title: Revealing hidden clonal complexity in Mycobacterium tuberculosis infection by qualitative and quantitative improvement of sampling.
abstract: The analysis of microevolution events, its functional relevance and impact on molecular epidemiology strategies, constitutes one of the most challenging aspects of the study of clonal complexity in infection by Mycobacterium tuberculosis. In this study, we retrospectively evaluated whether two improved sampling schemes could provide access to the clonal complexity that is undetected by the current standards (analysis of one isolate from one sputum). We evaluated in 48 patients the analysis by mycobacterial interspersed repetitive unit-variable number tandem repeat of M. tuberculosis isolates cultured from bronchial aspirate (BAS) or bronchoalveolar lavage (BAL) and, in another 16 cases, the analysis of a higher number of isolates from independent sputum samples. Analysis of the isolates from BAS/BAL specimens revealed clonal complexity in a very high proportion of cases (5/48); in most of these cases, complexity was not detected when the isolates from sputum samples were analysed. Systematic analysis of isolates from multiple sputum samples also improved the detection of clonal complexity. We found coexisting clonal variants in two of 16 cases that would have gone undetected in the analysis of the isolate from a single sputum specimen. Our results suggest that analysis of isolates from BAS/BAL specimens is highly efficient for recording the true clonal composition of M. tuberculosis in the lungs. When these samples are not available, we recommend increasing the number of isolates from independent sputum specimens, because they might not harbour the same pool of bacteria. Our data suggest that the degree of clonal complexity in tuberculosis has been underestimated because of the deficiencies inherent in a simplified procedure.
authors: Servicio Microbiología Clínica y Enfermedades Infecciosas, Hospital General Universitario Gregorio Marañón, Madrid, Spain; Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain; CIBER Enfermedades respiratorias, CIBERES, Spain.
authors: L
authors: L
authors: Pérez-Lago
authors: Unidad de Referencia Regional de Micobacterias, Servicio de Microbiología, Hospital Universitario Central de Asturias, Oviedo, Spain.
authors: J J
authors: JJ
authors: Palacios
authors: Servicio Microbiología Clínica y Enfermedades Infecciosas, Hospital General Universitario Gregorio Marañón, Madrid, Spain; Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain; CIBER Enfermedades respiratorias, CIBERES, Spain.
authors: M
authors: M
authors: Herranz
authors: Servicio Microbiología Clínica y Enfermedades Infecciosas, Hospital General Universitario Gregorio Marañón, Madrid, Spain; Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain; CIBER Enfermedades respiratorias, CIBERES, Spain.
authors: M J
authors: MJ
authors: Ruiz Serrano
authors: Servicio Microbiología Clínica y Enfermedades Infecciosas, Hospital General Universitario Gregorio Marañón, Madrid, Spain; Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain; CIBER Enfermedades respiratorias, CIBERES, Spain; Departamento de Medicina, Facultad de Medicina, Universidad Complutense de Madrid, Madrid, Spain.
authors: E
authors: E
authors: Bouza
authors: Servicio Microbiología Clínica y Enfermedades Infecciosas, Hospital General Universitario Gregorio Marañón, Madrid, Spain; Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain; CIBER Enfermedades respiratorias, CIBERES, Spain; CEI Campus Moncloa, UCM-UPM, Madrid, Spain. Electronic address: dgviedma2@gmail.com.
authors: D
authors: D
authors: García-de-Viedma
mesh_terms: D001980:Bronchi / Q000382:microbiology; D001992:Bronchoalveolar Lavage Fluid / Q000382:microbiology; D014644:Genetic Variation; D005838:Genotype; D006801:Humans; D020071:Interspersed Repetitive Sequences; D018598:Minisatellite Repeats; D058889:Molecular Typing; D009169:Mycobacterium tuberculosis / Q000145:classification* / Q000235:genetics*; D012189:Retrospective Studies; D013048:Specimen Handling / Q000379:methods*; D013183:Sputum / Q000382:microbiology; D014376:Tuberculosis / Q000382:microbiology*
keywords: Clonal complexity; hidden complexity; improved detection; sampling schemes; tuberculosis