Comparación y evaluación de métodos cuantitativos para determinar la susceptibilidad antimicrobiana de cepas del complejo Mycobacterium abscessus / Comparison and Evaluation of Quantitative Methods for Determining Antimicrobial Susceptibility of Complex Mycobacterium Abscessus Strains / Comparação e avaliação de métodos quantitativos para determinar a susceptibilidade antimicrobiana de cepas do complexo Mycobacterium abscessos

Resumen Introducción: el complejo Mycobacterium abscessus incluye especies patógenas emergentes multirresistentes, lo cual limita las opciones terapéuticas para tratar las infecciones causadas por dichos microorganismos. En este estudio se compararon las concentraciones inhibitorias mínimas (CIM) obtenidas mediante dos métodos cuantitativos, se establecieron los puntos de corte empleados en el micrométodo colorimétrico (MMC) y se evaluó la susceptibilidad antimicrobiana. Materiales y métodos: la CIM de nueve antibióticos fue determinada mediante el MMC y la microdilución en caldo (MDC) para 19 cepas del complejo M. abscessus. El test F de Snedecor se utilizó para establecer la diferencia significativa de las CIM entre los dos métodos y se determinaron los puntos de corte mediante la técnica de distribución de la probabilidad para el MMC. Resultados: se encontró una correlación de los resultados de la CIM del 50% entre MMC y MDC para los antibióticos ensayados. Probablemente esta discrepancia en los resultados se deba a diferencias en algunos parámetros técnicos de cada procedimiento. Todas las cepas fueron sensibles a la amikacina y resistentes a meropenem y ampicilina-sulbactam. Independientemente de la especie del complejo M. abscessus, las fluoroquinolonas mostraron una baja actividad inhibitoria (0-25%) sobre los aislados clínicos, resultados que son similares a los reportados por otros autores. Conclusión: Los patrones de multirresistencia observados en las cepas analizadas sugieren la necesidad de utilizar las pruebas de susceptibilidad como herramientas que permitan orientar y optimizar las conductas terapéuticas en infecciones producidas por M. abscessus.

Abstract Introduction: The Mycobacterium abscessus complex includes multidrug resistant emerging pathogens, which limit therapeutic options for treating infections caused by these microorganisms. In this study, the minimum inhibitory concentrations (MICS) obtained by 2 quantitative methods were compared, the cut-off points used in the colorimetric micromethod (CMM) were established and the antimicrobial susceptibility was evaluated. Materials and Methods: The MIC for nine antibiotics was determined by CMM and broth microdilution (BMD) for 19 strains of M. abscessus complex. The Snedecor F test was used to establish the significant difference in the CIM between the methods, cutoff points were determined by the probability distribution method for the CMM. Discussion: A correlation of 50% between CMM and BMD for antibiotics tested was found. Probably, this discrepancy in the results is due to differences in some technical parameters of each procedure. All strains were susceptible to amikacin and were resistant to meropenem and ampicillin-sulbactam. Independently of the species of M. abscessus complex, fluoroquinolones showed a low inhibitory activity (0-25%) on clinical isolates, results that are similar to those reported by other authors. Conclussion: The Multidrug resistance patterns observed in the strains tested suggest the need for susceptibility testing as tools to guide and optimize the therapeutic behavior in infections caused by M. abscessus.

