TRAINING

TOOLKIT Principles of
Drug-resistant
tuberculosis:
genotypic drug-
how to interpret
rapid molecular
susceptibility
test results
testing

European Laboratory
Initiative 2020
Module A version 1.0
Objectives of the training toolkit
European Laboratory
Initiative 2020

• Over the past decade, WHO has endorsed a number of genotypic drug-susceptibility
testing (gDST) assays to scale up DST for tuberculosis (TB) because they are easy
to use, fast and reduce the biosafety risks to laboratory staff.
• Since these endorsements, our understanding of the following factors has improved:
◦ What antibiotic resistance is on the phenotypic level.
◦ Which mutations cause resistance.
◦ The technical limitations of gDST and of phenotypic DST (pDST), including the
reasons for discordances between these approaches.
◦ The need to revise the reporting language of gDST assays.
• This toolkit covers the implications of these findings.
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Objectives of this module
European Laboratory
Initiative 2020

• This module outlines the principles of gDST and explains the changes made to the
interpretation of the various assays by the WHO Global Laboratory Initiative, which
have been adapted by the European Laboratory Initiative where necessary.
• Changes to the interpretation are underlined throughout this module for emphasis.

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Background: drug-resistant TB
European Laboratory
Initiative 2020

• Three main classes of resistances are recognized, with the following global rates:
◦ Isoniazid-resistant but rifampicin-susceptible (Hr) TB – 8% of TB.
◦ Rifampicin-resistant (RR) TB or multidrug-resistant (MDR) TB, which is resistant to
both rifampicin and isoniazid – 3.4% of new and 18% of previously treated cases.
◦ Extensively drug-resistant (XDR) TB (resistant to rifampicin, isoniazid, any
fluoroquinolone and one second-line injectable drug) – 6.2% of MDR-TB is XDR-TB.
• Treatment success globally depends on the degree of resistance:
◦ 85% for drug-susceptible TB.
◦ 56% for RR/MDR-TB.
◦ 39% for XDR-TB. 4
Background: DST
European Laboratory
Initiative 2020

• Given the increasing rates of antibiotic resistance, which are key predictors of
treatment outcome for TB, it is essential that all patients receive DST results –
ideally for all drugs that they are treated with.
• Owing to the slow growth rate of the causative agent of TB and other practical
reasons (e.g. the cost and need for specialized facilities for pDST), gDST represents
the only realistic approach to scale up and offer DST in a timely manner.

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Background: DST
European Laboratory
Initiative 2020

• WHO has endorsed five gDST assays so far:

◦ Cepheid GeneXpert MTB/RIF (Xpert) and GeneXpert MTB/RIF Ultra (Ultra) – for
rifampicin resistance (rpoB).
◦ Hain Lifescience GenoType MTBDRplus VER 2.0 (FL-LPA) and Nipro
NTM+MDRTB VER 2.0 (Nipro) – for ethionamide/prothionamide (inhA), isoniazid
(inhA and katG) and rifampicin (rpoB).
◦ Hain Lifescience GenoType MTBDRsl VER 2.0 (SL-LPA) – for fluoroquinolones
(gyrA and gyrB) and second-line injectable drugs (eis and rrs).
• The Nipro assay will not be covered in this toolkit as it is rarely used in the WHO
European Region.
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Background: DST
European Laboratory
Initiative 2020
FL-LPA SL-LPA

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Background: how do mutations confer drug
resistance? European Laboratory
Initiative 2020

• Reduced activation:
◦ katG – isoniazid.
• Increased inactivation:
◦ eis – kanamycin.
• Modification of the target:
◦ gyrA and gyrB – fluoroquinolones (i.e. levofloxacin and moxifloxacin).
◦ rpoB – rifampicin.
◦ rrs – second-line injectable drugs (i.e. amikacin, capreomycin and kanamycin).

