RESUMEN
Early detection of viral pathogens by DNA-sensors in clinical samples, contaminated foods, soil or water can dramatically improve clinical outcomes and reduce the socioeconomic impact of diseases such as COVID-19. Clustered regularly interspaced short palindromic repeat (CRISPR) and its associated protein Cas12a (previously known as CRISPR-Cpf1) technology is an innovative new-generation genomic engineering tool, also known as 'genetic scissors', that has demonstrated the accuracy and has recently been effectively applied as appropriate (E-CRISPR) DNA-sensor to detect the nucleic acid of interest. The CRISPR-Cas12a from Prevotella and Francisella 1 are guided by a short CRISPR RNA (gRNA). The unique simultaneous cis- and trans- DNA cleavage after target sequence recognition at the PAM site, sticky-end (5-7 bp) employment, and ssDNA/dsDNA hybrid cleavage strategies to manipulate the attractive nature of CRISPR-Cas12a are reviewed. DNA-sensors based on the CRISPR-Cas12a technology for rapid, robust, sensitive, inexpensive, and selective detection of virus DNA without additional sample purification, amplification, fluorescent-agent- and/or quencher-labeling are relevant and becoming increasingly important in industrial and medical applications. In addition, CRISPR-Cas12a system shows great potential in the field of E-CRISPR-based bioassay research technologies. Therefore, we are highlighting insights in this research direction.
Asunto(s)
Sistemas CRISPR-Cas/fisiología , ADN Viral/aislamiento & purificación , Técnicas de Amplificación de Ácido Nucleico , Animales , Técnicas Biosensibles/métodos , Técnicas Biosensibles/tendencias , COVID-19/virología , ADN Viral/análisis , Contaminantes Ambientales/análisis , Contaminantes Ambientales/aislamiento & purificación , Contaminación de Alimentos/análisis , Humanos , Tipificación Molecular/métodos , Tipificación Molecular/tendencias , Técnicas de Amplificación de Ácido Nucleico/métodos , Técnicas de Amplificación de Ácido Nucleico/tendencias , SARS-CoV-2/genética , Virología/métodos , Virología/tendencias , Virosis/clasificación , Virosis/diagnóstico , Virosis/virologíaRESUMEN
In recent years, approaches to tracking the spread of meticillin-resistant Staphylococcus aureus (MRSA) as part of outbreak management have used conventional DNA-based methods, including pulsed-field gel electrophoresis and spa typing. However, when a predominant clone is present, these methods may be insufficiently discriminatory. A literature search was undertaken to highlight how whole-genome sequencing (WGS) has revolutionized the investigation of outbreaks of MRSA, including intrahospital spread and MRSA in the community, and to review its future potential. WGS provides enhanced isolate discrimination, as it permits the entire genomic DNA sequence of isolates to be determined and compared rapidly. Software packages used for the analysis of WGS data are becoming increasingly available. To date, WGS has been more sensitive in confirming outbreaks, often persisting for prolonged periods, previously undetected by conventional molecular typing. The evolving dynamic of spread from the community to hospitals, within and between hospitals, and from hospitals to the community is only becoming clear with WGS studies, and is more complex and convoluted than widely appreciated. Also, WGS can exclude cross-transmission, when isolates are different. The challenges now are to make WGS technology more amenable for routine use, and to develop an evidence-based consensus for sequence difference thresholds for isolates that are deemed to be part of the same outbreak, including protracted outbreaks. Using such data in a timely way will provide increased sensitivity in detecting cross-transmission events at an earlier stage, with the potential to prevent outbreaks, and have a positive impact on infection prevention and control.
Asunto(s)
Brotes de Enfermedades , Staphylococcus aureus Resistente a Meticilina/aislamiento & purificación , Epidemiología Molecular/métodos , Tipificación Molecular/métodos , Infecciones Estafilocócicas/epidemiología , Infecciones Estafilocócicas/microbiología , Secuenciación Completa del Genoma , Transmisión de Enfermedad Infecciosa , Humanos , Staphylococcus aureus Resistente a Meticilina/clasificación , Staphylococcus aureus Resistente a Meticilina/genética , Epidemiología Molecular/tendencias , Tipificación Molecular/tendencias , Sensibilidad y Especificidad , Infecciones Estafilocócicas/transmisiónRESUMEN
High throughput sequencing has opened up new clinical opportunities moving towards a medicine of precision. Oncology, infectious diseases or human genomics, many applications have been developed in recent years. The introduction of a third generation of nanopore-based sequencing technology, addressing some of the weaknesses of the previous generation, heralds a new revolution. Portability, real time, long reads and marginal investment costs, these promising new technologies point to a new shift of paradigm. What are the perspectives opened up by nanopores for clinical applications?
