New simple decontamination method improves microscopic detection and culture of mycobacteria in clinical practice

This study was carried out at Dr. Cetrángolo Hospital, Buenos Aires, Argentina. The objective was to compare two digestion–decontamination procedures: the N-acetyl-L-cysteine–sodium citrate–NaOH (NALC-NaOH) and a combination of 7% NaCl plus NaOH, the hypertonic saline–sodium hydroxide (HS-SH) method, in detection and recovery of mycobacteria. Microscopy detection rates before and after concentration of specimens by both methods, were also compared. The study had two phases. Phase I: comparison of the gold standard NALC-NaOH and HS-SH on paired samples involving respiratory clinical specimens by means of receiver operating characteristic curve analysis. Phase II: blinded, randomized trial to assess the performance of HS-SH versus NALC-NaOH in clinical practice. Phase I: Positive microscopy rate was significantly increased in around 2.2% after concentration in comparison to that of specimens without concentration. The calculated sensitivity values for microscopy detection increased between 15.2% (HS-SH: 73.5%) to 16.7% (NALC-NaOH: 75.0%) over those without concentration (58.3%). Phase II: similar diagnostic rates by microscopy and cultures were obtained by either HS-SH or NALC-NaOH. The clinical performances were also very similar. These results and the low cost of the HS-SH procedure indicate the possibility of its implementation in clinical laboratories with high burden of tuberculosis cases and low resources

In vitro anti-tuberculosis activity of azole drugs against Mycobacterium tuberculosis clinical isolates

Background: Latent tuberculosis has been associated with the persistence of dormant Mycobacterium tuberculosis in the organism of infected individuals, who are reservoirs of the bacilli and the source for spreading the disease in the community. New active anti-TB drugs exerting their metabolic action at different stages and on latent/dormant bacilli are urgently required to avoid endogenous reactivations and to be part of treatments of multi- and extensively-drug resistant tuberculosis (M/XDR-TB). It was previously reported that azole drugs are active against M. tuberculosis. For that reason, the aims of this study were to determine the in vitro activity of azole drugs, imidazole (clotrimazole, CLO and econazole, ECO) and nitroimidazole (metronidazole, MZ and ipronidazole, IPZ), against a collection of MDR M. tuberculosis clinical isolates; and to analyze their potential use in both the LTB and the active forms of M/XDR-TB treatments. Methods: A total of 55 MDR M. tuberculosis isolates and H37Rv were included. MZ and IPZ activity against M. tuberculosis isolates were tested using anaerobic culture conditions. The activity of ECO and CLO was measured by the minimal inhibitory concentration (MIC) using a microdilution colorimetric method.Inst. de BiotecnologíaFil: Imperiale, Belen Rocio. Hospital Dr. Antonio A. Cetrángolo. Laboratorio de Referencia del Programa de Control de la Tuberculosis de la provincia de Buenos Aires; ArgentinaFil: Cataldi, Angel Adrian. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Morcillo, Nora. Hospital Dr. Antonio A. Cetrángolo. Laboratorio de Referencia del Programa de Control de la Tuberculosis de la provincia de Buenos Aires; Argentin

Plan de internacionalización de una agencia de marketing y comunicación política.

Mediante este trabajo, he analizado la evolución de una empresa de marketing y comunicación política en Colombia en el mercado online. Para esto, he estudiado las características culturales del país y he adaptado la empresa a este mercado.<br /

Detection of first- and second-line drug resistance in Mycobacterium tuberculosis clinical isolates by pyrosequencing

Conventional phenotypic drug susceptibility testing (DST) methods for Mycobacterium tuberculosis are laborious and very time-consuming. Early detection of drug-resistant tuberculosis (TB) is essential for prevention and control of TB transmission. We have developed a pyrosequencing method for simultaneous detection of mutations associated with resistance to rifampin, isoniazid, ethambutol, amikacin, kanamycin, capreomycin, and ofloxacin. Seven pyrosequencing assays were optimized for following loci: rpoB, katG, embB, rrs, gyrA, and the promoter regions of inhA and eis. The molecular method was evaluated on a panel of 290 clinical isolates of M. tuberculosis. In comparison to phenotypic DST, the pyrosequencing method demonstrated high specificity (100%) and sensitivity (94.6%) for detection of multidrug-resistant M. tuberculosis as well as high specificity (99.3%) and sensitivity (86.9%) for detection of extensively drug-resistant M. tuberculosis. The short turnaround time combined with multilocus sequencing of several isolates in parallel makes pyrosequencing an attractive method for drug resistance screening in M. tuberculosis.Fil: Engström, Anna. Karolinska Huddinge Hospital. Karolinska Institutet; Suecia. Swedish Institute for Communicable Disease Control; SueciaFil: Morcillo, Nora Susana. Provincia de Buenos Aires. Ministerio de Salud. Hospital "Dr. Antonio A. Cetrángolo"; ArgentinaFil: Imperiale, Belén Rocío. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Provincia de Buenos Aires. Ministerio de Salud. Hospital "Dr. Antonio A. Cetrángolo"; ArgentinaFil: Hoffner, Sven E.. Karolinska Huddinge Hospital. Karolinska Institutet; Suecia. Swedish Institute for Communicable Disease Control; SueciaFil: Juréen, Pontus. Karolinska Huddinge Hospital. Karolinska Institutet; Suecia. Swedish Institute for Communicable Disease Control; Sueci

