Method for determining the susceptibility of a strain of mycobacterium tuberculosis to pyrazinamide

Abstract

The invention relates to a method for determining the susceptibility of a strain of Mycobacterium tuberculosis to pyrazinamide, the method comprising the following steps: (i) determining the presence in the genome of this strain of at least one mutation in the rpoB gene between positions 761112 and 761182; and (ii) determining the sensitive or resistant nature of this strain depending on the presence or absence of the at least one mutation. The invention also relates to a kit comprising means for detecting at least one sequence chosen from among the sequences SEQ ID NO: 1 to 3 and to the use of this same kit for determining the susceptibility of a strain of Mycobacterium tuberculosis to pyrazinamide.

Description

[0001] METHOD FOR DETERMINING THE SUSCEPTIBILITY OF A STRAIN OF THE SPECIES MYCOBACTERIUM TUBERCULOSIS TO PYRAZINAMIDE

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to the technical field of molecular biology applied to bacterial genomics, and in particular to the field of determining resistance to antibiotics from the analysis of their genome. The invention particularly relates to a method for determining the resistance of a strain of the species Mycobacterium tuberculosis to a particular antibiotic, namely pyrazinamide.

[0004] TECHNOLOGICAL BACKGROUND

[0005] Tuberculosis (TB) is an infectious disease of the human respiratory tract that affects nearly one-third of the world's population and represents a global health problem.

[0006] Mycobacterium tuberculosis is the bacteria that causes tuberculosis, and although vaccines are available, their effectiveness diminishes over time, and infected patients are usually treated with antibiotics such as isoniazid, rifampicin, or pyrazinamide to control the disease.

[0007] Belonging to the genus of mycobacteria {Mycobacterium spp.) in the same way as the leprosy bacillus {Mycobacterium leprae or Hansen's bacillus), or the so-called atypical mycobacteria, it was discovered by Robert Koch in 1882 and its genome was sequenced in 1998.

[0008] In recent years, the development of multidrug-resistant, extensively drug-resistant, and completely drug-resistant Mycobacterium tuberculosis strains has been reported.

[0009] Multidrug resistance to antibiotics involves resistance of the microbial strain to any first-line anti-tuberculosis drug, including isoniazid and / or rifampicin, but may also involve resistance to any second-line drug, such as pyrazinamide.

[0010] Inadequate or delayed antibiotic therapy has a negative effect in that it promotes the selection of spontaneous mutations in favor of resistant strains through selection pressure, thus exacerbating the problem of antibiotic resistance. Therefore, the development of rapid and accurate detection of the resistance profile of Mycobacterium tuberculosis is of global public health interest.

[0011] The susceptibility of a bacterial strain to an antibiotic, that is, its sensitivity or resistance to treatment with the antibiotic administered to a human or animal, is not directly observable by a human being. Indeed, direct observation of the strain, even through microscopes, does not allow its behavior to be determined when faced with the antibiotic.

[0012] In vitro diagnosis in bacterial matters consists, by nature, in making this phenotypic character observable and therefore ultimately usable for a clinician. During the 20th century, in vitro diagnostic technologies essentially combined culture-based sample preparation techniques, in particular to make the bacterial strains present in the samples visible and manipulable, and optical measurement techniques of the behavior of the strains in the presence of an antibiotic.

[0013] For example, a typical workflow in a microbiological laboratory involves first spreading a sample taken from a patient suspected of having a bacterial infection on a culture medium in order to reveal, after incubation, bacterial colonies visible to a human operator or an automated system. Secondly, if the colonies are large enough, a technician or an automated system takes a colony, mixes it with an antibiotic at different concentrations and introduces the mixtures into a device that measures the optical density of each mixture and deduces the susceptibility to the antibiotic. Since optical density indicates bacterial proliferation, it unambiguously characterizes the sensitivity or resistance of the bacteria: if the density increases, this means that it proliferates despite the presence of the antibiotic, and therefore that it is resistant to it at the antibiotic concentration considered.

[0014] For example, document US7335485 describes a method for determining the susceptibility of a microorganism to an antibiotic, in which said microorganism is cultured in the presence of the antibiotic to be tested.

[0015] The combination of sample preparation technologies and optical density-based optical measurement technologies shows significant limitations in the face of rapid global evolution in the prokaryotic kingdom, namely the acquisition of multi-resistance to antibiotics, multi-resistance which is estimated to cause more deaths than cancer by 2050. Depending on the culture medium chosen, some strains will grow and others will not, so these technologies do not allow us to characterize the antibiotic susceptibility of all bacterial species. Secondly, these techniques are extremely slow because they are based on bacterial culture, which takes a lot of time. Thus, obtaining an antibiogram of a bacterium takes at least 30 hours from the time the sample is taken.This delay does not allow for effective treatment of patients who are routinely given a broad-spectrum antibiotic cocktail as a first-line treatment. In addition to the consequences for the patient, this inappropriate and massive administration of antibiotics increases the selection pressure for multi-resistant bacteria and therefore contributes to their spread.

[0016] To date, it is estimated that conventional in vitro diagnostic technologies are less and less suitable for treating patients and are, to a certain extent, one of the reasons for the emergence of multi-resistance.

[0017] More recently, sensitive technologies such as mass spectrometry are being applied to determine antibiotic resistance, but this still requires culturing the test microorganism in the presence of the test antibiotic. Furthermore, in all these techniques, each test microorganism must be tested against individual antibiotics or combinations of antibiotics, which requires extensive, time-consuming, and tedious testing.

[0018] The maturation of molecular biology technologies, particularly technologies for characterizing bacterial DNA and / or RNA, such as polymerase chain reaction (or PCR), DNA chips, or sequencing, is bringing about a paradigm shift in the analysis of antibiotic resistance in laboratories. First, they are more agnostic with respect to bacterial species. For example, metagenomic technology makes it possible to process bacterial DNA in a biological sample regardless of the bacterial species present. Second, they aim to provide a result in a few hours, with some, such as PCR, even providing a result in less than 20 minutes. In contrast, molecular techniques for characterizing antibiotic susceptibility rely on genomic signatures (absence / presence of genes, genetic mutations, prediction models, etc.) characterizing said susceptibility.

[0019] Without limitation, in the context of a microbiological workflow for the treatment of a patient suspected of a bacterial infection, there are two technologies for characterizing bacterial DNA, namely a PCR technology and a whole genome sequencing (or WGS) technology. Both flows begin with the collection of a biological sample from the patient, followed by the application of PCR technology or WGS technology, each returning a result of genomic signatures characterizing the susceptibility to one or more antibiotics, based on which an antibiotic treatment is chosen and then administered to the patient by a clinician.Conventionally according to the methods known to the person skilled in the art, each of the molecular technologies requires the preparation of the sample taken prior to the application of the PCR itself, for example a “nested” type PCR implemented by a FilmArray® platform from the company BioFire®, or to the application of the sequencing, for example an SBS (Sequencing By Synthesis) type sequencing implemented by a MiSeq platform from the company Illumina.

