GenoType MTBDRplus

VER 2.0

Instructions for Use

IFU-304A-06



for in vitro diagnostic use only

06/2015
GenoType MTBDRplus VER 2.0
Molecular Genetic Assay for Identification of the M. tuberculosis Complex and its Resistance to
Rifampicin and Isoniazid from Clinical Specimens and Cultivated Samples

Please read the instructions on hand completely and carefully before using the kit. Strictly adhere to the established procedure to obtain correct test
results.

Intended Use
The GenoType MTBDRplus VER 2.0 is a qualitative in vitro test for the identification of the Mycobacterium tuberculosis complex and its resistance to
rifampicin (RMP) and/or isoniazid (INH) from pulmonary smear-positive or -negative clinical specimens and cultivated samples. The following species are
included in the tuberculosis (TB)-causing M. tuberculosis complex: M. tuberculosis, M. africanum, M. bovis subsp. bovis, M. bovis subsp. caprae, M. bovis BCG,
M. microti, M. canettii, and M. pinnipedii. The identification of RMP resistance is enabled by the detection of the most significant associated mutations of the
rpoB gene (coding for the -subunit of the RNA polymerase). For detection of INH resistance, the katG gene (coding for the catalase peroxidase) and the
promoter region of the inhA gene (coding for the NADH enoyl ACP reductase) are examined.
The test is indicated as an aid for diagnosis and intended for use in medical laboratories.

Summary and Explanation

Tuberculosis (TB) is a bacterial infectious disease passed on by droplet infection. In 2013, there were an estimated 9.0 million incident cases of TB globally,
and an estimated 1.5 million deaths occurred [1]. TB treatment requires a therapy over several months. Emergence and spread of multidrug-resistant
tuberculosis (MDR-TB) is a major medical and public problem threatening global health. MDR-TB is defined as TB that is resistant at least to RMP and INH,
the two most important first-line anti-TB drugs [2]. MDR-TB is a challenge to TB control due to its complex diagnosis and obstacles in treatment. In 2013,
there were an estimated 480,000 cases of MDR-TB among the world’s 11 million prevalent cases of TB [1].
As long as MDR-TB is not verified, use of inadequate and hence ineffective antibiotics may lead to further spread of resistant bacteria and amplification of
resistance. Therefore, rapid diagnosis and identification of MDR-TB is a prerequisite for appropriate treatment.

Principles of the Procedure

The GenoType MTBDRplus test is based on the DNA•STRIP technology. The whole procedure is divided into three steps: (i) DNA extraction from clinical
specimens (pulmonary, decontaminated) or cultured material (solid/liquid medium) – the necessary reagents are not included in the kit, (ii) a multiplex
amplification with biotinylated primers, and (iii) a reverse hybridization.
All reagents needed for amplification such as polymerase and primers are included in the Amplification Mixes A and B (AM-A and AM-B) and are optimized
for this test. The membrane strips are coated with specific probes complementary to the amplified nucleic acids. After chemical denaturation, the single-
stranded amplicons bind to the probes (hybridization). Highly specific binding of complementary DNA strands is ensured by stringent conditions which
result from the combination of buffer composition and a certain temperature. Thus the probes reliably discriminate several sequence variations in the gene
regions examined. The streptavidin-conjugated alkaline phosphatase binds to the amplicons’ biotin via the streptavidin moiety. Finally, the alkaline
phosphatase transforms an added substrate into a dye which becomes visible on the membrane strips as a colored precipitate. A template ensures the
easy and fast interpretation of the banding pattern obtained.

Storage and Disposal of Kit Constituents

Kit Component 1 of 2

Kit Component 2 of 2

Store all constituents from Kit Component 1 at 2-8°C. Store all constituents from Kit Component 2 at –20°C and keep strictly separated from contaminating
DNA. Avoid repeated freezing and thawing of AM-A and AM-B; when processing only small sample numbers per run, aliquot AM-A and AM-B. Do not use
the reagents beyond their expiry date. Dispose of unused reagents and waste in accordance with federal, state, and local regulations.

Precautions for Handling Kit Constituents

Observe all federal, state, and local safety and environmental regulations. Always wear suitable protective clothing and gloves.
When handling kit reagents, the following special safety measures must be applied:

Hybridization Buffer (HYB) and Substrate Concentrate (SUB-C) are not classified as hazardous. Due to their ingredients, however, hazard statement
EUH210 applies: Safety data sheet available on request.

Denaturation Solution (DEN) contains <2% sodium hydroxide.

Warning!
H315: Causes skin irritation. H319: Causes serious eye irritation.
P280: Wear protective gloves/protective clothing/eye protection. P305+351+338: IF IN EYES: Rinse cautiously with water for several minutes.
Remove contact lenses, if present and easy to do. Continue rinsing. P313: Get medical advice/attention.

For additional information, please refer to the safety data sheets which can be downloaded from: www.hain-lifescience.com/products/msds.html

Quality Control
In order to control the correct performance of the test and the proper functioning of kit constituents, each strip includes 5 control zones:
– a Conjugate Control zone (CC) to check the binding of the conjugate on the strip and a correct chromogenic reaction
– an Amplification Control zone (AC) to check for a successful amplification reaction
– three Locus Control zones (rpoB, katG, and inhA) checking the optimal sensitivity of the reaction for each of the tested gene loci

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Observe the usual precautions for amplification setup. It is essential that all materials (such as pipette tips) coming in contact with the reagents are free
from DNases.
Do not interchange or pool Amplification Mixes or membrane strips from different kits unless the lots are identical.
A negative control sample for detection of possible contamination events containing water (molecular biology grade) instead of DNA should be part of each
test run; the respective test strip should show the bands CC and AC only.

Specimen Requirements
Decontaminated pulmonary smear-positive or -negative patient specimens such as sputum (induction or expectoration), bronchial material (e.g.
bronchoalveolar lavages), or aspirates (e.g. pleural aspirate) as well as cultivated samples (solid/liquid medium) can be used as starting material for DNA
extraction. Until the present edition of the instructions on hand, the performance of the test has not been validated with other sample materials than those
mentioned above.

Precautions for handling specimens

Patient specimens and cultures made from patient specimens must always be considered as potentially infectious and must be handled accordingly (e.g.
see [3] or [4]). Always wear suitable protective clothing and gloves. Samples from patients at risk (infected by pathogenic microorganisms including
Hepatitis B and Human Immunodeficiency Virus (HIV)) and cultures made from those samples must always be labeled and handled under suitable safety
conditions according to institutional guidelines.
Handling of potentially infectious specimens must be carried out in a class II safety cabinet. Potentially infectious samples must be centrifuged in a class II
safety cabinet or in an aerosol-tight rotor. Open aerosol-tight rotor in safety cabinet only. For inactivated samples, a standard rotor can be used for
centrifugation outside the safety cabinet.
Discard used pipette tips immediately after use in a container for biohazardous waste. After finishing the assay, discard all used disposables in a container
for biohazardous waste.

