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In this retrospective study, whole-genome sequencing (WGS) data generated on an Ion Torrent platform was used to predict phenotypic drug resistance profiles for first- and second-line drugs among Swedish clinical Mycobacterium tuberculosis isolates from 2016 to 2018. The accuracy was â¼99% for all first-line drugs and 100% for four second-line drugs. Our analysis supports the introduction of WGS into routine diagnostics, which might, at least in Sweden, replace phenotypic drug susceptibility testing in the future.
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Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla/genética , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Tuberculose/microbiologia , Sequenciamento Completo do Genoma , Humanos , Suécia , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológicoRESUMO
BACKGROUND: Pertussis (whooping cough) remains a public health problem despite extensive vaccination strategies. Better understanding of the host-pathogen interaction and the detailed B. pertussis (Bp) target recognition pattern will help in guided vaccine design. We characterized the specific epitope antigen recognition profiles of serum antibodies ('the reactome') induced by whooping cough and B. pertussis (Bp) vaccines from a case-control study conducted in 1996 in infants enrolled in a Bp vaccine trial in Sweden (Gustafsson, NEJM, 1996, 334, 349-355). METHODS: Sera from children with whooping cough, vaccinated with Diphtheria Tetanus Pertussis (DTP) whole-cell (wc), acellular 5 (DPTa5), or with the 2 component (a2) vaccines and from infants receiving only DT (n=10 for each group) were tested with high-content peptide microarrays containing 17 Bp proteins displayed as linear (n=3175) peptide stretches. Slides were incubated with serum and peptide-IgG complexes detected with Cy5-labeled goat anti-human IgG and analyzed using a GenePix 4000B microarray scanner, followed by statistical analysis, using PAM (Prediction Analysis for Microarrays) and the identification of uniquely recognized peptide epitopes. RESULTS: 367/3,085 (11.9%) peptides were recognized in 10/10 sera from children with whooping cough, 239 (7.7%) in DTPwc, 259 (8.4%) in DTPa5, 105 (3.4%) DTPa2, 179 (5.8%) in the DT groups. Recognition of strongly recognized peptides was similar between whooping cough and DPTwc, but statistically different between whooping cough vs. DTPa5 (p<0.05), DTPa2 and DT (p<0.001 vs. both) vaccines. 6/3,085 and 2/3,085 peptides were exclusively recognized in (10/10) sera from children with whooping cough and DTPa2 vaccination, respectively. DTPwc resembles more closely the whooping cough reactome as compared to acellular vaccines. CONCLUSION: We could identify a unique recognition signature common for each vaccination group (10/10 children). Peptide microarray technology allows detection of subtle differences in epitope signature responses and may help to guide rational vaccine development by the objective description of a clinically relevant immune response that confers protection against infectious pathogens.
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Anticorpos Antibacterianos/sangue , Bordetella pertussis/imunologia , Reconhecimento Automatizado de Padrão , Vacina contra Coqueluche/imunologia , Análise Serial de Proteínas/métodos , Coqueluche/sangue , Coqueluche/imunologia , Sequência de Aminoácidos , Criança , Humanos , Imunoglobulina G/sangue , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/imunologia , Proteoma/metabolismo , VacinaçãoRESUMO
Four molluscum contagiosum virus (MOCV) genotypes (MOCV1-4) and four subtype variants (MOCV1p, MOCV1va, MOCV1vb, and MOCV1vc) were partially characterized using restriction enzyme profiling in the early 1980s/1990s. However, complete genome sequences of only MOCV1 and MOCV2 are available. The evolutionary pathways of MOCV genotypes and subtype variants with unavailable sequences remain unclear, and also whether all MOCV genotypes/subtype variants can be reliably detected and appropriately categorized using available PCR-based protocols. We de novo fully characterized and functionally annotated 47 complete MOCV genomes, including two putative non-MOCV1/2 isolates, expanding the number of fully characterized MOCV genomes to 66. To ascertain the placement of any putative novel MOCV sequence into the restriction profiling typing scheme, we developed an original framework for extracting complete MOCV genome sequence-based restriction profiles and matching them with reference restriction profiles. We confirmed that two putative non-MOCV1/2 isolates represent the first complete genomes of MOCV3. Comprehensive phylogenomic, recombination, and restriction enzyme recognition site analysis of all 66 currently available MOCV genomes showed that they can be agglomerated into six phylogenetic subgroups (PG1-6), corresponding to the subtype variants from the pioneering studies. PG5 was a novel subtype variant of MOCV2, but no PGs corresponded to the subtype variants MOCV1vb or MOCV4. We showed that the phylogenetic subgroups may have diverged from the prototype MOCV genotype lineages following large-scale recombination events and hinted at partial sequence content of MOCV4 and direction of recombinant transfer in the events that spawned PG5 and the yet undetected subtype variant MOCV1vb.IMPORTANCEFour molluscum contagiosum virus (MOCV) genotypes (MOCV1-4) and four subtype variants were partially characterized using restriction enzyme profiling in the 1980s/1990s, but complete genome sequences of only MOCV1 and MOCV2 are available. The evolutionary pathways whereby genotypes/subtype variants with unavailable sequences emerged and whether all MOCVs can be detected using current diagnostic approaches remain unclear. We fully characterized 47 novel complete MOCV genomes, including the first complete MOCV3 genome, expanding the number of fully characterized genomes to 66. For reliably classifying the novel non-MOCV1/2 genomes, we developed and validated a framework for matching sequence-derived restriction maps with those defining MOCV subtypes in pioneering studies. Six phylogenetic subgroups (PG1-6) were identified, PG5 representing a novel MOCV2 subtype. The phylogenetic subgroups diverged from the prototype lineages following large-scale recombination events and hinted at partial sequence content of MOCV4 and direction of recombinant transfer in the events spawning PG5 and yet undetected MOCV1vb variant.