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Nanoporos , Análisis Mutacional de ADN/métodos , Farmacorresistencia Bacteriana/genética , Femenino , Genómica/métodos , Genómica/tendencias , Salud , Humanos , Terapia Molecular Dirigida/métodos , Terapia Molecular Dirigida/tendencias , Tipificación Molecular/métodos , Tipificación Molecular/tendencias , Pruebas de Farmacogenómica/métodos , Pruebas de Farmacogenómica/tendencias , Embarazo , Diagnóstico Prenatal/métodos , Diagnóstico Prenatal/tendenciasRESUMEN
BACKGROUND: Molecular diagnoses have become more widespread in many areas of laboratory medicine where qualitative or quantitative approaches are used to detect nucleic acids. The increasing number of assay methods and the targets for molecular diagnostics contribute to variability in the test results among clinical laboratories. Thus, reference materials (RMs) are required to enhance the comparability of results. METHODS: This review focuses on the definition of RMs as well as the production and characteristics of higher order RMs from different organizations and their future strategies. RESULTS: We describe the recent progress in RMs, including the definition of RMs by the Joint Committee for Guides in Metrology, as well as the production and characteristics of higher order RMs by international official bodies. CONCLUSIONS: There is an urgent need for RMs in nucleic acid testing, especially higher order RMs. To advance the harmonization and standardization of clinical nucleic acid detection, cooperation between the above organizations is proposed and different approaches to higher order RMs development are also needed.
Asunto(s)
Salud Global , Técnicas de Diagnóstico Molecular , Ácidos Nucleicos/metabolismo , Biomarcadores/sangre , Biomarcadores/metabolismo , Guías como Asunto , Humanos , Agencias Internacionales , Técnicas de Diagnóstico Molecular/normas , Técnicas de Diagnóstico Molecular/tendencias , Tipificación Molecular/normas , Tipificación Molecular/tendencias , Ácidos Nucleicos/sangre , Ácidos Nucleicos/normas , Estándares de Referencia , Terminología como Asunto , IncertidumbreRESUMEN
No disponible
Asunto(s)
Humanos , Bronquiectasia/microbiología , Fibrosis Quística/complicaciones , Tipificación Molecular/tendencias , Microbiota/inmunología , Antibacterianos/uso terapéuticoAsunto(s)
Infecciones por Actinomycetales/microbiología , Actinomycetales/clasificación , Actinomycetales/genética , Proteínas Bacterianas/genética , Chaperonina 60/genética , Tipificación Molecular/tendencias , Filogenia , Actinomycetales/aislamiento & purificación , Tipificación Molecular/normas , ARN Ribosómico 16S/genéticaRESUMEN
An American Society for Microbiology (ASM) conference titled the Conference on Rapid Next-Generation Sequencing and Bioinformatic Pipelines for Enhanced Molecular Epidemiological Investigation of Pathogens provided a venue for discussing how technologies surrounding whole-genome sequencing (WGS) are advancing microbiology. Several applications in microbial taxonomy, microbial forensics, and genomics for public health pathogen surveillance were presented at the meeting and are reviewed. All of these studies document that WGS is revolutionizing applications in microbiology and that the impact of these technologies will be profound. ASM is providing support mechanisms to promote discussions of WGS techniques to foster applications and interpretations.
