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.

Diagnóstico bacteriológico de la tuberculosis. Estado actual del conocimiento Primera parte / Bacteriologic Diagnosis of Tuberculosis. Current State of Knowledge First part

Uno de los principales desafíos para los Programas de Control de Tuberculosis (PCT) lo constituye la detección temprana de formas abiertas de la enfermedad. La Organización Mundial de la Salud (OMS) ha estimado que el desarrollo de un método de diagnóstico de tuberculosis (TB) que ofreciera un 85% de sensibilidad y un 95% de especificidad sobre muestras de esputo permitiría salvar unas 400000 vidas al año. En condiciones ideales, sería necesario, además, que un método accesible y preciso, aplicado en colectivos de mayor vulnerabilidad, pudiera aportar tanto a la identificación de especie como a su perfil de resistencia, en especial si este implicara un mayor riesgo de fracaso terapéutico. En la última década, el desarrollo del sistema de diagnóstico GeneXpert MTB/RIF ha significado un gran avance en ese sentido. A un costo de USD 9,98 por determinación (en los 145 países subsidiados), el método permitió acercarse a los objetivos mencionados, es decir, la detección precoz de la TB y la detección de resistencia a rifampicina, usualmente tomada como un indicador de fracaso terapéutico.

Recurrences of multidrug-resistant tuberculosis: Strains involved, within-host diversity, and fine-tuned allocation of reinfections.

Recurrent tuberculosis occurs due to exogenous reinfection or reactivation/persistence. We analysed 90 sequential MDR Mtb isolates obtained in Argentina from 27 patients with previously diagnosed MDR-TB that recurred in 2018 (1-10 years, 2-10 isolates per patient). Three long-term predominant strains were responsible for 63% of all MDR-TB recurrences. Most of the remaining patients were infected by strains different from each other. Reactivation/persistence of the same strain caused all but one recurrence, which was due to a reinfection with a predominant strain. One of the prevalent strains showed marked stability in the recurrences, while in another strain higher SNP-based diversity was observed. Comparisons of intra- versus inter-patient SNP distances identified two possible reinfections with closely related variants circulating in the community. Our results show a complex scenario of MDR-TB infections in settings with predominant MDR Mtb strains.

Five-year microevolution of a multidrug-resistant Mycobacterium tuberculosis strain within a patient with inadequate compliance to treatment.

BACKGROUND: Whole-genome sequencing has shown that the Mycobacterium tuberculosis infection process can be more heterogeneous than previously thought. Compartmentalized infections, exogenous reinfections, and microevolution are manifestations of this clonal complexity. The analysis of the mechanisms causing the microevolution -the genetic variability of M. tuberculosis at short time scales- of a parental strain into clonal variants with a patient is a relevant issue that has not been yet completely addressed. To our knowledge, a whole genome sequence microevolution analysis in a single patient with inadequate adherence to treatment has not been previously reported. CASE PRESENTATION: In this work, we applied whole genome sequencing analysis for a more in-depth analysis of the microevolution of a parental Mycobacterium tuberculosis strain into clonal variants within a patient with poor treatment compliance in Argentina. We analyzed the whole-genome sequence of 8 consecutive Mycobacterium tuberculosis isolates obtained from a patient within 57-months of intermittent therapy. Nineteen mutations (9 short-term, 10 fixed variants) emerged, most of them associated with drug resistance. The first isolate was already resistant to isoniazid, rifampicin, and streptomycin, thereafter the strain developed resistance to fluoroquinolones and pyrazinamide. Surprisingly, isolates remained susceptible to the pro-drug ethionamide after acquiring a frameshift mutation in ethA, a gene required for its activation. We also found a novel variant, (T-54G), in the 5' untranslated region of whiB7 (T-54G), a region allegedly related to kanamycin resistance. Notably, discrepancies between canonical and phage-based susceptibility testing to kanamycin were previously found for the isolate harboring this mutation. In our patient, microevolution was mainly driven by drug selective pressure. Rare short-term mutations fixed together with resistance-conferring mutations during therapy. CONCLUSIONS: This report highlights the relevance of whole-genome sequencing analysis in the clinic for characterization of pre-XDR and MDR resistance profile, particularly in patients with incomplete and/or intermittent treatment.

Trends of Two Epidemic Multidrug-Resistant Strains of Mycobacterium tuberculosis in Argentina Disclosed by Tailored Molecular Strategy.