Primera evaluación en Argentina de GenoType® MTBDRplus para la detección de Mycobacterium tuberculosis multidrogo-resistente desde aislamientos y especímenes clínicos

Tuberculosis (tB) and multidrug and extensively drug-resistant (dR) tB are important public health problems that are spreading worldwide. The aims of this study were to determine the sensitivity and specificity of the genotype® mtBdRplus assay from smear-positive clinical specimens and isolates and to explore its possible application in routine work. Clinical samples were previously decontaminated using naoH-n-acetyl-l-cystein or naoH-Clna hypertonic solution for Ziehl-neelsen staining and cultures. The leftover sediments of smear-positive samples were stored at -20 °C, 70 of which were selected to be included in this study according to their dR profile. thirty dR Mycobacterium tuberculosis isolates were also assessed. Sequencing was used as gold standard to detect mutations conferring isoniazid (InH) and rifampicin (RIF) resistance. Valid results were obtained in 94.0 % of the samples and 85.5 % (53/62) of the InH-R samples were properly identified. mutations in the katGS315t gene and inhA C-15t gene promoter region were present in 59.7 % (37/62) and 25.8 % (16/62) of the InH-R samples, respectively. the system could also identify 97.7 % (41/42) of the RIF-R samples; the mutations found were rpoBS531l (66.7 %, 28/42), d516V (19.0 %, 8/42), H526Y and S531p/W (4.8 %, 2/42 each one), and S522l/Q (2.4 %, 1/42). a 98.8 % concordance between the genotype assay and sequencing was obtained. genotype® mtBdRplus has demonstrated to be easy to implement and to perform in clinical laboratories and useful for a rapid detection of dR M. tuberculosis from decontaminated sputa and clinical isolates. Therefore, this assay could be applied as a rapid tool to predict InH-R and/or RIF-R in dR risk cases.La tuberculosis (TBC), y la TBC multi y extensivamente drogo-resistentes (DR) son importantes problemas de salud pública mundial. El objetivo de este estudio fue determinar la sensibilidad y especificidad del sistema GenoType® MTBDRplus a partir de esputos (baciloscopía positiva) y aislamientos clínicos de Mycobacterium tuberculosis, explorando su aplicación clínica. Previo a la tinción de Ziehl-Neelsen y al cultivo, las muestras fueron descontaminadas mediante NaOH-N-acetyl-L-cisteina o la solución hipertónica NaOH-NaCl. Los sedimentos remanentes se conservaron a –20 ºC, y 70 de ellos fueron incluidos en este estudio según su perfil de DR. Treinta cepas de M. tuberculosis DR fueron también evaluadas. La secuenciación fue utilizada como método de referencia para la detección de mutaciones que confieren resistencia a isoniacida (INH) y rifampicina (RIF). Se obtuvieron resultados válidos en el 94,0 % de las muestras, identificándose al 85,5 % (53/62) de las INH-R. La mutación katG S315T estuvo presente en 59,7 % (37/62), y la mutación C-15T del promotor del gen inhA en 25,8 % (16/62) de las mismas. El sistema identificó el 97,7 % (41/42) de las muestras RIF-R. Las mutaciones encontradas fueron rpoB S531L (66,7 %, 28/42), D516V (19,0 %, 8/42), H526Y y S531P/W (4,8 %, 2/42 cada una de ellas) y S522L/Q (2,4 %, 1/42). La concordancia entre el GenoType y la secuenciación fue del 98,8 %. El sistema GenoType® MTBDRplus resultó ser útil, fácil de realizar e implementar para la detección rápida de M. tuberculosis DR. Por lo tanto, este ensayo podría ser aplicado como una herramienta rápida para el diagnostico de TBC DR, principalmente en aquellos casos asociados a factores de riesgo.Fil: Imperiale, Belén Rocío. Provincia de Buenos Aires. Ministerio de Salud. Hospital ; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Zumárraga, Martín José. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Weltman, Gabriela. Provincia de Buenos Aires. Ministerio de Salud. Hospital ; ArgentinaFil: Zudiker, Roxana. Provincia de Buenos Aires. Ministerio de Salud. Hospital ; ArgentinaFil: Cataldi, Ángel Adrián. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Morcillo, Nora Susana. Provincia de Buenos Aires. Ministerio de Salud. Hospital ; Argentin