Asunto(s)
Enfermedades Transmisibles/diagnóstico , Genómica/métodos , Técnicas de Diagnóstico Molecular/métodos , Tipificación Molecular/métodos , Salud Pública , Enfermedades Transmisibles/epidemiología , Biología Computacional/métodos , Monitoreo Epidemiológico , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Técnicas de Diagnóstico Molecular/tendencias , Tipificación Molecular/tendencias , Análisis de Secuencia de ADN/métodosRESUMEN
Although the diagnosis of a diabetic foot infection is made based on clinical symptoms and signs, we also use blood laboratory, microbiological and radiological studies to make treatment decisions. All of these diagnostic studies have pitfalls that can lead to a delay in diagnosis. Such delays will likely lead to further tissue damage and to a higher chance of amputation. One of these pitfalls is that some clinicians rely on microbiological, rather than clinical data, to diagnose infection. Though subjective by nature, clinical signs predict outcome of foot infections accurately. Another pitfall is that microbiological data can be misleading. All wounds harbour microorganisms; therefore, a positive wound culture does not mean that a wound is infected. Furthermore, the outcome of cultures of wound swabs does not correlate well with culture results of tissue biopsies. Therapy guidance by wound swab will likely lead to overtreatment of non-pathogenic organisms. Genotyping might have a role in identifying previously unrecognized (combinations of) pathogens in diabetic foot infection, bacteria in sessile phenotype and non-culturable pathogens, e.g. in cases where antibiotics have already been administered. One more pitfall is that the diagnosis of osteomyelitis remains difficult. Although the result of percutaneous bone biopsy is the reference standard for osteomyelitis, some other diagnostic modalities can aid in the diagnosis. A combination of several of these diagnostic tests is probably a good strategy to achieve a higher diagnostic accuracy. Relying on a single test will likely lead to misidentification of patients with osteomyelitis with associated overtreatment and undertreatment.
Asunto(s)
Pie Diabético/microbiología , Medicina Basada en la Evidencia , Osteomielitis/diagnóstico , Medicina de Precisión , Enfermedades Cutáneas Infecciosas/diagnóstico , Infecciones de los Tejidos Blandos/diagnóstico , Biopelículas , Biopsia , Congresos como Asunto , Pie Diabético/complicaciones , Pie Diabético/patología , Bacterias Gramnegativas/clasificación , Bacterias Gramnegativas/crecimiento & desarrollo , Bacterias Gramnegativas/aislamiento & purificación , Bacterias Gramnegativas/fisiología , Bacterias Grampositivas/clasificación , Bacterias Grampositivas/crecimiento & desarrollo , Bacterias Grampositivas/aislamiento & purificación , Bacterias Grampositivas/fisiología , Humanos , Tipificación Molecular/tendencias , Osteomielitis/complicaciones , Osteomielitis/microbiología , Osteomielitis/patología , Enfermedades Cutáneas Infecciosas/complicaciones , Enfermedades Cutáneas Infecciosas/microbiología , Enfermedades Cutáneas Infecciosas/patología , Infecciones de los Tejidos Blandos/complicaciones , Infecciones de los Tejidos Blandos/microbiología , Infecciones de los Tejidos Blandos/patologíaRESUMEN
Twenty years ago, the publication of the first bacterial genome sequence, from Haemophilus influenzae, shook the world of bacteriology. In this Timeline, we review the first two decades of bacterial genome sequencing, which have been marked by three revolutions: whole-genome shotgun sequencing, high-throughput sequencing and single-molecule long-read sequencing. We summarize the social history of sequencing and its impact on our understanding of the biology, diversity and evolution of bacteria, while also highlighting spin-offs and translational impact in the clinic. We look forward to a 'sequencing singularity', where sequencing becomes the method of choice for as-yet unthinkable applications in bacteriology and beyond.
Asunto(s)
ADN Bacteriano/química , ADN Bacteriano/genética , Genoma Bacteriano , Análisis de Secuencia de ADN/historia , Análisis de Secuencia de ADN/métodos , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Epidemiología Molecular/historia , Epidemiología Molecular/métodos , Epidemiología Molecular/tendencias , Tipificación Molecular/historia , Tipificación Molecular/métodos , Tipificación Molecular/tendencias , Análisis de Secuencia de ADN/tendenciasRESUMEN
Sepsis is a rapidly progressing, severe inflammatory response to infection, causing more than 200,000 deaths per year. Rapid, specific pathogen identification is important to guide sepsis treatment. In this review, we describe and compare currently available commercial products for sepsis diagnosis and pathogen identification, based on microbiological, molecular, and mass spectrometric technologies. Microbiological techniques, the current "gold standard" in sepsis pathogen identification, include blood culture followed by subculturing and pathogen identification via biochemical or microscopic means. These methods have been automated but nevertheless require several days to generate results. Alternative technologies, including highly multiplexed PCR-based methods and mass spectrometric approaches, can decrease the required turnaround time. Matrix-assisted laser-desorption ionization time-of-flight-based systems have recently become an attractive option to rapidly identify a broad spectrum of sepsis pathogens with good sensitivity and specificity. Effectively integrating rapid sepsis pathogen identification into the hospital workflow can improve patient outcomes and can reduce the length of hospitalization and cost per patient.
