Method for determining sensitivity of mycobacterium tuberculosis strains to pyrazinamide

Abstract

The invention relates to a method for determining the sensitivity of a mycobacterium tuberculosis strain to pyrazinamide. The method comprises the following steps: (i) determining whether at least one mutation exists in the rpoB gene between 761112 and 761182 in the genome of the strain; and (ii) determining the sensitivity or drug resistance property of the strain according to the presence or absence of the at least one mutation. The invention also relates to a kit comprising a means for detecting at least one sequence selected from SEQ ID NO: 1-3; the invention also relates to application of the kit in determining the sensitivity of the mycobacterium tuberculosis strain to pyrazinamide.

Description

Field of the Invention

[0001] The present invention relates to the field of molecular biology applied to bacterial genomics, and more particularly to the field of determining antibiotic resistance through genomic analysis. In particular, the present invention relates to a method for determining the resistance of Mycobacterium tuberculosis strains to a specific antibiotic, namely pyrazinamide. Background Art

[0002] Tuberculosis (TB) is a human respiratory infectious disease that affects nearly one-third of the world's population and is a global health problem.

[0003] Mycobacterium tuberculosis is the bacterium that causes tuberculosis. Although a vaccine is available, its effectiveness decreases over time, and infected patients usually need to be treated with antibiotics such as isoniazid, rifampicin, or pyrazinamide to control the disease.

[0004] It belongs to the same genus as Mycobacterium leprae (Mycobacterium leprae, also known as Hansen's bacillus) or the so-called atypical mycobacteria (Mycobacterium spp.), which was discovered by Robert Koch in 1882 and whose genome was sequenced in 1998.

[0005] In recent years, there have been reports of the emergence of multidrug-resistant Mycobacterium tuberculosis strains, which are highly resistant or completely resistant to antibiotics and other drugs.

[0006] Multidrug antibiotic resistance includes resistance of an organism to any first-line anti-TB drug (including isoniazid and / or rifampicin) and may also include resistance to any second-line drug (such as pyrazinamide).

[0007] Inadequate or delayed antibiotic treatment has negative consequences because it can exacerbate the problem of antibiotic resistance by promoting spontaneous mutations that favor resistant strains through selective pressure. Therefore, the development of rapid and accurate methods to detect the resistance spectrum of Mycobacterium tuberculosis is of global public health significance.

[0008] The sensitivity of a bacterial strain to an antibiotic—that is, the strain's sensitivity or resistance to an antibiotic when used to treat humans or animals—cannot be directly observed by humans. In fact, even with a microscope, direct observation of the strain cannot determine its response to an antibiotic.

[0009] In the context of bacterial testing, in vitro diagnostics essentially make this phenotypic characteristic observable and ultimately available to clinicians. In the 20th century, in vitro diagnostics primarily combined culture-based sample preparation techniques (particularly to allow visualization and manipulation of the bacterial strains present in the sample) with optical measurement of the strain's response in the presence of antibiotics.

[0010] For example, the workflow of a traditional microbiology laboratory typically involves: first, a sample taken from a patient suspected of a bacterial infection is plated onto a culture medium. After incubation, bacterial colonies can be observed by a human operator or automated system. Second, when the colonies have grown large enough, a technician or automated system collects a colony, mixes it with different concentrations of antibiotics, and places the mixture into a device that measures the optical density of each mixture to infer the strain's sensitivity to the antibiotic. Because optical density reflects bacterial proliferation, it can clearly characterize bacterial sensitivity or resistance: an increase in optical density indicates that the bacteria can still proliferate in the presence of the antibiotic, meaning that the bacteria are resistant to the tested concentration of the antibiotic.

[0011] For example, document US Pat. No. 7,335,485 describes a method for determining the sensitivity of a microorganism to an antibiotic, wherein the microorganism is cultured in the presence of the antibiotic to be tested.

[0012] With the rapid global evolution of multidrug antibiotic resistance in the prokaryotic world—projected to cause more deaths than cancer by 2050—combinatorial sample preparation techniques and optical density-based measurement techniques have significant limitations. Depending on the chosen culture medium, some strains will grow while others will not, making these techniques unable to characterize the antibiotic susceptibility of all bacterial species. Furthermore, these techniques are extremely slow because they are based on time-consuming bacterial culture. Consequently, obtaining an antibiogram of bacteria can take at least 30 hours from the time the sample is collected. This delay prevents patients from receiving effective treatment, and physicians often begin by administering systemic cocktails of broad-spectrum antibiotics as first-line therapy. Beyond the impact on patients, this inappropriate and high-volume administration of antibiotics increases the selective pressure on multidrug-resistant bacteria, leading to their spread.

[0013] Therefore, traditional in vitro diagnostic technology is currently considered to be increasingly unsuitable for patient treatment, and to a certain extent has even become one of the reasons for the emergence of multidrug resistance.

[0014] In recent years, sensitive techniques such as mass spectrometry have been applied to the determination of antibiotic resistance, but these techniques still require the microorganisms to be cultured in the presence of the test antibiotics. Furthermore, in all of these techniques, each microorganism must be tested against a single antibiotic or a combination of antibiotics, which requires extensive, time-consuming, and tedious testing.

[0015] The maturity of molecular biology techniques (especially bacterial DNA and / or RNA characterization techniques such as polymerase chain reaction (PCR), DNA microarrays or sequencing) is bringing about a paradigm shift in the analysis of antibiotic resistance in the laboratory.

[0016] First, these techniques are more inclusive of bacterial species. For example, metagenomic techniques are able to process bacterial DNA in biological samples without being restricted to the bacterial species present in the sample. Second, these techniques aim to produce results within a few hours, with some techniques (such as PCR) even able to produce results within 20 minutes. On the other hand, molecular techniques used to characterize antibiotic susceptibility are based on genomic features that can characterize that sensitivity (presence / absence of genes, genetic mutations, predictive models, etc.).

[0017] Without limitation, in the microbiological process of treating patients suspected of bacterial infection, there are two technologies used to characterize bacterial DNA, namely PCR technology and whole genome sequencing (WGS) technology. Both processes start with the collection of patient biological samples, followed by the application of PCR or WGS technology, each of which will produce genomic feature results that can characterize the sensitivity to one or more antibiotics. Clinicians choose antibiotic treatment plans based on the results and administer them to patients. According to methods known to those skilled in the art, each molecular technology is traditionally used in the application of PCR (e.g., company's Before PCR ("nested" PCR) or sequencing (e.g., SBS-type sequencing (sequencing by synthesis) performed by Illumina's MiSeq platform), the collected samples need to be prepared.