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BACKGROUND: The kinetoplastid protozoan Leishmania tropica mainly causes cutaneous leishmaniasis in humans in the Middle East, and relapse or treatment failure after treatment are common in this area. L. tropica's digenic life cycle includes distinct stages in the vector sandfly and the mammalian host. Sexual reproduction and genetic exchange appear to occur more frequently than in other Leishmania species. Understanding these processes is complicated by chromosome instability during cell division that yields aneuploidy, recombination and heterozygosity. This combination of rare recombination and aneuploid permits may reveal signs of hypothetical parasexual mating, where diploid cells fuse to form a transient tetraploid that undergoes chromosomal recombination and gradual chromosomal loss. METHODOLOGY/PRINCIPAL FINDINGS: The genome-wide SNP diversity from 22 L. tropica isolates showed chromosome-specific runs of patchy heterozygosity and extensive chromosome copy number variation. All these isolates were collected during 2007-2017 in Sweden from patients infected in the Middle East and included isolates from a patient possessing two genetically distinct leishmaniasis infections three years apart with no evidence of re-infection. We found differing ancestries on the same chromosome (chr36) across multiple samples: matching the reference genome with few derived alleles, followed by blocks of heterozygous SNPs, and then by clusters of homozygous SNPs with specific recombination breakpoints at an inferred origin of replication. Other chromosomes had similar marked changes in heterozygosity at strand-switch regions separating polycistronic transcriptional units. CONCLUSION/SIGNIFICANCE: These large-scale intra- and inter-chromosomal changes in diversity driven by recombination and aneuploidy suggest multiple mechanisms of cell reproduction and diversification in L. tropica, including mitotic, meiotic and parasexual processes. It underpins the need for more genomic surveillance of Leishmania, to detect emerging hybrids that could spread more widely and to better understand the association between genetic variation and treatment outcome. Furthering our understanding of Leishmania genome evolution and ancestry will aid better diagnostics and treatment for cutaneous leishmaniasis caused by L.tropica in the Middle East.
