Un desafío terapéutico: la heterorresistencia en Mycobacterium tuberculosis / An emerging problem: heteroresistance in Mycobacterium tuberculosis

Resumen Se considera infección mixta por Mycobacterium tuberculosis (Mtb) a la coexistencia en forma simul tánea y en un mismo paciente de 2 cepas diferentes de Mtb o 2 variantes distintas de la misma cepa. Cuando una de las variantes selecciona mutaciones de resistencia, se denomina heterorresistencia (HTR) monoclonal; en caso de que sean 2 cepas diferentes, una sensible y una resistente (o cepas con diferentes patrones de resistencia), se denomina HTR policlo nal. Se presentan 3 pacientes, HIV/sida, todos con reiterados problemas de adherencia al tratamiento, en los cuales a través de la secuenciación genómica completa de Mtb se diagnosticó HTR monoclonal con coexistencia de 2 variantes de la misma cepa aisladas de muestras de pulmón y ganglios linfáticos, con diferentes perfiles de resistencia en cada uno de los casos. Es importante pensar en la posibilidad de HTR, principalmente en pacientes con múltiples intentos terapéuticos previos y altas poblaciones bacilares, como en el sida avanzado, dado que esta situación compromete potencialmente los resultados del tratamiento al coexistir cepas o variantes de ce pas sensibles y resistentes.

Abstract Mixed infection by Mycobacterium tuberculosis (Mtb) consists in the simultaneous coexistence in the same patient of two different strains of Mtb or 2 different variants of the same strain. When one of the variants selects for resistance mutations, it is called monoclonal heteroresistance (HTR); if there are 2 different strains, one sensitive and one resistant (or with different resis tance patterns), it is called polyclonal HTR. Three cases of HIV/AIDS patients are presented, all with repeated treatment adherence problems, in whom monoclonal HTR was diagnosed through Mtb complete genomic sequentiation with the coexistence of two variants of the same strain isolated from samples from lung and lymph nodes, with different resistance profiles in each case. It is important to consider the possibility of HTR, especially in patients with multiple previous therapeu tic attempts and high bacillary populations, such as in advanced AIDS, since this situation potentially com promises treatment results by coexisting sensitive and resistant variants of a strain (or strains).

[An emerging problem: heteroresistance in Mycobacterium tuberculosis]. / Un desafío terapéutico: la heterorresistencia en Mycobacterium tuberculosis.

Mixed infection by Mycobacterium tuberculosis (Mtb) consists in the simultaneous coexistence in the same patient of two different strains of Mtb or 2 different variants of the same strain. When one of the variants selects for resistance mutations, it is called monoclonal heteroresistance (HTR); if there are 2 different strains, one sensitive and one resistant (or with different resistance patterns), it is called polyclonal HTR. Three cases of HIV/AIDS patients are presented, all with repeated treatment adherence problems, in whom monoclonal HTR was diagnosed through Mtb complete genomic sequentiation with the coexistence of two variants of the same strain isolated from samples from lung and lymph nodes, with different resistance profiles in each case. It is important to consider the possibility of HTR, especially in patients with multiple previous therapeutic attempts and high bacillary populations, such as in advanced AIDS, since this situation potentially compromises treatment results by coexisting sensitive and resistant variants of a strain (or strains).

Se considera infección mixta por Mycobacterium tuberculosis (Mtb) a la coexistencia en forma simultánea y en un mismo paciente de 2 cepas diferentes de Mtb o 2 variantes distintas de la misma cepa. Cuando una de las variantes selecciona mutaciones de resistencia, se denomina heterorresistencia (HTR) monoclonal; en caso de que sean 2 cepas diferentes, una sensible y una resistente (o cepas con diferentes patrones de resistencia), se denomina HTR policlonal. Se presentan 3 pacientes, HIV/sida, todos con reiterados problemas de adherencia al tratamiento, en los cuales a través de la secuenciación genómica completa de Mtb se diagnosticó HTR monoclonal con coexistencia de 2 variantes de la misma cepa aisladas de muestras de pulmón y ganglios linfáticos, con diferentes perfiles de resistencia en cada uno de los casos. Es importante pensar en la posibilidad de HTR, principalmente en pacientes con múltiples intentos terapéuticos previos y altas poblaciones bacilares, como en el sida avanzado, dado que esta situación compromete potencialmente los resultados del tratamiento al coexistir cepas o variantes de cepas sensibles y resistentes.

Fluoromycobacteriophages Can Detect Viable Mycobacterium tuberculosis and Determine Phenotypic Rifampicin Resistance in 3-5 Days From Sputum Collection.

The World Health Organization (WHO) estimates that 40% of tuberculosis (TB) cases are not diagnosed and treated correctly. Even though there are several diagnostic tests available in the market, rapid, easy, inexpensive detection, and drug susceptibility testing (DST) of Mycobacterium tuberculosis is still of critical importance specially in low and middle-income countries with high incidence of the disease. In this work, we have developed a microscopy-based methodology using the reporter mycobacteriophage mCherrybomb ϕ for detection of Mycobacterium spp. and phenotypic determination of rifampicin resistance within just days from sputum sample collection. Fluoromycobacteriophage methodology is compatible with regularly used protocols in clinical laboratories for TB diagnosis and paraformaldehyde fixation after infection reduces biohazard risks with sample analysis by fluorescence microscopy. We have also set up conditions for discrimination between M. tuberculosis complex (MTBC) and non-tuberculous mycobacteria (NTM) strains by addition of p-nitrobenzoic acid (PNB) during the assay. Using clinical isolates of pre-XDR and XDR-TB strains from this study, we tested mCherrybomb Φ for extended DST and we compared the antibiotic resistance profile with those predicted by whole genome sequencing. Our results emphasize the utility of a phenotypic test for M. tuberculosis extended DST. The many attributes of mCherrybomb Φ suggests this could be a useful component of clinical microbiological laboratories for TB diagnosis and since only viable cells are detected this could be a useful tool for monitoring patient response to treatment.

Relation of Mycobacterium tuberculosis mutations at katG315 and inhA-15 with drug resistance profile, genetic background, and clustering in Argentina.

We analyzed 362 isoniazid-resistant clinical isolates of Mycobacterium tuberculosis obtained countrywide for the presence of mutation at katG315 and inhA-15 in relation to genotype, pattern of phenotypic resistance to other drugs, and ability to spread. We found the following mutation frequencies: katG315MUT/inhA-15wt 53.0%, katG315wt/inhA-15MUT 27.4%, katG315wt/inhA-15wt 19.3%, and katG315MUT/inhA-15MUT only 0.3%. Mutation at katG315 associated with the LAM superfamily; mutation at inhA-15 associated with the S family and the T1 Tuscany genotype; the combination katG315wt/inhA-15wt associated with the T1 Ghana genotype. Isolates harboring katG315MUT/inhA-15wt tended to accumulate resistance to other drugs and were more frequently found in cluster; isolates harboring katG315wt/inhA-15wt were more frequently found as orphan isolates. Although epidemiological and host factors could also be modulating the events observed, in Argentina, the systematic genotyping of drug resistant clinical isolates could help to predict an enhanced risk of transmission and a propensity to develop resistance to increasing numbers of drugs.