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Background: how do mutations confer drug
resistance? European Laboratory
Initiative 2020

• Overexpression of the target:

◦ inhA – ethionamide/prothionamide and isoniazid.
• Resistance is regarded to be clinically relevant if at least 1% of the bacterial
population is mutated (10% for pyrazinamide) – critical proportion.
• Heteroresistance occurs if the frequency of the resistant population is ≥1% but is
below 100%, which may be due to:
◦ An originally susceptible strain that is developing resistance.
◦ A mixed infection with strains with different susceptibilities to the same drug.
• Do not use the term "heteroresistance" in your clinical report.

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Background: bases, codons and amino acids
European Laboratory
Initiative 2020

• All gDST assays interrogate DNA, rather than RNA or proteins.

• Lower-case letters will be used for bases (i.e. adenine (a), cytosine (c), guanine (g)
and thymine (t)).
• 20 upper-case letters denote amino acids.
• An amino acid is encoded by three bases, which form a codon:
◦ gct, gcc, gca and gcg all encode alanine (A).
• A full codon table is provided in the documents section on the course website.

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Background: bases, codons and amino acids
European Laboratory
Initiative 2020

• For promoter mutations, the number between the wild type and mutated base
represents the distance in base pairs upstream of the start codon of inhA or eis:
◦ inhA c-15t
◦ eis c-14t
• For rrs resistance mutations, which fall into the 16S ribosomal RNA and are
consequently not translated into proteins, the number corresponds to the base
position relative to the start of the gene:
◦ rrs a1401g

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Background: bases, codons and amino acids
European Laboratory
Initiative 2020

• For katG and gyrA resistance mutations, the number between the wild type and
mutated amino acids is the amino acid position. The corresponding wild type and
mutated codons are separated by a slash:
◦ katG agc/acc S315T
◦ gyrA gcg/gtg A90V

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Background: bases, codons and amino acids
European Laboratory
Initiative 2020

• For rpoB mutations, the first amino acid position corresponds to the Escherichia coli
numbering, which has been traditionally used in TB, but the Mycobacterium
tuberculosis reference position should also be included in parentheses:
◦ rpoB gac/gtc D516V (D435V)
• For gyrB mutations, the first amino acid position corresponds to the so-called "1998"
numbering, which has been traditionally used by the SL-LPA, but the so-called
"2002" reference position should also be included in parenthesis:
◦ gyrB aac/gac N538D (N499D)

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Background: types of mutations in resistance
genes European Laboratory
Initiative 2020

• Neutral mutations do not confer antibiotic resistance:

◦ Synonymous mutations – base changes that do not change the amino acid (e.g.
gyrA ctg/cta L96L).
◦ Neutral non-synonymous mutations – base changes that alter the amino acid but
do not confer resistance (e.g. gyrA agc/acc S95T).
◦ Neutral base changes (e.g. in rrs or promoter regions).
• Resistance mutations:
◦ Usually single base changes.
◦ More rarely, insertions or deletions (including deletions of the entire resistance
gene).
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Detecting the causative agents of TB
European Laboratory
Initiative 2020

• A gDST result can only be reported if the Mycobacterium tuberculosis complex

(MTBC), the causative agent of TB, is detected by the assays.
• Each assay uses a different approach to detect MTBC, which explains why Ultra can
detect MTBC in samples with lower bacillary loads compared with Xpert or FL-LPA.

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Detecting the causative agents of TB
European Laboratory
Initiative 2020

• None of the assays can distinguish live from dead bacilli, nor differentiate the MTBC
species/ecotypes:
◦ M. tuberculosis – responsible for the vast majority of TB globally.
◦ M. africanum – found in up to 40% of TB patients in some countries in West Africa.
◦ M. canettii – causes up to 11% of TB in Djibouti.
◦ Animal variants (the chimpanzee bacillus, the dassie bacillus, M. bovis, M. bovis
BCG vaccine, M. caprae, M. microti, M. mungi, M. orygis, M. pinnipedii and M.
suricattae).

