Investigação de tuberculose pulmonar por infecção policlonal de Mycobacterium tuberculosis e possível associação com a resistência aos fármacos antimicobacterianos

Abstract

In clinical practice is widely accepted that an episode of active tuberculosis (TB) is caused by a single clone of Mycobacterium tuberculosis. However, several studies have shown that there is heterogeneity of clones and this can be much larger than expected. In this context, the aim of this study was to investigate pulmonary TB by M. tuberculosis polyclonal infection in newly diagnosed patients, from a sputum sample and before starting treatment. A total of 62 of sputum samples were select. By screening in 7H10 medium, simultaneous presence of M. tuberculosis clones phenotypically sensitive and resistant to INH and/or RIF in the same isolate was observed in 30.6% (n=19). Genotyping by DRE-PCR and DNA sequencing of katG, inhA and rpoB genes were carried out in 74 clones INH and RIF sensitive, 65 clones INH-resistant, a clone RIF-resistant and 3 clones INH/RIF-resistant. The presence of resistant and sensitive clones to INH and/or RIF as well as the occurrence of different genotypes indicating heterogeneity in the same isolated from M. tuberculosis. The frequency of polyclonal infection was superior to published studies that show percentages ranging from 0.4% to 19.0%. These results indicate that the presence of polyclonal infection is not a rare phenomenon in patients with pulmonary TB in this population. The partial sequencing of the katG gene revealed that only 4.4% of INH-resistant clones were present described mutations in this gene. For inhA gene mutations were not detected for INH-resistant clones. Clones resistant to RIF present mutations in the rpoB gene. In clones INH-resistant, 83.8% not show mutations in katG and inhA genes, while 11.7% had mutations in katG gene not yet described. The absence of mutations commonly associated with drug resistance in katG and inhA genes, besides detection of new mutations in M. tuberculosis INH-resistant clones, suggests the need to sequencing larger fragments of these genes to propose strategies for fast detection of M. tuberculosis resistance. The results of this survey show that polyclonal infection is present in approximately one third of patients newly TB diagnosed, either by genotypic or phenotypic characteristics of M. tuberculosis clones, although both are not strictly related.