Publications by authors named "Iñaki Comas"

87 Publications

Genomic analyses of Mycobacterium tuberculosis from human lung resections reveal a high frequency of polyclonal infections.

Nat Commun 2021 May 11;12(1):2716. Epub 2021 May 11.

Instituto de Biomedicina de Valencia IBV-CSIC, Valencia, Spain.

Polyclonal infections occur when at least two unrelated strains of the same pathogen are detected in an individual. This has been linked to worse clinical outcomes in tuberculosis, as undetected strains with different antibiotic resistance profiles can lead to treatment failure. Here, we examine the amount of polyclonal infections in sputum and surgical resections from patients with tuberculosis in the country of Georgia. For this purpose, we sequence and analyse the genomes of Mycobacterium tuberculosis isolated from the samples, acquired through an observational clinical study (NCT02715271). Access to the lung enhanced the detection of multiple strains (40% of surgery cases) as opposed to just using a sputum sample (0-5% in the general population). We show that polyclonal infections often involve genetically distant strains and can be associated with reversion of the patient's drug susceptibility profile over time. In addition, we find different patterns of genetic diversity within lesions and across patients, including mutational signatures known to be associated with oxidative damage; this suggests that reactive oxygen species may be acting as a selective pressure in the granuloma environment. Our results support the idea that the magnitude of polyclonal infections in high-burden tuberculosis settings is underestimated when only testing sputum samples.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-021-22705-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8113332PMC
May 2021

Local adaptation in populations of endemic to the Indian Ocean Rim.

F1000Res 2021 1;10:60. Epub 2021 Feb 1.

Institut Pasteur de Madagascar, Antananarivo, Madagascar.

Lineage 1 (L1) and 3 (L3) are two lineages of the complex (MTBC) causing tuberculosis (TB) in humans. L1 and L3 are prevalent around the rim of the Indian Ocean, the region that accounts for most of the world's new TB cases. Despite their relevance for this region, L1 and L3 remain understudied. We analyzed 2,938 L1 and 2,030 L3 whole genome sequences originating from 69 countries. We reconstructed the evolutionary history of these two lineages and identified genes under positive selection. We found a strongly asymmetric pattern of migration from South Asia toward neighboring regions, highlighting the historical role of South Asia in the dispersion of L1 and L3. Moreover, we found that several genes were under positive selection, including genes involved in virulence and resistance to antibiotics. For L1 we identified signatures of local adaptation at the locus, a gene coding for a secreted effector that targets the human endosomal sorting complex, and is included in several vaccine candidates. Our study highlights the importance of genetic diversity in the MTBC, and sheds new light on two of the most important MTBC lineages affecting humans.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.12688/f1000research.28318.2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7921886.2PMC
February 2021

Integrative transnational analysis to dissect tuberculosis transmission events along the migratory route from Africa to Europe.

J Travel Med 2021 Apr 5. Epub 2021 Apr 5.

Servicio de Microbiología Clínica y Enfermedades Infecciosas, Hospital General Universitario Gregorio Marañón, Madrid, Spain.

Background: Growing international migration has increased the complexity of tuberculosis transmission patterns. Italy's decision to close its borders in 2018 made of Spain the new European porte entrée for migration from the Horn of Africa (HA). In one of the first rescues of migrants from this region at the end of 2018, tuberculosis was diagnosed in eight subjects, mainly unaccompanied minors.

Methods: M. tuberculosis isolates from these recently arrived migrants were analyzed by Mycobacterial Interspersed Repetitive-Unit/Variable-Number of Tandem Repeat (MIRU-VNTR) and subsequent whole genome sequencing (WGS) analysis. Data were compared with those from collections from other European countries receiving migrants from the HA and a strain-specific PCR was applied for a fast searching of common strains. Infections in a cellular model were performed to assess strain virulence.

Results: MIRU-VNTR analysis allowed identifying an epidemiological cluster involving three of the eight cases from Somalia (0 single-nucleotide polymorphisms between isolates, HA cluster). Following detailed interviews revealed that two of these cases had shared the same migratory route in most of the trip and had spent a long time at a detention camp in Libya. To confirm potential en route transmission for the three cases, we searched the same strain in collections from other European countries receiving migrants from the HA. MIRU-VNTR, WGS, and a strain-specific PCR for the HA strain were applied. The same strain was identified in 12 cases from Eritrea diagnosed soon after their arrival in 2018 to the Netherlands, Belgium, and Italy. Intracellular replication rate of the strain did not reveal abnormal virulence.

Conclusions: Our study suggests a potential en route transmission of a pan-susceptible strain, which caused at least 15 tuberculosis cases in Somalian and Eritrean migrants diagnosed in four different European countries.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/jtm/taab054DOI Listing
April 2021

Overlapping prison/community tuberculosis outbreaks in Costa Rica revealed by alternative analysis of suboptimal material.

Transbound Emerg Dis 2021 Mar 9. Epub 2021 Mar 9.

Servicio de Microbiología Clínica y Enfermedades Infecciosas, Hospital General Universitario Gregorio Marañón, Madrid, Spain.

