Abordando la vigilancia trans-nacional de transmisión de tuberculosis

Investigación epidemiológica. Epidemiología. Estudio de contactos: Enfermería.Trabajadores sociales. Médicos de familia. Investigación epidemiológica: Alternativa desde el laboratorio. Epidemiología molecular. Métodos de genotipado actuales. Secuenciación de Genoma Completo. Identificación de clusters activos no controlados.N

The use of microbead-based spoligotyping for Mycobacterium tuberculosis complex to evaluate the quality of the conventional method: Providing guidelines for Quality Assurance when working on membranes

Fil: Abadia, Edgar. CNRS Université Paris-Sud 11 Universud. Institute of Genetics and Microbiology UMR8621; Francia.Fil: Zhang, Jian. CNRS Université Paris-Sud 11 Universud. Institute of Genetics and Microbiology UMR8621; Francia.Fil: Ritacco, Viviana. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas; Argentina.Fil: Kremer, Kristin. National Institute for Public Health and the Environment; Paises Bajos.Fil: Ruimy, Raymond. Université Paris- Diderot & Microbiology Laboratory; Francia.Fil: Rigouts, Leen. Prince Leopold Institute of Tropical Medicine. Mycobacteriology Unit; Bélgica.Fil: Gomes, Harrison Magdinier. Oswaldo Cruz Institute. Laboratory of Molecular Biology applied to Mycobacteria; Brasil.Fil: Elias, Atina Ribeiro. Oswaldo Cruz Institute. Laboratory of Molecular Biology applied to Mycobacteria; Brasil.Fil: Fauville-Dufaux, Maryse. Scientific Institute of Public Health. National Reference Centre of Tuberculosis and Mycobacteria; Bélgica.Fil: Stoffels, Karolien. Scientific Institute of Public Health. National Reference Centre of Tuberculosis and Mycobacteria; Bélgica.Fil: Rasolofo-Razanamparany, Voahangy. Institut Pasteur de Madagascar. Unité des Mycobactéries; Madagascar.Fil: Garcia de Viedma, Dario. Hospital Gregorio Marañón. Servicio de Microbiología Clínica y Enfermedades Infecciosas; España.Fil: Herranz, Marta. Hospital Gregorio Marañón. Servicio de Microbiología Clínica y Enfermedades Infecciosas; España.Fil: Al-Hajoj, Sahal. King Faisal Specialist Hospital and Research Center. Department of Comparative Medicine; Arabia Saudita.Fil: Rastogi, Nalin. Institut Pasteur de Guadeloupe. Unité de la Tuberculose et des Mycobactéries - WHO Supranational TB Reference Laboratory; Guadalupe.Fil: Garzelli, Carlo. Università di Pisa. Dipartimento di Patologia Sperimentale Biotecnologie Mediche Infettivologia ed Epidemiologia; Italia.Fil: Tortoli, Enrico. Careggi Hospital. Regional Reference Center for Mycobacteria; ItaliaFil: Suffys, Philip N. Oswaldo Cruz Institute. Laboratory of Molecular Biology applied to Mycobacteria; Brasil.Fil: van Soolingen, Dick. National Institute for Public Health and the Environment; Paises Bajos.Fil: Refregier, Guislaine. CNRS Université Paris-Sud 11 Universud. Institute of Genetics and Microbiology UMR8621; Francia.Fil: Sola, Christophe. CNRS Université Paris-Sud 11 Universud. Institute of Genetics and Microbiology UMR8621; Francia.Background: The classical spoligotyping technique, relying on membrane reverse line-blot hybridization of the spacers of the Mycobacterium tuberculosis CRISPR locus, is used world-wide (598 references in Pubmed on April 8th, 2011). However, until now no inter-laboratory quality control study had been undertaken to validate this technique. We analyzed the quality of membrane-based spoligotyping by comparing it to the recently introduced and highly robust microbead-based spoligotyping. Nine hundred and twenty-seven isolates were analyzed totaling 39,861 data points. Samples were received from 11 international laboratories with a worldwide distribution. Methods: The high-throughput microbead-based Spoligotyping was performed on CTAB and thermolyzate DNA extracted from isolated Mycobacterium tuberculosis complex (MTC) strains coming from the genotyping participating centers. Information regarding how the classical Spoligotyping method was performed by center was available. Genotype discriminatory analyses were carried out by comparing the spoligotypes obtained by both methods. The non parametric U-Mann Whitney homogeneity test and the Spearman rank correlation test were performed to validate the observed results. Results: Seven out of the 11 laboratories (63 %), perfectly typed more than 90% of isolates, 3 scored between 80-90% and a single center was under 80% reaching 51% concordance only. However, this was mainly due to discordance in a single spacer, likely having a non-functional probe on the membrane used. The centers using thermolyzate DNA performed as well as centers using the more extended CTAB extraction procedure. Few centers shared the same problematic spacers and these problematic spacers were scattered over the whole CRISPR locus (Mostly spacers 15, 14, 18, 37, 39, 40). Conclusions: We confirm that classical spoligotyping is a robust method with generally a high reliability in most centers. The applied DNA extraction procedure (CTAB or thermolyzate) did not affect the results in this study. However performance was center-dependent, suggesting that training is a key component in quality assurance of spoligotyping. Overall, no particular spacer yielded a higher degree of deviating results, suggesting that errors

