RESUMO
Next generation sequencing is in the process of evolving from a technology used for research purposes to one which is applied in clinical diagnostics. Recently introduced high throughput and benchtop instruments offer fully automated sequencing runs at a lower cost per base and faster assay times. In turn, the complex and cumbersome library preparation, starting with isolated nucleic acids and resulting in amplified and barcoded DNA with sequencing adapters, has been identified as a significant bottleneck. Library preparation protocols usually consist of a multistep process and require costly reagents and substantial hands-on-time. Considerable emphasis will need to be placed on standardisation to ensure robustness and reproducibility. This review presents an overview of the current state of automation of library preparation for next generation sequencing. Major challenges associated with library preparation are outlined and different automation strategies are classified according to their functional principle. Pipetting workstations allow high-throughput processing yet offer limited flexibility, whereas microfluidic solutions offer great potential due to miniaturisation and decreased investment costs. For the emerging field of single cell transcriptomics for example, microfluidics enable singularisation of tens of thousands of cells in nanolitre droplets and barcoding of the RNA to assign each nucleic acid sequence to its cell of origin. Finally, two applications, the characterisation of bacterial pathogens and the sequencing within human immunogenetics, are outlined and benefits of automation are discussed.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , RNA , Automação , Biblioteca Gênica , Humanos , Reprodutibilidade dos TestesRESUMO
Mycobacterial infection-related morbidity and mortality in patients following cardiopulmonary bypass surgery is high and there is a growing need for a consensus-based expert opinion to provide international guidance for diagnosing, preventing and treating in these patients. In this document the International Society for Cardiovascular Infectious Diseases (ISCVID) covers aspects of prevention (field of hospital epidemiology), clinical management (infectious disease specialists, cardiac surgeons, ophthalmologists, others), laboratory diagnostics (microbiologists, molecular diagnostics), device management (perfusionists, cardiac surgeons) and public health aspects.
Assuntos
Infecção Hospitalar , Infecções por Mycobacterium não Tuberculosas , Mycobacterium , Antibacterianos/uso terapêutico , Procedimentos Cirúrgicos Cardíacos/efeitos adversos , Procedimentos Cirúrgicos Cardíacos/métodos , Cardiologia , Ponte Cardiopulmonar , Doenças Transmissíveis , Infecção Hospitalar/diagnóstico , Infecção Hospitalar/tratamento farmacológico , Infecção Hospitalar/microbiologia , Infecção Hospitalar/prevenção & controle , Contaminação de Equipamentos , Humanos , Mycobacterium/isolamento & purificação , Infecções por Mycobacterium não Tuberculosas/diagnóstico , Infecções por Mycobacterium não Tuberculosas/tratamento farmacológico , Infecções por Mycobacterium não Tuberculosas/prevenção & controle , Fatores de Risco , Sociedades Médicas , Reino UnidoRESUMO
Assuntos
Antituberculosos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Clofazimina , Genótipo , Humanos , Testes de Sensibilidade Microbiana , Mutação , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Tuberculose Resistente a Múltiplos Medicamentos/microbiologiaRESUMO
Mycobacterium chimaera is involved in a worldwide alert due to contaminated heater-cooler units. A real-time polymerase chain reaction (RT-PCR)-based procedure was implemented to survey undetected cases of M. chimaera infection. PCR was negative in the 59 prosthetic heart valves from patients with PCR-16SrRNA-negative infective endocarditis. PCR identified M. chimaera in one of 15 clinically significant retrospective Mycobacterium avium-Mycobacterium intracellulare complex isolates, which corresponded to a patient who had undergone heart valve replacement in a different institution. Whole-genome sequencing demonstrated that he was the first case in Spain with involvement of the strain responsible for the global outbreak. These results highlight the relevance of retrospective tracking for undetected M. chimaera infections.