Storage and transport

All specimens should be collected and transported as recommended in the CDC publication “Public Health Mycobacteriology: A Guide for the Level III
Laboratory” [5], the “Clinical Microbiology Procedures Handbook” [6], or your laboratory procedure manual.
It must be ensured that until decontamination takes place, specimens are kept in sterile plastic containers at a temperature of 2-8°C. The transport of
specimens at room temperature has to be carried out as soon as possible and should be done within 1-2 days [7,8]. The specimens used for
decontamination must not be older than 4 days.
After decontamination and subsequent resuspension of the bacteria pellet with phosphate buffer, samples can be stored at –20°C or –80°C for a maximum
of 5 days until performing DNA extraction.

Preparation
Clinical specimens must be processed using the NALC/NaOH method according to the CDC publication “Public Health Mycobacteriology: A Guide for the
Level III Laboratory” [5]. After decontamination, the cell pellet should be resuspended in a maximum of 1 to 1.5 ml of phosphate buffer. When testing
patient specimens, higher volumes might hamper the sensitivity of the test. Due to the potential inhomogeneity of the specimen, the decontaminated
sample must be mixed before removing the aliquot to be analyzed; otherwise the sensitivity of the test might be influenced.
When the sample is to be cultivated, cultivation can be performed either on solid medium (e.g. Loewenstein-Jensen, Middlebrook) or in liquid medium (e.g.
MGIT (BD Diagnostics, Franklin Lakes, USA)).
Handling of potentially infectious specimens must be carried out in a class II safety cabinet.

DNA Extraction
Decontaminated patient samples as well as bacteria grown on solid medium (e.g. Loewenstein-Jensen, Middlebrook) or in liquid medium (e.g. MGIT (BD
Diagnostics, Franklin Lakes, USA)) may be used as starting material for DNA extraction. The working area must be free from contaminating DNA.
For DNA extraction from decontaminated clinical specimens or cultured material the GenoLyse® kit (see chapter Ordering Information) is used according
to protocol A. For automated DNA extraction from patient specimens, also the GenoXtract® instrument in combination with the GXT DNA/RNA Extraction
Kit (see chapter Ordering Information) can be used. For handling instructions, please refer to the respective instructions for use.

The methods described above were used for performance evaluation of the GenoType MTBDRplus test. Until the present edition of the instructions on hand,
the performance of the test has not been validated with other DNA extraction methods or sample materials.

Amplification
All reagents needed for amplification such as polymerase and primers are included in the Amplification Mixes A and B (AM-A and AM-B) and are optimized
for this test. After thawing, spin down AM-A and AM-B briefly and mix carefully by pipetting up and down. Pipette AM-A and AM-B only in a room free from
contaminating DNA. The DNA solution should be added in a separate working area.

Prepare for each sample:

After DNA extraction with GenoLyse® After DNA extraction with GXT DNA/RNA Extraction Kit
– 10 μl AM-A (see Kit Component 2) – 10 μl AM-A (see Kit Component 2)
– 35 μl AM-B (see Kit Component 2) – 35 μl AM-B (see Kit Component 2)
– 5 μl DNA solution – 10 μl DNA solution
Final volume: 50 μl Final volume: 55 μl

Determine the number of samples (number of samples to be analyzed plus control samples). Prepare the number of tubes needed. Prepare a master mix
containing AM-A and AM-B and mix carefully but thoroughly (do not vortex). Alternatively, the content of an AM-A reaction tube may completely be
transferred to an AM-B reaction tube. This will lead to 0.68 ml master mix for 12 amplification reactions (12 tests kit) or, respectively, 4x 1.35 ml for 4x 24
amplification reactions (96 tests kit). Please note that the master mix needs to be prepared freshly each time. Aliquot 45 μl into each of the prepared PCR
tubes and add 5 or 10 μl water (see above) to one aliquot (negative control). In a separate working area, add 5 or 10 μl DNA solution to each aliquot (except
for negative control).

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Amplification profile:
When using a thermal cycler from Hain Lifescience with the respective preinstallation, select protocol “MDR DIR” for clinical specimens or protocol “MDR
CUL” for cultivated samples.

Clinical specimens Cultivated samples

15 min 95°C 1 cycle 1 cycle

30 sec 95°C
2 min 65°C 20 cycles 10 cycles

25 sec 95°C
40 sec 50°C 30 cycles 20 cycles
40 sec 70°C

8 min 70°C 1 cycle 1 cycle

Heating rate ≤2.2°C/sec ≤2.2°C/sec

Amplification products can be stored at +8 to –20°C.

Hybridization
When using a hybridization instrument from Hain Lifescience, please refer to the document “Overview equipment programs” available on
www.hain-lifescience.com for the name of the hybridization protocol to be used.
The following protocol describes the manual hybridization using a water bath or a TwinCubator.

Preparation
Prewarm shaking water bath to 45°C (the maximum tolerated deviation from the target temperature is +/–1°C) or switch on TwinCubator. Prewarm
solutions HYB and STR to 37-45°C before use. The reagents must be free from precipitates (note, however, that solution CON-D is opaque). Mix if
necessary. Warm the remaining reagents with the exception of CON-C and SUB-C to room temperature. Using a suitable tube, dilute Conjugate
Concentrate (CON-C, orange) and Substrate Concentrate (SUB-C, yellow) 1:100 with the respective buffer (CON-C with CON-D, SUB-C with SUB-D) in the
amounts needed. Mix well and bring to room temperature. For each strip, add 10 μl concentrate to 1 ml of the respective buffer. Dilute CON-C before each
use. Diluted SUB-C is stable for 4 weeks if stored at room temperature and protected from light.

