Tuberculosis 134 (2022) 102199

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Tuberculosis
journal homepage: www.elsevier.com/locate/tube

Clinical evaluation of the cobas® MTB-RIF/INH reagent and the cobas®

6800 for the detection of isoniazid and rifampicin resistance
Akio Aono a, Yoshiro Murase a, Masaaki Minegishi b, Shuichi Ohtawa b, Masatoshi Yano b,
Kinuyo Chikamatsu a, Yoshiko Shimomura a, Makiko Hosoya a, Yuriko Igarashi a,
Yuta Morishige a, Hiroyuki Yamada a, Akiko Takaki a, Kenichi Togashi c, Mikako Hiura c,
Satoshi Mitarai a, *
a
Department of Mycobacterium Reference and Research, Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association, Kiyose, Tokyo, 2048533, Japan
b
Clinical laboratory, National Hospital Organization Tokyo Hospital, Kiyose, Tokyo, Japan
c
Roche Diagnostics KK, Shinagawa, Tokyo, Japan

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

Keywords: We aimed to validate the performance of a newly developed real-time PCR assay using cobas® MTB-RIF/INH
Isoniazid reagent on the cobas® 6800 system for detecting isoniazid (INH) and rifampicin (RIF) resistance, using Japa­
Rifampicin nese Mycobacterium tuberculosis (MTB) isolates. In total, 119 mock sputum specimens spiked with resistant MTB
Resistance
were tested using the cobas® MTB-RIF/INH reagent. The whole genomes of all MTB isolates were sequenced by
Mutation
MiSeq and analysed for mutations/indels causing drug resistance. All isolates were tested for phenotypic drug
Real-time polymerase chain reaction
susceptibility, then MTB negative sputa were collected and pooled to prepare mock sputum specimens for the
study. The sensitivity and specificity for INH resistance at a concentration equal to 3 × the limit of detection were
77.8% and 90.0%, respectively; those for RIF resistance were 91.8% and 93.5%, respectively. The sensitivities for
INH and RIF were statistically different (P = 0.014), but not the specificities (P = 0.624). Twenty-two false-
susceptible and two false-resistant results were obtained in INH; meanwhile, six false-susceptible and three false-
resistant results were obtained in RIF. False-resistance for INH and RIF was mainly due to disputed mutations.
The cobas® MTB-RIF/INH reagent showed better performance than other rapid molecular tests.

1. Introduction among cases of RR-TB in settings with high MDR-TB is high [4]. How­
ever, in some settings, the proportion of MDR-TB cases among new cases
Tuberculosis (TB) is a life-threatening disease. In 2019, 206,030 is 1%, and many cases of RIF mono-resistance are expected. Therefore, a
people with multidrug-resistant (MDR)-/rifampicin (RIF)-resistant (RR)- first-line LPA is necessary to confirm INH susceptibility where the
TB were notified, whereas 465,000 MDR-TB cases were estimated [1]. probability of MDR-TB among RR-TB cases is low.
The rate of bacteriological confirmation of MDR-TB is low; thus, In this study, a newly developed genetic diagnostic test for resistance
expansion of RIF and universal drug susceptibility testing is required. of Mycobacterium tuberculosis (MTB) against RIF and INH was evaluated
The World Health Organization (WHO) released consolidated using clinical MTB isolates. This diagnostic test involved the cobas®
guidelines on TB in 2020 and updated the use of rapid diagnostics for TB MTB-RIF/INH (Roche Diagnostics, Pleasanton, CA) reagent with the
detection [2]. In these modules, Xpert MTB/RIF and Ultra (Cepheid, cobas® 6800/8800 automated real-time nucleic acid amplification
Sunnyvale, CA) are recommended to initially presume TB [2]. According (NAA) and detection system (Roche Diagnostics, Pleasanton, CA). This
to the diagnostic algorithm released by the Global Laboratory Initiative system directly detects RIF resistance-associated mutations in rpoB and
in 2018, sputum specimens from RR-TB cases should be subjected for a INH resistance-associated mutations in katG and inhA of MTB in human
second-line line probe assay (LPA) [3]. Based on the results, a patient is respiratory specimens. This is the evaluation of cobas® MTB-RIF/INH
treated with different regimens for 9–20 months. In this algorithm, with the cobas® 6800 system, and the results will be useful for con­
isoniazid (INH) susceptibility is ignored, as the probability of MDR-TB firming MDR-TB in a single process.

