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Resumen El cultivo de micobacterias es la prueba de referencia, y la mas sensible, en el diagnostico microbiologico de la tuberculosis y de las infecciones por micobacterias no tuberculosas. Sin embargo, requiere al menos 2-3 semanas para ser positivo. La tincion, rapida, aunque poco sensible, actua como un complemento. Las pruebas de amplificacion genetica tienen una sensibilidad intermedia y obtienen resultados en 1-2 dias. Estas ultimas estan indicadas cuando el grado de sospecha es moderado o alto. En pacientes infectados por el VIH con inmunodepresion severa ( La identificacion de los aislamientos de los cultivos positivos es imprescindible para evaluar la significacion clinica y la orientacion terapeutica. Actualmente se dispone de un abanico de tecnicas de identificacion que facilitan resultados en periodos de solo 1-4 dias. El futuro del diagnostico pasa por un mayor desarrollo de las tecnicas de amplificacion genetica y por potenciar la busqueda de biomarcadores que permitan un nuevo enfoque del diagnostico de estas infecciones.

The advent of the Xpert® MTB/RIF technique was a revolution in the diagnosis of tuberculosis, especially in areas with high incidence and low resources. It allows the detection of Mycobacterium tuberculosis complex and simultaneously the most common resistance mutations to rifampicin in less than 2h. For respiratory samples the sensitivity is very high, but it decreases for extrapulmonary samples and children. Although it is faster and simpler than conventional methods, it presents some limitations and new and better techniques are needed to reduce the number of cases and deaths caused by tuberculosis. This review aims to assess the scientific evidence around the diagnostic performance of Xpert® MTB/RIF in different types of samples and populations, as well as analyse its strengths and limitations for TB diagnosis.

Resumen La aparicion de la tecnica Xpert® MTB/RIF supuso una revolucion en el diagnostico de la tuberculosis, especialmente en zonas con alta incidencia y pocos recursos. Permite la deteccion de Mycobacterium tuberculosis complex y, simultaneamente, de las mutaciones mas comunes de resistencia a rifampicina en menos de 2 h. Su sensibilidad en muestras respiratorias es muy alta, pero disminuye en muestras extrapulmonares y en ninos. Aunque mas rapida y simple que los metodos convencionales, presenta ciertas limitaciones y aun son necesarias nuevas y mejores herramientas diagnosticas que contribuyan a reducir los casos y las muertes por tuberculosis. Esta revision pretende compendiar la evidencia cientifica en torno al rendimiento diagnostico de Xpert® MTB/RIF en diferentes tipos de muestras y poblaciones, asi como analizar sus ventajas y limitaciones para el diagnostico de tuberculosis.

et al., [Objective]: Molecular epidemiology techniques in tuberculosis (TB) can identify high-risk strains that are actively transmitted. We aimed to implement a novel strategy to optimize the identification and control of multidrug-resistant (MDR) TB in a specific population. [Methods]: We developed a strain-specific PCR tailored from whole genome sequencing (WGS) data to track a specific MDR prevalent strain in Equatorial Guinea (EG-MDR). [Results]: The PCR was applied prospectively on remnants of GeneXpert reaction mixtures owing to the lack of culture facilities in Equatorial Guinea. In 147 (93%) of 158 cases, we were able to differentiate between infection by the EG-MDR strain or by any other strain and found that 44% of all rifampicin-resistant TB cases were infected by EG-MDR. We also analysed 93 isolates obtained from Equatorial Guinea 15 years ago, before MDR-TB had become the problem it is today. We found that two of the scarce historical MDR cases were infected by EG-MDR. WGS revealed low variability-six single nucleotide polymorphisms acquired by this strain over 15 years-likely because of the lack in the country of a specific program to treat MDR-TB. [Conclusions]: Our novel strategy, which integrated WGS analysis and strain-specific PCRs, represents a low-cost, rapid and transferable strategy that allowed a prospective efficient survey and fast historical analysis of MDR-TB in a population., This study was funded by Plan Estatal I+D+I 2013-2016, ISCIII (13/01207; 15/01554) and cofunded by the Fondo Europeo de Desarrollo Regional (FEDER): “Una manera de hacer Europa”. We also received funding from Fondation Merieux (DM/CL/cb15) and a Grant for International Cooperation Projects from the IiSGM (I and II-COOP-INT 2015). L.P. holds a Miguel Servet research grant (CP15/00075), MINECO research grant (SAF2013-43521-R); I.C. holds a European Research Council (ERC) grant (638553-TB-ACCELERATE). Funding was also received from the Spanish Network for Research in Infectious Diseases (REIPI, RD12/0015 to GT).

