Accepted Manuscript

Diagnostic performance of Anyplex II MTB/MDR/XDR for

detection of resistance to first and second line drugs in
Mycobacterium tuberculosis

F. Prez-Garca, M.J. Ruiz-Serrano, P. Lpez Roa, Fermn Acosta,

Laura Prez-Lago, D. Garca-De-Viedma, E. Bouza

PII: S0167-7012(17)30116-1
DOI: doi: 10.1016/j.mimet.2017.05.006
Reference: MIMET 5162
To appear in: Journal of Microbiological Methods
Received date: 9 December 2016
Revised date: 11 May 2017
Accepted date: 12 May 2017

Please cite this article as: F. Prez-Garca, M.J. Ruiz-Serrano, P. Lpez Roa, Fermn
Acosta, Laura Prez-Lago, D. Garca-De-Viedma, E. Bouza , Diagnostic performance of
Anyplex II MTB/MDR/XDR for detection of resistance to first and second line drugs in
Mycobacterium tuberculosis, Journal of Microbiological Methods (2017), doi: 10.1016/
j.mimet.2017.05.006

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 ACCEPTED MANUSCRIPT

Diagnostic performance of Anyplex II MTB/MDR/XDR for detection of

resistance to first and second line drugs in Mycobacterium tuberculosis

F. Prez-Garca1,* felipe.perez.garcia.87@gmail.com, M.J. Ruiz-Serrano1, 2,4, P. Lpez

Roa1, Fermn Acosta1,2, Laura Prez-Lago1,2,4, D. Garca-De-Viedma1,2,4, E. Bouza1,2,3,4,

PT
1
Department of Clinical Microbiology and Infectious Diseases, Hospital General
Universitario Gregorio Maran, Madrid, Spain

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2
Instituto de Investigacin Biomdica Gregorio Maran, Madrid, Spain
3

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Department of Medicine, Universidad Complutense de Madrid, Spain
4
CIBER Enfermedades Respiratorias (CB06/06/0058)
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*
Corresponding author at: Servicio de Microbiologa y Enfermedades Infecciosas,
Hospital General Universitario Gregorio Maran, Doctor Esquerdo, 46, 28007 Madrid,
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Spain.
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Abstract
Purpose
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Genotypic methods have considerably improved the diagnosis of

multidrug-resistant (MDR) tuberculosis. One of these tests is Anyplex II
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MTB/MDR/XDR (Anyplex). Our aim was to evaluate the diagnostic

performance of this multiplex PCR
Methods
We conducted our study on 47 MDR tuberculosis and 14 pan-susceptible
strains. We evaluated the ability of Anyplex to detect resistance mutations
in rpoB (rifampin [RIF]), katG and inhA (isoniazid [INH]), gyrA
(fluoroquinolones [FLQ]), and rrs and eis (aminoglycosides [AMG]). We

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used the agar proportion method as gold standard. We also studied

concordance with GenoType MTBDRplus (first line drugs) and MTBDRsl
(second line drugs). DNA sequencing was applied to clarify discrepancies.
Results
All pan-susceptible strains were susceptible by Anyplex. Sensitivity and
specificity of Anyplex for detection of resistance mutations were 97.9%
and 100%, respectively, for RIF, 91.5% and 100% for INH, 80% and 100%

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for FLQ, and 50% and 99.7% for AMG. Concordance with GenoType was

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perfect for RIF, INH, and FLQ (kappa score, k=1.0) and moderate for
AMG (k=0.48). Sensitivity and specificity for detection of MDR

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tuberculosis were 89.4% and 100%, respectively. DNA sequencing of the
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phenotypically resistant strains considered as susceptible by Anyplex,
confirmed no mutations in the corresponding genes.
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Conclusions
Anyplex is a reliable assay for the detection of MDR tuberculosis and
shows excellent concordance with GenoType. Anyplex reduces the time to
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diagnosis of MDR tuberculosis strains, as it is recommended by current

guidelines on control of tuberculosis.
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Keywords: Mycobacterium tuberculosis; molecular diagnostic methods; multiplex