Resumo Introdução: o complexo Mycobacterium abscessus inclui espécies patógenas emergentes multirresistentes, o qual limita as opções terapêuticas para tratar as infeções causadas por estes microrganismos. Neste estudo compararam-se as concentrações inibitórias mínimas (CIMS) obtidas mediante 2 métodos quantitativos, se estabeleceram os pontos de corte empregados no micrométodo colorimétrico (MMC) e se avaliou a susceptibilidade antimicrobiana. Materiais e métodos: a CIM de 9 antibióticos foi determinada mediante o MMC e microdiluição em caldo (MDC) para 19 cepas do complexo M. abscessus. O teste F de Snedecor utilizou-se para estabelecer a diferença significativa das CIMS entre os dois métodos e determinaram-se os pontos de corte mediante a técnica de distribuição da probabilidade para o MMC. Resultados: se encontrou uma correlação dos resultados da CIM do 50% entre MMC e MDC para os antibióticos testados. Provavelmente, esta discrepância nos resultados se deve a diferenças em alguns parâmetros técnicos de cada procedimento. Todas as cepas foram sensíveis à amikacina e resistentes a meropenem e ampicilina-sulbactam. Independentemente da espécie do complexo M. abscessos, as fluoroquinolonas mostraram uma baixa atividade inibitória (0-25%) sobre os isolados clínicos, resultados que são similares aos reportados por outros autores. Conclussão: Os patrões de multirresistência observados nas cepas analisadas, sugerem a necessidade de utilizar as provas de susceptibilidade como ferramentas que permitam orientar e otimizar as condutas terapêuticas em infeções produzidas por M. abscessus.

Relevancia clínica, diversidad y variabilidad genética de distintas especies del género Mycobacterium / Clinical Relevance, Diversity and Genetic Variability of Different Species within the Genus Mycobacterium

Introducción: El término micobacterias no tuberculosas (MNT) incluye distintas especies ambientales capaces de enfermar humanosy/o animales incluso mediante una probable transmisión zoonótica Objetivos. Determinar: la importancia clínica de varias especies del género Mycobacterium y la diversidad genética del Complejo M. avium (MAC), la sensibilidad bacteriana in vitro yel éxito del tratamiento especifico. Materiales y Métodos: Recolección de datos clínicos, epidemiológicos y aislamientos en el periodo 2009-2016; identificación molecular de los aislamientos; determinación de la sensibilidad bacteriana in vitro y de la diversidad genética del MAC; evaluación del tratamiento. Resultados: Fueron diagnosticados 225 casos de micobacteriosis, con prevalencia estable ≈ 6% por año, y 22 especies recuperadas: 4 de rápido desarrollo aisladas de 66 pacientes y 18 de lento desarrollo. MAC fue aislado en 95 casos, 40 M. avium hominissuis, 51 M. intracellulare, 3 M. chimaera, 1 M. colombiense. Se observó mayor probabilidad de enfermar por M. intracellulare en pacientes tratados previamente por tuberculosis (TB). Los pacientes HIV+ tuvieron riesgo incrementado de enfermedad causada por M. avium hominissuis. Los aminoglucósidos, fluoroquinolonas y macrólidos fueron las drogas más activas frente a la mayoría de las MNT. Aproximadamente la mitad de los casos curaron. Conclusiones: M. intracellulare, M. aviumhominissuis con una gran variabilidad genética, y M. abscessus fueron los patógenos más frecuentemente hallados. Un hallazgo importante fue el de casos de enfermedad mixta TB+MNT. Estos pacientes requirieron una terapia con agregado de drogas de segunda línea al esquema terapéutico para TB habiendo curado la mayoría de ellos.

Introduction: The term non-tuberculous mycobacteria (NTM) includes different ambient species capable of sickening humans and/or animals, even by means of a potential zoonotic transmission. Objectives: To determine: The clinical importance of several species within the genus Mycobacterium and the genetic diversity of the M. avium complex (MAC), the in vitro bacterial sensitivity and the success of the specific treatment. Materials and Methods: Collection of clinical and epidemiologic data and information about isolates of the 2009-2016 period; molecular identification of the isolates; determination of the in vitro bacterial sensitivity and genetic diversity of the MAC; treatment evaluation. Results: 225 mycobacteriosis cases were diagnosed, with a stable prevalence of ≈6% per year and 22 recovered species: 4 rapidly growing species isolated from 66 patients and 18 slowly growing species. The MAC was isolated in 95 cases, M. avium hominissuis - 40 cases, M. intracellulare - 51 cases, M. chimaera - 3 cases and M. colombiense - 1 case. We observed a greater probability of getting sick from M. intracellulare in patients previously treated for tuberculosis (TB). HIV-positive patients had a greater risk of falling ill from M. avium hominissuis. Aminoglycosides, fluoroquinolones and macrolides were the most active drugs against most NTM. Approximately half of the cases healed. Conclusions: M. intracellulare, M. aviumhominissuis with great genetic variability and M. abscessus were the most commonly found pathogens. The cases of TB+NTM mixed disease were an important finding. For treating these patients, it was necessary to add second line drugs to the therapeutic regimen for TB; and most of them healed.