[0018] For example, document WO2018 / 065830 describes a real-time quantitative PCR (qPCR) method for determining the antibiotic resistance spectrum of Mycobacterium tuberculosis.

[0019] Document CN101580879 describes a gene chip and method for detecting common genetic mutations in Mycobacterium tuberculosis, which can lead to resistance to antibiotics such as isoniazid, rifampicin, streptomycin, ethambutol or pyrazinamide.

[0020] Document IN201941006113 describes a method for detecting antibiotic resistance of a Mycobacterium tuberculosis strain from a clinical sample containing the strain, the method comprising the step of identifying single nucleotide variations (SNVs) from genes associated with antibiotic resistance of the strain.

[0021] More generally, in bacteria and humans, it is known that antibiotic resistance can be associated with genetic polymorphisms. Specific to genomic characterization, early approaches have been to identify previously identified antibiotic resistance markers in bacterial genomes, so-called “direct association” approaches. Although these approaches are effective when the genetic mechanisms of resistance are clear and simple, they also have significant shortcomings: the mechanisms of resistance are not fully understood in many species and for many antibiotics (e.g., reflected by incomplete databases), it is difficult to account for differences in the predictive power of markers, and the multifactorial nature of antibiotic susceptibility (e.g., epistasis, multiple mutation combinations, etc.).

[0022] To address these challenges, new approaches based on advanced computer technology (especially supervised machine learning techniques) can more effectively understand the genetic determinants of antibiotic susceptibility. The learning and application architecture can be summarized as follows:

[0023] A. For a set of training bacterial strains:

[0024] A.1 Sequence and phenotypically characterize each strain (e.g., measure its minimum inhibitory concentration and / or measure its susceptibility to one or more antibiotics—resistant, intermediate, or sensitive).

[0025] A.2 Based on genomic and phenotypic data, train a computer model for predicting antibiotic sensitivity. B. For a new strain whose sensitivity to an antibiotic in step (A.1) needs to be determined:

[0026] B.1 Sequence the strain;

[0027] B.2 Apply predictive computer models to their digitized genomes to determine their sensitivity.

[0028] For example, documents WO2021180768 and WO2021180771 in the name of the applicant describe such learning and prediction models.

[0029] Therefore, there is a need to develop new genomic signatures to enrich the arsenal of methods available for identifying antibiotic susceptibility, particularly pyrazinamide. Signatures are acquired through learning models, which are limited by the size and diversity of the genomic databases used to train them. Therefore, to determine antibiotic susceptibility with greater accuracy, it may be advantageous to use multiple signatures simultaneously. In this context, identifying new genomic signatures with sufficient performance (especially a specificity of at least 90%) for determining the susceptibility of Mycobacterium tuberculosis strains to pyrazinamide is of great importance. Summary of the Invention

[0030] After extensive research on the genome of Mycobacterium tuberculosis, the inventors have successfully identified a novel genomic signature that can be used to determine the susceptibility of strains of this species to pyrazinamide. Specifically, the susceptibility assay performed according to the present invention can identify whether strains of Mycobacterium tuberculosis are susceptible or resistant to pyrazinamide with over 90% specificity. Therefore, the present invention helps combat antibiotic resistance, particularly by avoiding the need for pyrazinamide treatment when strains are resistant.

[0031] Furthermore, mutations in the pncA gene are known to be associated with resistance to pyrazinamide in strains of Mycobacterium tuberculosis species. In contrast, and completely unexpectedly, the sensitivity or resistance of Mycobacterium tuberculosis strains to pyrazinamide can be determined by identifying the presence of mutations in genes not inherently associated with resistance to this antibiotic, such as the rpoB gene or the additional gyrA, embB, or katG genes.

[0032] Therefore, the first subject matter of the present invention relates to a method for determining the sensitivity of a Mycobacterium tuberculosis species strain to pyrazinamide, the method comprising the steps of determining whether there is at least one mutation between positions 761112-761182 of the rpoB gene in the genome of the strain, and determining the sensitivity or resistance of the strain based on the identified mutation.

[0033] Preferably, the method further comprises the following steps: determining whether there is at least one mutation between positions 1673413-1673454 of the fabG1 gene; optionally, determining whether there is at least one mutation between positions 7552-7582 of the gyrA gene; optionally, determining whether there is at least one mutation between positions 1165444-1165528 of the Rv1042c gene and its promoter region; optionally, determining whether there is at least one mutation between positions 1277873-1277957 of the Rv1149 gene and its promoter region; optionally, determining whether there is at least one mutation between positions 4247581-4247622 of the embB gene; optionally, determining whether there is at least one mutation between positions 2288853-2289239 of the pncA gene; and finally, optionally, determining whether there is at least one mutation between positions 2155164-2155205 of the katG gene.

[0034] Another subject of the present invention relates to a kit comprising means for detecting and / or amplifying at least one sequence selected from SEQ ID NOs: 1-3 (optionally, further comprising at least one other sequence selected from SEQ ID NOs: 4-12) in the genome of a Mycobacterium tuberculosis species strain, preferably in the rpoB gene of said strain; and also relates to the use of said kit in determining the sensitivity of said strain to pyrazinamide.

[0035] Since the method of the invention can be implemented by a computer, another final subject matter relates to a data processing device comprising:

[0036] (a) a tool for performing the method of the invention, in particular a tool for determining the presence of a mutation between positions 761112 and 761182 of the rpoB gene of a Mycobacterium tuberculosis strain, and / or a tool for comparing the sequence of said gene with the Mycobacterium tuberculosis H37Rv reference genome (reference number NC_000962.3), and / or a tool for identifying the presence of the sequences of SEQ ID NOs: 1 to 3 in the genome of a Mycobacterium tuberculosis strain, and / or a tool for providing output data regarding the presence or absence of said sequences when said tool is implemented or controlled by a computer; or

[0037] (b) A processor suitable for or configured to perform the computer-implemented method according to claim 16, in particular a processor suitable for or configured to perform said method steps. Detailed Description of the Invention

[0039] Pyrazinamide, a nicotinamide derivative, is also an antituberculosis drug. When used in vivo at the prescribed dose, it has a bactericidal effect against intracellular tuberculosis bacilli (which are then in an acidic environment, a prerequisite for pyrazinamide's effectiveness). Therefore, pyrazinamide is used in the management of tuberculosis. Mycobacterium bovis and atypical mycobacteria are naturally resistant to pyrazinamide. In contrast, Mycobacterium tuberculosis and its highly similar strain, Mycobacterium africanum, are generally susceptible to pyrazinamide.

[0040] As mentioned above, inadequate or delayed antibiotic treatment has negative consequences: it can exacerbate the problem of antibiotic resistance by promoting spontaneous mutations that favor resistant strains through selective pressure.

