Analysis of hypervariable DNA sequences by NGS technologies: QuasiFlow

The development of Next Generation Sequencing (NGS) technologies has allowed deep characterization of highly variable sequences such as viral or mitochondrial genomes. With respect to RNA and ssDNA viruses, their low replication fidelity generates viral populations consisting of complex mutant spectra termed viral quasispecies. Their study is of special interest as they can be considered a phenotypic reservoir1. Similarly, heteroplasmy of human mitochondrial genomes, in which different sequences are found within a single individual, might have important clinical consequences. For the analysis of the mutant spectrum of such hypervariable sequences from NGS data, we have developed QuasiFlow, a workflow designed in AutoFlow2 that uses Illumina reads. QuasiFlow provides information about DNA variability, such as SNPs, indels and recombination events (Figure 1). Furthermore, it allows haplotype reconstruction of viral quasispecies and characterization of its diversity through normalized Shannon index, nucleotide diversity and mutation networks. Quasiflow performs also a comparative study among samples, based on correlation, ANOVA and PCA analysis, in order to determine which parameters are affected by the experiment and how the samples behave according to their biological origin. In this work, we have applied QuasiFlow to analyze the population structure of the begomovirus Tomato yellow leaf curl virus (TYLCV) infectious clone inoculated in Arabidopsis thaliana plants, using HiSeq or MiSeq reads. Their analysis allowed detection of minor quasispecies variants with a frequency of 10-4 to 10-5 and reconstructed the haplotypes present in the sample. In addition, QuasiFlow was used to discover variants and recombinants in mixed infections of tomato plants. These results show the fast generation of recombinant genomes in geminivirus mixed infections and demonstrate the potential of QuasiFlow for the analysis of mutant spectra using Illumina MiSeq sequencing data. We have extended the use of QuasiFlow to the analysis of highly variable sequences such as the mitochondrial DNA. For that, we have analyzed DNA Illumina Miseq reads from 47 human mitochondrial samples from different cell lines obtained from the NCBI SRA database. Quasiflow generated automatically SNPs, SNP frequencies, indels and analyzed up to 23 variables using PCA analysis and performed an hierarchical clustering of the samples. Our analysis was able to detect pathological variants presented in a frequency lower than 1%.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. This research was funded by Junta de Andalucía and EU through the ERDF 2014-2020, Projects P10-CVI-6075 to M. G.C. and P10-CVI-6561 to A.G-P

Analysis of viral quasispecies by NGS technologies: quasiflow

We have developed QuasiFlow, a workflow designed in AutoFlow that takes advantage of NGS technologies to reconstruct quasispecies based in Illumina reads. QuasiFlow characterises and computes several key parameters, such as recombination events, SNPs, transitions, transversions, indels, quasispecies reconstruction, normalized Shannon index, nucleotide diversity and mutation networks. Moreover, it performs a comparative study of the samples comprising correlation, ANOVA and PCA analyses of the previously obtained virus population parameters. Using QuasiFlow we have analysed Illumina MiSeq reads from DNA samples obtained in mixed infections of ssDNA begomovirus in tomato plants amplified by rolling circle amplification. Further, we have extended the use of QuasiFlow to the analysis of the highly variable mitochondrial DNA. For that, we have used DNA Illumina MiSeq reads from 47 human mitochondrial samples from different cell lines obtained from the NCBI SRA databaseUniversidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Role of the Cys loop and transmembrane domain in the allosteric modulation of α4β2 nicotinic acetylcholine receptors

​Allosteric modulators of pentameric ligand gated ion channels (pLGICs) are thought to act on elements of the pathways that couple agonist binding to channel gating. Using α4β2 nicotinic acetylcholine receptors (nAChRs) and the α4β2-selective positive modulators 17β-estradiol (βEST) and desformylflustrabromine (dFBr), we have identified pathways that link the binding sites for these modulators to the Cys loop, a region that is critical for channel gating in all pLGICs. Previous studies have shown that the binding site for potentiating βEST is in the C-terminal (post-M4 region) of the α4 subunit. Here, using homology modelling in combination with mutagenesis and electrophysiology, we identified the binding site for potentiating dFBr on the top-half of a cavity between the third (M3) and fourth transmembrane (M4) α-helices of the α4 subunit. We found that the binding sites for βEST and dFBr communicate with the Cys loop, through interactions between the last residue of post-M4 and F170 of the conserved FPF sequence of the Cys loop, and that these interactions affect potentiating efficacy. In addition, interactions between a residue in M3 (Y309) and F167, a residue adjacent to the Cys loop FPF motif, also affect dFBr potentiating efficacy. Thus, the Cys loop acts as a key control element in the allosteric transduction pathway for potentiating βEST and dFBr. Overall, we propose that positive allosteric modulators that bind the M3-M4 cavity or post-M4 region increase the efficacy of channel gating through interactions with the Cys loop

