CUSTOMER SPOTLIGHT
PHILIP SUPPLY, PHD

Seeking the source ofbacterial drug resistance
A targeted deep sequencing assay identifies multidrugresistant tuberculosis strains responsible for silent outbreaks

Tuberculosis (TB) is often thought of as a disease of the past. Yet,this potentially lethal medical affliction, once known as “consumption,” remains a leading cause of death globally, especially in lowand lower-middle-income countries.1 The COVID-19 pandemic has
stalled and reversed progress made on access to TB diagnosis andtreatment over the past years.2 According to the World Health Organization (WHO), an estimated 10.6 million people fell ill with TB in
2021 and 1.6 million perished from the disease.2 Its current predominance stems in part from the bacterium’s ability to evolve, evenwhen faced with a six-month treatment regimen of four specificantimicrobial drugs. Such treatment has been used for decades
and, over time, many TB strains have developed resistance to once
effective first-line drugs such as isoniazid and rifampicin. In 2021,
an estimated 450,000 new TB cases were resistant to rifampicin
or to both isoniazid and rifampicin,2 representing multidrug resistance (MDR) and making the disease that much harder to treat andmore likely to spread. Early detection of TB outbreaks and effective
identification of new MDR-TB strains have become essential incombating the disease.
PHILIP SUPPLY, PHD IS A SENIORSCIENTIST AND RESEARCHDIRECTOR OF THE CNRS AT THE
CENTER FOR INFECTION AND
IMMUNITY AT THE INSTITUTPASTEUR DE LILLE AND A SCIENTIFIC
CONSULTANT AT GENOSCREEN.

Philip Supply, PhD, a senior scientist and research director of
the French National Center for Scientific Research (CNRS) at theCenter for Infection and Immunity at the Institut Pasteur de Lille,
has spent most of his career studying the bacterium that causes
TB, Mycobacterium tuberculosis. As a scientific consultant for
GenoScreen, a French biotech company that provides state-of-theart sequencing and genomic analysis services, Dr. Supply assisted
in the development of a novel targeted deep sequencing assay,
Deeplex Myc-TB.3 Sensitive and accurate, this assay can identify
drug-resistant TB strains directly from clinical sputum samples,
without the need for time-consuming bacterial cultures.3

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We spoke with Dr. Supply about the growing problem of MDR-TB,
the value that Illumina next-generation sequencing (NGS) systems
bring to their detection, development of the Deeplex Myc-TB
assay, and how it could be used to detect MDR-TB strains and limit
silent outbreaks.

"TB is a contagious disease
that is silently transmitted.
Patients can live without clear
clinical signs of TB infection
for quite some time before
being diagnosed. This makes
it very important to be able toproperly diagnose the disease
and trace its transmission."

Q: What sparked your interest in the bacterial genomics andmolecular epidemiology of TB?
Philip Supply (PS): Early in my career, in Belgium, I studied very
basic research questions related to yeast genetics. I wanted tochange and conduct research on something that benefited the
public directly. I contacted the head of a lab at the Institut Pasteurof Lille in northern France and he suggested that I work on TB.
The Institut Pasteur of Lille is where the original anti-TB vaccine,
the bacillus of Calmette-Guerin (BCG), was developed a century
ago, so there’s an extensive history of TB research here. During theconversation, I learned a lot about the contemporaneous extent of
the TB problem.

Q: Why has TB been so difficult to eradicate?
PS: TB is a contagious disease that is silently transmitted. Patients
can live without clear clinical signs of TB infection for quite sometime before being diagnosed. This makes it very important to beable to properly diagnose the disease and trace its transmission.
However, it is difficult to trace TB transmission in a population
due to the properties of the bacterium, including its highly clonal
population structure. The low diversity among TB strains results inthe need for very precise molecular tools to trace strains and theirspread in a population. The data that are obtained can be used tobetter identify routes of transmission among people with TB and all
the people that they’ve been in contact with, as well as track the
spread of epidemic strains to delineate outbreaks, in order to better
control and prevent further propagation.