PCR; resistance mutations; Anyplex II MTB/MDR/XDR
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Introduction
Tuberculosis (TB) is a leading cause of morbidity and mortality worldwide.
According to the World Health Organization (WHO) 2015 global
tuberculosis report, there were almost 10 million new TB patients and 1.5
million deaths from the disease in 2014 [1]. MDR-TB is defined as
resistance to at least the two most effective TB drugs, isoniazid and

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rifampin. It is estimated that about 3.3% of newly diagnosed patients and

20% of previously treated patients had MDR-TB in 2014 [1].
Early diagnosis of TB including detection of resistance is crucial for
the control of the disease and patient management. Culture remains the
gold standard for diagnosis. However, it is slow, as it can take several
weeks to obtain susceptibility results [2, 3].
In recent years, rapid molecular methods for detection of mutations

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conferring drug resistance have been developed to replace the conventional

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drug-susceptibility testing (DST) based on phenotypic techniques [4-7].
The most widely used commercial tests are GenXpert MTB/RIF, (Cepheid,

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Sunnyvale, CA, USA) and GenoType (Hain-LifeScience, Nehren,
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Germany).
Anyplex II MTB/MDR/XDR (Seegene, Korea, Seoul) (Anyplex) is a
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recently marketed multiplex PCR that enables the detection of resistance to

first and second line drugs in the same assay.
The aim of our study was to evaluate the diagnostic performance of
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Anyplex to detect mutations that confer resistance to first- and second-line

drugs in previously characterized MDR-TB strains. We also compared the
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results obtained using the new technique with those obtained using
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GenoType.
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Materials and methods

Settings and study design
We conducted the study in a large general teaching hospital with 1,550
beds and a catchment population of approximately 715,000 inhabitants in
Madrid, Spain.
We analyzed 47 strains of M. tuberculosis previously characterized as

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MDR by drug-susceptibility testing in our laboratory between 1992 and

2015. Twenty-six of these 47 strains had already been tested using
GenoType MTBDRsl, as we reported in a previous study [8].
We also included 13 strains of pan-susceptible M. tuberculosis by
phenotypic methods as external negative controls. These strains were
randomly selected from pan-susceptible strains that were isolated in 2016.
Genotypic methods

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Anyplex: We applied Anyplex (Seegene, Korea, Seoul) following the

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manufacturers instructions. This multiplex PCR takes 1 hour for DNA
extraction and 3 hours for DNA amplification; the results are automatically

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interpreted by its software and can be exported to external computers. The
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technique is based on dual priming oligonucleotide and tagging
oligonucleotide cleavage and extension technologies. Anyplex requires 5
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quality controls: 1) a negative control; 2) an internal control of

amplification; 3) a control of the presence of M. tuberculosis DNA; 4) a
wild type of sensitive M. tuberculosis control; and 5) a positive control,
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which is an extremely resistant (XDR) strain that harbors all of the

mutations covered by the assay. Anyplex enables simultaneous
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amplification of 18 RIF resistance mutations, 7 INH resistance mutations, 7

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fluoroquinolone (FLQ) resistance mutations, and 6 aminoglycoside (AMG)

resistance mutations. The PCR reaction occurs in 2 independent tubes: in
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the first, PCR (MTB/MDR) detects the presence of M. tuberculosis DNA

and resistance to INH and RIF; in the second (MTB/XDR) detects the
presence of M. tuberculosis DNA and resistance to FLQ and AMG.
GenoType: GenoType (Hain-LifeScience, Nehren, Germany) was applied
according to the manufacturers instructions [9, 10]. GenoType MTBDplus
v 2.0 and GenoType MTBDRsl v 1.0 were used for detection of resistance
to RIF and INH and resistance to FLQ and AMG, respectively. The
resistance mutations covered by these methods are summarized in Table 1.

