant 0 No mutation 62

pDST: Phenotypic drug susceptibility test.

Ψ: Low level resistant.
#: Kit does not target.
*: Phenotypic DST error.
§: Kit error.

rapid detection of MDR and XDR-TB directly from clinical samples. org/10.1016/j.jctube.2022.100303.
In conclusion, the sensitivities of TBMDR and XDRA TB drug resis­
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