Two Mycobacterium tuberculosis strains-M (sublineage 4.1) and Ra (sublineage 4.3)-have long prevailed in Argentina among patients with multidrug-resistant tuberculosis (MDR-TB). Recently, budget constraints have hampered the surveillance of MDR-TB transmission. Based on whole-genome sequence analysis, we used M- and Ra-specific single nucleotide polymorphisms to tailor two multiplex allele-specific polymerase chain reactions (PCRs), which we applied to 252 stored isolates (95% of all newly diagnosed MDR-TB cases countrywide, 2015-2017). Compared with the latest data available (2007-2009), the M strain has receded (80/324 to 20/252, P < 0.0001), particularly among cross-border migrants (12/58 to 0/53, P = 0.0003) and HIV-infected people (30/97 to 7/74, P = 0.0007), but it still accounts for 4/12 new cases of extensively drug-resistant TB. Differently, the Ra strain remained stable in frequency (39/324 to 33/252) and contributed marginally to the extensive drug-resistance load (1/12). Our novel strategy disclosed recent trends of the two major MDR-TB strains, providing meaningful data to allocate control interventions more efficiently.

Survival of an epidemic MDR strain of Mycobacterium tuberculosis and its non-prosperous variant within activated macrophages.

The fitness of a pathogen results from the interaction of multiple factors favoring either epidemiological success or failure. Herein, we studied the performance of the M strain, a highly successful multidrug resistant Mycobacterium tuberculosis genotype, and its non-prosperous variant, the 410 strain, in activated human monocyte-derived macrophages. Both strains showed comparable ability to induce necrotic cell death and to survive in apoptotic macrophages. Of the various macrophage activation conditions tested, none led to an enhanced control of the outbreak strain. The combination of 1,25(OH)2 vitaminD3 and IFN-γ favored significantly the control of the non-prosperous 410 strain. These observations indicate that the ability of the M strain to survive within the hostile intracellular milieu is conserved, and the overall fitness cost paid by this genotype would be low. Our results provide additional evidence on bacterial traits that may have contributed to the epidemiological success of the M strain.

Bedaquiline and linezolid MIC distributions and epidemiological cut-off values for Mycobacterium tuberculosis in the Latin American region.

Objectives: To describe the distributions of bedaquiline and linezolid MIC values for the Mycobacterium tuberculosis WT population and to define the corresponding epidemiological cut-offs (ECOFFs) in three Latin American countries. Methods: MICs of bedaquiline and linezolid were determined by the resazurin microtitre assay (REMA). In phase 1, interlaboratory reproducibility was assessed using a panel of 10 fully susceptible M. tuberculosis strains. Phase 2 involved MIC determination for 248 clinical isolates from Argentina (n = 58), Brazil (n = 100) and Peru (n = 90) from patients who were treatment-naive for bedaquiline and linezolid. We then determined the ECOFFs for bedaquiline and linezolid by the eyeball method and the ECOFFinder statistical calculator. Results: Phase 1: REMA MIC values in the three sites were either identical to each other or differed by one 2-fold dilution from the consensus value with the exception of a single value. Phase 2: the bedaquiline MIC range was 0.0039-0.25 mg/L for pan-susceptible and drug-resistant isolates combined. The linezolid MIC range was 0.062-0.5 mg/L for pan-susceptible isolates and 0.031-4 mg/L for drug-resistant isolates. ECOFFs were 0.125 mg/L for bedaquiline and 0.50 mg/L for linezolid. Conclusions: REMA is reproducible and robust for the determination of bedaquiline and linezolid MIC distributions and ECOFF values when applied in laboratories of medium/low-resource countries. We suggest that WT MIC distributions for both drugs should be used as a monitoring tool to control the possible rapid emergence of resistance.

Crystal violet decolorization assay for rapid detection of multidrug-resistant Mycobacterium tuberculosis isolates: A multicenter study.

Background: Effective control of tuberculosis is achieved by early diagnosis and drug susceptibility testing for initiation of appropriate treatment. The performance of crystal violet decolorization assay (CVDA) for susceptibility testing of Mycobacterium tuberculosis to isoniazid (INH) and rifampicin (RIF) was compared in a multicenter study. Methods: Seventy-two M. tuberculosis isolates were tested in two phases by CVDA. Results: In Phase I, the specificity, sensitivity, positive predictive value (PPV), negative predictive value (NPV), and agreement for INH were 100%, respectively. Specificity, sensitivity, PPV, NPV, and agreement for RIF were 98.2%, 100%, 94.1%, 100%, and 98.6%, respectively. In Phase II, specificity, sensitivity, PPV, NPV, and agreement were 98%, 100%, 95.4%, 100%, and 98.6% for INH, respectively. Specificity, sensitivity, PPV, NPV, and agreement for RIF were 96.3%, 88.2%, 88.2%, 96.3%, and 94.4%, respectively. Results in the study were obtained on average 10.9 ± 3.1 days in Phase I and 9.8 ± 2.2 days in Phase II. Conclusion: CVDA can be performed for drug susceptibility testing in developed and developing countries. In addition, further studies with larger sample size are needed for evaluation of this method.

Genotypic diversity of Mycobacterium tuberculosis in Buenos Aires, Argentina.