Mycobacterial culture has a high sensitivity and is the test of choice for the microbiological diagnosis of tuberculosis and nontuberculous mycobacterial infections. However, the results of this culture require at least 2-3 weeks to obtain positivity. Staining is rapid and can be used as a complementary study, although its sensitivity is low. Gene amplification tests have an intermediate sensitivity and obtain results in 1-2 days. These last tests are indicated in cases with moderate or high clinical suspicion. In HIV patients with severe immunodeficiency (

Mycobacteria are a large group of microorganisms, multiple species of which are major causes of morbidity and mortality, such as tuberculosis and leprosy. At present, the emergence and spread of multidrug-resistant strains of Mycobacterium tuberculosis complex are one of the most serious health problems worldwide. Furthermore, in contrast to M. tuberculosis and Mycobacterium leprae, non-tuberculous mycobacteria (NTM) are more frequently isolated and, in many cases, treatment is based on drug susceptibility testing. This article is a review of the different methods to determine the in vitro drug susceptibility of M. tuberculosis complex and the most relevant NTM isolates. The molecular techniques currently used for rapid detection of resistance of clinical specimens are also analysed.

Resumen Fundamento y objetivo Conocer el patron uveitico de nuestra area geografica. Los recientes cambios demograficos, ecologicos y cientificos pueden determinar cambios epidemiologicos en las uveitis, que nos hemos propuesto investigar. Pacientes y metodo Corte transversal desde el 1-01-2009 al 30-06-2010. Se incluyeron todos los pacientes atendidos por uveitis en nuestro centro. Se analizaron el tipo anatomico (IUSG & SUN), la etiologia (tests «a medida»), la edad, el sexo, la procedencia y la lateralidad. Los resultados entre las «primeras visitas» y los controles fueron comparados estadisticamente mediante los tests de ANOVA y ji al cuadrado. Resultados De 416 pacientes incluidos, 150 (36%) fueron uveitis anteriores, 128 (31%) posteriores, 101 (24%) panuveitis y 37 (9%) uveitis intermedias (UI). Un 56% (236) fueron bilaterales, la edad media fue de 46 anos (extremos 6-87) y un 58% fueron mujeres. El 14% (58) fueron pacientes extranjeros. Por etiologia, un 20% fueron inclasificables, un 31% infecciosas, un 26% asociadas a enfermedades sistemicas y un 23% oculares especificas. Entre las causas conocidas, la herpetica (11%), Toxoplasma (8%), Behcet (8%) y tuberculosis (6%) fueron las mas frecuentes. Las UI afectaron significativamente por primera vez a mas extranjeros (p = 0,018), afectaron a pacientes mas jovenes (p = 0,008) y fueron mas frecuentemente cronicas (p = 0,003) e inclasificables (p = 0,024). Conclusion En nuestro medio, un 80% de las uveitis pueden ser correctamente clasificadas. Las UI han incrementado su frecuencia en poblacion extranjera, suelen ser inclasificables y cronicas. La tuberculosis (6%) y la enfermedad de Birdshot (4%) deben ser tenidas en cuenta como causa de uveitis.