Clinical Relevance, Diversity and Genetic Variability of Different Species within the Genus Mycobacterium

Introduction: The term non-tuberculous mycobacteria (NTM) includes different ambient species capable of sickening humans and/or animals, even by means of a potential zoonotic transmission. Objectives: To determine: The clinical importance of several species within the genus Mycobacterium and the genetic diversity of the M. avium complex (MAC), the in vitro bacterial sensitivity and the success of the specific treatment. Materials and Methods: Collection of clinical and epidemiologic data and information about isolates of the 2009-2016 period; molecular identification of the isolates; determination of the in vitro bacterial sensitivity and genetic diversity of the MAC; treatment evaluation. Results: 225 mycobacteriosis cases were diagnosed, with a stable prevalence of ≈6% per year and 22 recovered species: 4 rapidly growing species isolated from 66 patients and 18 slowly growing species. The MAC was isolated in 95 cases, M. avium hominissuis - 40 cases, M. intracellulare - 51 cases, M. chimaera - 3 cases and M. colombiense - 1 case. We observed a greater probability of getting sick from M. intracellulare in patients previously treated for tuberculosis (TB). HIV-positive patients had a greater risk of falling ill from M. avium hominissuis. Aminoglycosides, fluoroquinolones and macrolides were the most active drugs against most NTM. Approximately half of the cases healed. Conclusions: M. intracellulare, M. aviumhominissuis with great genetic variability and M. abscessus were the most commonly found pathogens. The cases of TB+NTM mixed disease were an important finding. For treating these patients, it was necessary to add second line drugs to the therapeutic regimen for TB; and most of them healed

Evolución de la tuberculosis resistente en la Provincia de Buenos Aires en el período 2006 a 2014 / Drug-Resistant Tuberculosis Evolution in Buenos Aires Province During the Period 2016 to 2014

Introducción: En la Argentina la notificación de tuberculosis (TB) mantiene una tendencia descendente, lenta pero sostenida, siendo las formas resistentes las que complican el panorama epidemiológico actual. Objetivo: Describir la incidencia de TB multi y extensivamente resistente (TB M/XDR) en niños y adultos, analizar su transmisión y la relación con los re-tratamientos. Materiales y métodos: En el período 2006-2014 fue notificado un total 41.782 casos totales de TB de los cuales 35.862 (85.8%) eran pulmonares, 24.913 (69,5%) confirmados por bacteriología y, de éstos, 21.842 (87.7%) por examen directo. Un total de 5.712 (13,7%) casos correspondió a menores de 15 años y 35.843 (85,8%) a adultos. Resultados: Fueron registrados 636 casos de TB resistente a los fármacos: 445 (70,0%) con TB MDR, de los cuales 13 (2,9%) fueron niños; 18 (2,6%) casos presentaron TB XDR (2 niños) y 109 (24,5%) eran MDR con resistencia extendida a una fluoroquinolona o un agente inyectable (amicacina, kanamicina, capreomicina, Pre-XDR). Además, 64 casos presentaban mono o poli resistencia. En total 82 (18,4%) pacientes con TB M/XDR cursaban su primer episodio de TB siendo 20 de ellos contactos confirmados de un caso previo de TB. Un total de 19 adultos MDR fueron considerados casos índices de 31 contactos, 13 niños, que desarrollaron la misma forma de TB. Discusión: El contagio desde los casos índices a niños y adultos fue demostrado por epidemiología molecular y clásica. Los genotipos de los aislamientos y la fecha de diagnóstico de la enfermedad permitieron trazar la cadena epidemiológica del contagio.