Low Dose Aerosol Fitness at the Innate Phase of Murine Infection Better Predicts Virulence amongst Clinical Strains of Mycobacterium tuberculosis

Background: Evaluation of a quick and easy model to determine the intrinsic ability of clinical strains to generate active TB has been set by assuming that this is linked to the fitness of Mycobacterium tuberculosis strain at the innate phase of the infection. Thus, the higher the bacillary load, the greater the possibility of inducting liquefaction, and thus active TB, once the adaptive response is set. Methodology/Principal Findings: The virulence of seven clinical Mycobacterium tuberculosis strains isolated in Spain was tested by determining the bacillary concentration in the spleen and lung of mice at weeks 0, 1 and 2 after intravenous (IV) inoculation of 10 4 CFU, and by determining the growth in vitro until the stationary phase had been reached. Cord distribution automated analysis showed two clear patterns related to the high and low fitness in the lung after IV infection. This pattern was not seen in the in vitro fitness tests, which clearly favored the reference strain (H37Rv). Subsequent determination using a more physiological low-dose aerosol (AER) inoculation with 10 2 CFU showed a third pattern in which the three best values coincided with the highest dissemination capacity according to epidemiological data. Conclusions/Significance: The fitness obtained after low dose aerosol administration in the presence of the innate immune response is the most predictive factor for determining the virulence of clinical strains. This gives support to a mechanism o

Urgent Implementation in a Hospital Setting of a Strategy To Rule Out Secondary Cases Caused by Imported Extensively Drug-Resistant Mycobacterium tuberculosis Strains at Diagnosis

Current migratory movements require new strategies for rapidly tracking the transmission of high-risk imported Mycobacterium tuberculosis strains. Whole-genome sequencing (WGS) enables us to identify single-nucleotide polymorphisms (SNPs) and therefore design PCRs to track specific relevant strains. However, fast implementation of these strategies in the hospital setting is difficult because professionals working in diagnostics, molecular epidemiology, and genomics are generally at separate institutions. In this study, we describe the urgent implementation of a system that integrates genomics and molecular tools in a genuine high-risk epidemiological alert involving 2 independent importations of extensively drug resistant (XDR) and pre-XDR Beijing M. tuberculosis strains from Russia into Spain. Both cases involved commercial sex workers with long-standing tuberculosis (TB). The system was based on strain-specific PCRs tailored from WGS data that were transferred to the local node that was managing the epidemiological alert. The optimized tests were available for prospective implementation in the local node 33 working days after receiving the primary cultures of the XDR strains and were applied to all 42 new incident cases. An interpretable result was obtained in each case (directly from sputum for 27 stain-positive cases) and corresponded to the amplification profiles for strains other than the targeted pre-XDR and XDR strains, which made it possible to prospectively rule out transmission of these high-risk strains at diagnosis

Susceptibility and genotyping features from cases with subtle changes in the clonal composition of the isolates

