title: Hsp65 phylogenetic assay for molecular diagnosis of nontuberculous mycobacteria isolated in Mexico.
abstract: OBJECTIVE
  Nontuberculous mycobacteria (NTM) are mainly distributed as important emerging pathogens in patients with chronic or immunosuppressive diseases. Accurate identification of causative species is crucial for proper treatment and patient follow-up. However, several difficulties are associated with phenotypic and molecular diagnostic methods for precise identification at the species level due to shared metabolic and genetic characteristics. We undertook this study to evaluate the application of the phylogenetic method based on hsp65 gene into Telenti's PCR-restriction enzyme analysis (PRA) for molecular identification of NTM.
  
  
  METHODS
  The study population was comprised of 1646 Mycobacterium clinical isolates (AFB positive) collected from 2008-2011, of which 537 (32.6%) were MNT identified by PRA analysis. DNA sequencing of hsp65 in 53 isolates (10%) was performed. Sequence identification through NCBI-Basic Local Alignment Search Tool (BLAST) achieved correct identification in 23 isolates. Phylogenetic trees including hsp65 available GenBank sequences for all described genres of NTM and hsp65 obtained sequences were constructed using Mega 5.05 software. We compared sequence identification based on phylogenetic clustering and BLAST similarity search.
  
  
  RESULTS
  Phylogenetic clustering allowed more specific differentiation of closely related species and clearer identification in comparison with BLAST; 30 Mycobacterium species (this is the first report of isolation of some of these from clinical samples in Mexico) were identified in this way.
  
  
  CONCLUSIONS
  The proposed 440 bp hsp65 phylogenetic method allows a better identification tool to differentiate Mycobacterium species and is useful to complement diagnosis and epidemiological surveillance of NTM.
authors: Departamento de Biología Molecular y Validación de Técnicas, Instituto de Diagnóstico y Referencia Epidemiológicos (InDRE), Secretaría de Salud, México, D.F., Mexico.
authors: Noé
authors: N
authors: Escobar-Escamilla
authors: Departamento de Biología Molecular y Validación de Técnicas, Instituto de Diagnóstico y Referencia Epidemiológicos (InDRE), Secretaría de Salud, México, D.F., Mexico.
authors: José Ernesto
authors: JE
authors: Ramírez-González
authors: Departamento de Biología Molecular y Validación de Técnicas, Instituto de Diagnóstico y Referencia Epidemiológicos (InDRE), Secretaría de Salud, México, D.F., Mexico.
authors: Maribel
authors: M
authors: González-Villa
authors: Departamento de Biología Molecular y Validación de Técnicas, Instituto de Diagnóstico y Referencia Epidemiológicos (InDRE), Secretaría de Salud, México, D.F., Mexico.
authors: Pilar
authors: P
authors: Torres-Mazadiego
authors: Departamento de Biología Molecular y Validación de Técnicas, Instituto de Diagnóstico y Referencia Epidemiológicos (InDRE), Secretaría de Salud, México, D.F., Mexico.
authors: América
authors: A
authors: Mandujano-Martínez
authors: Laboratorio de Micobacterias, InDRE, Secretaría de Salud, México, D.F., Mexico.
authors: Candelaria
authors: C
authors: Barrón-Rivera
authors: Laboratorio de Micobacterias, InDRE, Secretaría de Salud, México, D.F., Mexico.
authors: Claudia Elena
authors: CE
authors: Bäcker
authors: Departamento de Biología Molecular y Validación de Técnicas, Instituto de Diagnóstico y Referencia Epidemiológicos (InDRE), Secretaría de Salud, México, D.F., Mexico.
authors: David Esaú
authors: DE
authors: Fragoso-Fonseca
authors: Departamento de Biología Molecular y Validación de Técnicas, Instituto de Diagnóstico y Referencia Epidemiológicos (InDRE), Secretaría de Salud, México, D.F., Mexico.
authors: Hiram
authors: H
authors: Olivera-Díaz
authors: Departamento de Biología Molecular y Validación de Técnicas, Instituto de Diagnóstico y Referencia Epidemiológicos (InDRE), Secretaría de Salud, México, D.F., Mexico.
authors: Patricia
authors: P
authors: Alcántara-Pérez
authors: Servicio de Neumología, Hospital General de México "Eduardo Liceaga", Secretaría de Salud, México, D.F., Mexico.
authors: Alejandro
authors: A
authors: Hernández-Solís
authors: Facultad de Medicina, Universidad Nacional Autónoma de México, México, D.F., Mexico.
authors: Raúl
authors: R
authors: Cícero-Sabido
authors: Departamento de Biología Molecular y Validación de Técnicas, Instituto de Diagnóstico y Referencia Epidemiológicos (InDRE), Secretaría de Salud, México, D.F., Mexico. Electronic address: iliancortes@yahoo.com.mx.
authors: Iliana Alejandra
authors: IA
authors: Cortés-Ortíz
mesh_terms: D000328:Adult; D001426:Bacterial Proteins / Q000235:genetics*; D018834:Chaperonin 60 / Q000235:genetics*; D005260:Female; D006801:Humans; D008297:Male; D008800:Mexico; D008875:Middle Aged; D009170:Nontuberculous Mycobacteria / Q000235:genetics* / Q000302:isolation & purification; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods; D015183:Restriction Mapping
chemical_list: D001426:Bacterial Proteins; D018834:Chaperonin 60; C074119:heat-shock protein 65, Mycobacterium
keywords: Molecular epidemiology; Nontuberculous mycobacteria; hsp65