Q: How is TB treated currently?
PS: TB is very difficult to treat compared to other bacterial infections and requires a very demanding treatment regimen. The
easiest course takes six months and works only in cases where
the patient’s TB strain is susceptible to current first-line antibiotics. In this regimen, the TB patient takes four drugs every day
for two months. During the following four months, they need tocontinue to take two of those four drugs to ensure a cure.

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Unfortunately, we have been using these first-line drugs for quite
a long time now and resistant strains have developed. Patientsafflicted with drug-resistant strains face a longer, even more
complicated regimen. Treatment for these patients can take up to
two years, with 5–7 different drugs that are less efficient and havemore side effects. This has consequences for the treatment’s rateof success and mortality. New treatment regimens that incorporate
novel anti-TB drugs have a shorter course, but resistance to these
new antibiotics, which begins to be seen for some of them, needs
to be closely monitored and diagnosed as well.
The WHO estimates that approximately 450,000 new patients are
suffering from rifampicin-resistant (RR) TB or MDR-TB each year.2
However, only one in three of these patients are diagnosed assuch and treated accordingly.2 This diagnostic gap represents ahuge problem.

Q: How do doctors know when they are dealing with a resistantstrain of the disease?
PS: The classical way to identify a drug-resistant TB strain is tocollect sputum from the infected patient, grow the specimen in
culture, and test the obtained isolate for antibiotic susceptibility.
If the strain is resistant to first-line TB drugs, like isoniazid andrifampicin, then additional cultures are done to test growth in thepresence of second-line drugs. The difficulty with this approach isthat these bacteria grow very slowly. It can take up to two months
before a complete diagnosis can be made.

"The WHO estimates thatapproximately 450,000 new
patients are suffering fromrifampicin-resistant (RR) TB or
MDR-TB each year. However,
only one in three of thesepatients are diagnosed as suchand treated accordingly."

Q: Are there new drugs available to combat MDR-TB?
PS: A few new drugs have been released in the last few years,
offering major progress in treating MDR-TB. Bedaquiline wasapproved in 2012, delamanid in 2014, and pretomanid in 2019 (inthe US, 2020 in the EU). The WHO has recently announced that asix-month treatment regimen incorporating bedaquiline and pretomanid in combination with linezolid may be used in eligible patientswith MDR/RR-TB, in place of longer treatments.4 Unfortunately, we
are already seeing resistance emerge against some of these antibiotics. Resistance to these latest compounds must also be closely
monitored and effectively detected.

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Q: How does the Deeplex Myc-TB assay identify drug-resistantstrains of TB?

"The Deeplex Myc-TB assay
can be applied directly to DNA
extracted from clinical samples
with minimal bacterial loads,
alleviating the need for culture
and expediting analysis."

PS: Deeplex Myc-TB is a targeted NGS-based assay, in contrast
to a whole-genome sequencing (WGS) assay. While a WGS assay
could provide a comprehensive picture of all the mutations that
occur in a TB strain, the large amount of DNA needed to run theassay is a problem. It can take between 7–10 days of TB culture
to obtain adequate amounts of DNA for a WGS assay. This is asignificant limitation that can delay identification of drug-resistant
TB strains.
We moved to a targeted sequencing method where we first amplify
the main drug resistance gene targets of the TB bacteria prior tosequencing. The Deeplex Myc-TB assay can be applied directly to
DNA extracted from clinical samples with minimal bacterial loads,
alleviating the need for culture and expediting analysis.
In addition to speed, another benefit of a targeted sequencing
approach is depth of sequencing. Deep sequencing is importantbecause it enables users to capture possible subpopulations ofstrains that carry drug-resistant mutations confidently. Even if
these subpopulations represent a minority of the bacterial population, they can cause drug resistance because they will be selectedunder antibiotic pressure. The Deeplex Myc-TB assay can detect abacterial subpopulation carrying a resistance mutation that represents as little as 1-3% of the sample.3
Another critical aspect of the success of the Deeplex Myc-TB
assay is that we successfully developed a fully parameterized andautomated web application for rapid, easy analysis and interpretation of the sequencing results. This makes the assay easily accessible to the many users who are not NGS specialists.