* Corresponding author.
E-mail address: mitarai@jata.or.jp (S. Mitarai).

https://doi.org/10.1016/j.tube.2022.102199
Received 11 October 2021; Received in revised form 9 March 2022; Accepted 15 March 2022
Available online 21 March 2022
1472-9792/© 2022 Published by Elsevier Ltd.
A. Aono et al. Tuberculosis 134 (2022) 102199

2. Materials and methods Madison, WI). The purified library was evaluated using the Qubit dsDNA
HS Assay Kit. Next generation sequencing was performed using the
2.1. Preparation of MTB-negative sputum specimen pool MiSeq reagent kit (600 cycles) with 350-mer and 250-mer paired-end
short reads following the manufacturer’s instructions, and the gener­
Sputum specimens were spiked with MTB with known drug- ated fastq data were obtained and analysed using CLC Genomics
resistance patterns. The sputum specimens were collected from pa­ Workbench ver. 6.0 (Qiagen, Venlo, Netherland) or MTBseq ver. 1.0.4
tients with presumed TB at the National Tokyo Hospital between March [8]. Finally, confident mutations and indels were identified using the list
and May 2020, pre-treated using the standard method with N-acetyl-L- in TB-Profiler ver. 2.8.6 [9].
cysteine–sodium hydroxide (NALC-NaOH) (MycoPrep, Becton Dick­
inson, Sparks, MD), and then concentrated by centrifugation at 3000×g 2.4. Preparation of mock MTB-spiked specimens
for 15 min [5]. After discarding the supernatant, the pellet was resus­
pended in 2 mL phosphate buffer (pH 6.8) (FujiFilm Wako Pure Chem­ In Japan, it is difficult to test clinical specimens of RIF mono-
ical Co., Tokyo, Japan). The suspension was subjected to acid-fast bacilli resistant- and MDR-TB, primarily due to a low prevalence of MTB
smear microscopy and inoculated in Mycobacteria Growth Indicator drug resistance. Therefore, spiked sputum specimens with known drug
Tube (MGIT, Becton Dickinson, Sparks, MD) at 37 ◦ C for 6 weeks. After resistance patterns were used. Pre-treated and pooled MTB-negative
confirming negative results using smear microscopy and MGIT, the sputum specimens (540 μL) were dispensed into 2.0 mL tubes. The
remaining suspension was used as the MTB-negative sputum specimen. suspensions were adjusted according to the concentration of each
Although 150 pre-treated specimens confirmed as MTB-negative were isolate: 256.6 colony forming units (CFU)/60 μL for INH mono-resistant
pooled to collect at least 50 mL of the suspension, the volume of the MTB strains (256.6 CFU/600 μL = 427.7 CFU/mL) and 600 CFU/60 μL
actual pooled MTB-negative pre-treated sputum specimens was 250 mL. for RIF mono-resistant and MDR MTB strains (600 CFU/600 μL = 1000
CFU/mL). These MTB concentrations were determined based on the
2.2. Preparation of MTB isolates limit of detection (LoD) of system (3 × the LoD) (internal data).