[0041] Therefore, in the management of tuberculosis patients, it is important to ensure that the strain of M. tuberculosis infecting the patient is not resistant to pyrazinamide.

[0042] Therefore, the first subject matter of the present invention relates to a method for determining the sensitivity of a Mycobacterium tuberculosis species strain to pyrazinamide, comprising the steps of determining the presence of at least one mutation between positions 761112-761182 of the rpoB gene in the genome of the strain, and determining the sensitivity or resistance of the strain based on the presence or absence of the mutation in the gene.

[0043] As previously mentioned, it was completely unexpected that the methods of the present invention were able to determine sensitivity to pyrazinamide by identifying mutations in genes that are not inherently associated with pyrazinamide resistance but are instead associated with resistance to other antibiotics. In fact, identification of mutations in the rpoB gene is often associated with resistance to rifamycins and their derivatives, such as rifampicin.

[0044] Unless otherwise specifically defined, the technical and scientific terms used in this specification have the same meanings as commonly understood by those skilled in the art to which this specification belongs.

[0045] The term "determining sensitivity" refers to determining the sensitivity or resistance of a strain to pyrazinamide.

[0046] In the remainder of this specification, the term "strain" refers to a strain of the species Mycobacterium tuberculosis unless the context clearly indicates that another species is being referred to.

[0047] Identify the presence of at least one mutation in the rpoB gene

[0048] The term "mutation" refers to a variation in a sequence relative to a reference sequence. For example, such a reference sequence may be a sequence determined in a primary wild-type organism, or a sequence determined in a reference organism such as a specific and known bacterial strain. A mutation is, for example, a deletion of one or more nucleotides, an insertion of one or more nucleotides, a substitution of one or more nucleotides, a duplication of one or more nucleotide sequences, a translocation of one or more nucleotide sequences, and in particular a single nucleotide polymorphism (SNP).

[0049] The genome of Mycobacterium tuberculosis was completely sequenced in 1998. This species possesses a circular chromosome of 4,411,529 base pairs in length (GC% = 65.6) containing 3,924 genes.

[0050] In the context of the present invention, the presence of mutations in one or more genes of a strain is determined with reference to the wild-type reference genome of Mycobacterium tuberculosis (i.e., Mycobacterium tuberculosis H37Rv). This wild-type reference genome is indexed in the NCBI database, specifically by the reference number NC_000962.3. Therefore, all genomic positions mentioned in this specification are given with reference to the NC_000962.3 genome.

[0051] The comparison can be performed according to methods known to the person skilled in the art for comparing two genomes (or more precisely, comparing specific genomic positions in two genomes), in particular by bioinformatics methods.

[0052] Therefore, according to the present invention, referring to the wild-type genome of Mycobacterium tuberculosis H37Rv, determining that there is at least one mutation between positions 761112-761182 of the rpoB gene in the strain genome can identify the sensitivity or resistance of the strain to pyrazinamide.

[0053] In the present invention, identifying or determining the sensitivity or drug resistance of a Mycobacterium tuberculosis strain should be understood as a prediction of the sensitivity or drug resistance with a certain associated error.

[0054] The presence of at least one mutation in one or more genes of a Mycobacterium tuberculosis strain can be determined using methods known to those skilled in the art, such as hybridization, amplification, or sequencing.

[0055] In one embodiment, hybridization techniques (preferably using hybridization microchips or using techniques such as The presence of the mutation can be determined by using amplification techniques (preferably PCR or qPCR) or sequencing techniques (preferably high-throughput sequencing or synthetic sequencing).

[0056] In a preferred embodiment, the method further comprises, before the step of determining the presence of mutations in the genes of the Mycobacterium tuberculosis strain, a preliminary step of characterizing the DNA of the strain, which is performed according to methods known to those skilled in the art, preferably by PCR or sequencing (e.g., WGS).

[0057] The method may further comprise the preliminary step of obtaining a biological sample from the subject, said sample potentially containing at least one strain of Mycobacterium tuberculosis species.

[0058] In a specific embodiment, the step of determining the presence of at least one mutation in the rpoB gene comprises determining the presence of at least one mutation between positions 761151-761182, and / or between positions 761112-761142, and / or between positions 761138-761173.

[0059] In a preferred variant of this embodiment, the step of determining the presence of at least one mutation in the rpoB gene of the strain comprises determining the presence of at least one sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, preferably SEQ ID NO: 2, and more preferably SEQ ID NO: 1-3. According to the method of the present invention, the sensitivity or resistance of the strain is determined based on the presence or absence of mutations between the aforementioned positions (particularly based on the presence or absence of one or more sequences selected from SEQ ID NO: 1-3). Table 1 of the exemplary embodiment summarizes various conclusions regarding the sensitivity or resistance of the strain based on the presence or absence of the aforementioned sequences.

[0060] Identify the presence of at least one mutation in one or more additional genes

[0061] The performance of the method of the invention may be improved by combining the determination of the presence of mutations in one or more additional genes of the M. tuberculosis strain.

[0062] Therefore, in a specific embodiment, the method further comprises the step of determining that there is at least one mutation in the fabG1 gene between positions 1673413-1673454, preferably between positions 1673415-1673454 and / or between positions 1673413-1673444 in the genome of the strain.

[0063] In a preferred variant of this embodiment, determining the presence of at least one mutation in the fabG1 gene between the above-mentioned genomic positions advantageously comprises determining the presence of SEQ ID NO: 4 and / or SEQ ID NO: 5, preferably both.

[0064] Advantageously, the method of the present invention thus includes the step of determining the presence of at least one mutation in the rpoB and fabG1 genes in the genome of a Mycobacterium tuberculosis strain, comprising determining the presence of sequences SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 in the rpoB gene, and SEQ ID NO:4 and SEQ ID NO:5 in the fabG1 gene, and determining susceptibility or drug resistance based on the presence or absence of these sequences. Table 2 of the exemplary embodiment summarizes various conclusions drawn regarding the susceptibility or drug resistance of the strain based on the presence or absence of these sequences.

[0065] In another specific embodiment, the method of the present invention further comprises the step of determining the presence of at least one mutation in the gyrA gene between positions 7552-7582 in the genome of the strain. In a preferred variant of this embodiment, determining the presence of at least one mutation in the gyrA gene between said genomic positions advantageously comprises determining the presence of the sequence of SEQ ID NO: 6.

[0066] Therefore, advantageously, the method of the present invention includes the steps of determining the presence of at least one mutation in the rpoB, fabG1, and gyrA genes in the genome of a Mycobacterium tuberculosis strain, comprising determining the presence of sequences of SEQ ID NOs: 1-6 in each of these genes, and determining the susceptibility or resistance of the strain based on the presence or absence of these sequences. Table 3 of the exemplary embodiment summarizes various conclusions regarding the susceptibility or resistance of the strain based on the presence or absence of these sequences.