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Detecting the causative agents of TB
European Laboratory
Initiative 2020

• FL-LPA and SL-LPA only give qualitative results (i.e. "MTBC detected" vs "MTBC not
detected"), whereas Xpert and Ultra provide a semiquantitative output:
◦ "MTBC detected high"
◦ "MTBC detected medium"
◦ "MTBC detected low"
◦ "MTBC detected very low"
◦ "MTBC trace detected" (Ultra only)
◦ "MTBC not detected"
• Please note that the Cepheid software reports "MTB" instead of "MTBC", which
should be corrected in the clinical report. 17
gDST assays cannot rule out drug resistance
European Laboratory
Initiative 2020

• For two main reasons, the sensitivity of the WHO-endorsed gDST assays is limited
compared with pDST (i.e. some resistance is missed by gDST).

1. The probes in the gDST assays cover only some the known regions involved in
resistance (i.e. mutations in other genes or other parts of the same gene are not
interrogated, as is the case for about 30% of rifampicin resistance in Eswatini that
is missed because of an unusual rpoB mutation).

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gDST assays cannot rule out drug resistance
European Laboratory
Initiative 2020

2. Some mutations that fall within the regions covered by the probes are missed due
to:
§ The low bacillary load (e.g. Ultra "trace" results are always "indeterminate").
§ The frequency of the mutation in the sample is below the limit of detection (LoD)
of the assay but ≥1%, the critical proportion used for pDST. The precise LoDs for
heteroresistance depend on the mutation but are approximately:
▫ 20–80% for Xpert and 20–70% for Ultra.
▫ 5–10% for mutation (MUT) probes vs >95% for wild type (WT) probes for
FL-LPA and SL-LPA.
§ The design of the assay (i.e. some mutations cannot be detected reliably even if
they occur at a frequency of 100% in a sample with a high bacterial load). 19
gDST assays cannot rule out drug resistance
European Laboratory
Initiative 2020

• Because of the limited sensitivity of gDST, resistance cannot be ruled out (i.e. a
sample can still be resistant even if no resistance mechanism is found).
• To emphasize this limitation to clinicians, "resistance to [drug] not detected" rather
than "susceptible" or "sensitive" should be reported:
◦ Already implemented with Xpert and Ultra software.
◦ A change to the current interpretation of the FL-LPA and SL-LPA.

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Resistance inferred vs detected for LPAs
European Laboratory
Initiative 2020

• All resistance loci are covered by WT probes that bind to the corresponding wild type
sequences:
◦ Hain has not disclosed where exactly the WT probes for eis, inhA and rrs bind.
◦ The approximate binding regions for the WT probes for gyrA (see above), gyrB and
rpoB are known.

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Resistance inferred vs detected for LPAs
European Laboratory
Initiative 2020

• Some, but not all, resistance mutations are interrogated by MUT probes:
◦ Hain has not disclosed where exactly the MUT probes bind (e.g. the gyrA MUT
probes above have been included for illustrative purposes only and the actual MUT
probes might bind to slightly different regions).

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Resistance inferred vs detected for LPAs
European Laboratory
Initiative 2020

• Currently, "resistance" is reported if a WT probe does not bind and/or MUT probe
binds, which is not correct as both probes provide different kinds of information.

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Resistance inferred vs detected for LPAs
European Laboratory
Initiative 2020

• MUT probes:
◦ MUT probes only bind if a specific base variant of a resistance mutation is present
(e.g. gyrA MUT1 only binds if the gyrA gcg/gtg A90V mutation is present).
◦ Additional mutations in the region targeted by a MUT probe may prevent binding
(e.g. the double base gcg/gta change results in the same gyrA A90V amino acid
change, but gyrA MUT1 does not bind).
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Resistance inferred vs detected for LPAs
European Laboratory
Initiative 2020