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Since GenoType MTBDRplus could only be performed in 17 of the 47

strains, the sensitivity and specificity of the assay could not be established.
DNA Sequencing
Sanger DNA sequencing was performed for katG, inhA promoter, rpoB,
gyrA, rrs and eis promoter by using the primers indicated in Table 2.
Thermal conditions for the PCR were: 95C for 15 min, 30 cycles of
denaturation at 95C for 1 min, annealing at 58C for 1 min, and elongation

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at 72C for 1.5 min, followed by a final extension step at 72C for 10 min.

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The PCR products were sequenced on an ABI 3130x1 DNA analyzer
according to the protocol supplied by the manufacturer. Sequencing data

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were analyzed by the software Finch TV Version 1.4.0.
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Phenotypic methods
Agar proportion method: The agar proportion method was performed as
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recommended by the Clinical Laboratory Standards Institute (CLSI) [11]

and was established as the gold standard for susceptibility testing results.
We used the breakpoints for susceptibility and resistance that are
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recommended by CLSI for each antibiotic.

Quality control and strain identification
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H37Rv M. tuberculosis was used as a control strain in both the genotypic

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and the phenotypic tests. M. tuberculosis strains were identified using

immunochromatography (BD MGIT TBc, Becton Dickinson, Franklin
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Lanes, NJ, USA) or by probe hybridization (Accuprobe Gen-Probe MTBC,

Biomerieux, Marcy LEtoile, France).
Technical and performance characteristics
We evaluated the technical requirements and performance characteristics of
Anyplex compared with GenoType. We studied the following: 1) presence
of cross-contamination; 2) quality controls; 3) amplified targets; 4) time of
manipulation; 5) time to results; 6) interpretation and exportability of
results; 7) maximum no. of samples per assay; 8) cost per assay.

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Statistical analysis
We assessed the sensitivity, specificity, major errors (ME), very major
errors (VME), and categorical agreement (CA) of Anyplex using the agar
proportion method as the gold standard. MEs were defined as false-
resistant results and VMEs as false-susceptible results. The CA for each
antibiotic was calculated by comparing the results of Anyplex with those of
GenoType MTBDRplus and MTBDRsl. Therefore, we studied the

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correlation between Anyplex and GenoType using the kappa score. Finally,

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we recorded the time to obtain the results of Anyplex from the moment the
MGIT culture was grown, as compared with the time to results by the agar

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proportion method. A p-value less than or equal to 0.05 was considered
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statistically significant. The statistical analysis was performed using
Stata/IC 13.1 (StataCorp, Texas, USA).
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Table 1. Resistance mutations covered by the genotypic assays

Assay Drug Resistance mutations

Anyplex II MTB/MDR/XDR RIF rpoB (L511P, Q513K, Q513L, Q513P, 3 amino acid-deletion
in 513-516, D516V, D516Y, S522L, S522Q, H526C, H526D,
H526L, H526N, H526R, H526Y, S531L, S531W, L533P)

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INH katG (S315, S315N, S315T, S315T], inhA promoter [-15[T],

-8[A], -8[C])
FLQ gyrA [A90V, S91P, D94A, D94G, D94H, D94N, D94Y]
AMG rrs (A1401G, 1402[T], G1484T), eis promoter (-37[T], -
14[T], -10[A])
GenoType MTBDRplus v 2.0 RIF rpoB (D516V, H526Y, H526D, S531L)
INH katG (S315T1, S315T2), inhA (C15T, A16G, T8C, T8A)

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GenoType MTBDRsl v 1.0 FLQ gyrA (A90V, S91P, D94A, D94G, D94H, D94N, D94Y)
AMG rrs (A1401G, G1484T)

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RIF: rifampin; INH: isoniazid; FLQ: fluoroquinolones; AMG: aminoglycosides

Table 2. PCR primers for DNA sequencing

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Locus Name Primer sequence (5 to 3) Product size (bp) Ref.
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Tb86 GAAACAGCGGCGCTGATCGT
katG 247 [12]
KatGR GCTCCCACTCGTAGCCGTACA
TB92 CCTCGCTGCCCAGAAAGGGA
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inhA promoter 248 [13]