Asunto(s)
Infecciones Bacterianas/diagnóstico , Hongos/aislamiento & purificación , Bacterias Gramnegativas/aislamiento & purificación , Bacterias Grampositivas/aislamiento & purificación , Micosis/diagnóstico , Sepsis/diagnóstico , Choque Séptico/diagnóstico , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Antifúngicos/farmacología , Antifúngicos/uso terapéutico , Automatización de Laboratorios , Infecciones Bacterianas/tratamiento farmacológico , Infecciones Bacterianas/microbiología , Diagnóstico Diferencial , Hongos/clasificación , Hongos/efectos de los fármacos , Hongos/crecimiento & desarrollo , Bacterias Gramnegativas/clasificación , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Gramnegativas/crecimiento & desarrollo , Bacterias Grampositivas/clasificación , Bacterias Grampositivas/efectos de los fármacos , Bacterias Grampositivas/crecimiento & desarrollo , Humanos , Pruebas de Sensibilidad Microbiana , Tipificación Molecular/tendencias , Técnicas de Tipificación Micológica/tendencias , Micosis/tratamiento farmacológico , Micosis/microbiología , Sepsis/tratamiento farmacológico , Sepsis/microbiología , Choque Séptico/tratamiento farmacológico , Choque Séptico/microbiología , Virus/clasificación , Virus/efectos de los fármacos , Virus/aislamiento & purificaciónRESUMEN
The hepatitis C virus (HCV) infects more than 200 million people globally, with increasing incidence, especially in developing countries. HCV infection frequently progresses to chronic liver disease, creating a heavy economic burden on resource-poor countries and lowering patient quality of life. Effective HCV diagnosis, treatment selection, and treatment monitoring are important in stopping disease progression. Serological assays, which detect anti-HCV antibodies in the patient after seroconversion, are used for initial HCV diagnosis. Qualitative and quantitative molecular assays are used to confirm initial diagnosis, determine viral load, and genotype the dominant strain. Viral load and genotype information are used to guide appropriate treatment. Various other biomarker assays are performed to assess liver function and enable disease staging. Most of these diagnostic methods are mature and routinely used in high-resource countries with well-developed laboratory infrastructure. Few technologies, however, are available that address the needs of low-resource areas with high HCV prevalence, such as Africa and Southeast Asia.
Asunto(s)
Hepacivirus/aislamiento & purificación , Hepatitis C/diagnóstico , Anticuerpos Antivirales/análisis , Antivirales/farmacología , Antivirales/uso terapéutico , Automatización de Laboratorios , Biomarcadores/sangre , Farmacorresistencia Viral , Hepacivirus/clasificación , Hepacivirus/efectos de los fármacos , Hepacivirus/inmunología , Hepatitis C/sangre , Hepatitis C/inmunología , Hepatitis C/terapia , Hepatitis C/virología , Humanos , Hígado/efectos de los fármacos , Hígado/fisiopatología , Hígado/virología , Tipificación Molecular/tendencias , Carga Viral/efectos de los fármacosRESUMEN
There have recently been significant changes in diagnostic practices for detecting enterovirus (EV) infections across England and Wales. Reports of laboratory-confirmed EV infections submitted by National Health Service (NHS) hospital laboratories to Public Health England (PHE) over a 12-year period (2000-2011) were analysed. Additionally, the PHE Virus Reference Department (VRD) electronic database containing molecular typing data from 2004 onwards was interrogated. Of the 13,901 reports, there was a decline from a peak of 2254 in 2001 to 589 in 2006, and then an increase year-on-year to 1634 in 2011. This increase coincided with increasing PCR-based laboratory diagnosis, which accounted for 36% of reported cases in 2000 and 92% in 2011. The estimated annual incidence in 2011 was 3.9/100,000 overall and 238/100,000 in those aged <3 months, who accounted for almost one-quarter of reported cases (n = 2993, 23%). During 2004-2011, 2770 strains were submitted for molecular typing to the VRD, who found no evidence for a predominance of any particular strain. Thus, the recent increase in reported cases closely reflects the increase in PCR testing by NHS hospitals, but is associated with a lower proportion of samples being submitted for molecular typing. The high EV rate in young infants merits further investigation to inform evidence-based management guidance.