Evaluation of the Performance of the IDvet IFN-Gamma Test for Diagnosis of Bovine Tuberculosis in Spain

In Spain, the national bovine tuberculosis (bTB) eradication program is based on yearly skin testing of every ≥6 weeks old animal using the single or comparative tuberculin test and parallel use of the interferon-gamma (IFN-γ) assay as an ancillary diagnostic test in infected herds. There are several versions of the latter. Recently, a new commercial IDvet IFN-γ assay has been authorized for use in the program, but there is limited scientific evidence about its performance in different epidemiological settings. Therefore, two studies to evaluate the performance of the IDvet assay were conducted. In study 1, a concordance analysis between the new IDvet and the Bovigam IFN-γ assay in use in Spain for over 10 years was conducted. In study 2, results from the IDvet assay when applied in tandem with a single intradermal tuberculin (SIT) test were used to evaluate the concordance between both tests and to estimate their sensitivity (Se) and specificity (Sp) using a Bayesian latent-class model. Field data from cattle herds located in Madrid and Castilla y Leon (Spain) were collected. For study 1, herd selection was based on a high expected prevalence of reactors to the IFN-γ assay, while herds were selected at random to estimate Se and Sp of the new IDvet assay in study 2. Agreement between the results obtained with both kits for IFN-γ assay was poor (Kappa = 0.20), and a receiver operating characteristic (ROC) analysis indicated a low Se of the new IDvet relative to the Bovigam in a heavily bTB infected population. The Bayesian latent-class analysis estimated the Se of the IDvet assay to be 36.7% [95% probability posterior interval (PPI) 14.7–78.8%] with estimated Sp close to 100% when the cut-off recommended by the manufacturer (35) was applied. At the alternative cut-off values of 16 and 4, the estimated Se of the IDvet assay increased to 49.0% (PPI: 24.8–94.1%) and 56.0% (PPI: 30.8–96.3%), respectively, while maintaining a high specificity. The results suggest that the new IDvet assay may have lower sensitivity than the Bovigam for diagnosis of bTB in cattle herds in Spain, and that adjusting its cut-off might be considered

Antimicrobial resistance among migrants in Europe: a systematic review and meta-analysis

BACKGROUND: Rates of antimicrobial resistance (AMR) are rising globally and there is concern that increased migration is contributing to the burden of antibiotic resistance in Europe. However, the effect of migration on the burden of AMR in Europe has not yet been comprehensively examined. Therefore, we did a systematic review and meta-analysis to identify and synthesise data for AMR carriage or infection in migrants to Europe to examine differences in patterns of AMR across migrant groups and in different settings. METHODS: For this systematic review and meta-analysis, we searched MEDLINE, Embase, PubMed, and Scopus with no language restrictions from Jan 1, 2000, to Jan 18, 2017, for primary data from observational studies reporting antibacterial resistance in common bacterial pathogens among migrants to 21 European Union-15 and European Economic Area countries. To be eligible for inclusion, studies had to report data on carriage or infection with laboratory-confirmed antibiotic-resistant organisms in migrant populations. We extracted data from eligible studies and assessed quality using piloted, standardised forms. We did not examine drug resistance in tuberculosis and excluded articles solely reporting on this parameter. We also excluded articles in which migrant status was determined by ethnicity, country of birth of participants' parents, or was not defined, and articles in which data were not disaggregated by migrant status. Outcomes were carriage of or infection with antibiotic-resistant organisms. We used random-effects models to calculate the pooled prevalence of each outcome. The study protocol is registered with PROSPERO, number CRD42016043681. FINDINGS: We identified 2274 articles, of which 23 observational studies reporting on antibiotic resistance in 2319 migrants were included. The pooled prevalence of any AMR carriage or AMR infection in migrants was 25·4% (95% CI 19·1-31·8; I2 =98%), including meticillin-resistant Staphylococcus aureus (7·8%, 4·8-10·7; I2 =92%) and antibiotic-resistant Gram-negative bacteria (27·2%, 17·6-36·8; I2 =94%). The pooled prevalence of any AMR carriage or infection was higher in refugees and asylum seekers (33·0%, 18·3-47·6; I2 =98%) than in other migrant groups (6·6%, 1·8-11·3; I2 =92%). The pooled prevalence of antibiotic-resistant organisms was slightly higher in high-migrant community settings (33·1%, 11·1-55·1; I2 =96%) than in migrants in hospitals (24·3%, 16·1-32·6; I2 =98%). We did not find evidence of high rates of transmission of AMR from migrant to host populations. INTERPRETATION: Migrants are exposed to conditions favouring the emergence of drug resistance during transit and in host countries in Europe. Increased antibiotic resistance among refugees and asylum seekers and in high-migrant community settings (such as refugee camps and detention facilities) highlights the need for improved living conditions, access to health care, and initiatives to facilitate detection of and appropriate high-quality treatment for antibiotic-resistant infections during transit and in host countries. Protocols for the prevention and control of infection and for antibiotic surveillance need to be integrated in all aspects of health care, which should be accessible for all migrant groups, and should target determinants of AMR before, during, and after migration. FUNDING: UK National Institute for Health Research Imperial Biomedical Research Centre, Imperial College Healthcare Charity, the Wellcome Trust, and UK National Institute for Health Research Health Protection Research Unit in Healthcare-associated Infections and Antimictobial Resistance at Imperial College London