Buenos Aires is an overpopulated port city historically inhabited by people of European descent. Together with its broader metropolitan area, the city exhibits medium tuberculosis rates, and receives migrants, mainly from tuberculosis highly endemic areas of Argentina and neighboring countries. This work was aimed to gain insight into the Mycobacterium tuberculosis population structure in two suburban districts of Buenos Aires which are illustrative of the overall situation of tuberculosis in Argentina. The Lineage 4 Euro-American accounted for >99% of the 816 isolates analyzed (one per patient). Frequencies of spoligotype families were T 35.9%, LAM 33.2%, Haarlem 19.5%, S 3.2%, X 1.5%, Ural 0.7%, BOV 0.2%, Beijing 0.2%, and Cameroon 0.2%. Unknown signatures accounted for 5.3% isolates. Of 55 spoligotypes not matching any extant shared international type (SIT) in SITVIT database, 22 fitted into 15 newly-issued SITs. Certain autochthonous South American genotypes were found to be actively evolving. LAM3, which is wild type for RDrio, was the predominant LAM subfamily in both districts and the RDrio signature was rare among autochthonous, newly created, SITs and orphan patterns. Two genotypes that are rarely observed in neighboring countries ̶ SIT2/H2 and SIT159/T1 Tuscany ̶ were conspicuously represented in Argentina. The infrequent Beijing patterns belonged to Peruvian patients. We conclude that the genotype diversity observed reflects the influence of the Hispanic colonization and more recent immigration waves from Mediterranean and neighboring countries. Unlike in Brazil, the RDrio type does not play a major role in the tuberculosis epidemic in Buenos Aires.

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.

Tuberculosis extensamente resistente (XDR-TB) en Argentina: aspectos destacables epidemiológicos, bacteriológicos, terapéuticos y evolutivos / Extensively resistant tuberculosis (XDR-TB) in Argentina: epidemiology, bacteriology, therapy and outcome

La XDR-TB (resistente a isoniazida, rifampicina, alguna fluoroquinolona y al menos una entre kanamicina, amikacina o capreomicina), ha causado efectos devastadores en pacientes con SIDA y es prácticamente incurable. Se presentan 12 casos de localización pulmonar en pacientes no SIDA. Se trataron con esquemas que incluyeron en todos linezolid y en 9 moxifloxacino, todos negativizaron el examen directo y cultivo del esputo. Nueve pacientes cumplieron criterios de curación, 1 está aún en tratamiento y 2 abandonaron. Ocho pacientes presentaron efectos adversos, en solo 1 caso debió suspenderse la tioridazina. La utilización de linezolid, moxifloxacina y tioridazina han contribuido a la evolución satisfactoria de estos pacientes. Estos fármacos son considerados de utilidad en la serie reportada, debiendo ser utilizados en centros especializados con experiencia en el manejo de la TB MR y XDR-TB.

The XDR-TB (resistant to isoniazid, rifampiN, fluorquinolone and at least of the following: kanamycina, amikacyna or capreomycin), has caused devastating effects in patients with AIDS and is practically incurable. Twelve cases of pulmonary XDR-TB in non AIDS are described. All were treated with schemes that included linezolid in all and moxifloxacin in 9, all respiratory specimens became negative. Nine patients fulfilled healing criteria, 1 is still under treatment and 2 abandoned the therapy. Eight patients presented adverse effects, thioridazine was stopped in only one patient. Linezolid, moxifloxacin and tioridazin contributed to the satisfactory evolution of these patients. These drugs were considered useful in the reported series of cases and should be used in specialized centres with experience in the management of MR TB and XDR-TB.

Multicenter evaluation of the nitrate reductase assay for drug resistance detection of Mycobacterium tuberculosis.

The performance of the nitrate reductase assay was evaluated in a multicenter laboratory study to detect resistance of Mycobacterium tuberculosis to the first-line anti-tuberculosis drugs rifampicin, isoniazid, ethambutol and streptomycin using a set of coded isolates. Compared with the gold standard proportion method on Löwenstein-Jensen medium, the assay was highly accurate in detecting resistance to rifampicin, isoniazid and ethambutol with an accuracy of 98%, 96.6% and 97.9%, respectively. For streptomycin, discrepant results were obtained with an overall accuracy of 85.3%. The assay proved easy to be implemented in countries with limited laboratory facilities.

In-house phage amplification assay is a sound alternative for detecting rifampin-resistant Mycobacterium tuberculosis in low-resource settings.

An in-house mycobacteriophage amplification assay for detecting rifampin-resistant Mycobacterium tuberculosis showed 100% sensitivity, 97.7% specificity, and 95.2% predictive value for resistance in a test of 129 isolates from a hot spot area of multidrug-resistant M. tuberculosis. The applicability of the test was demonstrated in the routine work flow of a low-resource reference laboratory.