[0067] In another embodiment, the method of the present invention further comprises the following steps: determining that there is at least one mutation between positions 1165444-1165528 of the Rv1042c gene and its promoter region in the strain genome, and / or at least one mutation between positions 1277873-1277957 of the Rv1149 gene and its promoter region.

[0068] Preferably, in this embodiment, the method comprises the following steps: determining that there is at least one mutation in the Rv1042c gene and its promoter region between positions 1165444-1165492 and / or between positions 1165497-1165528, and determining that there is at least one mutation in the Rv1149 gene and its promoter region between positions 1277873-1277904 and / or between positions 1277909-1277957.

[0069] In a first variant of this embodiment, the method comprises the steps of determining the presence of at least one mutation in the Rv1042c gene between positions 1165444 and 1165492, and at least one mutation in the Rv1149 gene between positions 1277909 and 1277957. In this variant, determining the presence of at least one mutation in said genes advantageously comprises determining the presence of the sequence of SEQ ID NO: 7.

[0070] In a second variant of this embodiment, the method comprises the steps of determining the presence of at least one mutation in the Rv1042c gene and its promoter region at positions 1165497-1165528, and at least one mutation in the Rv1149 gene and its promoter region at positions 1277873-1277904. In this variant, determining the presence of at least one mutation in said genes advantageously comprises determining the presence of the sequence of SEQ ID NO:8.

[0071] In a third preferred variant of this embodiment, determining the presence of at least one mutation between the Rv1042c and / or Rv1149 genes and their respective promoter regions at said genomic locations advantageously comprises determining the presence of sequence SEQ ID NO: 7 and / or sequence SEQ ID NO: 8, preferably both.

[0072] Therefore, in another preferred embodiment, the method of the present invention includes the step of determining the presence of at least one mutation in the rpoB, fabG1, gyrA, Rv1042c, and Rv1149 genes in the genome of a Mycobacterium tuberculosis strain, comprising determining the presence of a sequence of SEQ ID NOs: 1-8 in each of said genes, and determining the susceptibility or drug resistance of the strain based on the presence or absence of said sequence. Table 4 of the exemplary embodiment summarizes various conclusions regarding the susceptibility or drug resistance of the strain based on the presence or absence of said sequence.

[0073] In another embodiment, the method of the present invention further comprises the step of determining the presence of at least one mutation in the embB gene between positions 4247581-4247622 in the genome of the strain. Preferably, in this embodiment, determining the presence of at least one mutation in the embB gene between said genomic positions advantageously comprises determining the presence of the sequence SEQ ID NO:9.

[0074] Therefore, in another preferred embodiment, the method of the present invention includes the step of determining the presence of at least one mutation in the rpoB, fabG1, gyrA, Rv1042c, Rv1149, and embB genes in the genome of a Mycobacterium tuberculosis strain, comprising determining the presence of a sequence of SEQ ID NOs: 1-9 in each of said genes, and determining the susceptibility or drug resistance of the strain based on the presence or absence of said sequence. Table 5 of the exemplary embodiment summarizes various conclusions regarding the susceptibility or drug resistance of the strain based on the presence or absence of said sequence.

[0075] In another embodiment, the method of the present invention further comprises the step of determining that there is at least one mutation in the pncA gene between positions 2288853-2289239, preferably between positions 2288853-2288885 and / or between positions 2289209-2289239 in the genome of the strain.

[0076] In a preferred variant of this embodiment, determining the presence of at least one mutation in the pncA gene between said genomic positions advantageously comprises determining the presence of the sequence SEQ ID NO: 10 and / or the sequence SEQ ID NO: 11.

[0077] Therefore, in another preferred embodiment, the method of the present invention includes the step of determining the presence of at least one mutation in the rpoB, fabG1, gyrA, Rv1042c, Rv1149, embB, and pncA genes in the genome of a Mycobacterium tuberculosis strain, comprising determining the presence of a sequence of SEQ ID NOs: 1-11 in each of said genes, and determining the susceptibility or drug resistance of the strain based on the presence or absence of said sequence. Table 6 of the exemplary embodiment summarizes various conclusions regarding the susceptibility or drug resistance of the strain based on the presence or absence of said sequence.

[0078] In another embodiment, the method of the present invention further comprises the step of determining that there is at least one mutation between positions 2155164-2155205 of the katG gene in the genome of the strain.

[0079] In a preferred variant of this embodiment, determining the presence of at least one mutation in the katG gene between said genomic positions advantageously comprises determining the presence of the sequence SEQ ID NO:12.

[0080] In another preferred embodiment, the method for determining the susceptibility of a Mycobacterium tuberculosis species strain to pyrazinamide comprises the following steps:

[0081] - Determine whether there is at least one mutation between positions 761112-761182 in the rpoB gene;

[0082] - Determine whether there is at least one mutation between positions 1673413-1673454 in the fabG1 gene;

[0083] - Determine whether there is at least one mutation between positions 7552-7582 in the gyrA gene;

[0084] - Determining the presence of at least one mutation in the genomic region between positions 1165444-1165492 and / or between positions 1165497-1165528 of the Rv1042c gene, and determining the presence of at least one mutation in the genomic region between positions 1277909-1277957 and / or between positions 1277873-1277904 of the Rv1149 gene;

[0085] - Determine whether there is at least one mutation between positions 4247581-4247622 in the embB gene;

[0086] - Determine whether there is at least one mutation between positions 2288853-2289239 in the pncA gene;

[0087] - determining whether there is at least one mutation between positions 2155164-2155205 in the katG gene; and

[0088] - Determining the sensitivity or resistance of the strain based on the presence or absence of the mutation.

[0089] In another preferred embodiment, the method of the present invention comprises the following steps: (a) determining the presence of at least one mutation in the rpoB, fabG1, gyrA, Rv1042c, Rv1149, embB, pncA, and katG genes in the genome of a Mycobacterium tuberculosis strain, the step comprising determining the presence of the sequences of SEQ ID NOs: 1, 4, 6, 7, 9, 10, and 12, preferably SEQ ID NOs: 1-12, in each of the genes; and (b) determining the susceptibility or drug resistance of the strain based on the presence or absence of the sequences. Table 7 of the exemplary embodiment summarizes various conclusions drawn about the susceptibility or drug resistance of the strain based on the presence or absence of the sequences.

[0090] In a particular embodiment, prior to determining the presence of at least one mutation in the rpoB gene and optionally in additional genes, the method may comprise the step of obtaining the genome of said M. tuberculosis strain.