Primera evaluación en Argentina de GenoType® MTBDRplus para la detección de Mycobacterium tuberculosis multidrogo-resistente desde aislamientos y especímenes clínicos

Tuberculosis (tB) and multidrug and extensively drug-resistant (dR) tB are important public health problems that are spreading worldwide. The aims of this study were to determine the sensitivity and specificity of the genotype® mtBdRplus assay from smear-positive clinical specimens and isolates and to explore its possible application in routine work. Clinical samples were previously decontaminated using naoH-n-acetyl-l-cystein or naoH-Clna hypertonic solution for Ziehl-neelsen staining and cultures. The leftover sediments of smear-positive samples were stored at -20 °C, 70 of which were selected to be included in this study according to their dR profile. thirty dR Mycobacterium tuberculosis isolates were also assessed. Sequencing was used as gold standard to detect mutations conferring isoniazid (InH) and rifampicin (RIF) resistance. Valid results were obtained in 94.0 % of the samples and 85.5 % (53/62) of the InH-R samples were properly identified. mutations in the katGS315t gene and inhA C-15t gene promoter region were present in 59.7 % (37/62) and 25.8 % (16/62) of the InH-R samples, respectively. the system could also identify 97.7 % (41/42) of the RIF-R samples; the mutations found were rpoBS531l (66.7 %, 28/42), d516V (19.0 %, 8/42), H526Y and S531p/W (4.8 %, 2/42 each one), and S522l/Q (2.4 %, 1/42). a 98.8 % concordance between the genotype assay and sequencing was obtained. genotype® mtBdRplus has demonstrated to be easy to implement and to perform in clinical laboratories and useful for a rapid detection of dR M. tuberculosis from decontaminated sputa and clinical isolates. Therefore, this assay could be applied as a rapid tool to predict InH-R and/or RIF-R in dR risk cases.La tuberculosis (TBC), y la TBC multi y extensivamente drogo-resistentes (DR) son importantes problemas de salud pública mundial. El objetivo de este estudio fue determinar la sensibilidad y especificidad del sistema GenoType® MTBDRplus a partir de esputos (baciloscopía positiva) y aislamientos clínicos de Mycobacterium tuberculosis, explorando su aplicación clínica. Previo a la tinción de Ziehl-Neelsen y al cultivo, las muestras fueron descontaminadas mediante NaOH-N-acetyl-L-cisteina o la solución hipertónica NaOH-NaCl. Los sedimentos remanentes se conservaron a –20 ºC, y 70 de ellos fueron incluidos en este estudio según su perfil de DR. Treinta cepas de M. tuberculosis DR fueron también evaluadas. La secuenciación fue utilizada como método de referencia para la detección de mutaciones que confieren resistencia a isoniacida (INH) y rifampicina (RIF). Se obtuvieron resultados válidos en el 94,0 % de las muestras, identificándose al 85,5 % (53/62) de las INH-R. La mutación katG S315T estuvo presente en 59,7 % (37/62), y la mutación C-15T del promotor del gen inhA en 25,8 % (16/62) de las mismas. El sistema identificó el 97,7 % (41/42) de las muestras RIF-R. Las mutaciones encontradas fueron rpoB S531L (66,7 %, 28/42), D516V (19,0 %, 8/42), H526Y y S531P/W (4,8 %, 2/42 cada una de ellas) y S522L/Q (2,4 %, 1/42). La concordancia entre el GenoType y la secuenciación fue del 98,8 %. El sistema GenoType® MTBDRplus resultó ser útil, fácil de realizar e implementar para la detección rápida de M. tuberculosis DR. Por lo tanto, este ensayo podría ser aplicado como una herramienta rápida para el diagnostico de TBC DR, principalmente en aquellos casos asociados a factores de riesgo.Fil: Imperiale, Belén Rocío. Provincia de Buenos Aires. Ministerio de Salud. Hospital ; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Zumárraga, Martín José. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Weltman, Gabriela. Provincia de Buenos Aires. Ministerio de Salud. Hospital ; ArgentinaFil: Zudiker, Roxana. Provincia de Buenos Aires. Ministerio de Salud. Hospital ; ArgentinaFil: Cataldi, Ángel Adrián. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Morcillo, Nora Susana. Provincia de Buenos Aires. Ministerio de Salud. Hospital ; Argentin