Q: How did you select the markers that you use in the Deeplex
Myc-TB assay?
PS: We based our marker choices on our previous knowledge ofthe main targets of drug resistance in M. tuberculosis. We defineda marker set of 18 genes and included the known drug resistancemutations within those genes (Figure 1).
The assay can be upscaled easily. We are working to increase thenumber of markers to include targets for recently released drugs
such as delamanid and pretomanid.

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Figure 1: Deeplex Myc-TB Assay gene targets include markers for the detection of
known (red) and potential (blue) anti-TB drug resistance-associated mutations.

Q: Has the Deeplex Myc-TB assay uncovered MDR-TB strains?
PS: We conducted a study of TB-positive cultures from four South
African provinces where 1823 TB isolates had initially been identified only with isoniazid resistance using current molecular technologies, such as the GeneXpert MTB-RIF assay and line probe
assays.5 We randomly selected 277 of those isolates and screened
them with a rifampicin-resistant mutation-specific assay followedby the Deeplex Myc-TB assay and WGS using the MiSeq™ System
to evaluate patterns of extensive resistance, transmission, andevolution. In 15% of the tested samples, we identified a special
mutation that causes rifampicin resistance that is not captured in
the current WHO-endorsed GeneXpert MTB/RIF molecular test orother tests. In addition, we identified mutations causing resistanceto isoniazid and two other first-line drugs, thus reclassifying theisolates as MDR-TB strains resistant to all first-line antibiotics.
We also found that most of these strains were part of the sameoutbreak. As a result, these strains were escaping MDR surveil-

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lance in the country. We also identified four distinct mutations in
six isolates that are potentially associated with decreased bedaquiline sensitivity. It was completely unexpected. These patientswere not supposed to have been treated with that drug, yet we saw
probable signs of resistance emerging in that patient population.

"The Deeplex Myc-TB assay

Q: How could drug resistance to bedaquiline develop so quickly?

offers much more than the

PS: It’s a delicate question and, in this specific case, we don’t
have a definitive answer. Results from our phylogenetic analyses
suggest that these mutations arose after the start of the clinicalaccess program in the country for bedaquiline treatment of patientswith MDR-TB. At the same time, we know that there can be crossresistance between bedaquiline and an earlier anti-TB drug, clofazimine. The main gene that is associated with bedaquiline resistancein clinical isolates is also the main gene that is associated withclofazimine resistance. Additional data are needed to better answer
this question.

classical molecular methods...it
amplifies > 10,000 base pairs,
including the main TB drug
resistance targets. It covers
drug resistance—associatedmutations for 13 anti-TB drugclasses, comprising all those
now defining extensively
drug resistant TB (XDR-TB)
according to the WHO."

Q: How can the Deeplex Myc-TB assay identify mutations thatolder technologies cannot?
PS: The Deeplex Myc-TB assay offers much more than the classicalmolecular methods. It is more comprehensive because it amplifies> 10,000 base pairs, including the main TB drug resistance targets.
It covers drug resistance–associated mutations for 13 anti-TB drugclasses, comprising all those now defining extensively drug-resistant TB (XDR-TB) according to the WHO. In addition, our assay
includes targets for M. tuberculosis genotyping, which is usefulfor molecular tracing, and detects more than 100 other mycobacterial species, comprising pathogenic or opportunist organismsof important medical relevance as well. Current technologies,
including the GeneXpert MTB-RIF and line probe assays, can’t
match that.
The GeneXpert MTB-RIF assay is based on real-time PCR and
it’s fast, taking only two hours to produce a result. However, it
covers only a limited region of the rpoB gene, which is a hotspot
for mutations causing rifampicin resistance. That’s why it missed
identifying the new South African rifampicin-resistant strains.
These strains carry a mutation that is outside this region in rpoB.
The newer GeneXpert MTB-RIF XDR assay can detect certain resistance mutations to isoniazid, ethionamide, fluoroquinolones, andaminoglycosides, but does not provide information on other drugs,
such as bedaquiline and linezolid representing now priority antibiotics for treating MDR-TB.