Asunto(s)
Infecciones por Enterovirus/diagnóstico , Infecciones por Enterovirus/epidemiología , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/estadística & datos numéricos , Tipificación Molecular/métodos , Tipificación Molecular/estadística & datos numéricos , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Inglaterra/epidemiología , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Persona de Mediana Edad , Epidemiología Molecular/métodos , Tipificación Molecular/tendencias , Gales/epidemiología , Adulto JovenRESUMEN
La candidemia es una complicación infecciosa que afecta fundamentalmente a pacientes ingresados en unidades de cuidados intensivos así como en otros servicios hospitalarios, y cuya mortalidad puede alcanzar el 40%. La candidemia es una infección de adquisición típicamente hospitalaria, por lo que la transmisión horizontal de Candida spp. puede traducirse en la aparición de brotes nosocomiales. La caracterización genotípica de los aislamientos de Candida causantes de candidemia puede ayudar a esclarecer el origen de la infección, detectar los servicios hospitalarios con transmisión activa y, consecuentemente, mejorar su prevención. Diversas técnicas de tipificación molecular han sido empleadas para el estudio genotípico de aislamientos de Candida. Las técnicas basadas en microsatélites son reproducibles y presentan un alto poder de discriminación, lo que las convierte en opciones atractivas para el estudio de brotes de candidemia. La mayor parte de los brotes han sido descritos en pacientes ingresados en unidades de cuidados intensivos, fundamentalmente en neonatos. La presente revisión pretende discutir el papel de la caracterización genotípica de aislamientos de Candida causantes de candidemia para el estudio de brotes hospitalarios, así como describir las poblaciones más frecuentemente afectadas por las cepas epidémicas (AU)
Candidemia is an infectious complication mainly affecting hospitalized patients, particularly those admitted to intensive care units. Patient mortality can reach up to 40%. Candidemia is typically nosocomially-acquired, and horizontal transmission of Candida spp. can lead to the presence of outbreaks of candidemia. Genotyping of isolates of Candida causing candidemia can help us to understand the source of the infection, detect the hospital wards with active Candida spp. transmission and, consequently, improve the prevention of the infection. Several genotyping tools have been used for the molecular characterization of Candida isolates involved in outbreaks of candidemia. Genotyping procedures based on microsatellites are reproducible and show a high discriminatory power. Microsatellites are recommended for the study of outbreaks of candidemia. In most hospital outbreaks of candidemia, patients admitted to intensive care units are involved, mostly neonatal patients. The role of genotyping Candida isolates causing candidemia for the study of nosocomial outbreaks of candidemia is reviewed, as well as the patients more commonly affected by epidemic strains (AU)
Asunto(s)
Humanos , Masculino , Femenino , Tipificación Molecular/métodos , Tipificación Molecular/normas , Tipificación Molecular/tendencias , Candidemia/microbiología , Candida/aislamiento & purificación , Técnicas de Genotipaje/instrumentación , Técnicas de Genotipaje/métodos , Plantones/microbiología , Tipificación Molecular , Candidemia/diagnóstico , Candidemia/patología , Técnicas de Genotipaje , ADN/análisis , Infección Hospitalaria/complicaciones , Infección Hospitalaria/microbiología , Infección Hospitalaria/prevención & controlRESUMEN
Trypanosoma cruzi, agente etiológico causante del Mal de Chagas, es una especie formada por poblaciones multiclonales altamente divergentes. La comprensión de su estructura poblacional es relevante para determinar su asociación con las diversas manifestaciones clínicas y severidad de la enfermedad, las cuales presentan una distribución geográfica diferencial. Recientemente se ha reconocido que T. cruzi está conformado por seis Unidades Discretas de Tipificación (UDTs): TcI a TcVI. Aquí presentamos un panorama que resume el conocimiento disponible sobre la existencia de asociaciones entre los UDTs y las formas clínicas de la enfermedad de Chagas(AU)