Costa Rica has a low incidence of tuberculosis. Thus, identifying transmission hotspots is key to implement interventions. A tuberculosis outbreak was suspected in a prison in Costa Rica. Given the suboptimal quality of the samples received in our laboratory in Madrid, we applied alternative schemes for their analysis. In the first scheme, we bypassed the standard approach of applying systematic mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) and used a strain-specific polymerase chain reaction (PCR) that allowed identifying a cluster involving six cases (C1). The second scheme followed the canonical MIRU-VNTR path coupled with a whole-genomic amplification step, by which a second unsuspected overlapping cluster (C2), was detected in the same prison. These findings justified the implementation of a surveillance programme adapted to local resources based on a tailored multiplex allele-specific oligonucleotide (ASO)-PCR targeting C1 and C2. Presence of the C2 strain at a different prison was determined. ASO-PCR was applied extensively and alerted to the active circulation of one of the strains within and beyond prisons. Our study shows that alternative methodological strategies may provide useful data in settings with lack of resources for performing systematic standard molecular epidemiology programmes and/or with suboptimal material for analysis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/tbed.14063DOI Listing
March 2021

Whole genomic sequencing based genotyping reveals a specific X3 sublineage restricted to Mexico and related with multidrug resistance.

Sci Rep 2021 Jan 21;11(1):1870. Epub 2021 Jan 21.

Programa de Maestría en Ciencias de la Salud, Instituto de Ciencias de la Salud, Universidad Veracruzana, Xalapa, Veracruz, México.

Whole genome sequencing (WGS) has been shown to be superior to traditional procedures of genotyping in tuberculosis (TB), nevertheless, reports of its use in drug resistant TB (DR-TB) isolates circulating in Mexico, are practically unknown. Considering the above the main of this work was to identify and characterize the lineages and genomic transmission clusters present in 67 DR-TB isolates circulating in southeastern Mexico. The results show the presence of three major lineages: L1 (3%), L2 (3%) and L4 (94%), the last one included 16 sublineages. Sublineage 4.1.1.3 (X3) was predominant in 18 (27%) of the isolates, including one genomic cluster, formed by eleven multidrug resistant isolates and sharing the SIT 3278, which seems to be restricted to Mexico. By the use of WGS, it was possible to identify the high prevalence of L4 and a high number of sublineages circulating in the region, also was recognized the presence of a novel X3 sublineage, formed exclusively by multidrug resistant isolates and with restrictive circulation in Mexico for at least the past 17 years.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-020-80919-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7820219PMC
January 2021

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

Transbound Emerg Dis 2021 Jan 7. Epub 2021 Jan 7.

Hospital General Universitario Gregorio Marañón, Madrid, Spain.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/tbed.13982DOI Listing
January 2021

Emergence and spread of a SARS-CoV-2 variant through Europe in the summer of 2020.

medRxiv 2020 Nov 27. Epub 2020 Nov 27.

Following its emergence in late 2019, SARS-CoV-2 has caused a global pandemic resulting in unprecedented efforts to reduce transmission and develop therapies and vaccines (WHO Emergency Committee, 2020; Zhu ., 2020). Rapidly generated viral genome sequences have allowed the spread of the virus to be tracked via phylogenetic analysis (Hadfield ., 2018; Pybus ., 2020; Worobey ., 2020). While the virus spread globally in early 2020 before borders closed, intercontinental travel has since been greatly reduced, allowing continent-specific variants to emerge. However, within Europe travel resumed in the summer of 2020, and the impact of this travel on the epidemic is not well understood. Here we report on a novel SARS-CoV-2 variant, 20A.EU1, that emerged in Spain in early summer, and subsequently spread to multiple locations in Europe, accounting for the majority of sequences by autumn. We find no evidence of increased transmissibility of this variant, but instead demonstrate how rising incidence in Spain, resumption of travel across Europe, and lack of effective screening and containment may explain the variant's success. Despite travel restrictions and quarantine requirements, we estimate 20A.EU1 was introduced hundreds of times to countries across Europe by summertime travellers, likely undermining local efforts to keep SARS-CoV-2 cases low. Our results demonstrate how genomic surveillance is critical to understanding how travel can impact SARS-CoV-2 transmission, and thus for informing future containment strategies as travel resumes.

Caveats: . .
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/2020.10.25.20219063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7709189PMC
November 2020

Glucocorticoid-dependent transcription in skin requires epidermal expression of the glucocorticoid receptor and is modulated by the mineralocorticoid receptor.

Sci Rep 2020 11 3;10(1):18954. Epub 2020 Nov 3.

Instituto de Biomedicina de Valencia-Consejo Superior de Investigaciones Científicas (IBV-CSIC), Jaime Roig 11, 46010, Valencia, Spain.