[0020] For example, WO2018 / 065830 describes a real-time quantitative PCR (qPCR) method for determining the resistance profile of Mycobacterium tuberculosis to antibiotics.

[0021] Document CN101580879 describes a gene chip and a method for detecting common genetic mutations in Mycobacterium tuberculosis responsible for resistance to antibiotics such as isoniazid, rifampicin, streptomycin, ethambutol or pyrazinamide.

[0022] Document IN201941006113 describes a method for testing the antibiotic resistance of a Mycobacterium tuberculosis strain from a clinical sample containing said strain, by implementing a step of identifying single nucleotide variants (SNVs) from the genes associated with the antibiotic resistance of said strain.

[0023] More generally, in both bacteria and humans, it is known that antibiotic resistance can be associated with genetic polymorphism. Focusing more specifically on genomic signatures, the first approaches consisted of identifying previously identified antibiotic resistance markers in the bacterial genome, approaches called "direct association". While these approaches are effective when the genetic mechanisms leading to resistance are well known and simple, they can suffer from significant limitations: incomplete knowledge of resistance mechanisms in many species and antibiotics, resulting for example in incomplete databases, difficulty in taking into account differences in the predictive powers of markers and the multifactorial aspect of antibiotic susceptibility (e.g. epistasis, combination of multiple mutations, etc.), etc.Faced with these difficulties, the genetic determinism of susceptibility to antibiotics is understood more effectively by new approaches based on advanced computer technologies, and in particular by supervised machine learning technologies whose learning and application architecture can be summarized as follows:.

[0024] A. for a set of training bacterial strains:

[0025] - A.1 each strain is sequenced and phenotypically characterized (e.g. measurement of its minimum inhibitory concentration and / or measurement of its susceptibility - resistance, intermediate or sensitive - for one or more antibiotics).

[0026] - A.2. a computer model for predicting antibiotic susceptibility is learned based on genomes and phenotypic data.

[0027] B. for a new strain whose susceptibility to an antibiotic from step (A.1) is being investigated,

[0028] B.1. the strain is sequenced;

[0029] B.2. The computer prediction model is applied to its digital genome to determine its susceptibility.

[0030] Such learning and prediction models are for example described in documents WO2021180768 and WO2021180771 of which the Applicant is the owner.

[0031] Thus, there is a need to develop new genomic signatures to strengthen the arsenal available for identifying susceptibility to antibiotics, and in particular to pyrazinamide. Indeed, the signatures are obtained by learning models whose limitation lies in the size and diversity of the genomic databases used to train the model. Therefore, it may be advantageous to combine the simultaneous use of several signatures to determine even more precisely the susceptibility to an antibiotic. In this context, it is relevant to identify new genomic signatures with a sufficient level of performance, namely a specificity of at least 90%, to determine the susceptibility of a strain of the species Mycobacterium tuberculosis to pyrazinamide.

[0032] SUMMARY OF THE INVENTION

[0033] After extensive research on the genome of the species Mycobacterium tuberculosis, it is to the credit of the inventors to have identified a new genomic signature making it possible to determine the susceptibility of a strain of said species to pyrazinamide. In other words, the determination of susceptibility according to the present invention makes it possible to identify whether a strain of the species Mycobacterium tuberculosis is sensitive or resistant to pyrazinamide, with a specificity of more than 90%. Thus, the present invention makes it possible to combat antibiotic resistance by, in particular, avoiding the initiation of pyrazinamide treatment while the strain is resistant.

[0034] Furthermore, it is known that the presence of mutations in the pncA gene is correlated with the resistance of Mycobacterium tuberculosis strains to pyrazinamide. Conversely, and quite surprisingly, the determination of the sensitive or resistant character of a Mycobacterium tuberculosis strain to pyrazinamide is carried out from the identification of the presence or absence of mutations in genes that are not constitutively correlated with resistance to said antibiotic, such as the rpoB gene or the gyrA, embB or katG genes.

[0035] A first subject of the invention thus relates to a method for determining the susceptibility of a strain of the species Mycobacterium tuberculosis to pyrazinamide, said method comprising the following steps of determining the presence in the genome of said strain of at least one mutation in the rpoB gene between positions 761112 and 761182, and of determining the sensitive or resistant character of said strain on the basis of the mutation(s) identified.

[0036] Preferably, the method also comprises the following steps of determining the presence or absence of at least one mutation in the fabG1 gene between positions 1673413 and 1673454, optionally also in the gyrA gene between positions 7552 and 7582, optionally also in the Rv1042c gene and its promoter region between positions 1165444 and 1165528, optionally also in the Rv1149 gene and its promoter region between positions 1277873 and 1277957, optionally also in the embB gene between positions 4247581 and 4247622, ​​optionally also in the pncA gene between positions 2288853 and 2289239, and finally, optionally also in the katG gene between positions 2155164 and 2155205.

[0037] Another subject of the invention relates to a kit comprising means for detecting and / or amplifying at least one sequence chosen from the sequences SEQ ID NOs: 1 to 3, and optionally at least one other sequence chosen from the sequences SEQ ID NOs: 4 to 12, in the genome of a strain of the species Mycobacterium tuberculosis, preferably in the rpoB gene of said strain, as well as the use of said kit for determining the susceptibility of the strain to pyrazinamide.

[0038] The method according to the invention can be implemented by computer, another object finally concerns a data processing device comprising:

[0039] (a) means for implementing the method according to the invention, in particular means for determining the presence of mutations in the rpoB gene of the Mycobacterium tuberculosis strain between positions 761112 and 761182, and / or means for comparing the sequence of said gene with the reference genome Mycobacterium tuberculosis H37Rv (reference NC_000962.3), and / or means for identifying the presence of the sequences SEQ ID NOs 1 to 3 in the genome of the Mycobacterium tuberculosis strain, and / or means for providing output data of the presence or absence of said sequences, when said means are implemented or controlled by computer, or

[0040] (b) 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.

[0041] DETAILED DESCRIPTION OF THE INVENTION

[0042] A derivative of nicotinamide, pyrazinamide is an anti-tuberculosis drug that produces in vivo, at the indicated dosages, a bactericidal action on intracellular tuberculosis bacilli (thus found in an acidic environment, a necessary condition for the action of pyrazinamide). Pyrazinamide is therefore used in the treatment of tuberculosis. The species Mycobacterium bovis and atypical mycobacteria are naturally resistant to it. Conversely, the species Mycobacterium tuberculosis and a very closely related species, Mycobacterium africanum, are regularly sensitive to it.