Sporangiospore Size Dimorphism Is Linked to Virulence of Mucor circinelloides

Mucor circinelloides is a zygomycete fungus and an emerging opportunistic pathogen in immunocompromised patients, especially transplant recipients and in some cases otherwise healthy individuals. We have discovered a novel example of size dimorphism linked to virulence. M. circinelloides is a heterothallic fungus: (+) sex allele encodes SexP and (−) sex allele SexM, both of which are HMG domain protein sex determinants. M. circinelloides f. lusitanicus (Mcl) (−) mating type isolates produce larger asexual sporangiospores that are more virulent in the wax moth host compared to (+) isolates that produce smaller less virulent sporangiospores. The larger sporangiospores germinate inside and lyse macrophages, whereas the smaller sporangiospores do not. sexMΔ mutants are sterile and still produce larger virulent sporangiospores, suggesting that either the sex locus is not involved in virulence/spore size or the sexP allele plays an inhibitory role. Phylogenetic analysis supports that at least three extant subspecies populate the M. circinelloides complex in nature: Mcl, M. circinelloides f. griseocyanus, and M. circinelloides f. circinelloides (Mcc). Mcc was found to be more prevalent among clinical Mucor isolates, and more virulent than Mcl in a diabetic murine model in contrast to the wax moth host. The M. circinelloides sex locus encodes an HMG domain protein (SexP for plus and SexM for minus mating types) flanked by genes encoding triose phosphate transporter (TPT) and RNA helicase homologs. The borders of the sex locus between the three subspecies differ: the Mcg sex locus includes the promoters of both the TPT and the RNA helicase genes, whereas the Mcl and Mcc sex locus includes only the TPT gene promoter. Mating between subspecies was restricted compared to mating within subspecies. These findings demonstrate that spore size dimorphism is linked to virulence of M. circinelloides species and that plasticity of the sex locus and adaptations in pathogenicity have occurred during speciation of the M. circinelloides complex

Modifying effect of dual antiplatelet therapy on incidence of stent thrombosis according to implanted drug-eluting stent type

Aim To investigate the putative modifying effect of dual antiplatelet therapy (DAPT) use on the incidence of stent thrombosis at 3 years in patients randomized to Endeavor zotarolimus-eluting stent (E-ZES) or Cypher sirolimus-eluting stent (C-SES). Methods and results Of 8709 patients in PROTECT, 4357 were randomized to E-ZES and 4352 to C-SES. Aspirin was to be given indefinitely, and clopidogrel/ticlopidine for ≥3 months or up to 12 months after implantation. Main outcome measures were definite or probable stent thrombosis at 3 years. Multivariable Cox regression analysis was applied, with stent type, DAPT, and their interaction as the main outcome determinants. Dual antiplatelet therapy adherence remained the same in the E-ZES and C-SES groups (79.6% at 1 year, 32.8% at 2 years, and 21.6% at 3 years). We observed a statistically significant (P = 0.0052) heterogeneity in treatment effect of stent type in relation to DAPT. In the absence of DAPT, stent thrombosis was lower with E-ZES vs. C-SES (adjusted hazard ratio 0.38, 95% confidence interval 0.19, 0.75; P = 0.0056). In the presence of DAPT, no difference was found (1.18; 0.79, 1.77; P = 0.43). Conclusion A strong interaction was observed between drug-eluting stent type and DAPT use, most likely prompted by the vascular healing response induced by the implanted DES system. These results suggest that the incidence of stent thrombosis in DES trials should not be evaluated independently of DAPT use, and the optimal duration of DAPT will likely depend upon stent type (Clinicaltrials.gov number NCT00476957