Surveillance and Characterization of Drug‑Resistant Mycobacterium tuberculosis Isolated in a Reference Hospital from Argentina during 8 Years’ Period

Background: Argentina is considered a country with a middle tuberculosis (TB) incidence. However, according to the last national epidemiological report released in 2018, since 2013, the trends are steadily increasing. The aims of this study were to determine the drug‑resistance (DR), multi‑DR and extensively DR (MDR/XDR‑TB), and rifampicin resistance (RIF‑R) burden as a part of the local TB diagnosis (June 2010–August 2018); to detect the mutations associated to isoniazid (INH) and RIF‑R and their geographical distribution; and to analyze the lineage relationship among the genetic patterns of the isolates circulating in the community. Methods: Respiratory and extrapulmonary specimens were processed by Ziehl–Neelsen stain and cultured on specific media. Drug‑susceptibility testing of isolates was performed by the MGIT 960 and a colorimetric micro‑method. Mutations conferring DR were detected by Genotype and DNA sequencing. Results: The study showed a DR‑TB prevalence of approximately 20% of the isolated strains, while M/XDR‑TB‑and particularly RIF‑R‑affected more than 5.0% of the total amount of cases. DR geographical distribution revealed isolates carrying mutations in the inhA gene promoter region only constrained to three districts where it was also registered two same family relatives’ cases with the infrequent rpoB S522 L/Q mutation. The fact that most DR/MDR‑TB isolates were not grouped in genetic clusters suggested that these cases may mostly have occurred due to endogenous reactivation rather than recently transmission. Conclusion: According to the obtained results, it would be convenient, in highly MDR‑TB suspected individuals, to confirm phenotypically, the INH and RIF susceptibility detected by molecular tests.Fil: Imperiale, Belén Rocío. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Di Giulio, Ángela Beatríz. Provincia de Buenos Aires. Hospital "Petrona V. de Cordero"; ArgentinaFil: Mancino, María Belén. Provincia de Buenos Aires. Ministerio de Salud. Hospital "Dr. Antonio A. Cetrángolo"; ArgentinaFil: Zumárraga, Martín José. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigacion En Ciencias Veterinarias y Agronomicas. Instituto de Agrobiotecnologia y Biologia Molecular. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Agrobiotecnologia y Biologia Molecular; ArgentinaFil: Morcillo, Nora Susana. Provincia de Buenos Aires. Ministerio de Salud. Hospital "Dr. Antonio A. Cetrángolo"; Argentin

Susceptibilidad in vitro a los medicamentos anti-tuberculosos de aislados de cepas del complejo Mycobacterium tuberculosis obtenidos a partir de lobos marinos