[0043] As mentioned above, inadequate or delayed antibiotic therapy has a negative effect favoring the selection of spontaneous mutations in favor of resistant strains by selection pressure, thus aggravating the problem of antibiotic resistance.

[0044] Therefore, when managing a tuberculosis patient, it is essential to be able to ensure that the Mycobacterium tuberculosis strain with which he is infected does not present a resistance to pyrazinamide.

[0045] Thus, a first subject of the invention relates to a method for determining the susceptibility of a strain of the species Mycobacterium tuberculosis to pyrazinamide, said method comprising a step of determining the presence in the genome of said strain of at least one mutation in the rpoB gene between positions 761112 and 761182, and a step of determining the sensitive or resistant character of said strain depending on the presence or absence of mutations in said gene.

[0046] Quite surprisingly, as explained previously, the present method allows to determine the susceptibility to pyrazinamide from the identification of mutations in genes that are not constitutively associated with resistance to pyrazinamide but to other antibiotics. Indeed, the identification of mutations in the rpoB gene is normally associated with resistance to rifamycin and its derivatives such as rifampicin.

[0047] Unless explicitly stated, the technical and scientific terms used in this description have the same meaning as that commonly understood by the person skilled in the art of the field of this description.

[0048] The term "susceptibility determination" refers to the determination of whether the strain is sensitive or resistant to pyrazinamide.

[0049] For the remainder of the description, the term "strain(s)" refers to strains of the species Mycobacterium tuberculosis, unless the context clearly identifies another species.

[0050] Determination of the presence of at least one mutation in the rpoB gene

[0051] The term "mutation" refers to a variation in sequence from a reference sequence. Such a reference sequence may be a sequence determined in a predominantly wild-type organism or a reference organism such as a defined and known bacterial strain, for example. A mutation is, for example, a deletion of one or more nucleotides, an insertion of one or more nucleotides, or a substitution of one or more nucleotides, a duplication of one or a sequence of several nucleotides, a translocation of one or a sequence of several nucleotides, and, in particular, a single nucleotide polymorphism (SNP).

[0052] The genome of Mycobacterium tuberculosis was fully sequenced in 1998. This species has a circular chromosome of 4,411,529 base pairs (GC%=65.6) for 3924 genes. In the context of the present invention, the determination of the presence of mutations in one or more genes of the strain is carried out in relation to the wild reference genome of Mycobacterium tuberculosis, namely Mycobacterium tuberculosis H37Rv. This wild reference genome is notably indexed in the NCBI database under the reference NC_000962.3. Thus, all the genomic positions according to the present description are given in relation to said genome NC_000962.3.

[0053] The comparison can be carried out according to methods known to the person skilled in the art for comparing two genomes with each other, or more precisely, particular genomic positions between two genomes, namely in particular by bioinformatics.

[0054] Thus, according to the present invention, determining the presence in the genome of the strain of at least one mutation in the rpoB gene between positions 761112 and 761182 compared to the wild-type Mycobacterium tuberculosis H37Rv genome makes it possible to identify the sensitive or resistant character of said strain to pyrazinamide.

[0055] For the purposes of the present invention, the identification or determination of the sensitive or resistant character of a Mycobacterium tuberculosis strain is understood as the prediction, with a certain degree of associated error, of said character.

[0056] The determination of the presence of at least one mutation in one or more genes of a strain of Mycobacterium tuberculosis can be carried out according to methods known to the person skilled in the art. For example, it is possible to implement a hybridization, amplification or sequencing method.

[0057] According to a particular embodiment, the determination of the presence of mutations is carried out by a hybridization technique, preferably with hybridization microchips or by techniques of the NanoString® nCounter® type, by an amplification technique, preferably by PCR or qPCR, or by a sequencing technique, preferably by high-throughput sequencing or sequencing by synthesis.

[0058] According to a preferred embodiment, the method comprises, before the step of determining the presence of mutations in the genes of the Mycobacterium tuberculosis strain, a prior step of characterizing the DNA of said strain. This step is carried out according to methods known to those skilled in the art, preferably by PCR or sequencing (for example WGS).

[0059] The method may also comprise a step of first obtaining a biological sample from a subject, said sample being likely to contain at least one strain of the species Mycobacterium tuberculosis.

[0060] According to a particular 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 and 761182, and / or between positions 761112 and 761142, and / or between positions 761138 and 761173.

[0061] According to 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 in this gene the presence of at least one sequence chosen from the sequences SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, preferably the sequence SEQ ID NO: 2, and more preferably, the sequences SEQ ID NOs 1 to 3. According to the method of the invention, the sensitive or resistant character of the strain is determined as a function of the presence or absence of mutation(s) between the positions described above, and in particular, as a function of the presence or absence of one or more sequences chosen from the sequences SEQ ID NOs: 1 to 3. Table 1 in the embodiments summarizes the different conclusions as to the sensitive or resistant character of said strain as a function of the presence or absence of said sequences.

[0062] Determination of the presence of at least one mutation in one or more additional genes

[0063] The performance of the method according to the present invention can be improved by combining the determination of the presence of mutation(s) in one or more additional genes of the Mycobacterium tuberculosis strain.

[0064] Thus, according to a particular embodiment, the method further comprises a step of determining the presence in the genome of the strain of at least one mutation in the fabG1 gene between positions 1673413 and 1673454, and preferably, between positions 1673415 and 1673454 and / or between positions 1673413 and 1673444.

[0065] According to a preferred variant of this embodiment, the determination of the presence of at least one mutation in the fabG1 gene between said genomic positions advantageously comprises the determination of the presence of the sequence SEQ ID NO: 4 and / or SEQ ID NO: 5, preferably both.

[0066] Advantageously, the method according to the invention thus comprises a step of determining in the genome of a Mycobacterium tuberculosis strain the presence of at least one mutation in the rpoB and fabG1 genes, said step comprising determining the presence of the sequences SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 in the rpoB gene, and of the sequences SEQ ID NO: 4 and SEQ ID NO: 5 in the fabG1 gene, as well as a step of determining the sensitive or resistant character depending on the presence or absence of said sequences. Table 2 in the exemplary embodiments shows the different conclusions regarding the sensitive or resistant character of said strain depending on the presence or absence of said sequences.

[0067] According to another particular embodiment, the method according to the present invention further comprises a step of determining the presence in the genome of the strain of at least one mutation in the gyrA gene between positions 7552 and 7582. According to a preferred variant of this embodiment, the determination of the presence of at least one mutation in the gyrA gene between said genomic positions advantageously comprises the determination of the presence of the sequence SEQ ID NO: 6.