[0091] In the present specification, the term "subject" refers to a human being, and the subject is preferably a patient.

[0092] Thus, the method of the present invention may be an in vitro or ex vivo method for determining the susceptibility of a Mycobacterium tuberculosis species strain to pyrazinamide from a biological sample of a subject that may contain the strain, the method comprising the following steps:

[0093] - determining that there is at least one mutation between positions 761112-761182 in the rpoB gene in the genome of the strain; and

[0094] - Determining the sensitivity or resistance of the strain based on the presence or absence of the mutation.

[0095] Of course, the specific or preferred embodiments of the present invention described above also apply to the in vitro or ex vivo method that is one of the subjects of the present invention.Thus, at least one mutation in one of the additional genes described above may also be determined.

[0096] As used herein, "biological sample" refers to any sample from a subject, which may be of different nature, such as blood or its derivatives or sputum, in which traces of the strain may be found.

[0097] In a specific embodiment, the biological sample is a sputum sample, a blood sample or a blood-derived sample, wherein the blood-derived sample can be selected from whole blood (e.g., collected venously, i.e., containing white blood cells, red blood cells, platelets and plasma), plasma and serum.

[0098] Kits for implementing the methods of the present invention

[0099] To implement the method according to any of the above embodiments, a kit can be prepared. Specifically, another subject of the present invention relates to a kit capable of detecting and / or amplifying at least one sequence selected from the group consisting of SEQ ID NOs: 1 to 3. Advantageously, the kit comprises operating instructions.

[0100] Thus, the kit comprises means capable of detecting and / or amplifying and / or quantifying said sequence (e.g. in a biological sample suspected of containing a strain of Mycobacterium tuberculosis species). These means may be primers and / or probes specific for said sequence.

[0101] In a preferred embodiment, the kit comprises means for detecting the sequences SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3. Advantageously, the kit comprises all the means required for detecting the sequences SEQ ID NO: 1-3 by amplification, preferably by PCR, in particular by qPCR.

[0102] In a specific embodiment, the kit further comprises means, preferably primers and / or probes, capable of detecting at least one of the sequences SEQ ID NOs: 4-12 in a strain of Mycobacterium tuberculosis species. Preferably, the kit comprises primers and / or probes for detecting all of the sequences SEQ ID NOs: 4-12.

[0103] All specific or preferred embodiments described above in connection with the method according to the invention apply equally to the kit which is one of the subjects of the present invention.

[0104] A "primer" or "amplification primer" refers to a nucleotide fragment, which may be composed of 5-100 nucleotides, preferably 10-20 nucleotides, that has hybridization specificity with a target nucleotide sequence (i.e., one of the sequences SEQ ID NOs: 1-12) under conditions determined for initiating an enzymatic polymerization reaction (e.g., in an enzymatic amplification reaction of the target nucleotide sequence). Typically, a "primer pair" consisting of two primers is used. When it is desired to amplify multiple different biomarkers (e.g., from different genes), it is preferred to use multiple different primer pairs, each pair of primers having the ability to specifically hybridize preferentially with a different biomarker.

[0105] A person skilled in the art can therefore use the sequences of SEQ ID NOs: 1-12 to determine the primers and probes required for amplification.

[0106] "Probe" or "hybridization probe" refers to a nucleotide fragment, usually consisting of 5-100 nucleotides, preferably 10-90 nucleotides, more preferably 15-35 nucleotides, which has hybridization specificity under conditions determined for forming a hybridization complex with a target nucleotide sequence. The probe also contains a reporter molecule (such as a fluorophore, an enzyme or any other detection system) that enables detection of the target nucleotide sequence. In the present invention, the target nucleotide sequence is a sequence of SEQ ID NO: 1-3, preferably a sequence of SEQ ID NO: 1-12. Therefore, it is preferred to use a plurality of different probes, each probe preferably having the ability to specifically hybridize with a target sequence.

[0107] "Hybridization" refers to the process in which two nucleotide fragments (such as a hybridization probe and a target nucleotide fragment) form a double-stranded chain with stable and specific hydrogen bonds under appropriate conditions due to sufficient sequence complementarity. A nucleotide fragment that is "capable of hybridizing" with a polynucleotide refers to a fragment that can hybridize with the polynucleotide under hybridization conditions, and the hybridization conditions can be determined one by one in a manner known in the art. Hybridization conditions are determined by stringency (i.e., the stringency of the operating conditions). The higher the stringency of the hybridization reaction, the stronger the specificity of the hybridization. The definition of stringency is particularly based on the base composition of the probe / target double chain, and also depends on the degree of mismatch between the two nucleic acids. Stringency can also be based on reaction parameters, such as the concentration and type of ionic species in the hybridization solution, the nature and concentration of the denaturant, and / or the hybridization temperature. The stringency of the conditions required for the hybridization reaction depends primarily on the hybridization probe used. All of this information is known in the art, and those skilled in the art can determine appropriate conditions.

[0108] Typically, depending on the length of the hybridization probe used, the hybridization reaction temperature is about 20-70° C., especially 35-65° C., in a salt solution with a concentration of about 0.5-1 M. This is followed by a step of detecting the hybridization reaction.

[0109] The probes or primers that can be used in the methods of the present invention are typically short nucleic acid molecules, such as DNA oligonucleotides of 10 nucleotides or longer in length, which can be linked to complementary target nucleic acid molecules through nucleic acid hybridization, thereby forming a hybrid between the primer or probe and the target nucleic acid chain.

[0110] The probe or primer can be unlabeled or labeled (eg, with a FRET-like donor or acceptor) so that its binding to the target sequence can be detected.

[0111] Under the action of polymerase, the primer can be extended along the target nucleic acid molecule.

[0112] Therefore, the primers can be used to amplify a target nucleic acid molecule, such as one of the sequences SEQ ID NO: 1-12 and / or variant sequences thereof.

[0113] The specificity of a probe or primer increases with its length. For example, a probe or primer comprising 30 consecutive nucleotides will bind to a target sequence with greater specificity than a corresponding primer comprising only 15 nucleotides. Therefore, to achieve higher specificity, probes and primers comprising at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 or more consecutive nucleotides can be selected.

[0114] In a specific example, the primer is at least 15 nucleotides long, for example, at least 15 consecutive nucleotides complementary to the target nucleic acid molecule. Specific primer lengths that can be used to practice the methods of the present invention include primers having at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70 or more consecutive nucleotides complementary to the target nucleic acid molecule to be amplified, for example, 15-70 nucleotides, 15-60 nucleotides, 15-50 nucleotides, or 15-30 nucleotides.