Glucocorticoid (GC) actions are mediated through two closely related ligand-dependent transcription factors, the GC receptor (GR) and the mineralocorticoid receptor (MR). Given the wide and effective use of GCs to combat skin inflammatory diseases, it is important to understand the relative contribution of these receptors to the transcriptional response to topical GCs. We evaluated the gene expression profiles in the skin of mice with epidermal-specific loss of GR (GR), MR (MR), or both (double KO; DKO) in response to dexamethasone (Dex). The overall transcriptional response was abolished in GR and DKO skin suggesting dependence of the underlying dermis on the presence of epidermal GR. Indeed, the observed dermal GC resistance correlated with a constitutive decrease in GR activity and up-regulation of p38 activity in this skin compartment. Upon Dex treatment, more than 90% of differentially expressed genes (DEGs) in CO overlapped with MR. However, the number of DEGs was fourfold increased and the magnitude of response was higher in MR vs CO, affecting both gene induction and repression. Taken together our data reveal that, in the cutaneous transcriptional response to GCs mediated through endogenous receptors, epidermal GR is mandatory while epidermal MR acts as a chief modulator of gene expression.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-020-75853-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7609727PMC
November 2020

clinical isolates carry mutational signatures of host immune environments.

Sci Adv 2020 May 29;6(22):eaba4901. Epub 2020 May 29.

Shanghai Public Health Clinical Center, Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), Shanghai Medical College and School of Basic Medical Sciences, Fudan University, Shanghai, China.

() infection results in a spectrum of clinical and histopathologic manifestations. It has been proposed that the environmental and immune pressures associated with different contexts of infection have different consequences for the associated bacterial populations, affecting drug susceptibility and the emergence of resistance. However, there is little concrete evidence for this model. We prospectively collected sputum samples from 18 newly diagnosed and treatment-naïve patients with tuberculosis and sequenced 795 colony-derived isolates. Mutant accumulation rates varied considerably between different bacilli isolated from the same individual, and where high rates of mutation were observed, the mutational spectrum was consistent with reactive oxygen species-induced mutagenesis. Elevated bacterial mutation rates were identified in isolates from HIV-negative but not HIV-positive individuals, suggesting that they were immune-driven. These results support the model that mutagenesis of in vivo is modulated by the host environment, which could drive the emergence of variants associated with drug resistance in a host-dependent manner.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/sciadv.aba4901DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7259932PMC
May 2020

Mycobacterium tuberculosis associated with severe tuberculosis evades cytosolic surveillance systems and modulates IL-1β production.

Nat Commun 2020 04 23;11(1):1949. Epub 2020 Apr 23.

i3S-Instituto de Investigação e Inovação em Saúde, University of Porto, Porto, Portugal.

Genetic diversity of Mycobacterium tuberculosis affects immune responses and clinical outcomes of tuberculosis (TB). However, how bacterial diversity orchestrates immune responses to direct distinct TB severities is unknown. Here we study 681 patients with pulmonary TB and show that M. tuberculosis isolates from cases with mild disease consistently induce robust cytokine responses in macrophages across multiple donors. By contrast, bacteria from patients with severe TB do not do so. Secretion of IL-1β is a good surrogate of the differences observed, and thus to classify strains as probable drivers of different TB severities. Furthermore, we demonstrate that M. tuberculosis isolates that induce low levels of IL-1β production can evade macrophage cytosolic surveillance systems, including cGAS and the inflammasome. Isolates exhibiting this evasion strategy carry candidate mutations, generating sigA recognition boxes or affecting components of the ESX-1 secretion system. Therefore, we provide evidence that M. tuberculosis strains manipulate host-pathogen interactions to drive variable TB severities.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-020-15832-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7181847PMC
April 2020

Contaminant DNA in bacterial sequencing experiments is a major source of false genetic variability.

BMC Biol 2020 03 2;18(1):24. Epub 2020 Mar 2.

Institute of Biomedicine of Valencia, IBV-CSIC, St. Jaume Roig 11, 46010, Valencia, Spain.

Background: Contaminant DNA is a well-known confounding factor in molecular biology and in genomic repositories. Strikingly, analysis workflows for whole-genome sequencing (WGS) data commonly do not account for errors potentially introduced by contamination, which could lead to the wrong assessment of allele frequency both in basic and clinical research.

Results: We used a taxonomic filter to remove contaminant reads from more than 4000 bacterial samples from 20 different studies and performed a comprehensive evaluation of the extent and impact of contaminant DNA in WGS. We found that contamination is pervasive and can introduce large biases in variant analysis. We showed that these biases can result in hundreds of false positive and negative SNPs, even for samples with slight contamination. Studies investigating complex biological traits from sequencing data can be completely biased if contamination is neglected during the bioinformatic analysis, and we demonstrate that removing contaminant reads with a taxonomic classifier permits more accurate variant calling. We used both real and simulated data to evaluate and implement reliable, contamination-aware analysis pipelines.

Conclusion: As sequencing technologies consolidate as precision tools that are increasingly adopted in the research and clinical context, our results urge for the implementation of contamination-aware analysis pipelines. Taxonomic classifiers are a powerful tool to implement such pipelines.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12915-020-0748-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7053099PMC
March 2020

Use of next generation sequencing technologies for the diagnosis and epidemiology of infectious diseases.