Mycobacteria strains belonging to the Mycobacterium tuberculosis complex were isolated from seals found in the South Atlantic. The animals were received in Mundo Marino installations and treated for Mycobacterium tuberculosis complex by conventional therapy of intensive care and enriched food supply; however, in all cases treatment failed. Necropsies of all animals revealed extensive lesions compatible with tuberculosis involving lungs, liver, spleen and lymphatic nodes. Classical biochemical methods as well as molecular techniques using the IS6110 probes were performed for mycobacterial identification. Furthermore, the LCx M. tuberculosis assay (Abbott Laboratories) identified all strains as Mycobacterium tuberculosis complex members. The in vitro susceptibility pattern was examined in mycobacterial strains isolated from seven seals and in 3 reference strains--BCG, H37Rv (M. tuberculosis) and AN5 (Mycobacterium bovis)--to 4 medications--isoniazid, rifampin, streptomycin and ethambutol. Minimal inhibitory drug concentrations were determined by the Mycobacterial Growth Indicator Tube (BD Argentina) method and a microdilution and colorimetric assay using 3-(4-5 dimethyltiazol-2)-2,5 diphenyltetrazolium bromide. All the isolates and the reference strains BCG and AN5 were inhibited by MIC values similar to those of H37Rv with good agreement obtained by both techniques. These findings suggest that a therapeutic regimen aimed to seals diagnosed with tuberculosis play an important role in the prevention of tuberculosis transmission from infected animals to humans that are in routine contact with them.Se han hallado cepas de micobacterias aisladas de lobos marinos del Atlántico sur y pertenecen al complejo de Mycobacterium tuberculosis. Los animales se recibieron en las instalaciones del Oceanario Mundo Marino y fueron tratados apropiadamente para su recuperación con la terapia convencional, cuidados intensivos y suplemento alimentario pero no se observó mejoría en su estado general. Se practicaron necropsias en todos los animales y se observaron lesiones extensas compatibles con tuberculosis en pulmones, hígado, bazo y ganglios linfáticos. Para la identificación de las micobacterias, se realizaron pruebas bioquímicas y técnicas de biología molecular con la sonda IS6110. Además, se identificaron todas las cepas como pertenecientes al complejo M. tuberculosis mediante el equipo LCx M. tuberculosis Assay (Abbott Laboratories). El objetivo de este estudio fue determinar in vitro la sensibilidad de las cepas patrón BCG, H37Rv (M. tuberculosis) y AN5 (Mycobacterium bovis) y la de las siete aisladas de lobos marinos a isoniacida, rifampicina, estreptomicina y etambutol. La concentración inhibitoria mínima (CIM) de las drogas antituberculosas se llevó a cabo con el equipo Mycobacterial Growth Indicator Tube (MGIT, BD, Argentina) y la microdilución con el ensayo colorimétrico con bromuro de 3-(4-5 dimetiltiazol-2)-2,5 difeniltetrazolio. Todos los aislamientos y las cepas de referencia BCG y AN5 se inhibieron con valores CIM de los de H37Rv con buena concordancia entre los resultados obtenidos con ambas técnicas. Los hallazgos permiten sugerir que podrían ser una importante ayuda terapéutica en los lobos marinos con diagnóstico de tuberculosis y evaluar el posible papel sanitario en la prevención y transmisión de la tuberculosis de los animales a los humanos y el trabajo en conjunto

Rifampin-Isoniazid Oligonucleotide Typing: an Alternative Format for Rapid Detection of Multidrug-Resistant Mycobacterium tuberculosis

Fil: Hernandez-Neuta, Iván. Corporacion CorpoGen; Colombia.Fil: Varela, Andres. Corporacion CorpoGen; Colombia.Fil: Martin, Anandi. Institute of Tropical Medicine. Mycobacteriology Unit; Bélgica.Fil: von Groll, Andrea. Institute of Tropical Medicine. Mycobacteriology Unit; Bélgica.Fil: Jureen, Pontus. Swedish Institute for Infectious Disease Control; Suecia.Fil: López, Beatriz. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas; Argentina.Fil: Imperiale, Belen. Hospital Dr. Cetrángolo; Argentina.Fil: Skenders, Girts. State Agency of Tuberculosis and Lung Diseases; Letonia.Fil: Ritacco, Viviana. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas; Argentina.Fil: Hoffner, Sven. Swedish Institute for Infectious Disease Control; Suecia.Fil: Morcillo, Nora. Hospital Dr. Cetrángolo; Argentina.Fil: Palomino, Juan Carlos. Institute of Tropical Medicine. Mycobacteriology Unit; Bélgica.Fil: Del Portillo, Patricia. Corporacion CorpoGen; Colombia.on of rifampin and isoniazid resistance in clinical isolates of Mycobacterium tuberculosis was based on the same amplification/reverse hybridization principle of the widely used spoligotyping. The test involved probing nine DNA regions that are targets of common drug resistance-associated mutations in the genes rpoB, katG, and inhA. Addition of quaternary amine tetramethyl ammonium chloride to the hybridization buffer promoted multiple hybrid formations at a single annealing temperature irrespective of the different GC contents of probes. The assay was standardized using 20 well-documented strains from the Institute of Tropical Medicine (Belgium) and evaluated blindly in a central laboratory with 100 DNA samples that were obtained from cultured clinical isolates and shipped dried from three other countries. Compared with drug susceptibility testing, both sensitivity and specificity for rifampin resistance detection were 93.0% while for isoniazid the values were 87.7% and 97.7%, respectively. Compared with sequencing and GenoType MTBDRplus methods, sensitivity and specificity reached 96.4% and 95.5% for rifampin and 92.7% and 100% for isoniazid. Altogether, 40/45 (89%) multidrug-resistant isolates were correctly identified. Advantages of this in-house development include versatility, capacity to run up to 41 samples by triplicate in a single run, and reuse of the membrane at least 10 times. These features substantially reduce cost per reaction and make the assay an attractive tool for use in reference laboratories of countries that have a high burden of multidrug-resistant tuberculosis but that cannot afford expensive commercial tests because of limited resources