[0068] Thus, in a completely advantageous manner, the method according to the invention comprises a step of determining in the genome of a Mycobacterium tuberculosis strain the presence of at least one mutation in the rpoB, fabG1 and gyrA genes, said step comprising determining the presence of the sequences SEQ ID NOs: 1 to 6 in said respective genes, and a step of determining the sensitive or resistant character depending on the presence or absence of said sequences. Table 3 in the exemplary embodiments shows the different conclusions as to the sensitive or resistant character of said strain depending on the presence or absence of said sequences.

[0069] According to another embodiment, the method according to the present invention further comprises a step of determining the presence in the genome of the strain of at least one mutation in the Rv1042c gene and its promoter region between positions 1165444 and 1165528, and / or of at least one mutation in the Rv1149 gene and its promoter region between positions 1277873 and 1277957. Preferably according to this embodiment, the method comprises a step of determining the presence of at least one mutation in the Rv1042c gene and its promoter region between positions 1165444 and 1165492 and / or between positions 1165497 and 1165528, and of determining at least one mutation in the Rv1149 gene and its promoter region between positions 1277873 and 1277904 and / or between positions 1277909 and 1277957.

[0070] According to a first variant of this embodiment, the method comprises a step 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. According to this variant, the determination of the presence of at least one mutation in said gene advantageously comprises the determination of the presence of the sequence SEQ ID NO: 7

[0071] According to a second variant of this embodiment, the method comprises a step of determining the presence of at least one mutation in the Rv1042c gene and its promoter region between positions 1165497 and 1165528 and at least one mutation in the Rv1149 gene and its promoter region between positions 1277873 and 1277904. According to this variant, determining the presence of at least one mutation in said gene advantageously comprises determining the presence of the sequence SEQ ID NO: 8.

[0072] According to a third preferred variant of this embodiment, the determination of the presence of at least one mutation in the Rv1042c and / or Rv1149 gene, and their respective promoter regions, between said genomic positions advantageously comprises the determination of the presence of the sequence SEQ ID NO: 7 and / or the sequence SEQ ID NO: 8, preferably both.

[0073] Thus, according to another preferred embodiment, the method according to the invention comprises a step of determining in the genome of a Mycobacterium tuberculosis strain the presence of at least one mutation in the rpoB, fabG1, gyrA, Rv1042c and Rv1149 genes, said step comprising determining the presence of the sequences SEQ ID NO: 1 to SEQ ID NO: 8 in said respective genes, and a step of determining the sensitive or resistant character depending on the presence or absence of said sequences. Table 4 in the exemplary embodiments shows the different conclusions regarding the sensitive or resistant character of said strain depending on the presence or absence of said sequences.

[0074] According to another embodiment, the method according to the present invention further comprises a step of determining the presence in the genome of the strain of at least one mutation in the embB gene between positions 4247581 and 4247622. Preferably, according to this embodiment, the determination of the presence of at least one mutation in the embB gene between said genomic positions advantageously comprises the determination of the presence of the sequence SEQ ID NO: 9.

[0075] Thus, according to another preferred embodiment, the method according to the invention comprises a step of determining in the genome of a Mycobacterium tuberculosis strain the presence of at least one mutation in the rpoB, fabG1, gyrA, Rv1042c, Rv1149 and embB genes, said step comprising determining the presence of the sequences SEQ ID NO: 1 to SEQ ID NO: 9 in said respective genes, and a step of determining the sensitive or resistant character depending on the presence or absence of said sequences. Table 5 in the exemplary embodiments shows the different conclusions regarding the sensitive or resistant character of said strain depending on the presence or absence of said sequences.According to another embodiment, the method according to the present invention further comprises a step of determining the presence in the genome of the strain of at least one mutation in the pncA gene between positions 2288853 and 2289239, and preferably between positions 2288853 and 2288885 and / or between positions 2289209 and 2289239.

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

[0077] Thus, according to another preferred embodiment, the method according to the invention comprises a step of determining in the genome of a Mycobacterium tuberculosis strain the presence of at least one mutation in the rpoB, fabG1, gyrA, Rv1042c, Rv1149, embB and pncA genes, said step comprising determining the presence of the sequences SEQ ID NO: 1 to SEQ ID NO: 11 in said respective genes, and a step of determining the sensitive or resistant character depending on the presence or absence of said sequences. Table 6 in the exemplary embodiments shows the different conclusions as to the sensitive or resistant character of said strain depending on the presence or absence of said sequences.

[0078] According to another embodiment, the method according to the present invention further comprises a step of determining the presence in the genome of the strain of at least one mutation in the katG gene between positions 2155164 and 2155205.

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

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

[0081] - determination of the presence or absence of at least one mutation in the rpoB gene between positions 7611 12 and 761182,

[0082] - determination of the presence or absence of at least one mutation in the fabG1 gene between positions 1673413 and 1673454,

[0083] - determination of the presence or absence of at least one mutation in the gyrA gene between positions 7552 and 7582,

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

[0085] - determination of the presence or absence of at least one mutation in the embB gene between positions 4247581 and 4247622,

[0086] - determination of the presence or absence of at least one mutation in the pncA gene between positions 2288853 and 2289239, - determination of the presence or absence of at least one mutation in the katG gene between positions 2155164 and 2155205, and

[0087] - determination of the sensitive or resistant nature of said strain depending on the presence or absence of said mutations.

[0088] According to another preferred embodiment, the method according to the invention comprises a step (a) of determining in the genome of a Mycobacterium tuberculosis strain the presence of at least one mutation in the rpoB, fabG1, gyrA, Rv1042c, Rv1149, embB, pncA and katG genes, said step comprising determining the presence of the sequences SEQ ID NOs 1, 4, 6, 7, 9, 10 and 12, and preferably SEQ ID NOs: 1 to 12, in said respective genes, and a step (b) of determining the sensitive or resistant character depending on the presence or absence of said sequences. Table 7 in the embodiments shows the different conclusions as to the sensitive or resistant character of said strain depending on the presence or absence of said sequences.

[0089] According to a particular embodiment, the method may comprise a step of obtaining the genome of said Mycobacterium tuberculosis strain prior to determining the presence or absence of at least one mutation in the rpoB gene, and optionally in the additional genes.

[0090] For the purposes of this description, the term "subject" means a human being and preferably, the subject is a patient.

[0091] Thus, the method according to the invention may be an in vitro or ex vivo method for determining the susceptibility to pyrazinamide of a strain of the species Mycobacterium tuberculosis from a biological sample of a subject likely to contain said strain, said method comprising the following steps of:

[0092] - determination of the presence in the genome of said strain of at least one mutation in the rpoB gene between positions 761112 and 761182, and

[0093] - determination of the sensitive or resistant nature of said strain depending on the presence or absence of said mutation.