1. Dispense 20 μl of Denaturation Solution (DEN, blue) in a corner of each of the wells used.
2. Add to the solution 20 μl of amplified sample, pipette up and down to mix well and incubate at room temperature for 5 minutes.
Meanwhile, take strips out of the tube using tweezers and mark them with a pencil underneath the colored marker. Always wear gloves when handling
strips.
3. Carefully add to each well 1 ml of prewarmed Hybridization Buffer (HYB, green). Gently shake the tray until the solution has a homogenous color.
Take care not to spill solution into the neighboring wells.
4. Place a strip in each well.
The strips must be completely covered by the solution and the coated side (identifiable by the colored marker near the lower end) must face upward.
Using tweezers, turn over strips which might have turned when immersed in the solution. Carefully clean tweezers after each use to avoid
contamination. This also applies to all following steps.
5. Place tray in shaking water bath/TwinCubator and incubate for 30 minutes at 45°C.
Adjust the shaking frequency of the water bath to achieve a constant and thorough mixing of the solution. To allow adequate heat transfer, the tray must
be dipped into the water to at least 1/3 of its height.
6. Completely aspirate Hybridization Buffer.
For example, use a Pasteur pipette connected to a vacuum pump.
7. Add 1 ml of Stringent Wash Solution (STR, red) to each strip and incubate for 15 minutes at 45°C in shaking water bath/TwinCubator.
8. Work at room temperature from this step forward.
Completely remove Stringent Wash Solution.
Pour out Wash Solution in a waste container and remove all remaining fluid by turning the tray upside down and gently striking it on an absorbent paper.
This also applies to all other wash steps.
9. Wash each strip once with 1 ml of Rinse Solution (RIN) for 1 minute on shaking platform/TwinCubator (pour out RIN after incubation).
10. Add 1 ml of diluted Conjugate (see above) to each strip and incubate for 30 minutes on shaking platform/TwinCubator.
11. Remove solution and wash each strip twice for 1 minute with 1 ml of Rinse Solution (RIN) and once for 1 minute with approx. 1 ml of distilled water
(e.g. use wash bottle) on shaking platform/TwinCubator (pour out solution each time).
Make sure to remove any trace of water after the last wash.
12. Add 1 ml of diluted substrate (see above) to each strip and incubate protected from light without shaking.
Depending on the test conditions (e.g. room temperature), the substrate incubation time, i.e. the time until the bands are clearly visible, can vary
between 3 and 20 minutes. Extended substrate incubation times can lead to increased background staining and might impair interpretation of the
results.
13. Stop reaction as soon as bands are clearly visible by briefly rinsing twice with distilled water.
14. Using tweezers, remove strips from the tray and dry them between two layers of absorbent paper.

4
Evaluation and Interpretation of Results
Paste strips and store protected from light. An evaluation sheet is included in the kit. When using this evaluation sheet, paste the developed strips in the
designated fields by aligning the bands CC and AC with the respective lines on the sheet. For technical reasons the distances between single probes on the
strips may vary slightly. For an accurate evaluation therefore please use the provided template and align it – separately for each locus – with the
respective Locus Control band. Determine the resistance status and note down in the respective column. As a help for interpretation, evaluation examples
are given in the subsequent chapter. Each strip has a total of 27 reaction zones (see figure).

Conjugate Control (CC)

Amplification Control (AC)
M. tuberculosis complex (TUB)
rpoB Locus Control (rpoB)
rpoB wild type probe 1 (rpoB WT1)
rpoB wild type probe 2 (rpoB WT2)
rpoB wild type probe 3 (rpoB WT3)
rpoB wild type probe 4 (rpoB WT4)
rpoB wild type probe 5 (rpoB WT5)
rpoB wild type probe 6 (rpoB WT6)
rpoB wild type probe 7 (rpoB WT7)
rpoB wild type probe 8 (rpoB WT8)
rpoB mutation probe 1 (rpoB MUT1)
rpoB mutation probe 2A (rpoB MUT2A)
rpoB mutation probe 2B (rpoB MUT2B)
rpoB mutation probe 3 (rpoB MUT3)
katG Locus Control (katG)
katG wild type probe (katG WT)
katG mutation probe 1 (katG MUT1)
katG mutation probe 2 (katG MUT2)
inhA Locus Control (inhA)
inhA wild type probe 1 (inhA WT1)
inhA wild type probe 2 (inhA WT2)
inhA mutation probe 1 (inhA MUT1)
inhA mutation probe 2 (inhA MUT2)
inhA mutation probe 3A (inhA MUT3A)
inhA mutation probe 3B (inhA MUT3B)
colored marker

Note: The strip is not displayed in original size.

Conjugate Control (CC)

A line must develop in this zone, documenting the efficiency of conjugate binding and substrate reaction.

Amplification Control (AC)

When the test is performed correctly, a control amplicon will bind to the Amplification Control zone.
In case of a positive test result, the signal of the Amplification Control zone can be weak or even vanish totally. This might be due to competition of the
single reactions during amplification. In this case, the test was performed correctly and does not have to be repeated.
When only the CC and AC bands are developed, this represents a valid negative result. A missing AC band in case of a negative test result indicates
mistakes during setup and/or performance of the amplification reaction, or presence of amplification inhibitors. In this case, the test result is not valid and
the test has to be repeated with the respective sample.

M. tuberculosis complex (TUB)

This zone hybridizes, as far as is known, with amplicons generated from all members of the Mycobacterium tuberculosis complex. If the TUB zone is
negative while no evaluable resistance pattern is developed, the tested specimen does not contain bacteria belonging to the M. tuberculosis complex and
cannot be evaluated by this test system. In rare cases, the TUB zone may be negative while an evaluable resistance pattern is developed. If so, the presence
of a strain belonging to the M. tuberculosis complex must be suspected and the test should be repeated (see below, “special case” no. 3).

Locus Controls (rpoB, katG, and inhA)

The Locus Control zones detect a gene region specific for the respective locus. In case of a positive test result (evaluable wild type and mutation banding
pattern), the signals of the Locus Control bands may be weak.

Wild type probes

The wild type probes comprise the most important resistance regions of the respective genes (see figure 1, as well as tables 1, 2, and 3). When all wild type
probes of a gene stain positive, there is no detectable mutation within the examined regions. This indicates that the strain tested is sensitive to the
respective antibiotic. In case of a mutation, the respective amplicon cannot bind to the corresponding wild type probe. The absence of a signal for at least
one of the wild type probes indicates a resistance of the tested strain to the respective antibiotic.
Each pattern deviating from the wild type pattern indicates a resistance of the tested strain. The banding pattern obtained with the rpoB probes allows
drawing a conclusion about an RMP resistance of the strain tested, the katG and the inhA banding pattern about an INH resistance.

Mutation probes
The mutation probes detect some of the most common resistance-mediating mutations (see tables 1, 2, and 3). Compared to the other probes, positive
signals of the mutation probes rpoB MUT2A and MUT2B may show a lower signal strength.
In rare cases, when the rpoB MUT3 band is positive, weak staining may be detected at the rpoB WT8 band which is to be considered negative.
Each pattern deviating from the wild type pattern indicates a resistance of the tested strain. The banding pattern obtained with the rpoB probes allows
drawing a conclusion about an RMP resistance of the strain tested, the katG and the inhA banding pattern about an INH resistance.

Please note:
Only those bands whose intensities are about as strong as or stronger than that of the Amplification Control zone (AC) are to be considered.
Not all bands of a strip have to show the same signal strength.

