Sepsis, a leading cause of morbidity and mortality throughout the world, is a clinical syndrome with signs and symptoms relating to an infectious event and the consequent important inflammatory response. From a clinical point of view, sepsis is a continuous process ranging from systemic inflammatory response syndrome (SIRS) to multiple-organ-dysfunction syndrome (MODS). Blood cultures are the current "gold standard" for diagnosis, and they are based on the detection of viable microorganisms present in blood. However, on some occasions, blood cultures have intrinsic limitations in terms of sensitivity and rapidity, and it is not expected that these drawbacks will be overcome by significant improvements in the near future. For these principal reasons, other approaches are therefore needed in association with blood culture to improve the overall diagnostic yield for septic patients. These considerations have represented the rationale for the development of highly sensitive and fast laboratory methods. This review addresses non-culture-based techniques for the diagnosis of sepsis, including molecular and other non-culture-based methods. In particular, the potential clinical role for the sensitive and rapid detection of bacterial and fungal DNA in the development of new diagnostic algorithms is discussed.

The Era of Molecular and Other Non-Culture-Based Methods in Diagnosis of Sepsis

The present invention provides control methods, control systems, and control software for microfluidic devices that operate by moving discrete micro-droplets through a sequence of determined configurations. Such microfluidic devices are preferably constructed in a hierarchical and modular fashion which is reflected in the preferred structure of the provided methods and systems. In particular, the methods are structured into low-level device component control functions, middle-level actuator control functions, and high-level micro-droplet control functions. Advantageously, a microfluidic device may thereby be instructed to perform an intended reaction or analysis by invoking micro-droplet control function that perform intuitive tasks like measuring, mixing, heating, and so forth. The systems are preferably programmable and capable of accommodating microfluidic devices controlled by low voltages and constructed in standardized configurations. Advantageously, a single control system can thereby control numerous different reactions in numerous different microfluidic devices simply by loading different easily understood micro-droplet programs.

Methods and systems for control of microfluidic devices

Outbreaks of multidrug-resistant bacteria present a frequent threat to vulnerable patient populations in hospitals around the world. Intensive care unit (ICU) patients are particularly susceptible to nosocomial infections due to indwelling devices such as intravascular catheters, drains, and intratracheal tubes for mechanical ventilation. The increased vulnerability of infected ICU patients demonstrates the importance of effective outbreak management protocols to be in place. Understanding the transmission of pathogens via genotyping methods is an important tool for outbreak management. Recently, whole-genome sequencing (WGS) of pathogens has become more accessible and affordable as a tool for genotyping. Analysis of the entire pathogen genome via WGS could provide unprecedented resolution in discriminating even highly related lineages of bacteria and revolutionize outbreak analysis in hospitals. Nevertheless, clinicians have long been hesitant to implement WGS in outbreak analyses due to the expensive and cumbersome nature of early sequencing platforms. Recent improvements in sequencing technologies and analysis tools have rapidly increased the output and analysis speed as well as reduced the overall costs of WGS. In this review, we assess the feasibility of WGS technologies and bioinformatics analysis tools for nosocomial outbreak analyses and provide a comparison to conventional outbreak analysis workflows. Moreover, we review advantages and limitations of sequencing technologies and analysis tools and present a real-world example of the implementation of WGS for antimicrobial resistance analysis. We aimed to provide health care professionals with a guide to WGS outbreak analysis that highlights its benefits for hospitals and assists in the transition from conventional to WGS-based outbreak analysis.

Whole-Genome Sequencing of Bacterial Pathogens: the Future of Nosocomial Outbreak Analysis

The technology described herein generally relates to systems for extracting polynucleotides from multiple samples, particularly from biological samples, and additionally to systems that subsequently amplify and detect the extracted polynucleotides. The technology more particularly relates to microfluidic systems that carry out PCR on multiple samples of nucleotides of interest within microfluidic channels, and detect those nucleotides.

Integrated Apparatus for Performing Nucleic Acid Extraction and Diagnostic Testing on Multiple Biological Samples

The laboratory confirmation of meningococcal disease and characterization of Neisseria meningitidis isolates was improved considerably in England and Wales by the Meningococcal Reference Unit between epidemiological years 1993/94 and 2003/04 to meet the challenge of increasing numbers of cases of clinical disease and the requirement for enhanced surveillance. Improved case ascertainment was made possible by the rapid introduction of an innovative centralized reference service for non-culture PCR-based DNA detection of meningococci utilizing the ctrA and siaD PCR assays, complemented by consistent phenotypic characterization of submitted isolates from culture-proven cases. This allowed the increased prevalence of serogroup C disease in specific age groups and the apparent associated increase in mortality from 1995/96 to 1999/00 to be defined, thereby prompting accelerated intervention with the newly licensed meningococcal serogroup C conjugate (MCC) vaccines into the under-25-year UK population (in November 1999). The continued increase in and predominance of serogroup B cases (1993/94 to 2000/01) were observed in conjunction with their diverse and changing phenotypic characteristics. Trends observed to be associated with the predominant phenotypic combinations of serogroup, serotype and sero-subtype were: a decline of both C : 2b and B : 2b meningococci, and a decline of B : 15 : P1.7,16 with a concomitant increase of B : 4 : P1.4 over the 11-year period. Detailed routine surveillance rapidly confirmed the introduction of W135 : 2a : P1.5,2 meningococci into the UK during 2000 and 2001. The importance of continued detailed surveillance of this important pathogen cannot be overestimated, both to monitor the effectiveness of the MCC vaccine and to identify changes within the meningococcal population that can inform the design of anti-serogroup B vaccines.

Epidemiology of meningococcal disease in England and Wales 1993/94 to 2003/04: contribution and experiences of the Meningococcal Reference Unit.

The demand for molecular diagnostic tests in medical microbiology has highlighted the need for efficient methods of DNA extraction. In addition, it is preferable for these methods to be automated. An example of such a requirement is for the confirmation of meningococcal disease where rapid, sensitive, and specific procedures are required for public health management purposes. Previous studies have shown that whole blood is the preferred method for the isolation of bacterial DNA in meningococcal disease, and in this study, we compare five commercially available kits for the extraction of bacterial genomic DNA from whole-blood samples. These include kits in a 96-well binding plate, 96-well filter plate, and metallic bead formats. The method for all five kits is described, and the sensitivity, specificity, ease of automation, and overall efficiency are determined.

Comparison of Commercial DNA Extraction Kits for Extraction of Bacterial Genomic DNA from Whole-Blood Samples

Standard and fluorescence-based PCR assays were developed for the identification of serogroup A meningococci by detection of the mynA gene. This assay was evaluated using bacterial cultures but provides the sensitivity required for the detection of the mynA gene from bodily fluids during meningococcal disease.

K. Smith

Papers

Comparison of Commercial DNA Extraction Kits for Extraction of Bacterial Genomic DNA from Whole-Blood Samples

Evaluation of a Fluorescence-Based PCR Method for Identification of Serogroup A Meningococci