Objectives: To determine the proportion and type of mutations in Mycobacterium tuberculosis isolates resistant to streptomycin, and their relationship with the level of resistance and with the epidemiological molecular pattern of the isolates. Methods: Sixty-nine streptomycin-resistant isolates from a M. tuberculosis strain collection (1995– 2005) from Barcelona were studied. The MIC of streptomycin for each isolate was determined using the proportions method with Middlebrook 7H11 medium. The entire rpsL gene and two specific fragments of the rrs gene (the 530 loop and the 912 region) were sequenced. IS6110-restriction fragment length polymorphism and spoligotyping were performed in each isolate. Results: Twenty-six (26/69, 37.7%) streptomycin-resistant isolates presented a mutation in either the rpsL gene and/or the rrs530 loop, with no mutation in the rrs912 region. Seventeen (24.6%) isolates showed rpsL mutations (codons 43 and 88) associated with high MIC levels. Nine (13.0%) isolates had alterations in the rrs gene (A513T, A513C and C516T). Nineteen isolates (19/64, 29.7%) were classified into seven clusters (containing 2– 5 isolates per cluster). Nineteen different spoligotype patterns were found. All the LAM3 spoligotype isolates (10/67, 14.9%) were associated with a C491T change in the rrs gene, being also observed in all LAM3 streptomycin-susceptible isolates. Conclusions: Mutations in therpsL andrrs genes were detected in 37.7% of streptomycin-resistantM.tuberculosis isolates. High-level resistance was associated with mutations in therpsL gene, whereas wild-type isolates showed low MIC levels. The presence of the C491T substitution in the rrs gene in streptomycin-susceptible and -resistant isolates demonstrates that this change is an epidemiological marker associated with LAM3 sublineage.

Resumen Debe sospecharse tuberculosis pulmonar en pacientes con sintomas respiratorios durante mas de 2–3 semanas. La inmunodepresion puede modificar la presentacion clinica y radiologica. La radiografia de torax presenta manifestaciones muy sugerentes de tuberculosis, aunque en ocasiones atipicas. Las pruebas radiologicas complejas (tomografia computarizada, resonancia magnetica) son mas utiles en la tuberculosis extrapulmonar. En el diagnostico, siempre que sea posible, se utilizaran al menos 3 muestras seriadas representativas de la localizacion clinica. La baciloscopia y el cultivo con medios liquidos deben realizarse en todos los casos. Las tecnicas de amplificacion genetica son coadyuvantes en la sospecha moderada o alta de tuberculosis. En los casos nuevos de tuberculosis, se recomienda administrar isoniacida, rifampicina, etambutol y piracinamida (HREZ) durante 2 meses e isoniacida mas rifampicina durante 4 meses mas, con las excepciones de los casos de meningitis, en que se alargara hasta 12 meses, y de la tuberculosis espinal con afeccion neurologica y la silicosis, hasta 9 meses. Se recomiendan las formulaciones combinadas. En pacientes con infeccion por el VIH deben realizarse los ajustes necesarios con el tratamiento antirretroviral. Debe realizarse antibiograma para farmacos de primera linea a todos los aislamientos iniciales de pacientes nuevos. El control del tratamiento es una de las actividades mas importantes en el abordaje de la tuberculosis. En la infeccion tuberculosa la prueba de tuberculina (PT) es positiva cuando es ≥5 mm y los metodos de deteccion de produccion de interferon gamma (IGRA) se recomiendan en combinacion con la prueba de la tuberculina. La pauta estandar de tratamiento de la infeccion es de 6 meses con isoniacida. En la tuberculosis pulmonar se aplicara aislamiento respiratorio durante 3 semanas o hasta obtener 3 muestras con baciloscopia negativa.