• Most, but not all, mutations targeted by a WT probe prevent binding. For gyrA, WT3:
◦ Does not bind for the gac/cac D94H resistance mutation – true resistant result.
◦ Binds for the neutral agc/acc S95T mutation – true susceptible result.
◦ Binds for the synonymous ctg/cta L96L mutation – true susceptible result.
◦ Does not bind for the synonymous ctg/ttg L96L mutation – false resistant result
(globally rare, but can be frequent locally, e.g. in 7% of MDR-TB in Colombia). 25
Resistance inferred vs detected for LPAs
European Laboratory
Initiative 2020

• Because the precise mutation is not detected by WT probes, the following changes
to the reporting language have been made:
◦ Report "resistance to [drug] inferred" instead of "resistant" to emphasize the
possibility of systematic false resistance (e.g. "resistance to levofloxacin inferred"
for gyrA WT1).
◦ Report the corresponding codons instead of precise mutations (e.g. "gyrA
mutation(s) in codon(s) 85-90" instead of "gyrA G88A or G88C" for gyrA WT1). 26
Resistance inferred vs detected for LPAs
European Laboratory
Initiative 2020

• To emphasize that a specific base change of a known resistance mutation is

detected by MUT probes:
◦ Report "resistance to [drug] detected" instead of "resistant" (e.g. "resistance to
levofloxacin detected" for gyrA MUT1).
◦ For mutations that are translated into amino acids, it is optional to show the base
changes (e.g. "gyrA gcg/ctg A90V" for gyrA MUT1).
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Resistance inferred vs detected for LPAs
European Laboratory
Initiative 2020

• A MUT probe binds if the corresponding mutation is present at an approximate

frequency of at least 5% of the total population (e.g. gyrA MUT1 detects gcg/gtg
A90V).
• Conversely, a WT probe only does not bind if >95% of the total population is mutated
in the corresponding region. For example, gyrA WT2 will not bind if:
◦ Either gyrA A90V or S91P is present at 100% of the population.
◦ gyrA A90V and S91P are each present at 50% in different populations (i.e. 100% of
the population is mutated).
• Therefore, any mutation that is detected or inferred by the Hain LPAs must be
present at a frequency above the critical proportion of 1% and must be reported,
even if the sample is heteroresistant.
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Resistance inferred vs detected for LPAs
European Laboratory
Initiative 2020

• The same logic applies to the remaining resistance loci in FL- and SL-LPAs, with the
following exception:
◦ Because Hain has not disclosed the approximate binding regions for the eis, inhA
and rrs WT probes, the exact base/codon region cannot be reported.
◦ Therefore, report the corresponding region (e.g. "resistance to kanamycin inferred"
and "eis mutation(s) in -37 region" instead of "eis g-37t" for eis WT1).

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Drug resistance is inferred by Xpert and Ultra
European Laboratory
Initiative 2020

• Xpert and Ultra report "rifampicin resistance detected" even though:

◦ Xpert cannot distinguish any synonymous changes from resistance mutations in
rpoB.
◦ Ultra can distinguish some but not all synonymous changes from resistance
mutations in rpoB.
• As systematic false resistant results are possible, the result should be changed to
"resistance to rifampicin inferred".

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Stratifying the level of drug resistance
European Laboratory
Initiative 2020

• Resistance to isoniazid and moxifloxacin is now stratified into low- and high-level
resistance as dose adjustments are possible or under consideration in some
circumstances.
• Dose adjustments are not possible for any of the remaining drugs:
◦ Resistance to amikacin, capreomycin, ethionamide/prothionamide, levofloxacin
and rifampicin is not stratified.
◦ Hain cannot remove the term "low-level resistance" for eis from its instructions for
the SL-LPA without having to re-register this assay. Therefore, we suggest that you
manually correct "low-level resistance" to simply "resistance" for kanamycin and
emphasize to clinicians that the dose of kanamycin cannot be increased.

