Towards application of CRISPR-Cas12a in the design of modern viral DNA detection tools (Review).

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.

Contribution of whole-genome sequencing to understanding of the epidemiology and control of meticillin-resistant Staphylococcus aureus.

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.

[Prospects for applications in human health of nanopore-based sequencing]. / Séquençage par nanopores - Perspectives d'applications en santé humaine.

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?

Reference materials for molecular diagnostics: Current achievements and future strategies.

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.

The Future of Whole-Genome Sequencing for Public Health and the Clinic.

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.

Pitfalls in diagnosing diabetic foot infections.

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.

Twenty years of bacterial genome sequencing.

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.

Sepsis Pathogen Identification.

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.

Diagnosis and Management of Hepatitis C Virus Infection.

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.

Enterovirus infections in England and Wales, 2000-2011: the impact of increased molecular diagnostics.

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.

Bioinformatics in bacterial molecular epidemiology and public health: databases, tools and the next-generation sequencing revolution.

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.

[Classical and new approaches in laboratory diagnosis of viridans streptococci]. / Viridans streptokoklarin laboratuvar tanisinda klasik ve yeni yaklasimlar.

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.