[0094] The specific or preferred embodiments described above according to the invention apply, of course, to the in vitro or ex vivo methods also forming the subject of the invention. Thus, the determination of at least one mutation in one of the additional genes as described above is possible.

[0095] By "biological sample" we refer here to any sample from a subject, and which may be of different natures, such as blood or its derivatives or sputum, in which it is possible to find traces of the strain.

[0096] According to a particular embodiment, the biological sample is a sputum sample, a blood sample or a blood-derived sample, which may in particular be chosen from whole blood (as collected venously, i.e. containing white and red cells, platelets and plasma), plasma and serum.

[0097] Kit for implementing the method according to the invention

[0098] Kits may be prepared to implement the method according to any of the preceding embodiments. In particular, another subject of the invention relates to a kit allowing the detection and / or amplification of at least one sequence chosen from the sequences SEQ ID NO: 1-3. The kit may advantageously comprise instructions for use.

[0099] The kit thus comprises means for detecting and / or amplifying and / or quantifying said sequences, for example in a biological sample suspected of containing a strain of the species Mycobacterium tuberculosis. These may thus be primers and / or probes specific for said sequences.

[0100] According to 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 contains all the means necessary for implementing the detection of the sequences SEQ ID NOs: 1 to 3 by amplification, preferably by PCR, and in particular by qPCR.

[0101] According to a particular embodiment, the kit also comprises means, preferably primers and / or probes, making it possible to also detect at least one of the sequences SEQ ID NOs:

[0102] 4 to 12 within a strain of the species Mycobacterium tuberculosis. Preferably, the kit contains primers and / or probes for the detection of all of the sequences SEQ ID NOs 4 to 12.

[0103] All of the specific or preferred embodiments described above in connection with the methods according to the invention also apply to the kit which is the subject of the invention.

[0104] The term "primer" or "amplification primer" means a nucleotide fragment which may consist of 5 to 100 nucleotides, preferably 10 to 20 nucleotides, and which has a hybridization specificity with a target nucleotide sequence, namely one of the sequences SEQ ID NOs 1 to 12, under conditions determined for the initiation of an enzymatic polymerization, for example in an enzymatic amplification reaction of the target nucleotide sequence. Generally, "primer pairs" are used, consisting of two primers. When it is desired to carry out the amplification of several different biomarkers (e.g. genes), several different primer pairs are preferably used, each preferably having a capacity to hybridize specifically with a different biomarker.

[0105] The person skilled in the art is thus able, from the sequences SEQ ID NOs: 1 to 12, to determine the primers and probes necessary for the amplification.

[0106] The term "probe" or "hybridization probe" means a nucleotide fragment typically consisting of

[0107] 5 to 100 nucleotides, preferably 10 to 90 nucleotides, even more preferably 15 to 35 nucleotides, having a hybridization specificity under determined conditions to form a hybridization complex with a target nucleotide sequence. The probe also comprises a reporter (such as a fluorophore, an enzyme or any other detection system), which will allow the detection of the target nucleotide sequence. In the present invention, the target nucleotide sequences are the sequences SEQ ID NOs 1 to 3, and preferably the sequences SEQ ID NOs 1 to 12. Thus, several different probes are preferably used, each preferentially having a capacity to hybridize specifically with one of the target sequences.

[0108] By "hybridization" is meant the process during which, under appropriate conditions, two nucleotide fragments, such as for example a hybridization probe and a target nucleotide fragment, having sufficiently complementary sequences, are capable of forming a double strand with stable and specific hydrogen bonds. A nucleotide fragment "capable of hybridizing" with a polynucleotide is a fragment capable of hybridizing with said polynucleotide under hybridization conditions, which can be determined in each case in a known manner. The hybridization conditions are determined by the stringency, that is to say the rigor of the operating conditions. The hybridization is all the more specific as it is carried out at a higher stringency. Stringency is defined in particular as a function of the base composition of a probe / target duplex, as well as by the degree of mismatch between two nucleic acids.Stringency may also be a function of reaction parameters, such as the concentration and type of ionic species present in the hybridization solution, the nature and concentration of denaturing agents, and / or the hybridization temperature. The stringency of the conditions under which a hybridization reaction is to be carried out will depend primarily on the hybridization probes used. All of these data are well known, and appropriate conditions can be determined by those skilled in the art.

[0109] In general, depending on the length of the hybridization probes used, the temperature for the hybridization reaction is between about 20 and 70°C, in particular between 35 and 65°C in a saline solution at a concentration of about 0.5 to 1 M. A step of detecting the hybridization reaction is then carried out.

[0110] The probes or primers that may be used in the methods of the invention may typically be short nucleic acid molecules, for example, DNA oligonucleotides of 10 nucleotides or more in length, which may be linked to the complementary target nucleic acid molecule by nucleic acid hybridization to form a hybrid between the primer or probe and the target nucleic acid strand.

[0111] The probe or primers may be unlabeled or labeled so that their binding to a target sequence can be detected (e.g., with a FRET-type donor or acceptor label).

[0112] A primer can be extended along the target nucleic acid molecule by a polymerase enzyme.

[0113] Therefore, primers can be used to amplify the target nucleic acid molecule, such as one of the sequences SEQ ID NOs:1 to 12, and / or their variant sequences.

[0114] The specificity of a probe or primer increases with its length. For example, a probe or primer that is 30 consecutive nucleotides long will bind to a target sequence with greater specificity than a corresponding primer of only 15 nucleotides. Thus, to achieve greater specificity, probes and primers that are at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or more consecutive nucleotides can be chosen.

[0115] In particular examples, a primer may be at least 15 nucleotides in length, such as at least 15 contiguous nucleotides complementary to a target nucleic acid molecule. Particular primer lengths that may be used to practice the methods of the present disclosure 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 contiguous nucleotides complementary to the target nucleic acid molecule to be amplify, such as a primer of 15-70 nucleotides, 15-60 nucleotides, 15-50 nucleotides, or 15-30 nucleotides.A forward primer is a primer located 5' of a reference point on a nucleic acid sequence. A reverse primer is a primer located 3' of a reference point on a nucleic acid sequence. Typically, at least one forward primer and one reverse primer (the "primer pair") are included in an amplification reaction.

[0116] Nucleic acid probes and primers, or primer pairs, can be readily prepared based on the nucleic acid sequence of any of SEQ ID NOs 1-12. PCR primer pairs can be derived from said sequences using computer programs intended for this purpose such as Primer 3 (v. 0.4.0 Whitehead Institute for Biomedical Research, Steve Rozen, and Helen Skaletsky).