A pan-European epidemiological study reveals honey bee colony survival depends on beekeeper education and disease control

Reports of honey bee population decline has spurred many national efforts to understand the extent of the problem and to identify causative or associated factors. However, our collective understanding of the factors has been hampered by a lack of joined up trans-national effort. Moreover, the impacts of beekeeper knowledge and beekeeping management practices have often been overlooked, despite honey bees being a managed pollinator. Here, we established a standardised active monitoring network for 5 798 apiaries over two consecutive years to quantify honey bee colony mortality across 17 European countries. Our data demonstrate that overwinter losses ranged between 2% and 32%, and that high summer losses were likely to follow high winter losses. Multivariate Poisson regression models revealed that hobbyist beekeepers with small apiaries and little experience in beekeeping had double the winter mortality rate when compared to professional beekeepers. Furthermore, honey bees kept by professional beekeepers never showed signs of disease, unlike apiaries from hobbyist beekeepers that had symptoms of bacterial infection and heavy Varroa infestation. Our data highlight beekeeper background and apicultural practices as major drivers of honey bee colony losses. The benefits of conducting trans-national monitoring schemes and improving beekeeper training are discussed

Surgical site infection after gastrointestinal surgery in high-income, middle-income, and low-income countries: a prospective, international, multicentre cohort study

Background: Surgical site infection (SSI) is one of the most common infections associated with health care, but its importance as a global health priority is not fully understood. We quantified the burden of SSI after gastrointestinal surgery in countries in all parts of the world. Methods: This international, prospective, multicentre cohort study included consecutive patients undergoing elective or emergency gastrointestinal resection within 2-week time periods at any health-care facility in any country. Countries with participating centres were stratified into high-income, middle-income, and low-income groups according to the UN's Human Development Index (HDI). Data variables from the GlobalSurg 1 study and other studies that have been found to affect the likelihood of SSI were entered into risk adjustment models. The primary outcome measure was the 30-day SSI incidence (defined by US Centers for Disease Control and Prevention criteria for superficial and deep incisional SSI). Relationships with explanatory variables were examined using Bayesian multilevel logistic regression models. This trial is registered with ClinicalTrials.gov, number NCT02662231. Findings: Between Jan 4, 2016, and July 31, 2016, 13 265 records were submitted for analysis. 12 539 patients from 343 hospitals in 66 countries were included. 7339 (58·5%) patient were from high-HDI countries (193 hospitals in 30 countries), 3918 (31·2%) patients were from middle-HDI countries (82 hospitals in 18 countries), and 1282 (10·2%) patients were from low-HDI countries (68 hospitals in 18 countries). In total, 1538 (12·3%) patients had SSI within 30 days of surgery. The incidence of SSI varied between countries with high (691 [9·4%] of 7339 patients), middle (549 [14·0%] of 3918 patients), and low (298 [23·2%] of 1282) HDI (p < 0·001). The highest SSI incidence in each HDI group was after dirty surgery (102 [17·8%] of 574 patients in high-HDI countries; 74 [31·4%] of 236 patients in middle-HDI countries; 72 [39·8%] of 181 patients in low-HDI countries). Following risk factor adjustment, patients in low-HDI countries were at greatest risk of SSI (adjusted odds ratio 1·60, 95% credible interval 1·05–2·37; p=0·030). 132 (21·6%) of 610 patients with an SSI and a microbiology culture result had an infection that was resistant to the prophylactic antibiotic used. Resistant infections were detected in 49 (16·6%) of 295 patients in high-HDI countries, in 37 (19·8%) of 187 patients in middle-HDI countries, and in 46 (35·9%) of 128 patients in low-HDI countries (p < 0·001). Interpretation: Countries with a low HDI carry a disproportionately greater burden of SSI than countries with a middle or high HDI and might have higher rates of antibiotic resistance. In view of WHO recommendations on SSI prevention that highlight the absence of high-quality interventional research, urgent, pragmatic, randomised trials based in LMICs are needed to assess measures aiming to reduce this preventable complication