Trypanosoma cruzi, the etiologic agent of Chagas disease, is a species formed by highly divergent multiclonal populations. Understanding the population structure is relevant to determine its association with clinical manifestations and severity of the disease, which have a different geographical spread. Recently it has been recognized that T. cruzi consists of six Discrete Typing Units (UDTs) ; from Tc I to Tc VI. We present an overview that summarizes the available knowledge about the existence of associations between UDTs and clinical forms of Chagas disease(AU)
Asunto(s)
Humanos , Masculino , Femenino , Trypanosoma cruzi/genética , Trypanosoma cruzi/aislamiento & purificación , Trypanosoma cruzi/patogenicidad , Genotipo , Enfermedad de Chagas/etiología , Enfermedad de Chagas/genética , Reacción en Cadena de la Polimerasa/métodos , Reacción en Cadena de la Polimerasa , Técnicas de Genotipaje/métodos , Técnicas de Genotipaje , Tipificación Molecular/métodos , Tipificación Molecular/tendencias , Tipificación MolecularRESUMEN
Advances in typing methodologies have been the driving force in the field of molecular epidemiology of pathogens. The development of molecular methodologies, and more recently of DNA sequencing methods to complement and improve phenotypic identification methods, was accompanied by the generation of large amounts of data and the need to develop ways of storing and analysing them. Simultaneously, advances in computing allowed the development of specialised algorithms for image analysis, data sharing and integration, and for mining the ever larger amounts of accumulated data. In this review, we will discuss how bioinformatics accompanied the changes in bacterial molecular epidemiology. We will discuss the benefits for public health of specialised online typing databases and algorithms allowing for real-time data analysis and visualisation. The impact of the new and disruptive next-generation sequencing methodologies will be evaluated, and we will look ahead into these novel challenges.
Asunto(s)
Biología Computacional/métodos , Epidemiología Molecular , Tipificación Molecular/métodos , Salud Pública , Algoritmos , Biología Computacional/tendencias , Bases de Datos Factuales , Bases de Datos de Ácidos Nucleicos , Genómica/métodos , Humanos , Tipificación Molecular/tendenciasRESUMEN
Viridans group streptococci (VGS) are gram-positive microorganisms that can form alpha-hemolytic colonies on sheep blood agar. They reside as normal flora in oral cavity, respiratory, gastrointestinal, urogenital tract and on skin. They can cause bacteremia, endocarditis, meningitis and septicemia following dental procedures. The diagnosis of VGS are difficult since the taxonomic classification and species na-mes may change due in time. Viridans group streptococci are classified into 5 groups (Sanguinis, Mitis, Mutans, Salivarius, Anginosus) according to biochemical reactions and 16S rRNA sequencing. Since Streptococcus pneumoniae is a member of the Mitis group, the other important species in this group deserves investigation. Genetic exchange between Streptococcus mitis, Streptococcus oralis and S.pneumoniae by transformation and lysis mechanisms occur continously as they share the same anatomical region. These mechanisms play role in exchanging capsular and antibiotic resistance genes between these species. The cultivation of VGS usually starts with the inoculation of various patient specimens into sheep blood agar and the detection of alpha-hemolytic colonies. Observation of gram-positive cocci microscopically, the detection of optochin-resistant and bile insoluble colonies with few exceptions are the further important steps in laboratory diagnosis. VGS are then identified at species level by using biochemical reactions, automated diagnostic systems and molecular methods. The last step in the laboratory diagnosis of VGS is antibiotic susceptibility testing which is of outmost importance as penicillin and erythromycin resistance are on rise. In this review article, classification of VGS, similarities between S.pneumoniae and Mitis group streptococci and the laboratory diagnosis of VGS have been discussed.