[0117] According to a particular embodiment, the sequences SEQ ID NOs 1-12, or their amplified or transformed products (cDNA for example) are detected by specific hybridization of nucleic acid probes.

[0118] These probes may also be immobilized on a solid surface (such as nitrocellulose, glass, quartz, a fused silica slide) such as in an array, microarray, or microarray. One skilled in the art will recognize that the precise sequence of particular probes and primers may be modified from the target sequence to a degree to produce probes that are "substantially identical" or "substantially complementary" to a target sequence, while retaining the ability to specifically bind (i.e., specifically hybridize) to the same targets from which they are derived.

[0119] In the context of this disclosure, the terms "capable of hybridizing to" and "specifically binds to," which are used interchangeably, refer to a polynucleotide sequence that forms Watson-Crick bonds with a complementary sequence. Depending on the length of the polynucleotides, the length of the complementary region, and the stringency of the conditions, the skilled person understands that the percentage of complementarity does not necessarily have to be 100% for hybridization or specific binding to occur. For example, a primer or probe is at least 60%, 70%, 80%, 90%, 95%, 99%, or 100% complementary over the length of the complementary region.

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

[0121] Another subject of the invention relates to the use of the sequences SEQ ID NOs 1 to 3, and preferably also the sequences SEQ ID NOs 4 to 12, to determine the susceptibility of a Mycobacterium tuberculosis strain to pyrazinamide.

[0122] The specific or preferred embodiments described in connection with the methods according to the invention apply, of course, to the uses which are the subject of the invention.

[0123] Computer implementation

[0124] The method as described in any embodiment detailed above may well be implemented by computer. In this case, it may be executed, partially or totally, by computer means.

[0125] To this end, the invention also provides a data processing apparatus comprising: (a) means for implementing a computer-implemented method according to the invention, in particular means for determining the presence of mutations in the rpoB gene of the Mycobacterium tuberculosis strain, and / or means for comparing the sequences of said genes with the reference genome Mycobacterium tuberculosis H37Rv (reference NC_000962.3), and / or means for identifying the presence of the sequences SEQ ID NOs 1 to 3 in the genome of the Mycobacterium tuberculosis strain, and / or means for providing output data of the presence or absence of said sequences, when said means are implemented or controlled by computer

[0126] (b) a processor adapted or configured to execute a computer-implemented method according to the invention, in particular a processor adapted or configured to execute the steps of a computer-implemented method according to the invention.

[0127] Advantageously, the processing apparatus may also comprise means for determining the presence of mutations in the fabG1, gyrA, Rv1042c, Rv1149, embB, pncA and katG genes at the positions as defined above, and / or means for identifying the presence or absence of the sequences SEQ ID NOs: 4 to 12 in said genes to which they relate.

[0128] According to a particular embodiment, such a data processing device comprises:

[0129] (a) an input interface for receiving the genome of a Mycobacterium tuberculosis strain whose susceptibility to pyrazinamide is to be determined,

[0130] (b) a memory for storing at least instructions of a computer program which, when the program is executed by a computer or processor, causes the determination of the presence of mutations in the rpoB gene at the positions described above, and optionally in one or more additional genes as described above, to be carried out, c) a processor accessing the memory to read the aforementioned instructions and execute a computer-implemented method according to the invention

[0131] (d) an output interface for providing output values, in particular output values ​​corresponding to the conclusion of whether the strain is sensitive or resistant to pyrazinamide.

[0132] Other items in the description

[0133] Another subject of the present description relates to a method of treating a subject infected with a strain of the species Mycobacterium tuberculosis comprising a step of determining the susceptibility of said strain to pyrazinamide according to any one of the methods as described previously, and a step of treating said subject with pyrazinamide when it is concluded that said strain is sensitive.

[0134] In particular, processing may be initiated as soon as it is concluded to be sensitive.

[0135] Another object of the present description concerns a method comprising the following steps:

[0136] - obtain a biological sample from a subject infected with a strain of the species Mycobacterium tuberculosis, - bring said biological sample into contact with detection means or specific reagents making it possible to detect the presence of mutations in the rpoB gene of said strain between positions 7611 12 and 761182, and

[0137] - determine the presence of the sequences SEQ ID NOs: 1 to 3.

[0138] The reagents specific to the expression products are selected from amplification primers, hybridization probes and are as defined previously, and in particular make it possible to determine the presence of the sequences SEQ ID NOs: 1 to 3.

[0139] Preferably, the biological sample is also brought into contact with means for determining the presence of mutations in the fabG1, gyrA, Rv1042c, Rv1149, embB, pncA and katG genes at the positions as defined above and in particular makes it possible to identify the presence or absence of the sequences SEQ ID NOs: 4 to 12 in said genes to which they relate.

[0140] The present invention is illustrated in a non-limiting manner from the examples below.

[0141] EXAMPLES

[0142] Example 1: Determination of the susceptibility of a strain of Mycobacterium tuberculosis to pyrazinamide.

[0143] 1. Obtaining the models

[0144] A set of 3606 Mycobacterium tuberculosis genomes with pyrazinamide-resistant (R) or sensitive (S) phenotypes was used. Among these genomes, 3038 were used to train supervised machine learning models (training set) and 568 to evaluate the performance of these models (validation set).

[0145] Among the 568 genomes in the validation set, 133 are resistant to pyrazinamide and 435 are sensitive to it. The tables below present for each model the presence of each “Wild type” sequence of the model in these validation genomes. The sequences are named by the name of the gene to which they belong and are ordered by decreasing value of the coefficient in the model (given as absolute value, in parentheses).

[0146] The tables below highlight different haplotypes defined as combinations of observed mutated or non-mutated sequences. Haplotypes are ordered by decreasing prevalence (percentage of observations among the 568 genomes).

[0147] For each haplotype, the prediction made by the model is reported, as well as the number of resistant and susceptible genomes in which this haplotype was observed. Thus, it is possible to identify the number of correct predictions and the number of errors made by the model in the validation set.

[0148] These errors are described in terms of “Very Major Error” (VME) corresponding to a resistant genome predicted to be susceptible by the model (in other words, a false negative) and “Major Error” (ME) corresponding to a susceptible genome predicted to be resistant by the model (in other words, a false positive).

[0149] Legends for Tables 1 to 6: NbSR = number of resistant strains; NbSS = number of susceptible strains; Prev = prevalence; Pred = prediction; 1: presence of the sequence; 0: absence of the sequence. 2. Application of models for the determination of susceptibility to pyrazinamide

[0150] 2.1 rpoB model

[0151] Table 1 below presents the results of susceptibility by detection of the presence of mutations in the rpoB gene of the strain by highlighting the presence or absence of the sequences SEQ ID NOs: 1 to 3.