Pulmonary TB should be suspected in patients with respiratory symptoms longer than 2-3 weeks. Immunosuppression may modify clinical and radiological presentation. Chest X-ray shows very suggestive, albeit sometimes atypical, signs of TB. Complex radiological tests (CT scan, MR) are more useful in extrapulmonary TB. At least 3 serial representative samples of the clinical location are used for diagnosis whenever possible. Bacilloscopy and liquid medium cultures are indicated in all cases. Genetic amplification techniques are coadjuvant in moderate or high TB suspicion. Administration of isoniazid, rifampicin, ethambutol and pyrazinamide (HREZ) for 2 months and HR for 4 additional months is recommended in new cases of TB, except in cases of meningitis in which treatment should continue for up to 12 months and up to 9 months in spinal TB with neurological involvement, and in silicosis. Appropriate adjustments with antiretroviral treatment should be made in HIV patients. Combined therapy is recommended to avoid development of resistance. An antibiogram to first line drugs should be performed in all the initial isolations of new patients. Treatment control is one of the most important activities in TB management. The Tuberculin Skin Test (TST) is positive in TB infection when >or=5mm, and Interferon-Gamma Release Assays (IGRA) are recommended in combination with TT. The standard treatment schedule for infection is 6 months with isoniazid. In pulmonary TB, respiratory isolation is applied for 3 weeks or until 3 negative bacilloscopy samples are obtained.

Para la microbiologia clinica, Mycobacterium tuberculosis supone un microorganismo cuya manipulacion es dificil, ya que entrana el riesgo de exponerse a un patogeno de transmision principalmente aerea que requiere una contencion de nivel 3. En estos momentos, la mayoria de los accidentes en el laboratorio de micobacterias puede reducirse mediante la practica de procedimientos microbiologicos adecuados, el uso de dispositivos de contencion y proteccion y un diseno de las instalaciones apropiado. La legislacion actual contempla el grado de responsabilidad de las instituciones sanitarias y del personal del laboratorio, y define especificamente el nivel de bioseguridad requerido para el procesamiento de muestras para micobacterias. El respeto a las recomendaciones en contencion primaria y secundaria, asi como el control sistematico del personal de laboratorio y la elaboracion de planes de actuacion frente a accidentes, contribuyen a minimizar los riesgos de ser infectado y proteger a la comunidad.

Fundamento El objetivo fue conocer, mediante un estudio multicentrico y con la colaboracion de clinicos, microbiologos y epidemiologos, el nivel de resistencias de Mycobacterium tuberculosis en el area de Barcelona y su relacion con datos clinicos de los pacientes para establecer posibles grupos de riesgo. Pacientes y metodo Se incluyeron las cepas de M. tuberculosis aisladas de los pacientes diagnosticados bacteriologicamente desde octubre de 1995 a septiembre de 1997, revisandose su historial clinico. Se estudio la resistencia a isoniacida, rifampicina, estreptomicina, etambutol y pirazinamida mediante el sistema Bactec 460. Los factores asociados a resistencia se analizaron mediante regresion logistica. Resultados El total de pacientes fue de 1.749 (1.535 iniciales y 214 tratados anteriormente). La resistencia primaria fue 5,7% (isoniacida, 3,8%; rifampicina, 1,0%, estreptomicina, 2,1%, etambutol, 0,3% y pirazinamida, 1,0%). La resistencia adquirida alcanzo el 20,5% (isoniacidas, 17,3%, rifampi-cina, 9,8%, etambutol, 1,9%, estreptomicina, 4,7% y pirazinamida, 6,5%). La multirresistencia primaria fue 0,9%, y la adquirida 9,3%. Las resistencias primarias totales, a isoniacida y a pirazinamida se asociaron con inmigracion y se objetivaron algunas diferencias entre laboratorios atribuibles a diferencias de poblacion. Las resistencias adquiridas totales y a isoniacida se asociaron a mas de 60 anos y al sexo femenino. Conclusiones El bajo nivel de resistencias primarias a isoniacida permite tratar los casos iniciales con 3 farmacos, salvo en determinados grupos de inmigrantes. Se recomienda realizar pruebas de sensibilidad en todas las cepas aisladas y realizar estudios de farmacorresistencia en tuberculosis basados en trabajos coordinados para evitar sesgos de poblacion.