Enferm Infecc Microbiol Clin 2020 01;38 Suppl 1:32-38

CIBER en Epidemiología y Salud Pública, Valencia, España; Unidad Mixta "Infección y Salud Pública" FISABIO-Universitat de València, Instituto de Biología Integrativa de Sistemas, I2SysBio (CSIC-UV), Valencia, España.

For the first time, next generation sequencing technologies provide access to genomic information at a price and scale that allow their implementation in routine clinical practice and epidemiology. While there are still many obstacles to their implementation, there are also multiple examples of their major advantages compared with previous methods. Their main advantage is that a single determination allows epidemiological information on the causative microorganism to be obtained simultaneously, as well as its resistance profile, although these advantages vary according to the pathogen under study. This review discusses several examples of the clinical and epidemiological use of next generation sequencing applied to complete genomes and microbiomes and reflects on its future in clinical practice.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.eimc.2020.02.006DOI Listing
January 2020

Screening of inmates transferred to Spain reveals a Peruvian prison as a reservoir of persistent Mycobacterium tuberculosis MDR strains and mixed infections.

Sci Rep 2020 02 17;10(1):2704. Epub 2020 Feb 17.

Hospital General Universitario Gregorio Marañón. Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain.

It is relevant to evaluate MDR-tuberculosis in prisons and its impact on the global epidemiology of this disease. However, systematic molecular epidemiology programs in prisons are lacking. A health-screening program performed on arrival for inmates transferred from Peruvian prisons to Spain led to the diagnosis of five MDR-TB cases from one of the biggest prisons in Latin America. They grouped into two MIRU-VNTR-clusters (Callao-1 and Callao-2), suggesting a reservoir of two prevalent MDR strains. A high-rate of overexposure was deduced because one of the five cases was coinfected by a pansusceptible strain. Callao-1 strain was also identified in 2018 in a community case in Spain who had been in the same Peruvian prison in 2002-5. A strain-specific-PCR tailored from WGS data was implemented in Peru, allowing the confirmation that these strains were currently responsible for the majority of the MDR cases in that prison, including a new mixed infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-020-59373-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026066PMC
February 2020

Antibiotic resistance prediction for from genome sequence data with Mykrobe.

Wellcome Open Res 2019 2;4:191. Epub 2019 Dec 2.

European Bioinformatics Institute, Cambridge, UK.

Two billion people are infected with , leading to 10 million new cases of active tuberculosis and 1.5 million deaths annually. Universal access to drug susceptibility testing (DST) has become a World Health Organization priority. We previously developed a software tool, , which provided offline species identification and drug resistance predictions for from whole genome sequencing (WGS) data. Performance was insufficient to support the use of WGS as an alternative to conventional phenotype-based DST, due to mutation catalogue limitations.  Here we present a new tool, , which provides the same functionality based on a new software implementation. Improvements include i) an updated mutation catalogue giving greater sensitivity to detect pyrazinamide resistance, ii) support for user-defined resistance catalogues, iii) improved identification of non-tuberculous mycobacterial species, and iv) an updated statistical model for Oxford Nanopore Technologies sequencing data. is released under MIT license at https://github.com/mykrobe-tools/mykrobe. We incorporate mutation catalogues from the CRyPTIC consortium et al. (2018) and from Walker et al. (2015), and make improvements based on performance on an initial set of 3206 and an independent set of 5845 Illumina sequences. To give estimates of error rates, we use a prospectively collected dataset of 4362 . Using culture based DST as the reference, we estimate to be 100%, 95%, 82%, 99% sensitive and 99%, 100%, 99%, 99% specific for rifampicin, isoniazid, pyrazinamide and ethambutol resistance prediction respectively. We benchmark against four other tools on 10207 (=5845+4362) samples, and also show that gives concordant results with nanopore data.  We measure the ability of -based DST to guide personalized therapeutic regimen design in the context of complex drug susceptibility profiles, showing 94% concordance of implied regimen with that driven by phenotypic DST, higher than all other benchmarked tools.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.12688/wellcomeopenres.15603.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7004237PMC
December 2019

Tuberculosis in Liberia: high multidrug-resistance burden, transmission and diversity modelled by multiple importation events.

Microb Genom 2020 01;6(1)

CIBER of Epidemiology and Public Health (CIBERESP), Madrid, Spain.