To prepare the MTB-spiked specimens, a series of MTB isolates was 2.5. cobas® MTB-RIF/INH evaluation
prepared. These isolates were collected in Japan from 2007 to 2014 and
included isolates from National Drug Resistance surveys (2007 and The performance of the cobas® MTB-RIF/INH was evaluated ac­
2012) and other clinical isolates [6,7]. In total, 119 isolates were ob­ cording to the manufacturer’s instructions. The mock specimen was
tained, cultured on a solid medium (2% Ogawa, Kyokuto Pharmaceu­ considered to be pre-treated with NALC-NaOH. Briefly, 0.4 mL of the
ticals), and harvested for DNA extraction and conventional drug mock specimen was transferred to a barcode-labelled 5 mL poly­
susceptibility tests (DSTs). The conventional proportion method using propylene screw-capped tube containing 2 mL of cobas® Microbial
Löwenstein-Jensen media was used for each isolate according to the Inactivation Solution. After tightly closing the cap, the tube was vor­
standard proportion method [5]. The final DST results were obtained texed for 30–60 s for proper mixing. The mixed specimen was incubated
after 6 weeks of incubation at 37 ◦ C, and 53 MDR-TB, 20 RIF at an ambient temperature (22–25 ◦ C) for at least 60 min and then
mono-resistant, and 46 INH mono-resistant isolates were prepared. The shaken using vortex for 30 s. The specimen was sonicated using a tube
bacterial suspension for DST was diluted serially, and the absolute sonicator (TS 5, Rinco Ultrasonics AG, Romanshorn, Switzerland and
concentration of each isolate was confirmed. centrifuged for 1 min at 3000×g; then, the specimen tube was uncapped
The construction of MTB strains used in this study was unnatural, and loaded into a cobas® 6800 system. cobas® MTB-RIF/INH detected
because no pan-susceptible MTB was included. However, the general the RIF-resistance-associated mutations and INH resistance-associated
genetic DST specificity to pan-susceptible isolates is high, so that many mutations automatically in the specimens.
pan-susceptible isolates may automatically increase the specificity of the
system. Then, we tested drug resistant strains to isoniazid and/or 2.6. Statistical analysis
rifampicin, intensively. We tested H37Rv ATCC27294 as control.
The data obtained from the system were classified into four cate­
2.3. Whole-genome sequencing gories for each type of drug resistance tested: true positive, false posi­
tive, true negative, and false negative, using phenotypic DST (pDST)
DNA was extracted using a commercial kit (ISOPLANT II, FujiFilm results as the gold standard. Sensitivity and specificity were calculated
Wako Pure Chemicals, Japan), following the manufacturer’s protocol and compared between drugs using the chi-square test. Statistical sig­
with some modifications. In brief, five zirconia beads (3-mm size, TYZ-3, nificance was set at P < 0.05. The JMP ver. 15.1.0 (SAS Institute Inc.,
Nikkato Co., Ltd., Japan) were placed into a 2-mL DNA LoBind tube NC) was used for analyses.
(Eppendorf AG, Hamburg, Germany), and then 360 μL of Solution I of
ISOPLANT II containing RNase A (1 mg/mL) was transferred into the 2.7. Ethics considerations
tube. One loopful of MTB (10 mg) was inoculated into the tube, which
was then vortexed for 2 s. The dispersed MTB bacilli were incubated in Smear- and culture-negative pre-treated specimens were collected
the tube overnight at 37 ◦ C and then at 80 ◦ C for 20 min for sterilisation. from patients with presumed TB at the National Tokyo Hospital. The
Then, 180 μL of Solution II of ISOPLANT was added to the tube and study protocol was approved by the Institutional Review Board
incubated at 55 ◦ C for 15 min. Then, 180 μL of 7.5 M ammonium acetate (Approval # RIT/IRB 2019–23). The specimen collection process was a
was added, and the tube was centrifuged at 12,000×g at 4 ◦ C for 15 min. part of routine TB screening; therefore, no informed consent was
A 350 μL sample of the upper aqueous phase was transferred to a new required. Nonetheless, an opt-out strategy was adopted to protect the
LoBind tube, and 280 μL of isopropanol was added to precipitate the identity of the individuals.
DNA. The purified DNA was measured using a Qubit dsDNA HS assay kit
(Thermo Fisher Scientific, Waltham, MA), and the concentration was 3. Results
adjusted to 20 ng/μL. DNA library preparation for MiSeq sequencing
(Illumina) was performed using QIAseq FX DNA Library kit (Qiagen, The drug resistance patterns and numbers of specimens according to
Venlo, Netherland) and Agencourt AMPure XP Beads (Beckman phenotypic and whole-genome sequence DST and the analysis flowchart
Coulter), according to the manufacturers’ instructions. The library was are shown in Fig. 1. H37Rv control strain was phenotypically and
recovered using the Wizard SV Gel and PCR Clean-Up System (Promega, genetically susceptible to INH and RIF, then it was removed from the

2
A. Aono et al. Tuberculosis 134 (2022) 102199

Fig. 1. Flow of specimens and data analyses.