Introduction: During the last years, Argentina has been maintaining slow but progressive decreasing trends of tuberculosis (TB) incidence rates. In sanitary terms, the drug-resistant forms of the disease are complicating the global control of the disease. Objetive: The main objectives of this study were to describe the incidence of multi and extensive drug-resistant TB (M/XDR) in children and adults, to analyze its transmission within contacts and the relationship with previous treatments. Materials and methods: During 2006-2014 it was notifed to the Control Program of Buenos Aires Province 41,782 TB cases, 5.712 (13.7%) children <15 years old, 35.843 (85.8%) adults; 35,862 (85.8%) patients presented a respiratory form of the disease, 24,913 (69.5%) confrmed by bacteriology, 21,842 (87.7%) by microscopy examination. Results: They were diagnosed 636 cases of drug-resistant TB: 445 (70.0%) MDR TB, 13 (2.9%) children; 18 (2.6%) had a XDR pattern; from 109 (24.5%) individuals the bacteria showed a MDR pattern also resistant to fluoroquinolones or an injectable agent (amikacin, kanamicin or capreomicin). Besides, 64 cases suspected of being MDR were resistant to one or more drugs not accomplishing the MDR criterion. Eighty two (18.4%) patients with M/XDR had their first TB episode being 20 of them, 9 children, contacts of previous TB cases; 19 individuals were considered index cases of another 31 contacts that developed also M/XDR TB. Discussion: Transmission from index cases to contacts was demonstrated by molecular and classical epidemiology. The isolates fngerprinting and the diagnosis of TB dates were useful tools to trace the epidemiologic infection route.

Meta-análisis: diagnóstico de tuberculosis resistente por técnicas no comerciales de amplificación / Meta-analysis: drug-resistant tuberculosis diagnosis by non commercial amplification techniques / Meta-análise: diagnóstico de tuberculose resistente por técnicas não comerciais de amplificação

La emergencia de tuberculosis (TB) multidrogo y extensivamente-resistente reactivó la necesidad de contar con métodos rápidos para detectar resistencia a isoniacida (INH) y rifampicina (RIF). Por tal motivo, los objetivos de este trabajo fueron evaluar, mediante meta-análisis, la exactitud global y la posible utilidad de métodos caseros basados en PCR para la detección rápida de resistencia a INH y RIF en aislamientos clínicos de Mycobacterium tuberculosis. La búsqueda bibliográfica incluyó Medline/PubMed, BioMedLib. Para estimar la variabilidad entre los resultados de los estudios y el grado de exactitud diagnóstica de los métodos utilizados se realizaron gráficos "forest plot" y curvas SROC (summary receiver operating characteristic) mediante el software Meta-DiSc. Fueron seleccionados 15 estudios, conteniendo 1311 aislamientos resistentes a INH y 953 a RIF. Para la detección de resistencia a INH la sensibilidad y especificidad globales fueron: 84,0% y 96,0% respectivamente, mientras que para la detección de resistencia a RIF esos valores fueron 92,0% y 97,0%. Además, estos métodos mostraron alta exactitud diagnóstica, con áreas bajo la curva SROC>0,9. La alta sensibilidad y especificidad obtenidas con métodos moleculares caseros sugieren que algunos de ellos podrían ser aplicados para el diagnóstico rápido de resistencia a partir del aislamiento de M. tuberculosis.