<p><b>Copyright information:</b></p><p>Taken from "Rapid clonal analysis of recurrent tuberculosis by direct MIRU-VNTR typing on stored isolates"</p><p>http://www.biomedcentral.com/1471-2180/7/73</p><p>BMC Microbiology 2007;7():73-73.</p><p>Published online 30 Jul 2007</p><p>PMCID:PMC1988808.</p><p></p> Differences in the MIRUtypes are boxe

Susceptibility and genotyping features of the isolates from cases with reinfections

<p><b>Copyright information:</b></p><p>Taken from "Rapid clonal analysis of recurrent tuberculosis by direct MIRU-VNTR typing on stored isolates"</p><p>http://www.biomedcentral.com/1471-2180/7/73</p><p>BMC Microbiology 2007;7():73-73.</p><p>Published online 30 Jul 2007</p><p>PMCID:PMC1988808.</p><p></p> Differences in the MIRUtypes are boxe

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

Fil: Monteserin, Johana. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas; Argentina.Fil: Pérez-Lago, Laura. Hospital General Universitario Gregorio Marañón, Instituto de Investigación Sanitaria Gregorio Marañón, Madrid; España.Fil: Yokobori, Noemí. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas; Argentina.Fil: Paul, Roxana. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas; Argentina.Fil: Rodríguez Maus, Sandra. Hospital General Universitario Gregorio Marañón, Instituto de Investigación Sanitaria Gregorio Marañón, Madrid; España.Fil: Simboli, Norberto. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas; Argentina.Fil: Eldholm, Vegard. Norwegian Institute of Public Health, Oslo; Noruega.Fil: López, Beatriz. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas; Argentina.Fil: García de Viedma, Darío. Hospital General Universitario Gregorio Marañón, Instituto de Investigación Sanitaria Gregorio Marañón, Madrid; España.Fil: Ritacco, Viviana. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas; Argentina.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

Low Dose Aerosol Fitness at the Innate Phase of Murine Infection Better Predicts Virulence amongst Clinical Strains of Mycobacterium tuberculosis

Altres ajuts: The research leading to these results has received funding from the European Community's 7th Framework Programme HEALTH-2007-2.3.2-2 (FP7/2007-2013: TOPLATENT-TB project under grant agreement 200999). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Evaluation of a quick and easy model to determine the intrinsic ability of clinical strains to generate active TB has been set by assuming that this is linked to the fitness of Mycobacterium tuberculosis strain at the innate phase of the infection. Thus, the higher the bacillary load, the greater the possibility of inducting liquefaction, and thus active TB, once the adaptive response is set. The virulence of seven clinical Mycobacterium tuberculosis strains isolated in Spain was tested by determining the bacillary concentration in the spleen and lung of mice at weeks 0, 1 and 2 after intravenous (IV) inoculation of 10 4 CFU, and by determining the growth in vitro until the stationary phase had been reached. Cord distribution automated analysis showed two clear patterns related to the high and low fitness in the lung after IV infection. This pattern was not seen in the in vitro fitness tests, which clearly favored the reference strain (H37Rv). Subsequent determination using a more physiological low-dose aerosol (AER) inoculation with 10 2 CFU showed a third pattern in which the three best values coincided with the highest dissemination capacity according to epidemiological data. The fitness obtained after low dose aerosol administration in the presence of the innate immune response is the most predictive factor for determining the virulence of clinical strains. This gives support to a mechanism of the induction of active TB derived from the dynamic hypothesis of latent tuberculosis infection

Fitness in the <i>in vivo</i> models.

<p>Bacillary load in the lung from aerosol-infected mice and lung and spleen from IV-infected mice on day 0, week 1 and week 2. The H37Rv strain is indicated as St. Dotted lines show the inocula used to infect each mouse.</p

Fitness results.

<p>Fitness results from <i>in vivo</i> and <i>in vitro</i> studies. Top: fitness values for lung aerosol and IV experiments; bottom: <i>in vitro</i> assay and spleen IV infection values. Dotted lines indicate the fitness value for the H37Rv strain (shown as “St”).</p