Evaluation of a new line probe assay for rapid identification of gyrA mutations in Mycobacterium tuberculosis

Federico Giannoni, Elisabetta Iona, Federica Sementilli, Lara Brunori, Manuela Pardini, Giovanni Battista Migliori, Graziella Orefici, Lanfranco Fattorini

Research output: Contribution to journalArticle

47 Citations (Scopus)

Abstract

Resistance of Mycobacterium tuberculosis to fluoroquinolones (FQ) results mostly from mutations in the gyrA gene. We developed a reverse hybridization-based line probe assay in which oligonucleotide probes carrying the wild-type gyrA sequence, a serine-to-threonine (S95T) polymorphism, and gyrA mutations (A90V, A90V-S95T, S91P, S91P-S95T, D94A, D94N, D94G-S95T, D94H-S95T) were immobilized on nitrocellulose strips and hybridized with digoxigenin-labeled PCR products obtained from M. tuberculosis strains. When a mutated PCR product was used, hybridization occurred to the corresponding mutated probe but not to the wild-type probe. A panel of M. tuberculosis complex strains including 19 ofloxacin-resistant (OFL-R) and 9 ofloxacin-susceptible (OFL-S) M. tuberculosis strains was studied for detection and identification of gyrA mutations by the line probe assay and nucleotide sequencing, in comparison with testing of in vitro susceptibility to FQ. Results were 100% concordant with those of nucleotide sequencing. The S95T polymorphism, which is not related to FQ resistance, was found in 5 OFL-S and 2 OFL-R strains; the other 17 OFL-R strains harbored single mutations associated with serine or threonine at codon 95. No mutations were found in the other OFL-S strains. Overall, on the basis of the MICs on solid medium, the new line probe assay correctly identified all OFL-S and 17 out of 19 (89.5%) OFL-R strains. A nested-PCR protocol was also evaluated for the assay to amplify PCR products from M. tuberculosis-spiked sputa, with a good specificity and a sensitivity of 2 × 103 M. tuberculosis CFU per ml of sputum.

Original languageEnglish
Pages (from-to)2928-2933
Number of pages6
JournalAntimicrobial Agents and Chemotherapy
Volume49
Issue number7
DOIs
Publication statusPublished - Jul 2005

Fingerprint

Ofloxacin
Mycobacterium tuberculosis
Mutation
Fluoroquinolones
Polymerase Chain Reaction
Threonine
Sputum
Serine
Nucleotides
Digoxigenin
Collodion
Oligonucleotide Probes
Codon
Sensitivity and Specificity

ASJC Scopus subject areas

  • Pharmacology (medical)

Cite this

Evaluation of a new line probe assay for rapid identification of gyrA mutations in Mycobacterium tuberculosis. / Giannoni, Federico; Iona, Elisabetta; Sementilli, Federica; Brunori, Lara; Pardini, Manuela; Migliori, Giovanni Battista; Orefici, Graziella; Fattorini, Lanfranco.

In: Antimicrobial Agents and Chemotherapy, Vol. 49, No. 7, 07.2005, p. 2928-2933.

Research output: Contribution to journalArticle

Giannoni, Federico ; Iona, Elisabetta ; Sementilli, Federica ; Brunori, Lara ; Pardini, Manuela ; Migliori, Giovanni Battista ; Orefici, Graziella ; Fattorini, Lanfranco. / Evaluation of a new line probe assay for rapid identification of gyrA mutations in Mycobacterium tuberculosis. In: Antimicrobial Agents and Chemotherapy. 2005 ; Vol. 49, No. 7. pp. 2928-2933.
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abstract = "Resistance of Mycobacterium tuberculosis to fluoroquinolones (FQ) results mostly from mutations in the gyrA gene. We developed a reverse hybridization-based line probe assay in which oligonucleotide probes carrying the wild-type gyrA sequence, a serine-to-threonine (S95T) polymorphism, and gyrA mutations (A90V, A90V-S95T, S91P, S91P-S95T, D94A, D94N, D94G-S95T, D94H-S95T) were immobilized on nitrocellulose strips and hybridized with digoxigenin-labeled PCR products obtained from M. tuberculosis strains. When a mutated PCR product was used, hybridization occurred to the corresponding mutated probe but not to the wild-type probe. A panel of M. tuberculosis complex strains including 19 ofloxacin-resistant (OFL-R) and 9 ofloxacin-susceptible (OFL-S) M. tuberculosis strains was studied for detection and identification of gyrA mutations by the line probe assay and nucleotide sequencing, in comparison with testing of in vitro susceptibility to FQ. Results were 100{\%} concordant with those of nucleotide sequencing. The S95T polymorphism, which is not related to FQ resistance, was found in 5 OFL-S and 2 OFL-R strains; the other 17 OFL-R strains harbored single mutations associated with serine or threonine at codon 95. No mutations were found in the other OFL-S strains. Overall, on the basis of the MICs on solid medium, the new line probe assay correctly identified all OFL-S and 17 out of 19 (89.5{\%}) OFL-R strains. A nested-PCR protocol was also evaluated for the assay to amplify PCR products from M. tuberculosis-spiked sputa, with a good specificity and a sensitivity of 2 × 103 M. tuberculosis CFU per ml of sputum.",
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AU - Giannoni, Federico

AU - Iona, Elisabetta

AU - Sementilli, Federica

AU - Brunori, Lara

AU - Pardini, Manuela

AU - Migliori, Giovanni Battista

AU - Orefici, Graziella

AU - Fattorini, Lanfranco

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