To describe the uveitis pattern in our geographic area. Recent demographic, environmental and scientific changes can determine uveitis pattern changes, which we aim to investigate.A cross-sectional study between 1(st) January 2009 and 30(th) June 2010 was done. All uveitis patients visited at our institution were included. Anatomical (IUSGSUN) and aetiological patterns (by "tailored" tests), age, sex, origin and laterality were analyzed. Results from first visits and controls were compared statistically, by chi-square and ANOVA tests.From 416 patients included, 150 (36%) were anterior uveitis, 128 (31%) posterior, 101 (24%) panuveitis and 37 (9%) intermediate uveitis (IU). Fifty-six percent (236) were bilateral, mean age was 46 years (6-87) and 58% were women. Fourteen percent (58) were from non-Spanish origin. By aetiology, 20% were unclassifiable, 31% infectious, 26% associated with systemic immune diseases and 23% were ocular specific syndromes. Among classified causes, herpes virus (11%), Toxoplasma (8%), Behçet (8%) and tuberculosis (6%) were the most common. IU were diagnosed more frequently at first time in foreign patients (P=.018); they affected younger patients (P=.008), were more chronic (P=.003) and unclassifiable (P=.024).In our area, 80% of uveitis can be correctly classified. IU have an increased frequency among foreigners, and they use to be unclassifiable and chronic. Tuberculosis (6%) and Birdshot chorio-retinopathy (4%) must be kept in mind as uveitis causes.

Objectives: We analysed the ability of Mycobacterium tuberculosis clinical isolates to penetrate and grow inside murine macrophages as a surrogate of fitness. Methods: Thirty-five drug-resistant and 10 drug-susceptible M. tuberculosis isolates were studied in a murine macrophage model from the J774.2 cell line in a 6 day protocol, performing semi-quantitative counts in Middlebrook 7H11 medium. The mycobacterial penetration index (MPI) after infection and the mycobacterial growth ratio (MGR) inside the macrophages were determined to evaluate the fitness of isolates. Results: Isolates with the katG S315T mutation and multidrug-resistant (MDR) isolates had a significantly lower MGR compared with drug-susceptible isolates. The MPI of the isolates with the katG S315T mutation showed a significant decrease compared with the MPI of those without this mutation. A trend to significantly lower values was also observed on comparing the MPI of the MDR isolates with that of the drug-susceptible isolates and the isolates resistant to isoniazid. Conclusions: The isoniazid-resistant and MDR isolates with mutations in the katG gene showed decreased multiplication inside murine macrophages, suggesting a lower fitness of M. tuberculosis with these resistance patterns.