performance analysis. 3.3. Comparison of cobas® MTB-RIF/INH and whole-genome sequence

results
3.1. Detection of INH resistance using phenotypic DST as reference
Genotypic DST (gDST) results are shown in Table 2, as whole-
Among 119 mock specimens containing 99 INH-resistant MTB iso­ genome sequencing was performed for discordant specimens. The sen­
lates (53 MDR and 46 INH mono-resistant), 79 positive (INH resistance sitivities of cobas® 6800 for INH and RIF resistance were 84.0% (95%
detected) and 40 negative (no INH resistance detected) specimens were CI, 75.3–90.1) and 93.3% (95% CI, 85.3–97.1), respectively. The spec­
obtained using cobas® 6800 with cobas® MTB-RIF/INH. The sensitivity ificities of cobas® 6800 for INH and RIF resistance were 100% (95% CI:
and specificity were 77.8% (95% confidence interval [CI]: 68.6–84.8) 86.7–100) and 100% (95% CI: 92.0–100), respectively.
and 90.0% (95% CI: 69.9–97.2), respectively. The overall agreement
was 79.8% (95% CI: 71.7–86.1) (Table 1). 3.4. Discrepancy between phenotypic and genotypic results

3.2. Detection of RIF resistance using phenotypic DST as reference Twenty-four discrepant results were obtained in INH resistance: 22
false-susceptible and two false-resistant results. The two INH false-
Among 119 mock specimens containing 73 RR and MTB isolates (53 resistant specimens contained MTB with C-15T in the fabG1-inhA
MDR and 20 RIF mono-resistance), 79 positive (RIF resistance detected) operon. Meanwhile, 22 false-susceptible specimens contained MTB with
and 40 negative (no RIF resistance detected) results were obtained using mutations other than the targets of the cobas® MTB-RIF/INH reagent
cobas® 6800 with cobas® MTB-RIF/INH. The sensitivity and specificity (Table 3).
were 91.8% (95% CI: 83.2–96.2) and 93.5% (95% CI: 82.5–97.8), Six false-susceptible and three false-resistant results were obtained
respectively. The overall agreement was 92.4% (95% CI: 86.2–96.0). for RIF resistance. Three false-resistant specimens contained MTB with
The sensitivities for the detection of INH and RIF resistance were sta­ the amino acid substitutions H445D, L430P, and H445 N in the rpoB
tistically different (P = 0.014), but the specificities were not (P = 0.624) gene, and all were MDR. Meanwhile, six false-susceptible specimens
(Table 1). contained MTB with the amino acid substitutions A345V, H445 T/R,
V170P, and S450W in rpoB, V517E in rpoC, and other unknown muta­
tions (Table 3).

Table 1 Table 2
Sensitivity and specificity of cobas® MTB-RIF/INH according to phenotypic Sensitivity and specificity of cobas® MTB-RIF/INH according to genotypic drug
drug susceptibility results. susceptibility results.
Target drug Judgement Phenotypic drug Target drug Judgement WGS drug susceptibility
susceptibility testing testing result
result
Resistant Susceptible
Resistant Susceptible
cobas MTB-RIF/INH Isoniazid Resistant 79 0
cobas MTB-RIF/INH Isoniazid Resistant 77 2 result Susceptible 15 25
result Susceptible 22 18 Rifampicin Resistant 70 0
Rifampicin Resistant 67 3 Susceptible 5 44
Susceptible 6 43
WGS: whole genome sequence.

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A. Aono et al. Tuberculosis 134 (2022) 102199

Table 3
Summary of discrepant results with target substitutions.
Specimen ID Phenotypic DST result cobas® MTB-RIF/INH result Target genes Missense mutations

Sequence identified Target sequence of the test

JATA077 Susceptible Positive rpoB H526D H526D

JATA078 Susceptible Positive rpoB L511P L511P
JATA079 Susceptible Positive rpoB H526 N H526 N
JATA004 Resistant Negative rpoB A426V –
JATA016 Resistant Negative rpoC V517E –
JATA056 Resistant Negative rpoB V251P –
JATA067 Resistant Negative Other None –
JATA032 Resistant Negative rpoB H526 T/R H526R
JATA060 Resistant Negative rpoB S531W S531W
JATA062 Susceptible Positive katG_promoter C-15T C-15T
JATA066 Susceptible Positive katG_promoter C-15T C-15T
JATA004 Resistant Negative katG_promoter -2C –
JATA009 Resistant Negative ahpC K192E –
JATA013 Resistant Negative Other None –
JATA015 Resistant Negative katG T380I –
JATA016 Resistant Negative Other P334A –
JATA019 Resistant Negative Other None –
JATA023 Resistant Negative katG M216I –
JATA026 Resistant Negative katG S315R –
JATA028 Resistant Negative Other A247P –
JATA030 Resistant Negative ahpC_promoter C-52T –
JATA038 Resistant Negative ahpC_promoter G-48A –
katG K152Q –
JATA039 Resistant Negative ahpC_promoter C-54T –
JATA053 Resistant Negative Other None –
JATA081 Resistant Negative Other None –
JATA095 Resistant Negative katG N138D –
JATA106 Resistant Negative Other None –
JATA110 Resistant Negative Other None –
JATA112 Resistant Negative katG N138H –
R463L –
JATA118 Resistant Negative ahpC_promoter G-74A –
JATA119 Resistant Negative Other None –
JATA078 Resistant Negative katG S315T S315T
JATA101 Resistant Negative fabG1_promoter C-15T C-15T