Assuntos
Infecções por Mycobacterium não Tuberculosas/diagnóstico , Micobactérias não Tuberculosas/isolamento & purificação , Infecções Relacionadas à Prótese/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real , Idoso , Animais , Próteses Valvulares Cardíacas/efeitos adversos , Humanos , Masculino , Infecções por Mycobacterium não Tuberculosas/microbiologia , Micobactérias não Tuberculosas/genética , Infecções Relacionadas à Prótese/microbiologia , Estudos Retrospectivos , Espanha/epidemiologia , Sequenciamento Completo do GenomaRESUMO
According to the World Health Organization (WHO), tuberculosis is the leading cause of death attributed to a single microbial pathogen worldwide. In addition to the large number of patients affected by tuberculosis, the emergence of Mycobacterium tuberculosis drug-resistance is complicating tuberculosis control in many high-burden countries. During the past 5 years, the global number of patients identified with multidrug-resistant tuberculosis (MDR-TB), defined as bacillary resistance at least against rifampicin and isoniazid, the two most active drugs in a treatment regimen, has increased by more than 20% annually. Today we experience a historical peak in the number of patients affected by MDR-TB. The management of MDR-TB is characterized by delayed diagnosis, uncertainty of the extent of bacillary drug-resistance, imprecise standardized drug regimens and dosages, very long duration of therapy and high frequency of adverse events which all translate into a poor prognosis for many of the affected patients. Major scientific and technological advances in recent years provide new perspectives through treatment regimens tailor-made to individual needs. Where available, such personalized treatment has major implications on the treatment outcomes of patients with MDR-TB. The challenge now is to bring these adances to those patients that need them most.
RESUMO
Whole genome sequencing (WGS) can help to relate Mycobacterium tuberculosis genomes to one another to assess genetic relatedness and infer the likelihood of transmission between cases. The same sequence data are now increasingly being used to predict drug resistance and susceptibility. Controlling the spread of tuberculosis and providing patients with the correct treatment are central to the World Health Organization's target to 'End TB' by 2035, for which the global prevalence of drug-resistant tuberculosis remains one of the main obstacles to success. So far, WGS has been applied largely to drug-susceptible strains for the purposes of understanding transmission, leaving a number of analytical considerations before transferring what has been learnt from drug-susceptible disease to drug-resistant tuberculosis. We discuss these potential problems here, alongside some of the challenges to characterizing the Mycobacterium tuberculosis 'resistome'-the optimal knowledge-base required for WGS-based assays to successfully direct individualized treatment regimens through the prediction of drug resistance and susceptibility in the future.
Assuntos
Técnicas de Tipagem Bacteriana/métodos , Testes de Sensibilidade Microbiana/métodos , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/efeitos dos fármacos , Análise de Sequência de DNA/métodos , Tuberculose Resistente a Múltiplos Medicamentos/epidemiologia , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Genoma Bacteriano , Genótipo , Humanos , Epidemiologia Molecular/métodos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificaçãoRESUMO
In East Greenland, a dramatic increase of tuberculosis (TB) incidence has been observed in recent years. Classical genotyping suggests a genetically similar Mycobacterium tuberculosis (Mtb) strain population as cause, however, precise transmission patterns are unclear. We performed whole genome sequencing (WGS) of Mtb isolates from 98% of culture-positive TB cases through 21 years (n = 182) which revealed four genomic clusters of the Euro-American lineage (mainly sub-lineage 4.8 (n = 134)). The time to the most recent common ancestor of lineage 4.8 strains was found to be 100 years. This sub-lineage further diversified in the 1970s, and massively expanded in the 1990s, a period of lowered TB awareness in Greenland. Despite the low genetic strain diversity, WGS data revealed several recent short-term transmission events in line with the increasing incidence in the region. Thus, the isolated setting and the uniformity of circulating Mtb strains indicated that the majority of East Greenlandic TB cases originated from one or few strains introduced within the last century. Thereby, the study shows the consequences of even short interruptions in TB control efforts in previously TB high incidence areas and demonstrates the potential role of WGS in detecting ongoing micro epidemics, thus guiding public health efforts in the future.
Assuntos
Mycobacterium tuberculosis/genética , Tuberculose Pulmonar/transmissão , Adolescente , Adulto , Criança , Feminino , Genótipo , Groenlândia/epidemiologia , Humanos , Incidência , Masculino , Tipagem Molecular , Estudos Retrospectivos , Tuberculose Pulmonar/epidemiologia , Tuberculose Pulmonar/microbiologia , Sequenciamento Completo do Genoma , Adulto JovemRESUMO
Culturing before DNA extraction represents a major time-consuming step in whole-genome sequencing of slow-growing bacteria, such as Mycobacterium tuberculosis. We report a workflow to extract DNA from frozen isolates without reculturing. Prepared libraries and sequence data were comparable with results from recultured aliquots of the same stocks.