Tuberculosis (TB) surveillance is scarce in most African countries, even though it is the continent with the greatest disease incidence according to the World Health Organization. Liberia is within the 30 countries with the highest TB burden, probably as a consequence of the long civil war and the recent Ebola outbreak, both crippling the health system and depreciating the TB prevention and control programmes. Due to difficulties working in the country, there is a lack of resistance surveys and bacillus characterization. Here, we use genome sequencing of clinical isolates to fill this gap. Our results highlight that the bacillus population structure is dominated by lineage 4 strains that harbour an outstanding genetic diversity, higher than in the rest of Africa as a whole. Coalescent analyses demonstrate that strains currently circulating in Liberia were introduced several times beginning in the early year 600 CE until very recently coinciding with migratory movements associated with the civil war and Ebola epidemics. A higher multidrug-resistant (MDR)-TB frequency (23.5 %) than current estimates was obtained together with non-catalogued drug-resistance mutations. Additionally, 39 % of strains were in genomic clusters revealing that ongoing transmission is a major contribution to the TB burden in the country. Our report emphasizes the importance of TB surveillance and control in African countries where bacillus diversity, MDR-TB prevalence and transmission are coalescing to jeopardize TB control programmes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1099/mgen.0.000325DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7067037PMC
January 2020

Characterization of Polymorphisms Associated with Multidrug-Resistant Tuberculosis by Whole Genomic Sequencing: A Preliminary Report from Mexico.

Microb Drug Resist 2020 Jul 23;26(7):732-740. Epub 2019 Dec 23.

Laboratorio de Farmacogenómica, Instituto Nacional de Medicina Genómica, Ciudad de México, México.

Whole genome sequencing (WGS) has been proposed as a tool for the diagnosis of drug resistance in tuberculosis (TB); however, there have been few studies on its effectiveness in countries with significantly high drug resistance rates. This study therefore aimed to evaluate the effectiveness of WGS to identify mutations related to drug resistance in TB isolates from an endemic region of Mexico. The results showed that, of 35 multidrug-resistant isolates analyzed, the values of congruence found between the phenotypic drug susceptibility testing and polymorphisms were 94% for isoniazid, 97% for rifampicin, 90% for ethambutol, and 82% for pyrazinamide. It was also possible to identify eight isolates as potential pre-extensive drug resistant (XDR) and one as XDR. Twenty nine isolates were classified within L4 and two transmission clusters were identified. The results show the potential utility of WGS for predicting resistance against first- and second-line drugs, as well as providing a phylogenetic characterization of TB drug-resistant isolates circulating in Mexico.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/mdr.2019.0054DOI Listing
July 2020

Influence of Gut Microbiota on Progression to Tuberculosis Generated by High Fat Diet-Induced Obesity in C3HeB/FeJ Mice.

Front Immunol 2019 18;10:2464. Epub 2019 Oct 18.

Experimental Tuberculosis Unit (UTE), Fundació Institut Germans Trias i Pujol (IGTP), Badalona, Spain.

The administration of a high fat content diet is an accelerating factor for metabolic syndrome, impaired glucose tolerance, and early type 2 diabetes. The present study aims to assess the impact of a high fat diet on tuberculosis progression and microbiota composition in an experimental animal model using a C3HeB/FeJ mouse strain submitted to single or multiple consecutive aerosol infections. These models allowed us to study the protection induced by Bacillus Calmette-Guérin vaccination as well as by the natural immunity induced by chemotherapy after a low dose infection. Our results show that a high fat diet is able to trigger a pro-inflammatory response, which results in a faster progression toward active tuberculosis and an impaired protective effect of BCG vaccination, which is not the case for natural immunity. This may be related to dysbiosis and a reduction in the Firmicutes/Bacteroidetes ratio in the gut microbiota caused by a decrease in the abundance of the Porphyromonadaceae family and, in particular, the genus. It should also be noted that a high fat diet is also related to an increase in the genera , and , which have previously been related to dysbiotic processes. As diabetes mellitus type 2 is a risk factor for developing tuberculosis, these findings may prove useful in the search for new prophylactic strategies for this population subset.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2019.02464DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6813253PMC
November 2020

High-resolution mapping of tuberculosis transmission: Whole genome sequencing and phylogenetic modelling of a cohort from Valencia Region, Spain.

PLoS Med 2019 10 31;16(10):e1002961. Epub 2019 Oct 31.

Subdirección General de Epidemiología y Vigilancia de la Salud, Dirección General de Salud Pública, Valencia, Spain.

Background: Whole genome sequencing provides better delineation of transmission clusters in Mycobacterium tuberculosis than traditional methods. However, its ability to reveal individual transmission links within clusters is limited. Here, we used a 2-step approach based on Bayesian transmission reconstruction to (1) identify likely index and missing cases, (2) determine risk factors associated with transmitters, and (3) estimate when transmission happened.

