title: Mycobacterium tuberculosis pyrazinamide resistance determinants: a multicenter study.
abstract: Pyrazinamide (PZA) is a prodrug that is converted to pyrazinoic acid by the enzyme pyrazinamidase, encoded by the pncA gene in Mycobacterium tuberculosis. Molecular identification of mutations in pncA offers the potential for rapid detection of pyrazinamide resistance (PZA(r)). However, the genetic variants are highly variable and scattered over the full length of pncA, complicating the development of a molecular test. We performed a large multicenter study assessing pncA sequence variations in 1,950 clinical isolates, including 1,142 multidrug-resistant (MDR) strains and 483 fully susceptible strains. The results of pncA sequencing were correlated with phenotype, enzymatic activity, and structural and phylogenetic data. We identified 280 genetic variants which were divided into four classes: (i) very high confidence resistance mutations that were found only in PZA(r) strains (85%), (ii) high-confidence resistance mutations found in more than 70% of PZA(r) strains, (iii) mutations with an unclear role found in less than 70% of PZA(r) strains, and (iv) mutations not associated with phenotypic resistance (10%). Any future molecular diagnostic assay should be able to target and identify at least the very high and high-confidence genetic variant markers of PZA(r); the diagnostic accuracy of such an assay would be in the range of 89.5 to 98.8%. Importance: Conventional phenotypic testing for pyrazinamide resistance in Mycobacterium tuberculosis is technically challenging and often unreliable. The development of a molecular assay for detecting pyrazinamide resistance would be a breakthrough, directly overcoming both the limitations of conventional testing and its related biosafety issues. Although the main mechanism of pyrazinamide resistance involves mutations inactivating the pncA enzyme, the highly diverse genetic variants scattered over the full length of the pncA gene and the lack of a reliable phenotypic gold standard hamper the development of molecular diagnostic assays. By analyzing a large number of strains collected worldwide, we have classified the different genetic variants based on their predictive value for resistance which should lead to more rapid diagnostic tests. This would assist clinicians in improving treatment regimens for patients.
authors.affiliation: Emerging Bacterial Pathogens Unit, Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan, Italy miotto.paolo@hsr.it.
authors.forename: Paolo
authors.initials: P
authors.lastname: Miotto
authors.affiliation: Emerging Bacterial Pathogens Unit, Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan, Italy.
authors.forename: Andrea M
authors.initials: AM
authors.lastname: Cabibbe
authors.forename: Silke
authors.initials: S
authors.lastname: Feuerriegel
authors.forename: Nicola
authors.initials: N
authors.lastname: Casali
authors.forename: Francis
authors.initials: F
authors.lastname: Drobniewski
authors.affiliation: Samara TB Service, Samara, Russian Federation.
authors.forename: Yulia
authors.initials: Y
authors.lastname: Rodionova
authors.affiliation: Department of Immunology and Cell Biology, Institute of Biotechnology, Vilnius University, Vilnius, Lithuania.
authors.forename: Daiva
authors.initials: D
authors.lastname: Bakonyte
authors.affiliation: Department of Immunology and Cell Biology, Institute of Biotechnology, Vilnius University, Vilnius, Lithuania.
authors.forename: Petras
authors.initials: P
authors.lastname: Stakenas
authors.affiliation: National Tuberculosis Reference Laboratory, Infectious Diseases and Tuberculosis Hospital, Vilnius University Hospital Santariskiu Klinikos, Vilnius, Lithuania.
authors.forename: Edita
authors.initials: E
authors.lastname: Pimkina
authors.affiliation: Department of Microbiology, National Tuberculosis and Lung Diseases Research Institute, Warsaw, Poland.
authors.forename: Ewa
authors.initials: E
authors.lastname: Augustynowicz-Kopeć
authors.affiliation: Biocrystallography Unit, Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan, Italy.
authors.forename: Massimo
authors.initials: M
authors.lastname: Degano
authors.affiliation: Vita-Salute San Raffaele University, Milan, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Ambrosi
authors.affiliation: Public Health Agency of Sweden, Solna, and Department of Microbiology, Tumor and Cell Biology (MTC), Karolinska Institute, Stockholm, Sweden.
authors.forename: Sven
authors.initials: S
authors.lastname: Hoffner
authors.affiliation: Public Health Agency of Sweden, Solna, and Department of Microbiology, Tumor and Cell Biology (MTC), Karolinska Institute, Stockholm, Sweden.
authors.forename: Mikael
authors.initials: M
authors.lastname: Mansjö
authors.affiliation: Public Health Agency of Sweden, Solna, and Department of Microbiology, Tumor and Cell Biology (MTC), Karolinska Institute, Stockholm, Sweden.
authors.forename: Jim
authors.initials: J
authors.lastname: Werngren
authors.affiliation: National Reference Center for Mycobacteria, Research Center Borstel, Borstel, Germany.
authors.forename: Sabine
authors.initials: S
authors.lastname: Rüsch-Gerdes
authors.forename: Stefan
authors.initials: S
authors.lastname: Niemann
authors.affiliation: Emerging Bacterial Pathogens Unit, Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan, Italy.
authors.forename: Daniela M
authors.initials: DM
authors.lastname: Cirillo
mesh_terms: D000581:Amidohydrolases / Q000235:genetics* / Q000378:metabolism*; D000995:Antitubercular Agents / Q000494:pharmacology*; D024881:Drug Resistance, Bacterial; D014644:Genetic Variation; D006801:Humans; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics / Q000302:isolation & purification; D010802:Phylogeny; D011718:Pyrazinamide / Q000494:pharmacology*; D017422:Sequence Analysis, DNA; D014376:Tuberculosis / Q000382:microbiology
chemical_list: D000995:Antitubercular Agents; D011718:Pyrazinamide; D000581:Amidohydrolases; C487496:PncA protein, Mycobacterium tuberculosis