Note the following special cases:

1. There is a possibility that the specimen tested contains a heteroresistant strain. In case of a heteroresistance, a mutated as well as a wild-type
sequence can be detected in the respective strain; hence, one of the mutation probes as well as the corresponding wild type probe may stain positive on
the respective strip. Whether the respective resistance becomes phenotypically evident depends on the ratio of mutated and nonmutated sequences at
investigation.
2. There is a possibility that the tested specimen contains more than one M. tuberculosis complex strain (due to mixed culture or contamination). If at least
one of these strains harbors a mutation, one of the mutation probes as well as the corresponding wild type probe may stain positive. Whether the
respective resistance becomes phenotypically evident, depends on the ratio of resistant and sensitive strain at investigation.

5
3. There is a possibility that due to a mixed infection the tested specimen contains both an M. tuberculosis complex strain and a nontuberculous
mycobacterium. In rare cases, the TUB band may be missing due to competition of the single amplification reactions during PCR. However, when an
evaluable resistance pattern is developed, the presence of a strain belonging to the M. tuberculosis complex must be suspected and the test should be
repeated.
4. In rare cases, all bands of a gene locus (including the Locus Control band) may be missing completely on a test strip. If this result is generated from a
clinical specimen, possible reasons could be, but are not limited to, a DNA concentration in the sample below the limit of detection or the presence of
interfering substances in the sample. Such a banding pattern cannot be evaluated and the test must be repeated.
If a cultivated sample generates a result with the complete katG locus missing, this indicates an INH resistance of the strain tested.

Resistance regions and common resistance-mediating mutations

rpoB WT2 rpoB WT4 rpoB WT6

rpoB WT1 rpoB WT3 rpoB WT5 rpoB WT7 rpoB WT8

505 508 509 510 511 513 514 515 516 518 522 526 531 533

rpoB MUT1 (D516V) rpoB MUT2B (H526D)

rpoB MUT3 (S531L)
Figure 1: RMP resistance region of the rpoB gene
rpoB WT1-8: rpoB wild type probes; rpoB MUT1-3: rpoB mutation probes. The numbers specify the positions of the amino acids (codons) for all mutations
listed in the table. The codons for which mutation probes were designed are highlighted.

Table 1: Mutations in the rpoB gene and the corresponding wild type and mutation bands (according to [9])

Failing Codons Developing

wild type band(s) analyzed mutation band Mutation
rpoB WT1 505-509 F505L
T508A
S509T
rpoB WT2 510-513 E510H
L511P*
rpoB WT2/WT3 510-517 Q513L*
Q513P
del514-516
rpoB WT3/WT4 513-519 rpoB MUT1 D516V
D516Y
del515
rpoB WT4/WT5 516-522 del518*
N518I
rpoB WT5/WT6 518-525 S522L
S522Q
rpoB WT7 526-529 rpoB MUT2A H526Y
rpoB MUT2B H526D
H526R
H526P*
H526Q*
H526N
H526L
H526S
H526C
rpoB WT8 530-533 rpoB MUT3 S531L
S531Q*
S531W
L533P

* These rare mutations have only been detected theoretically (in silico).

Table 2: Mutations in the katG gene and the corresponding wild type and mutation bands

Failing Codon Developing

wild type band analyzed mutation band Mutation
katG WT 315 katG MUT1 S315T1
katG MUT2 S315T2

Table 3: Mutations in the inhA promoter region and the corresponding wild type and mutation bands

Failing Analyzed nucleic Developing

wild type band acid position mutation band Mutation
inhA WT1 –15 inhA MUT1 C–15T
–16 inhA MUT2 A–16G
inhA WT2 –8 inhA MUT3A T–8C
inhA MUT3B T–8A

6
Evaluation Examples
katG
RMP INH

rpoB MUT

katG MUT

inhA MUT
rpoB MUT2B
rpoB MUT2A
#

inhA MUT3B
inhA MUT3A

katG WT

inhA WT
rpoB MUT1

rpoB MUT3

katG MUT1
katG MUT2

inhA MUT1
inhA MUT2

sensitive

sensitive
resistant

resistant
rpoB WT1
rpoB WT2
rpoB WT3
rpoB WT4
rpoB WT5
rpoB WT6
rpoB WT7
rpoB WT8

inhA WT1
inhA WT2
katG WT

TUB
rpoB

katG
TUB

inhA
CC
AC

1 ++ -+ - +- + +
2 + - + - ++ - + +
3 ++- + - -+ + +
4 + -+- + -+ + +
5 +- -- -+- + +
rpoB
inhA
Figure 2: Examples for banding patterns and their evaluation with respect to RMP and/or INH resistance

If all wild type bands display a signal, this is classified as positive and marked in the WT column of the respective gene as “+”. If at least one of the wild type
bands is absent, this is classified as negative and marked in the WT column as “–“. Negative entries are only made to the mutation columns when none of
the mutation bands displays a coloration. If at least one of the mutation bands displays a coloration, this is classified as positive and the MUT column of the
respective gene is marked with a “+”.

Below, two of the examples shown above are explicated:

Example 1 shows the wild type banding pattern. All wild type probes, but none of the mutation probes display a signal; hence, the evaluation chart shows “+”
in the three wild type columns and “–” in the three mutation columns. Accordingly, the boxes “RMP sensitive” and “INH sensitive” are marked with a “+”.

In example 5, one of the rpoB and the katG wild type probes are missing; hence, the boxes for “rpoB WT” and “katG WT” are marked with a “–”. As none of
the mutation probes are developed, these boxes are also marked with a “–”. The inhA promoter region does not deviate from the wild type pattern. The
strain is evaluated as resistant to RMP and INH.

Limitations
Strictly adhere to the established protocols and procedures in order to obtain correct test results and to avoid contaminations.
Use of this assay is limited to qualified personnel well trained in the test procedure and familiar with molecular biological methods.

As with any DNA-based assay, this test only screens the nucleic acid sequence and not the amino acid sequence. Therefore, it is possible that mutations in
the probe region that do not cause an amino acid exchange (silent mutations) will still produce the absence of one of the wild type bands. A silent mutation
in codon 514 of the rpoB gene leading to the absence of the rpoB WT3 band was observed in rare cases [10]. Hence, if an RMP resistance is detected solely
by a missing rpoB WT3 band, results of phenotypic drug susceptibility testing should be considered.

Additional mutations within the tested rpoB gene region causing RMP resistance have been published [11]. As these mutations are very rare, they were not
accessible for validation purposes of this test system but were only detected in silico.

The GenoType MTBDRplus only detects those resistances that have their origins in the rpoB, katG, and inhA regions examined here. Resistances originating
from mutations of other genes or gene regions as well as other RMP and INH resistance mechanisms will not be detected by this test.