TB93 ATCCCCCGGTTTCCTCCG
TR9 TCGCCGCGATCAAGGAGT
157
rpoB [14]
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TR-8 TGCACGTCGCGGACCTCCA
gyrAF GTGCTCTATGCAATGTTCGATT
gyrA 263 This study
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gyrAR AGCATCTCCATCGCCAAC
rrsf GATCGGGGTCTGCAACTC -3
rrs 240 This study
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rrsr GAAAGGAGGTGATCCAGCC
eisF TTCGTCGCTGATTCTCGCA
eis promoter 224 This study
eisR GCTGATTCAGGGCCGATGA
Results
Application of the agar proportion method showed that all of the 47 strains
were resistant to RIF and INH; therefore, they were all defined as MDR
strains. Five strains were also resistant to FLQ, and a further 4 strains were
resistant to AMG. Since none of the 47 MDR strains was resistant to both

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FLQ and AMG, we did not find any extremely drug-resistant (XDR)
strains.
The agar proportion method showed that the remaining 13 M. tuberculosis
strains and also the H37Rv strain were pan-susceptible, so they could be
used as external quality controls. Anyplex did not find resistance mutations
in these strains.
Diagnostic performance data are summarized in Table 3. The sensitivity

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and specificity of Anyplex for detection of resistance mutations were

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97.9% and 100% for RIF, 91.5% and 100% for INH, 80% and 100% for
FLQ, and 50% and 99.7% for AMG. There were 8 VMEs: 1 corresponding

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to RIF, 4 to INH, 1 to FLQ, and 2 to AMG. There was only 1 ME for
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AMG, namely, a single strain that had a mutation in the eis promoter but
was not associated with AMG resistance. GenoType MTBDRsl showed
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equal sensitivity and specificity for FLQ and a sensitivity of 25% and
specificity of 100% for AMG. Concordance of Anyplex with the
GenoType for RIF, INH, and FLQ was 100% (kappa=1.0, perfect
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correlation). Concordance for AMG was 95.1% (kappa=0.48, moderate

correlation). Furthermore, compared with the agar proportion method, the
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accuracy of Anyplex for diagnosis of an MDR strain (defined as the

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simultaneous presence of resistance mutations for RIF and INH) was

89.4% (sensitivity) and 100% (specificity).
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Of the 5 FLQ-resistant strains that were diagnosed by the agar proportion

method, only 4 were detected using genotypic tests (both Anyplex and
GenoType). Of the 4 AMG-resistant strains, Anyplex detected 2 and
GenoType detected only 1. AMG-resistant strains that were detected by
genotypic tests presented mutations in the rrs gene.
DNA sequencing was performed for the eight MDR isolates with
discrepancies between Anyplex and the phenotypic methods. No mutations
were detected for katG, inhA promoter, rpoB, rrs and eis promoter. A SNP

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was identified in gyrA (S95T, [AGC to ACC]) which has been described as
not associated with fluoroquinolone resistance [15, 16].

Table 3. Diagnostic performance of Anyplex II MTB/MDR/XDR and

GenoType

GenoType

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No of Anyplex II MTB/MDR/XDR
MTBDRsl CA Kappa
resistant
Drug Sn Sp VME ME Sn Sp (%) score

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isolates a
(%) (%) (n) (n) (%) (%)

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RIF 47/61 97.9 100 1 0 NA NA 100b 1.00
INH 47/61 91.5 100 4 0 NA NA 100b 1.00
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FLQ 5/61 80 100 1 0 80 100 100 1.00
AMG 4/61 50 99.7 2 1 25 100 95.1 0.48
MDR-TB 47/61 89.4 100 5 0 NA NA NA NA
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RIF: rifampin; INH: isoniazid; FLQ: fluoroquinolones; AMG: aminoglycosides; MDR-

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TB: multidrug-resistant tuberculosis; Sn: sensitivity; Sp: specificity; VME: very major
errors; ME: major errors; NA: not applicable; CA: categorical agreement of Anyplex II
MTB/MDR/XDR with GenoType.
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a
According to the agar proportion method (gold standard).
b
Categorical agreement of Anyplex II MTB/MDR/XDR with GenoType MTBDRplus
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is based on only 17 of the 47 tested strains.