Due to the emergency of multidrug and extensively-drug resistant tuberculosis, molecular methods for a rapid detection of isoniazid (INH) and rifampicin (RIF) resistance are urgently needed. For that reason, the objectivesof this study were to asses through a meta-analysis the global accuracy and the utility of the home-made molecular methods based in PCR for INH and RIF resistance rapid detection from Mycobacterium tuberculosis clinical isolates. The articles were searched using Medline/PubMed, BioMedLib. The variability among different studies results and the diagnostic accuracy of the used methods were estimated by forest plot and summary receiver operating characteristic (SROC) curves performed with software Meta-DiSc. Fifteen studies were chosed: 1311 containing INH resistant and 953 RIF resistant isolates. The pooled sensitivity and specificity for INH resistance detection was 84.0% and 96.0% respectively, while 92.0% and 97.0% were the pooled values for RIF resistance detection. Besides, these methods showed a high diagnostic accuracy, with the area under the SROC curve >0.9. Due to the high sensitivity and specificity obtained with the home-made molecular methods, some of these tests could be applied for a rapid detection of M. tuberculosis drug resistance in clinical practice.

A emergência de tuberculose (TB) multidrogas e extensivamente-resistente reativou a necessidade de contar com métodos rápidos para detectar resistência à isoniazida (INH) e rifampicina (RIF). Por isso, o objetivo deste trabalho foi a avaliação através da meta-análise, da exatidão global e da possível utilidade de métodos caseiros baseados em PCR para detectar rapidamente a resistência a INH e RIF em isolamentos clínicos de Mycobacterium tuberculosis. A pesquisa bibliográfica incluiu Medline/PubMed, Bio MedLib. Para estimar a variabilidade entre os resultados dos estudos e o grau de exatidão diagnóstica dos métodos utilizados, foram realizados gráficos "forest plot" e curvas SROC (summary receiver operating characteristic) com o software Meta-Disc. Foram selecionados 15 estudos, contendo 1311 isolamentos resistentes a INH e 953 a RIF. Para a detecção de resistência a INH, a sensibilidade e especificidade globais foram 84,0% e 96,0% respectivamente, enquanto que para a detecção de resistência a RIF esses valores foram de 92,0% e 97,0%. Alem disso, os mesmos métodos mostraram elevada exatidão diagnóstica, com áreas inferiores à curva SROC>0,9. A elevada sensibilidade e especificidade obtida através de métodos moleculares caseiros sugere que alguns deles poderiam ser aplicados para o diagnóstico rápido de resistência a partir do isolamento de M. tuberculosis.

Disease caused by non-tuberculous mycobacteria: diagnostic procedures and treatment evaluation in the North of Buenos Aires Province

Non-tuberculous mycobacteria (NTM) have emerged as pathogens frequently associated to HIV co-infection. The aims of this study were to describe the clinical importance of NTM in patients from the North of Buenos Aires Province and the drug-susceptibility patterns in relation with the therapy used. A total of 23,624 clinical specimens were investigated during the period 2004-2010. Ziehl-Neelsen stain and cultures were used for diagnosis. Molecular and biochemical tests were performed to identify the mycobacteria. TB and mycobacterioses cases were 2 118 and 108 respectively. Sixteen NTM species were found: Mycobacterium avium and Mycobacterium intracellulare as the main causative agents. Infections produced by more than one species at the same time were confirmed (4 cases). Macrolides and fluoroquinolones were the most active in vitro drugs. Treatment evaluation showed that 68.0 % of the cases completed the therapy, 20 % died; and 12 % were relapses. The cases in which the treatment outcome was evaluated received an individual tailor-made therapeutic scheme including those drugs showing in vitro activity and presumed in vivo usefulness. More than a quarter of the patients had HIV co-infection and the majority of the deaths were associated with this co-infection.