To evaluate the risk of developing active tuberculosis (TB) in a cohort of HIV-1-infected patients.Prospective longitudinal follow-up of 839 HIV-infected patients, of whom 505 (60%) were parenteral drug users and 269 (32%) homosexual men. Tuberculin skin tests were performed at baseline and annually thereafter. Prophylaxis with isoniazid (300 mg daily for 9 months) was offered to those with a positive tuberculin test (indurationor = 5 mm). Diagnosis of TB was accepted if it could be confirmed microbiologically (acid-fast bacilli seen in Ziehl-Neelsen stains or grown in Lowenstein-Jensen cultures) or pathologically (presence of caseating granulomas) and patients had consistent clinical manifestations.Active TB developed in 23 out of the 733 (3.1%) patients with a negative tuberculin skin test after a mean follow-up of 16 +/- 11 months (range, 2-52 months), with an estimated cumulative probability of 1.5 and 7% after 1 and 3 years, respectively (or 2.4 per 100 patient-years). None of the 87 patients with a negative tuberculin test but a positive Multitest developed TB. Conversely, 106 patients had a positive tuberculin skin test (97 at baseline and nine who converted during follow-up). Active TB developed in seven out of the 26 not receiving prophylaxis or in whom prophylaxis had to be discontinued (16.2 per 100 patient-years), in four out of 61 patients 3-27 months after having completed 9 months of prophylaxis with isoniazid (8.9 per 100 patient-years) and in none of the 19 still receiving isoniazid. When TB was diagnosed, the mean CD4 lymphocyte count of the 34 patients who developed it during follow-up was 77 +/- 103 x 10(6)/l (range, 1-400 x 10(6)/l).Among HIV-infected patients in whom the tuberculin skin test is negative, the risk of developing active TB is sufficient to consider prophylaxis if the CD4 count falls below 400 x 10(6)/l, at least in those patients with skin anergy living in high-risk geographical areas such as Spain. When the tuberculin skin test was positive, isoniazid (9 months) provided a 45% protection beyond the period of its administration.This study sought to evaluate the risk of developing active tuberculosis (TB) in a cohort of HIV-1-infected patients. A prospective longitudinal follow-up was carried out on 839 HIV-infected patients, of whom 505 (60%) were parenteral drug users and 269 (32%) were homosexual men. Tuberculin skin tests were performed at baseline and annually thereafter. Prophylaxis with isoniazid (300 mg daily for 9 months) was offered to those with a positive tuberculin test (induration or= 5 mm.). Diagnosis of TB was accepted if it could be confirmed microbiologically (acid-fast bacilli seen in Ziehl-Neelsen stains or grown in Lowenstein-Jensen cultures) or pathologically (presence of caseating granulomas) and patients had consistent clinical manifestations. Active TB developed in 23 of the 733 (3.1%) patients with a negative tuberculin skin test after a mean follow-up of 16 +or- 11 months (range, 2-52 months), with an estimated cumulative probability of 1.5 and 7% after 1 and 3 years, respectively (or 2.4 per 100 patient-years). None of the 87 patients with a negative tuberculin test, but a positive Multitest, developed TB. Conversely, 106 patients had a positive tuberculin skin test (97 at baseline and 9 who converted during follow-up). Active TB developed in 7 of the 26 not receiving prophylaxis or in whom prophylaxis had to be discontinued (16.2 per 100 patient-years), in 4 of 61 patients 3-27 months after having completed 9 months of prophylaxis with isoniazid (8.9 per 100 patient-years), and in none of the 19 still receiving isoniazid. When TB was diagnosed, the mean CD4 lymphocyte count of the 34 patients who developed it during follow-up was 77 +or- 103 x 106/L (range, 1-400 x 106/L). Among HIV-infected patients in whom the tuberculin skin test is negative, the risk of developing active TB is sufficient to consider prophylaxis if the CD4 count falls below 400 x 106/L, at least in those patients with skin anergy living in high-risk geographical areas such as Spain. When the tuberculin skin test was positive, isoniazid (9 months) provided a 45% protection beyond the period of its administration.

IS6110, a Mycobacterium tuberculosis complex species-specific insertion element, is targeted primarily for DNA fingerprinting of M. tuberculosis strains. The number and chromosomal positions of copies of this element have been found to be highly variable between unrelated strains and have been exploited for molecular epidemiological purpose but the utility of IS6110 as an informative marker of strain phylogeny has yet to be demonstrated. In the current study, a recently proposed IS6110-targetting PCR based typing methodology, fluorescent amplified fragment length polymorphism (fAFLP) was applied to a global panel of 166 of the clinically more predominant ‘modern’ strains characterised by spoligotype and, where available, Variable Number Tandem Repeat (VNTR) to identify potentially evolutionarily informative common fragments that could define strains as belonging to established genetic lineages. These common fragments are hereby proposed to be ancestral insertion sites present in common ancestors of these strains rather than preferential insertion sites or ‘hot spots’. Results indicate that the exact same spoligotype and VNTR-defined lineages are reflected in the fragment patterns but with greater resolution and are able to clearly define the very distinct Haarlem, LAM, X and also the currently ill-defined T and S and lineages and spoligotypes designated as U, or unknown, without ambiguity. The biogeographical patterns generated reflect the migration of mankind across the globe and indicate that only four successful clones (or individual bacteria) gave rise to virtually all of the tuberculosis in Europe and the Americas. Potential lies in the application of the data to determine IS6110 evolutionary events that have occurred during the evolution of these lineages.