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Line probe assays use PCR together with hybridization of online
probes. These probes contain only a limited set of common resistance mutations for some, but not all, anti-TB drugs.
Representing another approach, the Oxford Nanopore systemcurrently uses WGS, which captures more targets. As I indicatedpreviously, a WGS assay requires a culturing step to obtain sufficient DNA amounts. Hence, the system is slow and the depth ofcoverage is usually lower. The level of sequencing accuracy, as
far as we know, is not as high when compared to Illumina NGS
systems. This is a significant limitation, especially for detecting
mutations in minority populations. Available data indicate that thesame limitation of accuracy with WGS applies to targeted sequencing on the Oxford Nanopore system.6

Q: Are international organizations using the Deeplex
Myc-TB assay?

"The WHO is using our assay
for TB drug resistance surveys
and it was mentioned in theorganization’s 2018 Technical
Guide."

PS: The WHO is using our assay for TB drug resistance surveys andit was mentioned in the organization’s 2018 Technical Guide.7 WHO
survey data have already been published, including several thatinvolved data mining the levels of TB drug resistance in Djibouti,
the Democratic Republic of the Congo, and Eritrea. Culturing is
not performed systematically in these countries. As a result, theDeeplex Myc-TB assay was particularly useful because the assay
could be run directly on clinical samples with no culturing required.

Q: Has the inclusion of the Deeplex Myc-TB assay in the WHO
technical guide sparked an increase in health agencies using
NGS assays for surveillance?
PS: It has increased the use of our assay for surveillance, as well as
for rapid detection of TB drug resistance in individual patients. Surveillance can provide an accurate picture of TB epidemiology, thatis the number of cases and prevalence of drug resistance in the
total population, and an NGS-based assay can make a significant
difference here. Both WGS and targeted NGS assays can provide
extensive data on the prevalence of drug resistance in a patient
population, based on genotypic information obtained from isolates(WGS) and specimens (targeted NGS) from individual patients.
As a targeted assay, Deeplex Myc-TB can also be applied in nearreal-time conditions as soon as a clinical sample is obtained. Suchan assay could provide relevant information for rapid clinicaldecisions on the most appropriate treatment of a patient. In
contrast, a WGS assay has the advantage of most precisely
determining the genetic relatedness of isolates at a genome-wide

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level. Therefore, it can also provide valuable information about theexistence of outbreak strains in the population. As a result, targeted
NGS and WGS assays are now used on an increasingly large scaleby the WHO and many other health agencies.

"...Illumina NGS systems...
are well known for offeringhigh accuracy for detecting
mutations. This is very
important because we need
to be able to detect evenminority TB populations withina sample that are carrying drug
resistance mutations."

Q: Why did you and the GenoScreen team choose Illumina NGS
systems for the assay?
PS: We work with Illumina NGS systems, including the MiSeq,
NextSeq™ 500, MiniSeq™, and iSeq™ 100 systems, because theseplatforms are well known for offering high accuracy for detecting
mutations. This is very important because we need to be able
to detect even minority TB populations within a sample that arecarrying drug resistance mutations. The Illumina NGS systems arealso the most used worldwide. If we develop the assay and validate
it on these platforms, it gives us an important advantage because it
will favor a more rapid and efficient roll out of the assay.

Q: Do you believe that the Deeplex Myc-TB assay will be useful
in developing countries for TB surveillance and other kinds of
medical work?
PS: We hope that the Deeplex Myc-TB assay will be a valuable tool
in use throughout the world. GenoScreen and Illumina just engaged
in a partnership to increase accessibility of this solution globally.
As of today, the assay is being deployed in more than 35 countries
on five continents, including in developing countries with high TBburdens and high MDR rates. An evaluation for clinical use is also
being performed in cooperation with the Foundation for Innovative
New Diagnostics (FIND). FIND obtained a grant from Unitaid specifically to test new targeted NGS-based tools for TB diagnostics inlow- and middle-income countries.