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Genoma de Protozoário , Leishmania tropica/genética , Leishmaniose Cutânea/parasitologia , Afeganistão , Cromossomos/genética , Variações do Número de Cópias de DNA , DNA de Protozoário/genética , Variação Genética , Humanos , Irã (Geográfico) , Leishmania tropica/classificação , Leishmania tropica/isolamento & purificação , Filogenia , Polimorfismo de Nucleotídeo Único , Recombinação Genética , SíriaRESUMO
UNLABELLED: Helicobacter pylori chronically infects the gastric mucosa in more than half of the human population; in a subset of this population, its presence is associated with development of severe disease, such as gastric cancer. Genomic analysis of several strains has revealed an extensive H. pylori pan-genome, likely to grow as more genomes are sampled. Here we describe the draft genome sequence (63 contigs; 26× mean coverage) of H. pylori strain B45, isolated from a patient with gastric mucosa-associated lymphoid tissue (MALT) lymphoma. The major finding was a 24.6-kb prophage integrated in the bacterial genome. The prophage shares most of its genes (22/27) with prophage region II of Helicobacter acinonychis strain Sheeba. After UV treatment of liquid cultures, circular DNA carrying the prophage integrase gene could be detected, and intracellular tailed phage-like particles were observed in H. pylori cells by transmission electron microscopy, indicating that phage production can be induced from the prophage. PCR amplification and sequencing of the integrase gene from 341 H. pylori strains from different geographic regions revealed a high prevalence of the prophage (21.4%). Phylogenetic reconstruction showed four distinct clusters in the integrase gene, three of which tended to be specific for geographic regions. Our study implies that phages may play important roles in the ecology and evolution of H. pylori. IMPORTANCE: Helicobacter pylori chronically infects the gastric mucosa in more than half of the human population, and while most of the infected individuals do not develop disease, H. pylori infection doubles the risk of developing gastric cancer. An abundance and diversity of viruses (phages) infect microbial populations in most environments and are important mediators of microbial diversity. Our finding of a 24.6-kb prophage integrated inside an H. pylori genome and the observation of circular integrase gene-containing DNA and phage-like particles inside cells upon UV treatment demonstrate that we have discovered a viable H. pylori phage. The additional finding of integrase genes in a large proportion of screened isolates of diverse geographic origins indicates that the prevalence of prophages may have been underestimated in H. pylori. Since phages are important drivers of microbial evolution, the discovery should be important for understanding and predicting genetic diversity in H. pylori.
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Bacteriófagos/genética , DNA Bacteriano/genética , DNA Viral/genética , Genoma Bacteriano , Helicobacter pylori/genética , Helicobacter pylori/virologia , Bacteriófagos/isolamento & purificação , Bacteriófagos/ultraestrutura , Análise por Conglomerados , Mucosa Gástrica/microbiologia , Infecções por Helicobacter/complicações , Infecções por Helicobacter/microbiologia , Helicobacter pylori/isolamento & purificação , Helicobacter pylori/efeitos da radiação , Humanos , Linfoma/complicações , Lisogenia , Dados de Sequência Molecular , Filogenia , Análise de Sequência de DNA , Homologia de Sequência , Raios UltravioletaRESUMO
INTRODUCTION: Peptide microarray slides usually contain positive control spots to gauge for antibody binding. Unlike the good response on earlier prototype microarrays, human immunoglobulin controls do not function consistently on newer generation slides. This may be due to technical problems in high-density printing or degradation. Our objective was to identify and print reliable control peptides that did not suffer from the same problems as proteins. METHODS: Peptide microarray slides containing 10,000-23,000 synthetic peptides spanning proteins involved in M. tuberculosis or Bordatella pertussis were incubated with secondary antibody in sample dilution buffer. After removal of artefacts due to slide architecture, we identified peptides that gave a high mean response and low co-efficient of variation across all replicates. These control peptides were tested for their performance on newly manufactured slides. RESULTS: We selected three peptides on the TB slides and three peptides on the B. pertussis slides that had a mean response index of at least 5 and a coefficient of variation less than 15%. When used as controls in newly-designed slides, these peptides gave consistently high responses: the median index ranged from 4.5 to 9.5 in the absence of patient serum and was of a somewhat lesser magnitude when incubated with patient serum. We illustrate the use of these control responses to normalize the peptide responses on a set of slides prepared with human serum. CONCLUSIONS: Our work shows that it is possible to identify control peptides that can be used when protein controls do not function consistently. This has important consequences for the storage of peptide-microarrays and their use in the field: protein chips need to be kept at +4 degrees C while peptide chips can be kept at room temperature. Although we focused on TB and B. pertussis, our methodology has relevance for any disease or disorder where peptide arrays are used to assess immune response.
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Anticorpos/metabolismo , Antígenos de Bactérias/metabolismo , Peptídeos/metabolismo , Análise Serial de Proteínas/métodos , Sequência de Aminoácidos , Anticorpos/imunologia , Antígenos de Bactérias/imunologia , Bordetella pertussis/imunologia , Humanos , Dados de Sequência Molecular , Mycobacterium tuberculosis/imunologia , Peptídeos/imunologia , Reprodutibilidade dos Testes , SoftwareRESUMO
Studies performed in several countries have demonstrated the recent emergence and subsequent dominance of circulating Bordetella pertussis strains harboring pertactin and pertussis toxin variants not included in pertussis vaccines. Determination of the pertactin gene variants is commonly performed using a time-consuming and expensive sequence analysis. We developed a simple and reliable pertactin typing algorithm suitable for large-scale screening. The assay correctly identified all pertactin alleles in representative strains. The typing of 231 clinical strains of B. pertussis routinely isolated in Belgium showed that this algorithm was adequate to identify less-frequent prn types like prn9 and prn11.