[0152] Table 1

[0153] Interpretation of results

[0154] In this model, the majority haplotype is the one in which the presence of the sequences SEQ ID NOs: 1 to 3 is observed. This haplotype is observed in 398 genomes (391 + 7), or 70.07% of the 568 validation genomes. Consequently, the determination of the presence of the sequences SEQ ID NOs: 1 to 3 leads to the correct determination (or prediction) of the “sensitive” character for 391 genomes, but with 7 VMEs.

[0155] Conversely, the absence of the said sequences SEQ ID NOs: 1 to 3 represents the minority haplotype and it is observed in a single genome, i.e. 0.18% of the validation genomes. Therefore, the determination of the absence of the sequences SEQ ID NOs: 1 to 3 leads to the correct determination (or prediction) of the “resistant” character without ME or VME.

[0156] Model performance

[0157] Overall, this model makes it possible to predict, based on the presence or absence of the sequences SEQ ID NOs: 1 to 3, the sensitive or resistant character of a Mycobacterium tuberculosis strain with a specificity of 88.72%, a sensitivity of 93.56% and an error rate of 7.57% (ME and VME).

[0158] 2.2 Model rpoB-fabG1

[0159] Table 2 below presents the results of susceptibility by detecting the presence of mutations in the rpoB and fabG1 genes of the strain by highlighting the presence or absence of the sequences SEQ ID NOs: 1 to 5. Table 2

[0160] Interpretation of results

[0161] In this model, the majority haplotype is the one in which the presence of the sequences SEQ ID NOs: 1 to 5 is observed. This haplotype is observed in 365 genomes (359+6), or 64.26% of the 568 validation genomes. Consequently, the determination of the presence of the sequences SEQ ID NOs: 1 to 5 leads to the correct determination (or prediction) of the “sensitive” character for 359 genomes, but with 6 VMEs.

[0162] Model performance

[0163] Overall, this model makes it possible to predict, based on the presence or absence of the sequences SEQ ID NOs: 1 to 5, the sensitive or resistant character of a Mycobacterium tuberculosis strain with a specificity of 90.23%, a sensitivity of 93.56% and an error rate of 7.21% (ME and VME).

[0164] 2.3 rpoB-fabG1-gyrA model

[0165] Table 3 below presents the results of susceptibility by detecting the presence of mutations in the rpoB, fabG1 and gyrA genes of the strain by highlighting the presence or absence of the sequences SEQ ID NOs: 1 to 6. Table 3

[0166] Interpretation of results

[0167] In this model, the majority haplotype is the one in which the presence of the sequences SEQ ID NOs: 1 to 6 is observed. This haplotype is observed in 362 genomes (357+5), or 63.73% of the 568 validation genomes. Therefore, the determination of the presence of the sequences SEQ ID NOs: 1 to 6 leads to the correct determination (or prediction) of the “susceptible” character for 357 genomes, but with 5 VMEs. Model performance

[0168] Overall, this model makes it possible to predict, based on the presence or absence of the sequences SEQ ID NOs: 1 to 6, the sensitive or resistant character of a Mycobacterium tuberculosis strain with a specificity of 94.25%, a sensitivity of 88.72% and an error rate of 7.04% (ME and VME).

[0169] 2.4 Model rpoB-fabG1-gyrA-Rv1042c / Rv1149

[0170] Table 4 below presents the results of susceptibility by detection of the presence of mutations in the rpoB, fabG1, gyrA, Rv 1042c and Rv1149 genes of the strain by highlighting the presence or absence of the sequences SEQ ID NOs: 1 to 8.

[0171] Table 4

[0172]

[0173] Interpretation of results

[0174] In this model, the majority haplotype is the one in which we detect the presence of the sequences SEQ ID NOs: 1 to 6 and SEQ ID NO: 7 and the absence of the sequence SEQ ID NO: 8. This haplotype is observed in 187 genomes (186+1), or 32.92% of the 568 validation genomes.

[0175] Therefore, the determination of the presence of the sequences SEQ ID NOs: 1 to 6 and SEQ ID NO: 7 and the absence of the sequence SEQ ID NO: 8, leads to the correct determination (or prediction) of the “sensitive” character for 186 genomes and with only 1 VME.

[0176] Similarly, the haplotype in which the presence of the sequences SEQ ID NOs: 1 to 6 and the absence of the sequences SEQ ID NOs: 7 and 8 are detected is observed in 86 genomes, or 14.14% of the validation genomes. Determining the presence and absence of said sequences thus makes it possible to predict the “sensitive” character for 85 genomes and also with only 1 VME.

[0177] Model performance

[0178] Overall, this model makes it possible to predict, based on the presence or absence of the sequences SEQ ID NOs: 1 to 8, the sensitive or resistant character of a Mycobacterium tuberculosis strain with a specificity of 88.72%, a sensitivity of 94.71% and an error rate of 6.69% (ME and VME).

[0179] 2.5 Model rpoB-fabG1-gyrA-Rv1042c / Rv1149-embB

[0180] Table 5 below presents the results of susceptibility by detection of the presence of mutations in the rpoB, fabG1, gyrA, Rv1042c, Rv1149 and embB genes of the strain by highlighting the presence or absence of the sequences SEQ ID NOs: 1 to 9.

[0181] Table 5

[0182] Interpretation of results

[0183] In this model, the majority haplotype is the one in which the presence of the sequences SEQ ID NOs: 1 to 6, 9 and SEQ ID NO: 7 and the absence of the sequence SEQ ID NO: 8 are detected. This haplotype is observed in 186 genomes (185+1), or 32.75% of the 568 validation genomes.

[0184] Therefore, determining the presence and absence of said sequences in the genome of a Mycobacterium tuberculosis strain leads to the correct determination (or prediction) of the “susceptible” character for 185 genomes and with only 1 VME.

[0185] Similarly, the haplotype in which the presence of all sequences SEQ ID NOs: 1 to 9 is detected is observed in 85 genomes, or 14.96% of the validation genomes. Determining the presence and absence of said sequences thus makes it possible to predict the “sensitive” character for 82 genomes, and with 3 VMEs.

[0186] Model performance

[0187] Overall, this model makes it possible to predict, based on the presence or absence of the sequences SEQ ID NO: 1 to 9, the sensitive or resistant character of a Mycobacterium tuberculosis strain with a specificity of 87.22%, a sensitivity of 96.55% and with an error rate of 5.63% (ME and VME).