Q: What challenges must be overcome for NGS-based TB assays
to gain widespread use for TB surveillance and diagnostics?
PS: We’ll need to make progress in developing the molecularbiology infrastructure and skills in different countries. It might beeasier to equip and train TB reference centers and labs that will
use the NGS-based Deeplex Myc-TB assay than it would be tobuild capacity for bacterial culture–based assays. Indeed, thelatter require special biosafety infrastructures that are not required
for tests that can be directly applied on clinical samples. We are
already organizing onsite trainings and videos that review thetechnical steps of the Deeplex Myc-TB assay workflow. This will
ensure that the assay is well received and used most efficiently.

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Another area of focus will be to train staff on assay data interpretation based on the detection of different mutations. To facilitate
data interpretation, our web application for automated analysisof the sequencing data has a very user-friendly design. Finally,
support and maintenance of the sequencing platforms needs
also to be further improved for sustainable use in lower resource
settings. All of these challenges will be addressed through thepartnership between GenoScreen and Illumina.

Q: What are the next steps in the development of the Deeplex
Myc-TB assay?
PS: In addition to adding new targets to the Deeplex Myc-TB
assay to detect resistance to the latest TB antimicrobial drugs,
we envision extending the assay to other mycobacteria. We now
have an assay for Mycobacterium leprae, the mycobacterium thatcauses leprosy (Hansen’s disease). Not unlike TB, many people
think that leprosy is a disease of the past. While it is uncommon,it still exists in some parts of the world, including Africa, Brazil,
India, and China. Similarly to Deeplex Myc-TB, Deeplex Myc-Lep
detects drug resistance and identifies the strain genotype involved
for use in diagnosis and surveillance. While it does not cover thecomplete genome, this targeted assay can differentiate strain
types and drug-susceptible or drug-resistant strains that circulate
in patient populations.

"We hope that the Deeplex
Myc-TB assay will be avaluable tool in use throughout
the world...As of today, theassay is being deployed in
more than 35 countries onfive continents, including indeveloping countries with highTB burdens and high MDR
rates."

Learn more
MiSeq System, illumina.com/miseq
NextSeq 550 System, illumina.com/nextseq550
MiniSeq System, illumina.com/miniseq
iSeq 100 System, illumina.com/iseq
Deeplex Myc-TB assay, deeplex.com

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World Health Organization. The top 10 causes of death. https://www.who.int/
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2. World Health Organization. Global Tuberculosis Report 2022. https://www.who.int/
teams/global-tuberculosis-programme/tb-reports/global-tuberculosisreport-2022. Published October 27, 2022. Accessed December 20, 2022.
3. Jouet A, Gaudin C, Badalato N, et al. Deep amplicon sequencing for culture-free
prediction of susceptibility or resistance to 13 anti-tuberculous drugs [published correction appears in Eur Respir J. 2022 Nov 10;60(5):]. Eur Respir J.
2021;57(3):2002338. Published 2021 Mar 18. doi:10.1183/13993003.02338-2020
4. World Health Organization. Rapid communication: Key changes to the treatment ofdrug-resistant tuberculosis. https://www.who.int/publications/i/item/WHO-UCNTB-2022-2. Published May 2022. Accessed January 11, 2023.
5. Makhado NA, Matabane E, Faccin M, et al. Outbreak of multidrug-resistanttuberculosis in South Africa undetected by WHO-endorsed commercial tests: anobservational study. Lancet Infect Dis. 2018; 18:1350-1359.
6. Mariner-Llicer C, Goig GA, Zaragoza-Infante L, et al. Accuracy of an amplicon-sequencing nanopore approach to identify variants in tuberculosis drug-resistance-associated genes. Microb Genom. 2021;7(12):000740. doi:10.1099/
mgen.0.000740
7.

World Health Organization. The use of next-generation sequencing technologies for the detection of mutations associated with drug resistance in Mycobacterium tuberculosis complex: technical guide. https://apps.who.int/iris/handle/10665/274443. License: CC BY-NC-SA 3.0 IGO. Published 2018. Accessed
January 11, 2023.

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