Enfermedad causada por micobacterias no tuberculosas: diagnóstico y evaluación del tratamiento en el norte del Gran Buenos Aires. Las micobacterias no tuberculosas (MNT) emergieron como patógenos frecuentemente asociados a la co-infección con el HIV. EL objetivo del estudio fue describir la importancia clínica de las MNT en pacientes de la región norte de la provincia de Buenos Aires y los patrones de drogo-sensibilidad en relación con la terapia empleada. Se investigó un total de 23.624 especímenes clínicos durante, el período 2004-2010. La tinción de Ziehl-Neelsen y los cultivos se utilizaron para diagnóstico. Las micobacterias fueron identificadas mediante pruebas bioquímicas y moleculares. Los casos de tuberculosis y micobacteriosis fueron 2 118 y 108, respectivamente. Se encontraron 16 especies de MNT, siendo las principales, Mycobacterium avium y Mycobacterium intracellulare. En 4 casos se confirmaron infecciones producidas por más de una especie al mismo tiempo. Los macrólidos y las fluoroquinolonas tuvieron mayor actividad in vitro. La evaluación del tratamiento confirmó que el 68 % de los casos completó la terapia; 20 % murió y el 12 % recayó. Los casos en los que se evaluó el tratamiento recibieron un esquema terapéutico individual incluyendo aquellas drogas que mostraron actividad in vitro. Más de un cuarto de los pacientes tuvieron co-infeccion con el HIV y la mayoría de las muertes estuvieron asociadas con esta co-infección.

Tuberculosis pediátrica, MDR-TB y XDR-TB en la provincia de Buenos Aires durante el período 2002-2007 / Paediatric tuberculosis, MDR-TB and XDR-TB in Buenos Aires province during the period 2002-2007

Los objetivos de este estudio fueron: describir la incidencia de tuberculosis (TB), MDR-TB Y XDR-TB en niños y adultos, y analizar la transmisión entre contactos comprobados. Fue realizado un análisis retrospectivo de los casos notificados al Programa de Control de Tuberculosis de Buenos Aires (PCTB). Durante este lapso se notificaron 30784 casos nuevos de TB, 4276 (14.0%) niños y 26508 adultos. El 4.9% (14/284) y el 71 % (146/ 2042) de los niños y adultos tenían MDR-TB, 5.1 % (13/256) de los niños y 5.3% (86/ 1616) de los adultos eran vírgenes de tratamiento. Se registraron 12 casos de XDR-TB en adultos, 3 sin tratamiento previo. Además 25.0% (40/160) de los casos MDR pertenecían a 9 familias o eran trabajadores de salud. La transmisión en estos casos fue verificada por pruebas moleculares. La evaluación del tratamiento para casos con MDR-TB o XDR-TB pudo realizarse en 47.5% de los casos ya que de 76 adultos y 6 niños no se obtuvo información. Asimismo, se pone de manifiesto la necesidad de contar con una notificación exhaustiva y un riguroso estudio de cohortes que permitan conocer y evaluar la situación real de la TB en la provincia. Debería asimismo recomendarse el incremento en la realización de cultivos y pruebas de sensibilidad que permitan determinar el perfil de resistencia bacteriana para tratar apropiadamente la TBP y la MDR- TB, indicadores de transmisión activa y del impacto de las actividades del PCTB en la comunidad.

The aims of the study were to describe the incidence of tuberculosis (TB), multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) in children and adults and to analyze the transmission of infection among proved contacts. The method was a retrospective analysis of notified cases to the TB Control Programme of Buenos Aires province. During the period 2002-2007, 30784 TB cases were notified; 4276 (14.0%) were children and 26508 adults. The prevalence of MDR-TB was 4.9% (14/ 284) among children and 7.1 % (146/2042) among adults. The proportions of MDR-TB cases who had not had any previous TB treatment were 5.1 % (13/256) in children and 5.3% (86/1616) in adults. A total of 12 XDR-TB cases were notified in adults; 3 of them without any previous TB treatment. Among the 160 MDR-TB cases, 40 belonged to 9 families or were health workers. Transmission in these cases was traced by molecular epidemiology tests. Data on evaluation of treatment outcomes were only available for 47.5% of MDR-TB cases; no information was registered in 76 adults and 6 children. Intensive notification and strict follow-up of cohort of cases are needed in arder to know and assess the actual TB situation in the province. It is also recommended to increase the use of cultures and drug susceptibility tests to have better estimates of the prevalence of drug-resistant TB, prescribe more effective treatment of TB and MDR- TB, improve indicators of infection transmission and better assess the impact of the TB control activities in the community.