Theoretically, a resistance can exist in spite of a wild type pattern. If the sample contains a strain that has developed a heteroresistance and the resistance
is caused by a mutation not covered by the mutation probes, the wild type pattern will appear. Similarly, if the sample contains more than one
M. tuberculosis complex strain (due to mixed culture or contamination) and one of these harbors a mutation not covered by the mutation probes, the wild
type pattern will also appear.

As any DNA detection method the test system on hand detects DNA from viable and nonviable bacteria. Therefore, the GenoType MTBDRplus may not be
used for monitoring the progression or success of treatment of patients with antimicrobial therapy.
The GenoType MTBDRplus generates qualitative results. The intensities of the bands on a strip do not give information about the number of cells in a
positive sample.
The presence of multiple bacterial species in the sample to be analyzed might hamper the interpretation of the test.
The members of the M. tuberculosis complex cannot be differentiated.

The test only works within the limits of the genomic regions the primers and probes were chosen from.
As with any detection system based on hybridization, the test system on hand bears the possibility that sequence variations in the genomic regions the
primers and probes were chosen from but the detection of which the test was not designed for may lead to false results. Due to the high variability of
bacterial genomes, it is possible that certain subtypes might not be detected. The test reflects the current state of knowledge of Hain Lifescience.

Performance evaluation of this assay was carried out using the GenoLyse® kit for DNA extraction from decontaminated pulmonary smear-positive and
smear-negative clinical specimens as well as from cultivated samples and using the GXT DNA/RNA Extraction Kit for automated DNA extraction from
decontaminated clinical specimens. Until the present edition of the instructions on hand, the performance of the test has not been validated with other DNA
extraction methods or sample materials.

The results of this test may only be interpreted in combination with additional laboratory and clinical data available to the responsible physician. In
addition, results of phenotypic drug susceptibility testing have to be considered in certain cases.
The user must have or acquire information about the local mutation distribution pattern of the genes investigated with this test. Confirmation of the
results by phenotypic drug susceptibility testing may be necessary.

7
Troubleshooting
Overall weak or no signals (including Conjugate Control zone)
– Room temperature too low or reagents not equilibrated to room temperature.
– No or too little amount of CON-C and/or SUB-C used.
Repeat reverse hybridization.

Weak or no signals except for Conjugate Control zone

– Quality of extracted DNA does not allow an efficient amplification. Repeat extraction.
– Amplification Mixes (AM-A and AM-B) not mixed properly, interchanged, or added in wrong amounts. Prepare a new master mix and repeat
amplification.
– Incubation temperature too high. Repeat reverse hybridization.

No homogeneous staining
– Strips were not completely immersed during incubation steps.
– Tray was not shaken properly.
Repeat reverse hybridization.

High background color

– CON-C and/or SUB-C used too concentrated.
– Washing steps were not performed with the necessary care.
– Wash solutions too cold.
Repeat reverse hybridization.

Unexpected result
– Wrong incubation temperature.
– Hybridization Buffer and/or Stringent Wash Solution were not properly prewarmed or mixed.
– Contamination of neighboring wells by spillage during addition of Hybridization Buffer.
Repeat reverse hybridization.
– Contamination of extracted DNA with previously extracted or amplified DNA. Repeat extraction.
– Contamination of amplification reagents. In this case, a negative control sample shows additional bands besides CC and AC. Repeat amplification using
fresh reagents.
– Depending on the amount of amplified DNA used and on the specific reaction conditions, a strong and fast color development may occur. In such cases,
discontinue the substrate incubation as soon as the signals are clearly visible in order to prevent the development of cross-hybridizing bands.
– No pure culture as starting material. Re-culture in order to exclude contamination.
– Improper sampling, storage, transport, or preparation of specimen. Request new specimen and repeat test.
– Error during DNA extraction. Repeat extraction.

Material Required but not Included in the Kit

– Absorbent paper
– Adjustable pipettes for 10, 20, 200, and 1000 μl
– Class II safety cabinet
– Disposable gloves
– Disposable sterile pipette tips with filter
– DNA extraction kit (GenoLyse® or GXT DNA/RNA Extraction Kit, see chapter Ordering Information) as well as necessary equipment
– Graduated cylinder
– PCR tubes, DNase and RNase free
– Reagents for cultivation of mycobacteria as well as necessary equipment (when cultivated samples are to be used)
– Sample decontamination reagents as well as necessary equipment
– Shaking water bath + shaking platform or TwinCubator (instrument for manual hybridization) or automated hybridization instrument
– Thermal cycler
– Timer
– Tweezers
– Water (distilled)
– Water (molecular biology grade, for negative controls)

8
Kit Contents
Order no. 304A 30496A
Tests 12 96

Kit Component 1 of 2 (store at 2-8°C)

Membrane strips coated with specific probes (MTBDRplus VER 2.0 STRIPS) 12 2x 48

Denaturation Solution (DEN)

contains <2% NaOH, dye 0.3 ml 2x 1.2 ml

Hybridization Buffer (HYB)

contains <10% anionic tenside, dye 20 ml 120 ml

Stringent Wash Solution (STR)

contains >25% of a quaternary ammonium compound,
<1% anionic tenside, dye 20 ml 120 ml

Rinse Solution (RIN)

contains buffer, <1% NaCl, <1% anionic tenside 50 ml 3x 120 ml

Conjugate Concentrate (CON-C)

contains streptavidin-conjugated alkaline phosphatase, dye 0.2 ml 1.2 ml

Conjugate Buffer (CON-D)

contains buffer, 1% blocking reagent, <1% NaCl 20 ml 120 ml

Substrate Concentrate (SUB-C)

contains <70% dimethyl sulfoxide, <10% 4-nitro blue tetrazolium chloride,
<10% 5-bromo-4-chloro-3-indolyl phosphate 0.2 ml 1.2 ml

Substrate Buffer (SUB-D)

contains buffer, <1% MgCl2, <1% NaCl 20 ml 120 ml

Tray, evaluation sheet 1 of each 4 of each

Instructions for use, template 1 of each 1 of each

Kit Component 2 of 2 (store at –20°C)

Amplification Mix A (AM-A GT MTBDRplus VER 2.0)

contains buffer, nucleotides, Taq polymerase 0.15 ml 4x 0.3 ml

Amplification Mix B (AM-B GT MTBDRplus VER 2.0)

contains salts, specific primers, dye 0.53 ml 4x 1.05 ml

Ordering Information Order no.