Methods And Findings: We developed our transmission reconstruction method using genomic and epidemiological data from a population-based study from Valencia Region, Spain. Tuberculosis (TB) incidence during the study period was 8.4 cases per 100,000 people. While the study is ongoing, the sampling frame for this work includes notified TB cases between 1 January 2014 and 31 December 2016. We identified a total of 21 transmission clusters that fulfilled the criteria for analysis. These contained a total of 117 individuals diagnosed with active TB (109 with epidemiological data). Demographic characteristics of the study population were as follows: 80/109 (73%) individuals were Spanish-born, 76/109 (70%) individuals were men, and the mean age was 42.51 years (SD 18.46). We found that 66/109 (61%) TB patients were sputum positive at diagnosis, and 10/109 (9%) were HIV positive. We used the data to reveal individual transmission links, and to identify index cases, missing cases, likely transmitters, and associated transmission risk factors. Our Bayesian inference approach suggests that at least 60% of index cases are likely misidentified by local public health. Our data also suggest that factors associated with likely transmitters are different to those of simply being in a transmission cluster, highlighting the importance of differentiating between these 2 phenomena. Our data suggest that type 2 diabetes mellitus is a risk factor associated with being a transmitter (odds ratio 0.19 [95% CI 0.02-1.10], p < 0.003). Finally, we used the most likely timing for transmission events to study when TB transmission occurred; we identified that 5/14 (35.7%) cases likely transmitted TB well before symptom onset, and these were largely sputum negative at diagnosis. Limited within-cluster diversity does not allow us to extrapolate our findings to the whole TB population in Valencia Region.

Conclusions: In this study, we found that index cases are often misidentified, with downstream consequences for epidemiological investigations because likely transmitters can be missed. Our findings regarding inferred transmission timing suggest that TB transmission can occur before patient symptom onset, suggesting also that TB transmits during sub-clinical disease. This result has direct implications for diagnosing TB and reducing transmission. Overall, we show that a transition to individual-based genomic epidemiology will likely close some of the knowledge gaps in TB transmission and may redirect efforts towards cost-effective contact investigations for improved TB control.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pmed.1002961DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6822721PMC
October 2019

Development and application of affordable SNP typing approaches to genotype Mycobacterium tuberculosis complex strains in low and high burden countries.

Sci Rep 2019 10 25;9(1):15343. Epub 2019 Oct 25.

Instituto de Biomedicina de Valencia (IBV-CSIC), Valencia, Spain.

The Mycobacterium tuberculosis complex (MTBC) comprises the species that causes tuberculosis (TB) which affects 10 million people every year. A robust classification of species, lineages, and sub-lineages is important to explore associations with drug resistance, epidemiological patterns or clinical outcomes. We present a rapid and easy-to-follow methodology to classify clinical TB samples into the main MTBC clades. Approaches are based on the identification of lineage and sub-lineage diagnostic SNP using a real-time PCR high resolution melting assay and classic Sanger sequencing from low-concentrated, low quality DNA. Thus, suitable for implementation in middle and low-income countries. Once we validated our molecular procedures, we characterized a total of 491 biological samples from human and cattle hosts, representing countries with different TB burden. Overall, we managed to genotype ~95% of all samples despite coming from unpurified and low-concentrated DNA. Our approach also allowed us to detect zoonotic cases in eight human samples from Nigeria. To conclude, the molecular techniques we have developed, are accurate, discriminative and reproducible. Furthermore, it costs less than other classic typing methods, resulting in an affordable alternative method in TB laboratories.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-019-51326-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6814786PMC
October 2019

Evolutionary Dissection of the Dot/Icm System Based on Comparative Genomics of 58 Legionella Species.

Genome Biol Evol 2019 09;11(9):2619-2632

Institut Pasteur, Departement of Microbiology, Biologie des Bactéries Intracellulaires, Paris, France.

The Dot/Icm type IVB secretion system of Legionella pneumophila is essential for its pathogenesis by delivering >300 effector proteins into the host cell. However, their precise secretion mechanism and which components interact with the host cell is only partly understood. Here, we undertook evolutionary analyses of the Dot/Icm system of 58 Legionella species to identify those components that interact with the host and/or the substrates. We show that high recombination rates are acting on DotA, DotG, and IcmX, supporting exposure of these proteins to the host. Specific amino acids under positive selection on the periplasmic region of DotF, and the cytoplasmic domain of DotM, support a role of these regions in substrate binding. Diversifying selection acting on the signal peptide of DotC suggests its interaction with the host after cleavage. Positive selection acts on IcmR, IcmQ, and DotL revealing that these components are probably participating in effector recognition and/or translocation. Furthermore, our results predict the participation in host/effector interaction of DotV and IcmF. In contrast, DotB, DotO, most of the core subcomplex elements, and the chaperones IcmS-W show a high degree of conservation and not signs of recombination or positive selection suggesting that these proteins are under strong structural constraints and have an important role in maintaining the architecture/function of the system. Thus, our analyses of recombination and positive selection acting on the Dot/Icm secretion system predicted specific Dot/Icm components and regions implicated in host interaction and/or substrate recognition and translocation, which will guide further functional analyses.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/gbe/evz186DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6761968PMC
September 2019

Genome-wide mutational biases fuel transcriptional diversity in the Mycobacterium tuberculosis complex.

Nat Commun 2019 09 5;10(1):3994. Epub 2019 Sep 5.

Instituto de Biomedicina de Valencia, IBV-CSIC, Valencia, Spain.