[0188] 2.6 Model rpoB-fabG1-gyrA-Rv1042c / Rv1149-embB-pncA-katG

[0189] Table 6 below presents the results of susceptibility by detection of the presence of mutations in the rpoB, fabG1, gyrA, Rv1042c, Rv1149, embB, pncA and katG genes in the strain via the detection of the presence or absence of the sequences SEQ ID NOs: 1 to 12.

[0190] Table 6

[0191]

[0192]

[0193] Interpretation of results

[0194] In this model, the majority haplotype is the one in which we detect the presence of the sequences SEQ ID NOs: 1 to 6, 9 to 12 and SEQ ID NO: 7 and the absence of the sequence SEQ ID NO: 8. This haplotype is observed in 164 genomes (163+1), or 28.87% of the 568 validation genomes.

[0195] Therefore, determining the presence and absence of said sequences in the genome of a Mycobacterium tuberculosis strain leads to the correct determination (or prediction) of the “susceptible” character for 163 genomes and with only 1 VME.

[0196] Similarly, the haplotype in which the presence of all sequences SEQ ID NOs: 1 to 12 is detected is observed in 75 genomes, or 13.2% of the validation genomes. Determining the presence and absence of said sequences thus makes it possible to predict the “sensitive” character for 72 genomes, and with 3 VMEs.

[0197] Model performance

[0198] Overall, this model makes it possible to predict, based on the presence or absence of the sequences SEQ ID NOs: 1 to 12, the sensitive or resistant character of a Mycobacterium tuberculosis strain with a specificity of 88.72%, a sensitivity of 97.01% and with an error rate of 5.28% (ME and VME).

[0199] Example 2: Description of the sequences useful for implementing the method according to the invention

Claims

CLAIMS 1. Method for determining the susceptibility of a strain of the species Mycobacterium tuberculosis to pyrazinamide, said method comprising the following steps: determining the presence in the genome of said strain of at least one mutation in the rpoB gene between positions 761112 and 761182, determining the sensitive or resistant character of said strain depending on the presence or absence of said mutation.

2. Method according to claim 1, characterized in that it further comprises determining the presence in the genome of said strain of at least one mutation in the fabG1 gene between positions 1673413 and 1673454.

3. Method according to claim 1 or 2, characterized in that it further comprises determining the presence in the genome of said strain of at least one mutation in the gyrA gene between positions 7552 and 7582.

4. Method according to one of claims 1 to 3, characterized in that it further comprises determining the presence in the genome of said strain of at least one mutation in the Rv1042c gene and its promoter region between positions 1165444 and 1165528 and / or of at least one mutation in the Rv1 149 gene and its promoter region between positions 1277873 and 1277957.

5. Method according to claim 4, characterized in that it further comprises a step 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 and / or a step of determining the presence of at least one mutation in the Rv1042c gene and its promoter region between positions 1165497 and 1165528 and at least one mutation in the Rv1149 gene and its promoter region between positions 1277873 and 1277904.

6. Method according to one of claims 1 to 6, characterized in that it further comprises the following steps of: determining the presence in the genome of said strain of at least one mutation in the embB gene between positions 4247581 and 4247622, ​​and / or determining the presence in the genome of said strain of at least one mutation in the pncA gene between positions 2288853 and 2289239, and / or determining the presence in the genome of said strain of at least one mutation in the katG gene between positions 2155164 and 2155205.

7. Method according to one of claims 1 to 6, characterized in that the determination of the presence of at least one mutation in the rpoB gene comprises the determination of the presence of at least one sequence chosen from the sequences SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO:

3.

8. Method according to one of claims 2 to 7, characterized in that the determination of the presence of at least one mutation in the fabG1 gene comprises the determination of the presence of at least one sequence chosen from the sequences SEQ ID NO: 4 and SEQ ID NO:

5.

9. Method according to one of claims 3 to 8, characterized in that the determination of the presence of at least one mutation in the gyrA gene comprises the determination of the presence of the sequence SEQ ID NO:

6.

10. Method according to one of claims 4 to 9, characterized in that the determination of the presence of at least one mutation in the genomic region between positions 1165444 and 1165528 and / or of at least one mutation in the genomic region between positions 1277873 and 1277957 comprises the determination of the presence of at least one sequence chosen from the sequences SEQ ID NO: 7 and SEQ ID NO:

8.

11. Method according to one of claims 6 to 10, characterized in that the determination of the presence of at least one mutation in the embB gene comprises the determination of the presence of the sequence SEQ ID NO:

9.

12. Method according to one of claims 6 to 11, characterized in that: the determination of the presence of at least one mutation in the pncA gene consists of determining the presence of at least one of the sequences SEQ ID NO: 10 and SEQ ID NO: 11, the determination of the presence of at least one mutation in the katG gene consists of determining the presence of the sequence SEQ ID NO:

12.

13. Method according to claim 12, characterized in that it comprises a step of determining the presence of the sequences SEQ ID NOs: 1 to 12 in the respective genes and of determining the sensitive or resistant character of said strain depending on the presence or absence of said sequences.

14. Method according to any one of claims 1 to 13, characterized in that the steps of determining the presence of mutations are carried out in relation to the wild reference genome of Mycobacterium tuberculosis H37Rv (reference NC_000962.3).

15. Method according to any one of claims 1 to 14, wherein the step of determining the presence of at least one mutation in said genes is carried out by a hybridization, amplification or sequencing method.

16. Method according to any one of claims 1 to 15, characterized in that it is implemented by a computer.

17. Data processing apparatus comprising: means for implementing the method according to claim 16, in particular means for determining the presence of mutations in the rpoB gene of the Mycobacterium tuberculosis strain between positions 761112 and 761182, and / or means for comparing the sequence of said gene with the reference genome Mycobacterium tuberculosis H37Rv (reference NC_000962.3), and / or means for identifying the presence of the sequences SEQ ID NOs 1 to 3 in the genome of the Mycobacterium tuberculosis strain, and / or means for providing output data of the presence or absence of said sequences, when said means are implemented or controlled by computer, or a processor adapted to or configured to execute the computer-implemented method according to claim 16, in particular a processor adapted to or configured to execute the steps of said method.

18. Kit comprising means for detecting at least one sequence chosen from the sequences SEQ ID NO: 1 to 3, and optionally at least one other sequence chosen from the sequences SEQ ID NO: 4 to 12, said means preferably being primers and / or probes.

19. Use of the kit according to claim 18 for determining the susceptibility of a Mycobacterium tuberculosis strain to pyrazinamide.

20. Use of the sequences SEQ ID NOs 1 to 3, and preferably also of the sequences SEQ ID NOs 4 to 12, for determining the susceptibility of a Mycobacterium tuberculosis strain to pyrazinamide.

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