GenoType MTBDRplus VER 2.0 (kit for analysis of 12 samples) 304A

GenoType MTBDRplus VER 2.0 (kit for analysis of 96 samples) 30496A

GenoType MTBDRplus VER 2.0 and GenoLyse® (kit for analysis

of 12 samples and kit for manual DNA extraction of 12 samples) 304AM

GenoType MTBDRplus VER 2.0 and GenoLyse® (kit for analysis

of 96 samples and kit for manual DNA extraction of 96 samples) 30496AM

GenoType MTBDRplus VER 2.0 and GXT DNA/RNA Extraction Kit (kit for analysis
of 96 samples and kit for automated DNA extraction of 96 samples using the GenoXtract®) 30496AA
®
GenoLyse (kit for manual DNA extraction of 12 samples) 51612
®
GenoLyse (kit for manual DNA extraction of 96 samples) 51610

GXT DNA/RNA Extraction Kit (kit for automated DNA/RNA extraction

of 96 samples using the GenoXtract®) 12.01.02

GenoXtract® (instrument for DNA extraction of up to 12 samples) 8.31.01

9
Performance Characteristics
Diagnostic performance
Pulmonary clinical specimens
Diagnostic performance characteristics of the GenoType MTBDRplus VER 2.0 were determined in a study [12] with 338 specimens (including sputum,
bronchoalveolar lavages, and pleural aspirates) compared to culture (successful cultivation on Loewenstein-Jensen solid medium or in MGIT (BD
Diagnostics, Franklin Lakes, USA)) and subsequent speciation using the GenoType Mycobacterium CM VER 1.0) and phenotypic drug susceptibility testing
(DST). Additionally, the samples were examined by microscopy. Clinical data of the patients were included in the evaluation.
The study site was located in a high MDR-TB burden country. Microscopy and cultivation methods were conducted on site. Aliquots of the NALC-
decontaminated sputum specimens were shipped to a second laboratory to perform DNA extraction and the GenoType MTBDRplus. Manual DNA extraction
was performed with the GenoLyse® kit (162 of the 338 sputum samples), automated DNA extraction was carried out on the GenoXtract® instrument using
the GXT DNA/RNA Extraction Kit (176 of the 338 sputum samples) according to the respective instructions for use.

A congruent GenoType MTBDRplus positive, clinical positive result was defined either by positivity of culture and GenoType MTBDRplus or when only
GenoType MTBDRplus was positive and culture negative, but TB was indicated by previous culture-based findings of the respective patient. A discrepant
result (GenoType MTBDRplus positive and culture negative) does not exclude in all cases a TB infection of the patient as for some patients’ histories of a
probable TB infection were not available.

Table 1: Performance characteristics of the GenoType MTBDRplus for detection of MTBC from pulmonary clinical specimens compared to
culture/GenoType Mycobacterium CM (GT Myco CM) and clinical findings

Smear-positive Smear-negative
Culture/GT Myco CM Culture/GT Myco CM
and clinic Sens: 100% and clinic Sens: 80.3%
Positive Negative Spec: /* Positive Negative Spec: 98.4%
GenoLyse® Positive 39 0 PPV: 100% Positive 49 1 PPV: 98.0%
GenoType MTBDRplus
Negative 0 1 NPV: /* Negative 12 60 NPV: 83.3%

Culture/GT Myco CM Culture/GT Myco CM

and clinic Sens: 97.5% and clinic Sens: 78.3%
Positive Negative Spec: /* Positive Negative Spec: 96.0%
GXT Positive 39 1 PPV: 97.5% Positive 47 3 PPV: 94.0%
GenoType MTBDRplus
Negative 1 0 NPV: /* Negative 13 72 NPV: 84.7%

Sens, diagnostic sensitivity; Spec, diagnostic specificity; PPV, positive predictive value; NPV, negative predictive value
* no value due to low sample number

For evaluation of resistance detection, the 156 samples (78 GenoLyse® isolates and 78 GXT isolates) were used which were MTBC-positive in both culture
and GenoType MTBDRplus.

Table 2: Performance characteristics of the GenoType MTBDRplus for detection of RMP resistance from pulmonary clinical specimens compared to
culture/DST

Smear-positive Smear-negative
Culture/DST Sens: 100% Culture/DST Sens: 96.0%
RMP-R RMP-S Spec: 92.3% RMP-R RMP-S Spec: 93.3%
GenoLyse® RMP-R 25 1 PPV: 96.2% RMP-R 24 1 PPV: 96.0%
GenoType MTBDRplus
RMP-S 0 12 NPV: 100% RMP-S 1 14 NPV: 93.3%

Culture/DST Sens: 96.3% Culture/DST Sens: 86.2%

RMP-R RMP-S Spec: 100% RMP-R RMP-S Spec: 100%
GXT RMP-R 26 0 PPV: 100% RMP-R 25 0 PPV: 100%
GenoType MTBDRplus
RMP-S 1 12 NPV: 92.3% RMP-S 4 10 NPV: 71.4%

Sens, diagnostic sensitivity; Spec, diagnostic specificity; PPV, positive predictive value; NPV, negative predictive value;
RMP-R, resistant to rifampicin; RMP-S, sensitive to rifampicin

Table 3: Performance characteristics of the GenoType MTBDRplus for detection of INH resistance from pulmonary clinical specimens compared to
culture/DST

Smear-positive Smear-negative
Culture/DST Sens: 96.7% Culture/DST Sens: 96.7%
INH-R INH-S Spec: 87.5% INH-R INH-S Spec: 90.0%
GenoLyse® INH-R 29 1 PPV: 96.7% INH-R 29 1 PPV: 96.6%
GenoType MTBDRplus
INH-S 1 7 NPV: 87.5% INH-S 1 9 NPV: 90.0%

Culture/DST Sens: 100% Culture/DST Sens: 90.6%

INH-R INH-S Spec: 100% INH-R INH-S Spec: 71.4%
GXT INH-R 28 0 PPV: 100% INH-R 29 2 PPV: 93.5%
GenoType MTBDRplus
INH-S 0 11 NPV: 100% INH-S 3 5 NPV: 62.5%

Sens, diagnostic sensitivity; Spec, diagnostic specificity; PPV, positive predictive value; NPV, negative predictive value;
INH-R, resistant to isoniazid; INH-S, sensitive to isoniazid

10
Cultured material
The diagnostic performance characteristics of the GenoType MTBDRplus were determined in a study with 74 cultured samples compared to GenoType
Mycobacterium CM VER 1.0 and phenotypic drug susceptibility testing (DST). The study site was located in a low MDR-TB burden country. Manual DNA
extraction was performed using the GenoLyse® kit according to the instructions for use. From 74 cultures, 49 were positive for M. tuberculosis complex
(MTBC) and 25 cultures showed growth of nontuberculous mycobacteria. Hence, for resistance detection in cultured material, 49 isolates were available.