[0115] A "sense" (or forward) primer refers to a primer located 5' relative to a reference point on a nucleic acid sequence. An "antisense" (or reverse) primer refers to a primer located 3' relative to a reference point on a nucleic acid sequence. Typically, an amplification reaction includes at least one sense primer and one antisense primer (i.e., a "primer pair").

[0116] Nucleic acid probes and primers or primer pairs can be readily prepared based on any of the nucleic acid sequences in SEQ ID NOs: 1 to 12. PCR primer pairs can be derived from the sequences using specialized computer programs (e.g., Primer 3, version 0.4.0, Whitehead Institute for Biomedical Research, Steve Rozen and Helen Skaletsky).

[0117] In a specific embodiment, the sequences of SEQ ID NOs: 1-12 or amplified or converted products thereof (eg, cDNA) are detected by specific hybridization of a nucleic acid probe.

[0118] These probes can also be immobilized on a solid surface (e.g., nitrocellulose membrane, glass, quartz, fused silica slide), for example, on an array, microarray, or DNA chip. Those skilled in the art will appreciate that the precise sequence of specific probes and primers can be modified to some extent based on the target sequence to produce probes that are "substantially identical" or "substantially complementary" to the target sequence while retaining the ability to specifically bind (i.e., specifically hybridize) to the same target sequence from which it is derived.

[0119] Throughout this specification, the terms "capable of hybridizing to" and "specifically binding" are used interchangeably to refer to a polynucleotide sequence capable of forming a Watson-Crick bond with a complementary sequence. Those skilled in the art will appreciate that, depending on the length of the polynucleotide, the length of the region of complementarity, and the stringency of the reaction conditions, hybridization or specific binding does not necessarily require 100% complementarity. For example, a primer or probe may be at least 60%, 70%, 80%, 90%, 95%, 99%, or 100% complementary along the length of the region of complementarity.

[0120] Another subject of the present invention relates to the use of the kit defined above for determining the sensitivity of a Mycobacterium tuberculosis strain to pyrazinamide.

[0121] Another subject of the present invention relates to the use of sequences SEQ ID NOs: 1 to 3, preferably also sequences SEQ ID NOs: 4 to 12, for determining the sensitivity of Mycobacterium tuberculosis strains to pyrazinamide.

[0122] Of course, the specific or preferred embodiments described in connection with the method according to the invention apply also to these applications which are one of the subjects of the present invention.

[0123] Computer implementation

[0124] The method described in any of the detailed embodiments can certainly be implemented by a computer. In this case, the method can be performed in part or in whole by computer means.

[0125] To this end, the present invention further provides a data processing device, comprising:

[0126] (a) a means for performing the computer-implemented method of the present invention, specifically comprising a means for determining the presence of a mutation in the rpoB gene of a Mycobacterium tuberculosis strain and / or a means for comparing the sequence of said gene with the reference genome of Mycobacterium tuberculosis H37Rv (reference number NC_000962.3), and / or a means for identifying the presence of the sequences SEQ ID NOs: 1-3 in the genome of a Mycobacterium tuberculosis strain, and / or a means for providing output data regarding the presence or absence of said sequences when said means are implemented or controlled by a computer;

[0127] (b) A processor suitable for or configured to perform the computer-implemented method of the present invention, in particular a processor suitable for or configured to perform each step of the computer-implemented method of the present invention.

[0128] Advantageously, the processing device may also include means for determining the presence of mutations in the fabG1, gyrA, Rv1042c, Rv1149, embB, pncA and katG genes at the above-defined positions, and / or means for identifying the presence of sequences SEQ ID NO: 4-12 in each of the relevant genes.

[0129] In a specific embodiment, such data processing equipment comprises:

[0130] (a) Input interface: for receiving the genome of the Mycobacterium tuberculosis strain to be tested for sensitivity to pyrazinamide;

[0131] (b) a memory for storing at least instructions for a computer program, which, when executed by a computer or a processor, is capable of performing a determination on the presence of a mutation in the rpoB gene at the aforementioned position, and optionally on the presence of a mutation in the aforementioned one or more additional genes;

[0132] (c) a processor configured to access a memory to read the instructions and execute the computer-implemented method of the present invention;

[0133] (d) Output interface: used to provide output values, especially output values ​​corresponding to the conclusion of the strain's sensitivity or resistance to pyrazinamide.

[0134] Other topics in the manual

[0135] Another subject of the present specification relates to a method for treating a subject infected with a strain of Mycobacterium tuberculosis species, comprising the steps of determining the susceptibility of the strain to pyrazinamide by any of the above methods, and treating the subject with pyrazinamide after reaching a conclusion on the susceptibility of the strain.

[0136] Specifically, once conclusions about sensitivity have been reached, processing can begin.

[0137] Another subject of this specification relates to a method comprising the following steps:

[0138] - Obtaining biological samples from subjects infected with Mycobacterium tuberculosis species strains;

[0139] - contacting the biological sample with a detection tool or a specific reagent, wherein the detection tool or the specific reagent is capable of detecting a mutation between positions 761112-761182 of the rpoB gene of the strain; and

[0140] - Determine the presence of the sequences SEQ ID NO: 1-3.

[0141] The reagents specific for the expression products are selected from amplification primers and hybridization probes, as defined above, and can in particular be used to determine the presence of the sequences SEQ ID NO: 1-3.

[0142] Preferably, the biological sample is also contacted with tools for determining the presence of mutations in the fabG1, gyrA, Rv1042c, Rv1149, embB, pncA and katG genes at the above-defined positions, these tools being particularly capable of identifying the presence of sequences SEQ ID NOs: 4-12 in each of the relevant genes.

[0143] The following examples illustrate the present invention in a non-limiting manner. Example

[0144] Example 1: Determination of the sensitivity of Mycobacterium tuberculosis strains to pyrazinamide

[0145] 1. Obtain the model

[0146] A set of 3606 Mycobacterium tuberculosis genomes, representing strains resistant (R) or susceptible (S) to pyrazinamide, was used. Of these genomes, 3038 were used to train supervised machine learning models (training set) and 568 were used to evaluate the performance of these models (validation set).

[0147] Of the 568 genomes in the validation set, 133 were resistant to pyrazinamide and 435 were sensitive. The table below shows, for each model, the presence of each wild-type sequence in these validation genomes. Sequences are named after the gene to which they belong and are sorted by descending coefficient value in the model (absolute values ​​are given in parentheses).

[0148] The table below shows the different haplotypes, defined as the observed combinations of mutant or non-mutant sequences. Haplotypes are sorted by decreasing prevalence (prevalence refers to the percentage observed in the 568 genomes).