DST, drug susceptibility test.

4. Discussion that harboured the same mutation were detected successfully. It may
have been be a maldistribution of the target gene or an epistatic effect of
The cobas® 6800/8800 is an automated real-time PCR system, while other mutation(s).
the cobas® MTB-RIF/INH is a newly developed reagent kit for the mo­ The discrepant results for INH between pDST and gDST were
lecular detection of mutations that confer resistance to INH and RIF. To observed in 20% of cases. Two isolates with false-resistant gDST result
our knowledge, this is the first study to evaluate the performance of the harboured C-15T in the fabG1-inhA operon, and this mutation often
cobas® MTB-RIF/INH reagent. This automated high-throughput NAA renders a low MIC value and is susceptible to pDST. It is common in
will be useful to confirm true MDR-TB, not the estimation from RIF Japanese isolates and is one of the limitations of INH gDST [15].
resistance only. However, MGIT AST is well-correlated with gDST, indicating a problem
For RIF resistance, the cobas® MTB-RIF/INH showed comprehensive because of the phenotypic DST method [16]. Other discrepancies and
sensitivity and specificity. Three MTB mutants with false-resistant re­ false-susceptible results in this system were mainly because of mutations
sults on the system contained “disputed mutations” and showed that were out of the target range of cobas® MTB-RIF/INH, except for two
discrepant results between pDST and gDST [10,11]. In 2018, the WHO isolates. One isolate with C-15T in the fabG1-inhA operon may not have
conducted a large meta-analysis on the interpretation of mutations and been detected because of maldistribution of the target gene or an
indels for each anti-tuberculosis drug and released a guide for deter­ epistatic effect of other mutation(s), whereas another isolate harboured
mining the confidence level of gDST. According to the list of rpoB a wild-type sequence in furA-katG, fabG1-inhA, ndh, kasA, and ahpC-­
mutations/indels, although H445 N and L430P are categorised as mu­ promoter. Moreover, INH resistance may have been because of other
tations with minimal confidence, H445D is categorised with high con­ unknown mutations.
fidence [12]. The minimum inhibitory concentration (MIC) of the MTB Japanese MTB isolates showed a low sensitivity to the INH gDST
isolate with H445D was 0.125 μg/mL, which is high even among assays. Chikamatsu et al. reported 65.9% using MTBDRplus (Hain Life­
pan-susceptible MTB isolates (0.06–0.125 μg/mL) [13], and it was cat­ science GmbH, Nehren, Germany) [17], and Igarashi et al. reported
egorised into the indeterminate range. Perdigão et al. categorised 69.1% using Anyplex II MTB/MDR (Seegene, Seoul, Korea) [18]. The
H445D as a lower penetrance allele [14]. Therefore, although it may be cobas® MTB-RIF/INH demonstrated a similar or higher sensitivity than
a rare phenotypic variant, it can be considered resistant MTB. The MTBDRplus, the major gDST assay used for INH and RIF. This may be an
false-susceptible results were obtained mainly because of the non-target advantage of cobas® MTB-RIF/INH.
mutations observed in cobas® MTB-RIF/INH. However, two MTB iso­ This study has several limitations. First, the specimens were spiked
lates harboured H445R and S450W mutations: one isolate contained sputa, which may not have been representative of the actual sputum
both H445R and H445T mutations; therefore, the proportion of H445R specimens of active TB patients. However, spiked specimens are useful
may have been lower and not detected. The reason for the lack of in validation experiments [19]. For a validation study of cobas®
detection of H450W in one isolate is unknown because other isolates MTB-RIF/INH, this was considered appropriate. Second, not all MTB

4
A. Aono et al. Tuberculosis 134 (2022) 102199

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