Table 4: Performance characteristics of the GenoType MTBDRplus for detection of MTBC from cultured material compared to culture/GenoType
Mycobacterium CM (GT Myco CM)

Culture/GT Myco CM Sens: 100%

Positive Negative Spec: 100%
Positive 49 0 PPV: 100%
GenoType MTBDRplus
Negative 0 25 NPV: 100%

Sens, diagnostic sensitivity; Spec, diagnostic specificity; PPV, positive predictive value; NPV, negative predictive value

Table 5: Performance characteristics of the GenoType MTBDRplus for detection of RMP resistance from cultured material compared to culture/DST

Culture/DST Sens: /*
RMP-R RMP-S Spec: 100%
RMP-R 0 0 PPV: /*
GenoType MTBDRplus
RMP-S 0 49 NPV: 100%

Sens, diagnostic sensitivity; Spec, diagnostic specificity; PPV, positive predictive value; NPV, negative predictive value;
RMP-R, resistant to rifampicin; RMP-S, sensitive to rifampicin
* no value due to low sample number

Table 6: Performance characteristics of the GenoType MTBDRplus for detection of INH resistance from cultured material compared to culture/DST

Culture/DST Sens: /*
INH-R INH-S Spec: 100%
INH-R 3 0 PPV: /*
GenoType MTBDRplus
INH-S 0 46 NPV: 100%

Sens, diagnostic sensitivity; Spec, diagnostic specificity; PPV, positive predictive value; NPV, negative predictive value;
INH-R, resistant to isoniazid; INH-S, sensitive to isoniazid
* no value due to low sample number

Analytical performance
Analytical specificity
The specificity of the GenoType MTBDRplus VER 2.0 is ensured by the accurate design of specific primers and probes which considers, among others,
homology comparisons of the sequences published in gene databases, and by stringent reaction conditions.
The analytical specificity was determined with 61 DNA isolates including the following MTBC strains: M. tuberculosis, M. africanum, M. bovis, M. canettii,
M. microti, and M. pinnipedii (all RMP- and INH-sensitive). The following strains not detectable with the test system were analyzed: Actinomyces naeslundii,
Aggregatibacter actinomycetemcomitans, Bacillus cereus, Corynebacterium ammoniagenes, C. bovis, C. durum, Escherichia coli, Gordona rubropertinctus,
Klebsiella oxytoca, Mycobacterium abscessus, M. alvei, M. asiaticum, M. avium, M. celatum, M. chelonae, M. chimaera, M. fortuitum (2 sequevars),
M. frederiksbergense, M. gastri, M. genavense, M. goodii, M. gordonae, M. heckeshornense, M. immunogenum, M. interjectum, M. intermedium, M. intracellulare,
M. lentiflavum, M. marinum, M. mucogenicum, M. palustre, M. peregrinum, M. scrofulaceum, M. shimoidei, M. simiae, M. smegmatis, M. szulgai, M. triplex,
M. ulcerans, M. xenopi, MRSA, Nocardia abscessus, N. africana, N. amarae, N. asteroides, N. farcinica, Porphyromonas gingivalis, Prevotella intermedia,
Rhodococcus erythropolis, Saccharomonospora glauca, Tannerella forsythia, Treponema denticola, Tsukamurella inchonensis, T. pulmonis.
The six MTBC isolates were correctly identified as RMP- and INH-sensitive MTBC strains. All other 55 isolates displayed no TUB band and no evaluable
band pattern for RMP and INH resistances. Hence, an analytical specificity of 100% was achieved.

Analytical sensitivity
For determination of analytical sensitivity of the GenoType MTBDRplus for clinical samples, ten parallel BCG cultures were diluted and spiked into MTBC-
negative sputum samples (final concentrations in sputum samples: 1000, 500, 160, and 100 bacteria/ml). Including a negative control, DNA was extracted
once using the GenoLyse® kit and once using the GXT DNA/RNA Extraction Kit, and analyzed with the GenoType MTBDRplus applying the “MDR DIR” PCR
protocol. A limit of detection of 160 bacteria/ml was determined with both extraction methods.
For determination of analytical sensitivity of the GenoType MTBDRplus for culture samples, four BCG cultures (RMP- and INH-sensitive, 1.6x 104, 1.6x 103,
1.6x 102, and 100 bacteria/ml) were set up in triplicate. Including a negative control, DNA was extracted using the GenoLyse® kit and analyzed with the
GenoType MTBDRplus applying the “MDR CUL” PCR protocol. A limit of detection of 1.6x 104 bacteria/ml was determined.

Reproducibility
Intra-assay precision
In order to determine the intra-assay precision of the GenoType MTBDRplus, two BCG cultures (RMP- and INH-sensitive, 1,500 and 150 bacteria/ml) and an
M. avium culture (10,000 bacteria/ml) were set up in triplicate and spiked into negative sputum specimens. These samples and a negative control were
tested with the GenoType MTBDRplus under identical conditions, applying the “MDR DIR” PCR protocol. DNA extraction was performed once using the
GenoLyse® DNA extraction kit, and once using the GXT DNA/RNA Extraction Kit. All parallels showed identical and correct banding patterns and
comparable signal strengths. Additionally, signal strengths between the two DNA extraction methods and between different dilutions of the same samples
were comparable. Hence, an intra-assay precision of 100% was achieved.

11
Inter-assay precision
In order to determine the inter-assay precision of the GenoType MTBDRplus, two BCG cultures (RMP- and INH-sensitive, 1,500 and 150 bacteria/ml) and an
M. avium culture (10,000 bacteria/ml) were set up in triplicate and spiked into negative sputum specimens. These samples and a negative control were
tested in nine runs: on three different days, using three different sets of instruments, and conducted by three different operators. DNA extraction was
performed once using the GenoLyse® DNA extraction kit and once using the GXT DNA/RNA Extraction Kit. The “MDR DIR” PCR protocol was applied for
PCR. Apart from the varied parameter, all other testing conditions were identical. No deviations were detected between parallel samples, that is between
runs banding patterns were identical and correct and signal strengths were comparable. Moreover, signal strengths were comparable between different
DNA extraction methods and different bacterial concentrations. Hence, the inter-assay precision was 100%.

Interfering substances
There are substances that may inhibit PCR reactions. Such inhibitors may, for example, originate from the culture medium. In order to assess if the
medium influences the GenoType MTBDRplus, 6 different M. tuberculosis complex samples (4 RMP- and INH-resistant, 2 RMP- and INH-sensitive) were
cultured in 4 different media (solid media: Loewenstein-Jensen, Stonebrink, and Middlebrook-7H10, liquid medium: MGIT (BD Diagnostics, Franklin Lakes,
USA)). DNA was extracted from the culture samples using the GenoLyse® DNA extraction kit and then tested with the GenoType MTBDRplus.
All M. tuberculosis complex samples showed the same correct results. Hence, it can be excluded that the tested media import inhibitors into the GenoType
MTBDRplus test.

Interfering substances may also be carried over from the sample material. Hence, the substances indicated in table 7 were tested in order to assess a
potential interference of the GenoType MTBDRplus. Defined BCG culture dilutions above and at the detection limit were spiked with various amounts of the
potential inhibitors. From all samples, DNA extraction was performed using the GenoLyse® DNA extraction kit. Then the culture dilutions were tested with
the GenoType MTBDRplus.