[0149] For each haplotype, the prediction made by the model is recorded, along with the number of resistant and sensitive genomes observed for that haplotype. This allows the identification of the number of correct and incorrect predictions made by the model in the validation set.

[0150] These errors are divided into "very serious errors (VME)" and "serious errors (ME)". "Very serious errors" correspond to the model predicting the resistant genome as sensitive (i.e., false negatives); "serious errors" correspond to the model predicting the sensitive genome as resistant (i.e., false positives).

[0151] Legend for Tables 1 to 6: NoRS = number of resistant strains; NoSS = number of susceptible strains; Prev = prevalence; Pred = prediction; 1 = sequence present; 0 = sequence not present.

[0152] 2. Application of the model for determining sensitivity to pyrazinamide

[0153] 2.1 rpoB model

[0154] Table 1 below shows the sensitivity results by demonstrating the presence or absence of mutations in the rpoB gene of the strain being detected.

[0155] Table 1

[0156]

[0157]

[0158] Interpretation of the results

[0159] In this model, the predominant haplotype is the one observed to contain the sequences SEQ ID NOs: 1-3. This haplotype was observed in 398 genomes (391+7), representing 70.07% of the 568 validated genomes. Therefore, confirming the presence of the sequences SEQ ID NOs: 1-3 correctly determined the sensitivity (or prediction) for the 391 genomes, but with 7 VMEs.

[0160] In contrast, the haplotype in which SEQ ID NOs: 1-3 are absent is a minor haplotype, observed in only one genome, accounting for 0.18% of the validated genome. Therefore, the absence of SEQ ID NOs: 1-3 can be used to correctly determine (or predict) drug resistance, without any ME or VME.

[0161] Model performance

[0162] Overall, the model could predict the susceptibility or resistance of Mycobacterium tuberculosis strains based on the presence or absence of sequences SEQ ID NO: 1-3 with a specificity of 88.72%, a sensitivity of 93.56%, and an error rate of 7.57% (ME and VME).

[0163] 2.2 rpoB-fabG1 model

[0164] Table 2 below shows the results of the sensitivity test by demonstrating the presence or absence of mutations in the rpoB and fabG1 genes of the strains tested.

[0165] Table 2

[0166] Interpretation of the results

[0167] In this model, the predominant haplotype is the one observed to contain the sequences SEQ ID NOs: 1-5. This haplotype was observed in 365 genomes (359 + 6), representing 64.26% of the 568 validated genomes. Therefore, determining the presence of the sequences SEQ ID NOs: 1-5 correctly determined the sensitivity (or prediction) for 359 genomes, but with 6 VMEs.

[0168] Model performance

[0169] Overall, the model could predict the susceptibility or resistance of Mycobacterium tuberculosis strains based on the presence or absence of sequences SEQ ID NO: 1-5 with a specificity of 90.23%, a sensitivity of 93.56%, and an error rate of 7.21% (ME and VME).

[0170] 2.3 rpoB-fabG1-gyrA model

[0171] Table 3 below shows the sensitivity results of detecting mutations in the rpoB, fabG1 and gyrA genes of the strains by demonstrating the presence or absence of sequences SEQ ID NOs: 1-6.

[0172] Table 3

[0173] Interpretation of the results

[0174] In this model, the predominant haplotype is the one in which the presence of SEQ ID NOs: 1-6 was observed. This haplotype was observed in 362 genomes (357 + 5), representing 63.73% of the 568 validated genomes. Therefore, determining the presence of SEQ ID NOs: 1-6 correctly determined the sensitivity of the 357 genomes, but with 5 VMEs.

[0175] Model performance

[0176] Overall, the model could predict the susceptibility or resistance of Mycobacterium tuberculosis strains based on the presence or absence of sequences SEQ ID NO: 1-6 with a specificity of 94.25%, a sensitivity of 88.72%, and an error rate of 7.04% (ME and VME).

[0177] 2.4 rpoB-fabG1-gyrA-Rv1042c / Rv1149 model

[0178] Table 4 below shows the sensitivity results by demonstrating the presence or absence of mutations in the rpoB, fabG1, gyrA, Rv1042c and Rv1149 genes of the strains.

[0179] Table 4

[0180]

[0181]

[0182] Interpretation of the results

[0183] In this model, the major haplotype is the one observed with the presence of sequences SEQ ID NOs: 1-6 and SEQ ID NO: 7 and the absence of sequence SEQ ID NO: 8. This haplotype was observed in 187 genomes (186+1), representing 32.92% of the 568 validated genomes.

[0184] Thus, determining the presence of sequences SEQ ID NOs: 1-6 and SEQ ID NO: 7 and the absence of sequence SEQ ID NO: 8 allowed correct calls (or predictions) of "sensitivity" to be made for 186 genomes, with only 1 VME.

[0185] Similarly, the haplotype with the presence of SEQ ID NOs: 1-6 and the absence of SEQ ID NOs: 7 and 8 was observed in 86 genomes, representing 14.14% of the validated genomes. Determining the presence or absence of these sequences allowed for a "sensitive" prediction in 85 genomes, with only one VME present.

[0186] Model performance

[0187] Overall, the model could predict the susceptibility or resistance of Mycobacterium tuberculosis strains based on the presence or absence of sequences SEQ ID NO: 1-8 with a specificity of 88.72%, a sensitivity of 94.71%, and an error rate of 6.69% (ME and VME).

[0188] 2.5rpoB-fabG1-gyrA-Rv1042c / Rv1149-embB model

[0189] Table 5 below shows the sensitivity results by demonstrating the presence or absence of mutations in the rpoB, fabG1, gyrA, Rv1042c, Rv1149 and embB genes of the strains.

[0190] Table 5

[0191]

[0192]

[0193]

[0194] Interpretation of the results

[0195] In this model, the major haplotype is the one observed with the presence of SEQ ID NOs: 1-6, 9, and 7, and the absence of SEQ ID NO: 8. This haplotype was observed in 186 genomes (185+1), representing 32.75% of the 568 validated genomes.

[0196] Therefore, determining the presence or absence of the sequence in the genome of M. tuberculosis strains allowed a correct determination (or prediction) of "sensitivity" for 185 genomes, with only 1 VME present.

[0197] Likewise, the presence of all sequences of SEQ ID NO: 1-9 was observed in 85 genomes, representing 14.96% of the validated genomes. The presence or absence of these sequences allowed a "sensitivity" prediction to be made for 82 genomes, with 3 VMEs present.