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Stratifying the level of isoniazid resistance
European Laboratory
Initiative 2020

• Isoniazid cannot be used except in the standardized shorter MDR-TB regimen:

◦ "High-level resistance to isoniazid detected" – at least one katG MUT probe binds.
◦ "High-level resistance to isoniazid inferred" – at least one katG WT probe does not
bind or all katG probes (i.e. locus control, WT and MUT) do not bind when FL-LPA is
used on positive culture.
• High-dose isoniazid (up to 15 mg/kg) can be used in circumstances that are currently
being clarified by WHO:
◦ "At least low-level resistance to isoniazid detected" – at least one inhA MUT probe
binds.
◦ "At least low-level resistance to isoniazid inferred" – at least one inhA WT probe
does not bind. 32
Stratifying the level of moxifloxacin
resistance European Laboratory
Initiative 2020

• Moxifloxacin cannot be used, irrespective of dose, where:

◦ "High-level resistance to moxifloxacin detected" – gyrA MUT3B, MUT3C or MUT3D
probe binds.
• A high dose (up to 800 mg daily to adults) can be used as part of the longer MDR-TB
regimen only (i.e. this is an exclusion criterion for the standardized shorter MDR-TB
regimen, even though it uses high-dose moxifloxacin), where:
◦ "At least low-level resistance to moxifloxacin detected" – gyrA MUT1, MUT2 or
MUT3A, or gyrB MUT1 or MUT2 probe binds.
◦ "At least low-level resistance to moxifloxacin inferred" – at least one gyrA WT or
gyrB WT probe does not bind.
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Report the most significant result
European Laboratory
Initiative 2020

• If more than one probe provides information for the same antibiotic, report the most
significant result.
• For amikacin, capreomycin, ethionamide/prothionamide, levofloxacin and rifampicin,
the results in decreasing order of significance are:
◦ "Resistance to [drug] detected"
◦ "Resistance to [drug] inferred"
◦ "Resistance to [drug] not detected"

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Report the most significant result
European Laboratory
Initiative 2020

• For kanamycin "low-level resistance" is equivalent to "resistance" resulting in the

following hierarchy:
◦ "(Low-level) resistance to kanamycin detected"
◦ "(Low-level) resistance to kanamycin inferred"
◦ "Resistance to kanamycin not detected"

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Report the most significant result
European Laboratory
Initiative 2020

• For isoniazid, the results in decreasing order of significance are:

◦ "High-level resistance to isoniazid detected"
◦ "High-level resistance to isoniazid inferred"
◦ "At least low-level resistance to isoniazid detected"
◦ "At least low-level resistance to isoniazid inferred"
◦ "Resistance to isoniazid not detected"

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Report the most significant result
European Laboratory
Initiative 2020

• For moxifloxacin, the results in decreasing order of significance are:

◦ "High-level resistance to moxifloxacin detected"
◦ "At least low-level resistance to moxifloxacin detected"
◦ "At least low-level resistance to moxifloxacin inferred"
◦ "Resistance to moxifloxacin not detected"

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Other changes to LPAs
European Laboratory
Initiative 2020

• Remaining resistance genes (i.e. gyrA, gyrB and rrs) can be interpreted if an eis
deletion is inferred from positive culture (i.e. the locus control, all WT probes and the
MUT probe for eis do not bind).
• If rrs WT1 probe does not bind (and none of the corresponding MUT probes bind),
"resistance inferred" should be reported for all second-line injectable drugs, including
amikacin, to avoid missing resistance (previously "resistance" was only reported for
capreomycin and kanamycin).

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What about capreomycin and kanamycin?
European Laboratory
Initiative 2020

• WHO no longer recommends capreomycin and kanamycin for the treatment of TB

as their use correlated with worse treatment outcomes in a large meta-analysis. You
should still report DST results for these drugs during the transition period, but should
stop this as soon as these drugs are no longer used in your setting.

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European Laboratory Initiative 2020

All reasonable precautions have been taken by the authors to verify the information contained in this
online course. However, the published material is being distributed without warranty of any kind, either
expressed or implied. Responsibility for the interpretation and use of the material lies with the reader.
In no event shall the authors be liable for damages arising from its use.