Table 7: Tested potential interferents of the GenoType MTBDRplus

Substance/class Description/active ingredient Test concentration(s)

Allergy relief medicines Tea tree oil 0.008 % v/v to 0.5 % v/v
Anesthetics (endotracheal intubation) Lidocaine HCl 4% 20% v/v; 30% v/v
Anesthetics (oral) Benzocaine 20% 5% w/v
Antibiotics (nasal ointment) Mupirocin 1.2 mg/ml; 2.4 mg/ml
Antibiotics (systemic) Amoxicillin 2.2 μg/ml
Anti-tuberculosis drugs Isoniazid 1 mg/ml 50 μg/ml
Anti-tuberculosis drugs Rifampicin 1 mg/ml 25 μg/ml
Anti-tuberculosis drugs Pyrazinamide 10 mg/ml 100 μg/ml
Anti-tuberculosis drugs Ethambutol 1 mg/ml 5 μg/ml; 50 μg/ml
Anti-tuberculosis drugs Streptomycin 1 mg/ml 25 μg/ml
Anti-viral drugs Zanamivir 800 μg/ml
Blood Whole blood 0.2% v/v to 5% v/v
Blood Hemoglobin 0.05% v/v to 0.3% v/v
Bronchodilators Theophylline 222 pmol/ml
DNA (human) 10 μg/ml
Expectorants (oral) Guaifenesin 400 mg/pill 2.5 mg/ml; 5 mg/ml
Gastric acid 0.5% HCl, 0.1 M KCl, 0.1 M NaCl, pH 1-2 5% v/v
Influenza vaccine as nasal spray (FluMist®) Live attenuated influenza vaccine 5% v/v
Inhaled bronchodilators Salbutamol sulfate 2.5 mg/3 ml 50 μg/ml; 100 μg/ml
Mouthwash/gargle solutions Listerine (eucalyptol 0.029%, menthol 0.042%, methyl- 20% v/v
salicylate 0.06%, thymol 0.064%, denatured alcohol 20%)
Mucin: bovine submaxillary gland, type I-S Purified mucin protein 5% w/v 1.5% w/v; 5% w/v
Nasal corticosteroids Dexamethasone 1.52 pmol/ml
Nasal gel (homeopathic) Sulfur 5% w/v
Nasal sprays or drops Phenylephrine 0.5% 25% v/v; 100% v/v
Nebulizing solutions (hypertonic saline) NaCl 3% w/v; 5% w/v
Physiologic saline NaCl (0.9%) 0.9% w/v
Pneumocystis jiroveci medications Pentamidine 300 ng/ml
Pus 0.2% v/v to 5% v/v
Specimen processing reagents NALC-NaOH (N-acetyl-l-cysteine-sodium hydroxide) 0.5% v/v; 1% v/v
Tobacco Nicogel (40% tobacco extract) 0.5% w/v

Inhibition of the GenoType MTBDRplus VER 2.0 (invalid test result) was observed in the presence of the following substances in the concentrations as
indicated: whole blood at 0.6%, hemoglobin at 0.1%, pus at 2%, lidocaine at 30%, mupirocin at 2.4 mg/ml, tea tree oil at 0.5%, and guaifenesin at 5 mg/ml.

Stability
Shelf life of the GenoType MTBDRplus test kit when stored as recommended: see box label.
Stability is determined according to DIN EN ISO 23640.

12
References
1. World Health Organization. Global tuberculosis report 2014. WHO/HTM/TB/2014.08. World Health Organization, Geneva, Switzerland 2014.
2. Zhang Y, Yew WW. Mechanisms of drug resistance in Mycobacterium tuberculosis. Int J Tuberc Lung Dis 2009; 13: 1320–1330.
3. Biosafety in microbiological and biomedical laboratories, 5th edition. U.S. Department of Health and Human Services, Centers for Disease Control and
Prevention, Atlanta, USA 2009.
4. Protection of laboratory workers from occupationally acquired infections. Approved guideline. Clinical and Laboratory Standards Institute (formerly
National Committee for Clinical Laboratory Standards), USA, Document M29 (please refer to the latest version).
5. Kent PT, Kubica GP. Public health mycobacteriology: a guide for the level III laboratory. U.S. Department of Health and Human Services, Centers for
Disease Control and Prevention, Atlanta, USA 1985.
6. Isenberg HD. Clinical microbiology procedures handbook. American Society for Microbiology, Washington, D.C., USA 1992.
7. Richter E, Beer J, Diel R, Hillemann D, Hoffmann H, Klotz M, Mauch H, Rüsch-Gerdes S. MiQ 5, Tuberkulose, Mykobakteriose. In: Podbielski A,
Herrmann M, Kniehl E, Mauch H, Rüssmann H (eds): Mikrobiologisch-infektiologische Qualitätsstandards. Elsevier, Munich, Germany 2010.
8. DIN, Deutsches Institut für Normung e.V. (ed). DIN 58943-4:2009-02: Medical microbiology - Diagnosis of tuberculosis - Part 4: Primary samples for the
diagnosis of tuberculosis and mycobacteria – Qualitative and quantitative requirements, extraction, transport and storage. Beuth, Berlin, Germany 2009.
9. Telenti A, Imboden P, Marchesi F, Lowrie D, Cole S, Colston MJ, Matter L, Schopfer K, Bodmer T. Detection of rifampicin-resistance mutations in
Mycobacterium tuberculosis. Lancet 1993, 341: 647-650.
10. Alonso M, Palacios JJ, Herranz M, Penedo A, Menéndez A, Bouza E, García de Viedma D. Isolation of Mycobacterium tuberculosis strains with a silent
mutation in rpoB leading to potential misassignment of resistance category. J Clin Microbiol 2011; 49: 2688-2690.
11. Musser JM. Antimicrobial agent resistance in mycobacteria: molecular genetic insights. Clin Microbiol Rev 1995; 8: 496-514.
12. Crudu V, Stratan E, Romancenco E, Allerheiligen V, Hillemann A, Moraru N. First evaluation of an improved assay for molecular genetic detection of
tuberculosis as well as RMP and INH resistances. J Clin Microbiol 2012; Epub ahead of print, doi:10.1128/JCM.05903-11.

Important Changes in IFU-304A-06

Chapter Change
Precautions for Handling Kit Classification of the kit reagents according to GHS criteria (Regulation (EC) No. 1272/2008)
Constituents

13
 304A-06-02

Hain Lifescience GmbH

Hardwiesenstraße 1, 72147 Nehren, Germany
www.hain-lifescience.de, +49 (0) 74 73- 94 51- 0