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The median time to obtain results was 1 day (interquartile range: IQR, 1-2
days) for Anyplex, compared with 28 days (IQR, 22-34 days) by the agar
proportion method. This difference was statistically significant (p<0.0001).
The results of the comparison of technical and performance characteristics
between the genotypic methods are summarized in Table 4. Anyplex
performed better than GenoType in all the evaluated parameters. It required
only 1 PCR reaction to cover first and second line resistance mutations. In

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order to detect those resistance mutations using GenoType, 2 different

amplification reactions were needed. GenoType was more laborious than
Anyplex. We experienced some cross-contamination events with
GenoType that did not occur with Anyplex. Thus, Anyplex has a higher
number of quality controls and enables automatic interpretation of results,
which are directly exportable to laboratory information systems. GenoType
results are usually interpreted and exported manually through the reading of

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the bands of hybridization. However, they could also be interpreted through

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an electronic device called GenoScan (Hain-LifeScience, Nehren,
Germany). This device is sold separately from both GenoType kits

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(MTBDRplus and MTBDRsl). NU
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Table 4. Comparison of technical and performance characteristics of

genotypic methods

GenoType MTBDRplus
Evaluated parameter Anyplex II MTB/MDR/XDR
and MTBDRsl

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Cross-contaminations Yes
1. Hybridization control
2. Amplification control
Quality controls
3. M. tuberculosis DNA control
4. Negative control
Amplified targets Sensitive and resistant
No
1. Amplification control
2. M. tuberculosis DNA control
3. Negative control
4. WT control (sensitivity control)
5. XDR control (positive control)
Resistant

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Time of manipulation 1 hour 30 minutes
Time to results 7-8 hours 4.5 hours

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Interpretation of results Manual* Automatic
Exportation of results Manual* Automatic exportation to LIS

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No. of samples per assay
Up to 19 Up to 45
(maximum)
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100 (45 for MTBDRplus 30 (together to evaluate first and second
Cost per determinationa
and 55 for MTBDRsl) line drugs)
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LIS: laboratory information system; WT: wild type; XDR: extremely drug-resistant.
a
Established cost at our hospital.
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*Interpretation and exportation of results can be done automatically through an

electronic device called GenoScan (Hain, LifeScience, Nehren, Germany)
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Discussion
Early detection of resistance is one of the key approaches to prevent the
emergence of MDR-TB [1, 17, 18]. Although drug susceptibility testing
remains the gold standard for assessing resistance to antituberculosis drugs,

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these techniques often take several weeks to yield results. Molecular

techniques offer considerable advantages and may be superior to current
reference standards. A number of studies have demonstrated the excellent
performance of the GenXpert MTB/RIF assay for detection of RIF
resistance [5, 7]. Sufficient data have also been published on GenoType
MTBDRplus [6, 9] and GenoType MTBDRsl [8, 10].
To the best of our knowledge, only 2 studies have assessed the diagnostic

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performance of Anyplex [19, 20]. Causse et al. [19] evaluated the assay on

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susceptible and resistant M. tuberculosis strains and compared the results
with those obtained using GenoType MTBDRplus and GenoType

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MTBDRsl. They reported excellent agreement between the tests and better
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sensitivity and specificity results for second line drugs (95% and 99%,
respectively, for FLQ and 100% and 100%, respectively, for AMG), but
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poorer results for INH (61% and 98%, respectively). Molina-Moya et al.
[20] performed a study on sensitive and resistant M. tuberculosis strains
and also on respiratory samples. Their results were very similar to those of
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Causse et al. in terms of sensitivity and specificity for first and second line
drugs. It should be noted that the results of Causse et al. were particularly
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good in direct samples.