The Mycobacterium tuberculosis complex (MTBC) members display different host-specificities and virulence phenotypes. Here, we have performed a comprehensive RNAseq and methylome analysis of the main clades of the MTBC and discovered unique transcriptional profiles. The majority of genes differentially expressed between the clades encode proteins involved in host interaction and metabolic functions. A significant fraction of changes in gene expression can be explained by positive selection on single mutations that either create or disrupt transcriptional start sites (TSS). Furthermore, we show that clinical strains have different methyltransferases inactivated and thus different methylation patterns. Under the tested conditions, differential methylation has a minor direct role on transcriptomic differences between strains. However, disruption of a methyltransferase in one clinical strain revealed important expression differences suggesting indirect mechanisms of expression regulation. Our study demonstrates that variation in transcriptional profiles are mainly due to TSS mutations and have likely evolved due to differences in host characteristics.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-019-11948-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6728331PMC
September 2019

Whole genome sequencing of Mycobacterium tuberculosis: current standards and open issues.

Nat Rev Microbiol 2019 09;17(9):533-545

Foundation for Innovative New Diagnostics, Geneva, Switzerland.

Whole genome sequencing (WGS) of Mycobacterium tuberculosis has rapidly progressed from a research tool to a clinical application for the diagnosis and management of tuberculosis and in public health surveillance. This development has been facilitated by drastic drops in cost, advances in technology and concerted efforts to translate sequencing data into actionable information. There is, however, a risk that, in the absence of a consensus and international standards, the widespread use of WGS technology may result in data and processes that lack harmonization, comparability and validation. In this Review, we outline the current landscape of WGS pipelines and applications, and set out best practices for M. tuberculosis WGS, including standards for bioinformatics pipelines, curated repositories of resistance-causing variants, phylogenetic analyses, quality control and standardized reporting.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41579-019-0214-5DOI Listing
September 2019

Whole genomic sequencing as a tool for diagnosis of drug and multidrug-resistance tuberculosis in an endemic region in Mexico.

PLoS One 2019 5;14(6):e0213046. Epub 2019 Jun 5.

Instituto de Salud Pública, Universidad Veracruzana, Veracruz, México.

Background: Whole genome sequencing (WGS) has been proposed as a tool for diagnosing drug resistance in tuberculosis. However, reports of its effectiveness in endemic countries with important numbers of drug resistance are scarce. The goal of this study was to evaluate the effectiveness of this procedure in isolates from a tuberculosis endemic region in Mexico.

Methods: WGS analysis was performed in 81 tuberculosis positive clinical isolates with a known phenotypic profile of resistance against first-line drugs (isoniazid, rifampin, ethambutol, pyrazinamide and streptomycin). Mutations related to drug resistance were identified for each isolate; drug resistant genotypes were predicted and compared with the phenotypic profile. Genotypes and transmission clusters based on genetic distances were also characterized.

Findings: Prediction by WGS analysis of resistance against isoniazid, rifampicin, ethambutol, pyrazinamide and streptomycin showed sensitivity values of 84%, 96%, 71%, 75% and 29%, while specificity values were 100%, 94%, 90%, 90% and 98%, respectively. Prediction of multidrug resistance showed a sensitivity of 89% and specificity of 97%. Moreover, WGS analysis revealed polymorphisms related to second-line drug resistance, enabling classification of eight and two clinical isolates as pre- and extreme drug-resistant cases, respectively. Lastly, four lineages were identified in the population (L1, L2, L3 and L4). The most frequent of these was L4, which included 90% (77) of the isolates. Six transmission clusters were identified; the most frequent was TC6, which included 13 isolates with a L4.1.1 and a predominantly multidrug-resistant condition.

Conclusions: The results illustrate the utility of WGS for establishing the potential for prediction of resistance against first and second line drugs in isolates of tuberculosis from the region. They also demonstrate the feasibility of this procedure for use as a tool to support the epidemiological surveillance of drug- and multidrug-resistant tuberculosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0213046PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6550372PMC
February 2020

Cryptic Resistance Mutations Associated With Misdiagnoses of Multidrug-Resistant Tuberculosis.

J Infect Dis 2019 06;220(2):316-320

Instituto de Biomedicina de Valencia-Consejo Superior de Investigaciones Científicas.

Understanding why some multidrug-resistant tuberculosis cases are not detected by rapid phenotypic and genotypic routine clinical tests is essential to improve diagnostic assays and advance toward personalized tuberculosis treatment. Here, we combine whole-genome sequencing with single-colony phenotyping to identify a multidrug-resistant strain that had infected a patient for 9 years. Our investigation revealed the failure of rapid testing and genome-based prediction tools to identify the multidrug-resistant strain. The false-negative findings were caused by uncommon rifampicin and isoniazid resistance mutations. Although whole-genome sequencing data helped to personalize treatment, the patient developed extensively drug-resistant tuberculosis, highlighting the importance of coupling new diagnostic methods with appropriate treatment regimens.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/infdis/jiz104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6581888PMC
June 2019

Large genomics datasets shed light on the evolution of the Mycobacterium tuberculosis complex.

Infect Genet Evol 2019 08 26;72:10-15. Epub 2019 Feb 26.