[0198] Model performance

[0199] Overall, the model can predict the sensitivity or resistance of Mycobacterium tuberculosis strains based on the presence or absence of sequences SEQ ID NO: 1-9 with a specificity of 87.22%, a sensitivity of 96.55%, and an error rate of 5.63% (ME and VME). 2.6rpoB-fabG1-gyrA-Rv1042c / Rv1149-embB-pncA-katG model

[0200] Table 6 below shows the sensitivity results by demonstrating the presence or absence of sequences SEQ ID NOs: 1-12 in detecting mutations in the rpoB, fabG1, gyrA, Rv1042c, Rv1149, embB, pncA and katG genes of the strains.

[0201]

[0202]

[0203]

[0204] Interpretation of the results

[0205] In this model, the major haplotype is the one observed with the presence of SEQ ID NOs: 1-6, 9-12, and SEQ ID NO: 7, and the absence of SEQ ID NO: 8. This haplotype was observed in 164 genomes (163+1), representing 28.87% of the 568 validated genomes.

[0206] Thus, determining the presence or absence of the sequence in the genome of M. tuberculosis strains allowed a correct call (or prediction) of "sensitivity" for 163 genomes, with only 1 VME present.

[0207] Likewise, the presence of all sequences of SEQ ID NO: 1-12 was detected in 75 genomes, representing 13.2% of the validated genomes. The presence or absence of these sequences allowed a "sensitivity" prediction for 72 genomes, with 3 VMEs present.

[0208] Model performance

[0209] Overall, the model could predict the susceptibility or resistance of Mycobacterium tuberculosis strains based on the presence or absence of sequences SEQ ID NO: 1-12 with a specificity of 88.72%, a sensitivity of 97.01%, and an error rate of 5.28% (ME and VME).

[0210] Example 2: Description of sequences used to implement the method of the invention

[0211]

Claims

1. A method for determining the sensitivity of a Mycobacterium tuberculosis species strain to pyrazinamide, the method comprising the following steps: - determining that there is at least one mutation between positions 761112-761182 in the rpoB gene in the genome of the strain; - Determining the sensitivity or resistance of the strain based on the presence or absence of the mutation.

2. The method according to claim 1, wherein The method also includes determining that there is at least one mutation between positions 1673413-1673454 of the fabG1 gene in the genome of the strain.

3. The method according to claim 1 or 2, wherein: The method also includes determining that there is at least one mutation between positions 7552-7582 of the gyrA gene in the genome of the strain.

4. The method according to any one of claims 1 to 3, wherein The method also includes determining that there is at least one mutation between positions 1165444-1165528 of the Rv1042c gene and its promoter region in the genome of the strain, and / or at least one mutation between positions 1277873-1277957 of the Rv1149 gene and its promoter region.

5. The method according to claim 4, wherein The method also includes the following steps: determining that there is at least one mutation between positions 1165444-1165492 of the Rv1042c gene and at least one mutation between positions 1277909-1277957 of the Rv1149 gene; and / or determining that there is at least one mutation between positions 1165497-1165528 of the Rv1042c gene and its promoter region and at least one mutation between positions 1277873-1277904 of the Rv1149 gene and its promoter region.

6. The method according to any one of claims 1 to 6, wherein The method further comprises the following steps: - determining that there is at least one mutation between positions 4247581-4247622 in the embB gene in the genome of the strain; and / or - determining that there is at least one mutation between positions 2288853-2289239 in the pncA gene of the strain genome; and / or - Determine that there is at least one mutation between positions 2155164-2155205 in the katG gene of the strain genome.

7. The method according to any one of claims 1 to 6, wherein Determining the presence of at least one mutation in the rpoB gene comprises determining the presence of at least one sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:

3.

8. The method according to any one of claims 2 to 7, wherein Determining the presence of at least one mutation in the fabG1 gene comprises determining the presence of at least one sequence selected from the group consisting of SEQ ID NO:4 and SEQ ID NO:

5.

9. The method according to any one of claims 3 to 8, wherein Determining the presence of at least one mutation in the gyrA gene includes determining the presence of the sequence SEQ ID NO:

6.

10. The method according to any one of claims 4 to 9, wherein Determining the presence of at least one mutation between genomic region 1165444-1165528 and / or at least one mutation between genomic region 1277873-1277957 comprises determining the presence of at least one sequence selected from the group consisting of SEQ ID NO:7 and SEQ ID NO:

8.

11. The method according to any one of claims 6 to 10, wherein: Determining the presence of at least one mutation in the embB gene comprises determining the presence of the sequence SEQ ID NO:

9.

12. The method according to any one of claims 6 to 11, characterized in that: - determining the presence of at least one mutation in the pncA gene, i.e., determining the presence of at least one of the sequences SEQ ID NO: 10 and SEQ ID NO: 11; - Determining the presence of at least one mutation in the katG gene, ie, determining the presence of the sequence SEQ ID NO:

12.

13. The method according to claim 12, wherein: The method comprises the following steps: determining the presence of the sequence SEQ ID NO: 1-12 in each gene, and determining the sensitivity or drug resistance of the strain according to the presence or absence of the sequence.

14. The method according to any one of claims 1 to 13, wherein The steps to determine the presence of mutations were all performed with reference to the Mycobacterium tuberculosis H37Rv wild-type reference genome (reference number NC_000962.3).

15. The method according to any one of claims 1 to 14, wherein The step of determining the presence of at least one mutation in said gene is performed by hybridization, amplification or sequencing methods.

16. The method according to any one of claims 1 to 15, wherein The method is implemented by a computer.

17. A data processing device comprising: - a means for carrying out the method according to claim 16, in particular a means for determining the presence of a mutation in the rpoB gene of a Mycobacterium tuberculosis strain between positions 761112-761182, and / or a means for comparing the sequence of said gene with the reference genome of Mycobacterium tuberculosis H37Rv (reference number NC_000962.3), and / or a means for identifying the presence of the sequences SEQ ID NO: 1-3 in the genome of a Mycobacterium tuberculosis strain, and / or a means for providing output data on the presence or absence of said sequences when said means is implemented or controlled by a computer; or - A processor adapted or configured to perform the computer-implemented method according to claim 16, in particular a processor adapted or configured to perform the steps of said method.

18. A kit comprising means for detecting at least one sequence selected from the group consisting of SEQ ID NOs: 1-3 and optionally means for detecting at least one further sequence selected from the group consisting of SEQ ID NOs: 4-12, said means preferably being primers and / or probes.

19. Use of the kit according to claim 18 in determining the sensitivity of Mycobacterium tuberculosis strains to pyrazinamide.

20. Use of sequences comprising SEQ ID NOs: 1 to 3, and preferably also comprising sequences comprising SEQ ID NOs: 4 to 12, in determining the sensitivity of Mycobacterium tuberculosis strains to pyrazinamide.

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