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We found that Anyplex was very able to diagnose MDR-TB. We obtained

higher sensitivity and specificity for RIF (97.9% and 100%, respectively)
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and also INH (91.4% and 100%, respectively). Anyplex showed a perfect
correlation with GenoType for RIF, INH, and FLQ. For AMG, Anyplex
showed better results that GenoType. Anyplex also performed better than
GenoType, leading to a faster, easier, and more inexpensive diagnosis.
However, the fact that GenoType amplifies resistant and sensitive regions
is an advantage over Anyplex in cases of co-infection by resistant and
sensitive strains, which, although very uncommon, has been reported [21].
Our study is subject to a series of limitations. We could only provide data

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for 17 GenoType MTBDRplus tests. However, the high sensitivity and

specificity of GenoType MTBDRplus has been widely reported in the
literature [9, 22]. Moreover, the perfect correlation between GenoType
MTBDRplus and Anyplex for first line drugs in our study was consistent
with previously published data. Furthermore, we have a limited number of
MDR strains that were also resistant to second line drugs and no XDR
strains. Thus, our results for second line drugs have to be interpreted with

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caution. Discrepancies between some of the resistant strains and the

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genotypic data were clarified not to be limitations of the tests, because in
all cases mutations in the targeted regions were ruled out by DNA

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sequencing. Therefore, the discrepancies were due to the obvious
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limitations of any genotypic test which do not include all the potential
regions where resistance mutations could map [23, 24]. Finally, ours was a
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retrospective study that was performed in a single center. Prospective

multicenter studies are needed to establish the clinical impact of Anyplex,
as has been established for GenoType and GenXpert MTB/RIF.
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Although the present study was performed in culture isolates, the test can
also be performed in respiratory samples with similar diagnostic capacity
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[20]. We were able to perform the test over 3 sputum samples from patients
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admitted to our hospital with suspicion of MDR-TB. When a highly

positive baciloscopy result (over 50 acid-fast bacilli/field) was confirmed,
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we performed Anyplex directly on the sputum samples. Two MDR strains

(with RIF and INH mutations) and a sensitive strain were diagnosed using
Anyplex II MTB/MDR/XDR and confirmed in all cases by phenotypic
methods. Although the data reported here are limited, they show that
Anyplex II MTB/MDR/XDR can be performed directly on respiratory
samples with a high bacillary load.
In conclusion, Anyplex is a reliable assay for detection of MDR-TB and
has an excellent correlation with GenoType and better performance

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characteristics. Anyplex can reduce the time to diagnosis of MDR-TB to

only 1 day. Although phenotypic tests remain the gold standard for
detection of resistance of M. tuberculosis to drugs, genotypic methods such
as Anyplex offer particular advantages, as they lead to a faster diagnosis of
MDR-TB, which is recommended by all current guidelines.

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Acknowledgements

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We thank Thomas OBoyle for his help with the preparation of the
manuscript.

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Compliance with Ethical Standards
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Funding
F. Acosta is supported by Programa de Becas IFARHU-SENACYT de
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Panam (270-2016-293).
L. PrezLago was supported by M. Servet CP15/00075.
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Conflict of interest
The authors declare that they have no conflicts of interest.
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Ethical approval
This study was evaluated and approved by the local ethics committee
(Comit tico de Investigacin Clnica Hospital General Universitario
Gregorio Maran rea 1).

Informed consent
Since the present study is retrospective, informed consent was not required.

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Highlights
Anyplex II MTB/MDR/XDR (Anyplex) is a reliable assay for
detection of MDR-TB.
Anyplex has excellent correlation results with GenoType
MTBDRplus and MTBDRsl.
Anyplex has better performance characteristics than GenoType
MTBDRplus and MTBDRsl.

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Anyplex can reduce the time to diagnosis of MDR-TB to only 1 day.

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