CIBER en Epidemiología y Salud Pública, Valencia, Spain; Instituto de Biomedicina de Valencia, IBV-CSIC, Valencia, Spain. Electronic address:

Two strains of Mycobacterium tuberculosis complex can be separated as much as 2500 single nucleotide differences (Coscolla and Gagneux, 2014). In that limited amount of diversity, we find an astonishing range of clinical, epidemiological and biological phenotypes. The most striking is the strong host preferences depending on the infecting strain while more subtle differences can be found looking at different human tuberculosis isolates. Those subtle differences are the most difficult to spot given that analysis methods for so little diversity are limited and phenotypes like virulence are difficult to define and measure. Recent genomics advances allow to study the pathogen diversity at a resolution not available before from comparative species level, to global diversity to transmission in local settings. Here, we will review some of these recent results to highlight how population genomics approaches can aid not only to understand how MTBC evolved but also to identify relevant biomedical targets.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.meegid.2019.02.028DOI Listing
August 2019

The Future of TB Resistance Diagnosis: The Essentials on Whole Genome Sequencing and Rapid Testing Methods.

Arch Bronconeumol 2019 08 20;55(8):421-426. Epub 2019 Feb 20.

Biomedicine Institute of Valencia (CSIC), Valencia, Spain. Electronic address:

Tuberculosis resistance diagnostics have vastly improved in recent years thanks to the development of standardised phenotypic and molecular testing methods. However, these methods are either slow or limited in the number of resistant genotypes they can detect. With the advent of next-generation sequencing (NGS) we can sidestep all those problems, as we can sequence whole tuberculosis genomes at increasingly smaller costs and requiring less and less DNA. In this review, we explain how accumulated knowledge in the field has allowed us to go from phenotypic testing to molecular methods to Whole Genome Sequencing (WGS) for resistance diagnostics. We compare current diagnostic methods with WGS as to their efficacy in detecting resistant cases, and show how forthcoming advances in NGS technologies will be crucial in widespread implementation of WGS as a diagnostic tool.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.arbres.2019.01.002DOI Listing
August 2019

Simplified Model to Survey Tuberculosis Transmission in Countries Without Systematic Molecular Epidemiology Programs.

Emerg Infect Dis 2019 03;25(3):507-514

Systematic molecular/genomic epidemiology studies for tuberculosis surveillance cannot be implemented in many countries. We selected Panama as a model for an alternative strategy. Mycobacterial interspersed repetitive unit-variable-number tandem-repeat (MIRU-VNTR) analysis revealed a high proportion (50%) of Mycobacterium tuberculosis isolates included in 6 clusters (A-F) in 2 provinces (Panama and Colon). Cluster A corresponded to the Beijing sublineage. Whole-genome sequencing (WGS) differentiated clusters due to active recent transmission, with low single-nucleotide polymorphism-based diversity (cluster C), from clusters involving long-term prevalent strains with higher diversity (clusters A, B). Prospective application in Panama of 3 tailored strain-specific PCRs targeting marker single-nucleotide polymorphisms identified from WGS data revealed that 31.4% of incident cases involved strains A-C and that the Beijing strain was highly represented and restricted mainly to Colon. Rational integration of MIRU-VNTR, WGS, and tailored strain-specific PCRs could be a new model for tuberculosis surveillance in countries without molecular/genomic epidemiology programs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3201/eid2503.181593DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6390753PMC
March 2019

Whole genome sequencing-based analysis of tuberculosis (TB) in migrants: rapid tools for cross-border surveillance and to distinguish between recent transmission in the host country and new importations.

Euro Surveill 2019 Jan;24(4)

CIBER Enfermedades respiratorias (CIBERES), Spain.

BackgroundThe analysis of transmission of tuberculosis (TB) is challenging in areas with a large migrant population. Standard genotyping may fail to differentiate transmission within the host country from new importations, which is key from an epidemiological perspective.AimTo propose a new strategy to simplify and optimise cross-border surveillance of tuberculosis and to distinguish between recent transmission in the host country and new importationsMethodsWe selected 10 clusters, defined by 24-locus mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR), from a population in Spain rich in migrants from eastern Europe, north Africa and west Africa and reanalysed 66 isolates by whole-genome sequencing (WGS). A multiplex-allele-specific PCR was designed to target strain-specific marker single nucleotide polymorphisms (SNPs), identified from WGS data, to optimise the surveillance of the most complex cluster.ResultsIn five of 10 clusters not all isolates showed the short genetic distances expected for recent transmission and revealed a higher number of SNPs, thus suggesting independent importations of prevalent strains in the country of origin. In the most complex cluster, rich in Moroccan cases, a multiplex allele-specific oligonucleotide-PCR (ASO-PCR) targeting the marker SNPs for the transmission subcluster enabled us to prospectively identify new secondary cases. The ASO-PCR-based strategy was transferred and applied in Morocco, demonstrating that the strain was prevalent in the country.ConclusionWe provide a new model for optimising the analysis of cross-border surveillance of TB transmission in the scenario of global migration.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2807/1560-7917.ES.2019.24.4.1800005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6351995PMC
January 2019