13 research outputs found
Burn Injury Reduces Neutrophil Directional Migration Speed in Microfluidic Devices
Thermal injury triggers a fulminant inflammatory cascade that heralds shock, end-organ failure, and ultimately sepsis and death. Emerging evidence points to a critical role for the innate immune system, and several studies had documented concurrent impairment in neutrophil chemotaxis with these post-burn inflammatory changes. While a few studies suggest that a link between neutrophil motility and patient mortality might exist, so far, cumbersome assays have prohibited exploration of the prognostic and diagnostic significance of chemotaxis after burn injury. To address this need, we developed a microfluidic device that is simple to operate and allows for precise and robust measurements of chemotaxis speed and persistence characteristics at single-cell resolution. Using this assay, we established a reference set of migration speed values for neutrophils from healthy subjects. Comparisons with samples from burn patients revealed impaired directional migration speed starting as early as 24 hours after burn injury, reaching a minimum at 72–120 hours, correlated to the size of the burn injury and potentially serving as an early indicator for concurrent infections. Further characterization of neutrophil chemotaxis using this new assay may have important diagnostic implications not only for burn patients but also for patients afflicted by other diseases that compromise neutrophil functions
Harmonization of pulsed-field gel electrophoresis protocols for epidemiological typing of strains of methicillin-resistant Staphylococcus aureus: a single approach developed by consensus in 10 European laboratories and its application for tracing the spread of related strains
Pulsed-field gel electrophoresis (PFGE) is the most common genotypic
method used in reference and clinical laboratories for typing
methicillin-resistant Staphylococcus aureus (MRSA). Many different
protocols have been developed in laboratories that have extensive
experience with the technique and have established national databases.
However, the comparabilities of the different European PFGE protocols
for MRSA and of the various national MRSA clones themselves had not been
addressed until now. This multinational European Union (EU) project has
established for the first time a European database of representative
epidemic MRSA (EMRSA) strains and has compared them by using a new
“harmonized” PFGE protocol developed by a consensus approach that
has demonstrated sufficient reproducibility to allow the successful
comparison of pulsed-field gels between laboratories and the tracking of
strains around the EU. In-house protocols from 10 laboratories in eight
European countries were compared by each center with a “gold
standard” or initial harmonized protocol in which many of the
parameters had been standardized. The group found that it was not
important to standardize some elements of the protocol, such as the type
of agarose, DNA block preparation, and plug digestion. Other elements
were shown to be critical, namely, a standard gel volume and
concentration of agarose, the DNA concentration in the plug, the ionic
strength and volume of running buffer used, the running temperature, the
voltage, and the switching times of electrophoresis. A new harmonized
protocol was agreed on, further modified in a pilot study in two
laboratories, and finally tested by all others. Seven laboratories’ gels
were found to be of sufficiently good quality to allow comparison of the
strains by using a computer software program, while two gels could not
be analyzed because of inadequate destaining and DNA overloading.
Good-quality gels and inclusion of an internal quality control strain
are essential before attempting intercenter PFGE comparisons. A number
of clonally related strains have been shown to be present in multiple
countries throughout Europe. The well-known Iberian clone has been
demonstrated in Belgium, Finland, France, Germany, and Spain (and from
the wider HARMONY collection in Portugal, Slovenia, and Sweden). Strains
from the United Kingdom (EMRSA-15 and -16) have been identified in
several other countries, and other clonally related strains have also
been identified. This highlights the need for closer international
collaboration to monitor the spread of current epidemic strains as well
as the emergence of new ones
Evaluation of molecular typing methods in characterizing a European collection of epidemic methicillin-resistant Staphylococcus aureus strains: The HARMONY collection
We analyzed a representative sample of methicillin-resistant Staphylococcus aureus (MRSA) from 11 European countries (referred to as the HARMONY collection) using three molecular typing methods used within the HARMONY group to examine their usefulness for large, multicenter MRSA surveillance networks that use these different laboratory methodologies. MRSA isolates were collected based on their prevalence in each center and their genetic diversity, assessed by pulsed-field gel electrophoresis (PFGE). PFGE groupings (≤3 bands difference between patterns) were compared to those made by sequencing of the variable repeats in the protein A gene spa and clonal designations based on multilocus sequence typing (MLST), combined with PCR analysis of the staphylococcal chromosome cassette containing the mec genes involved in methicillin resistance (SCCmec). A high level of discrimination was achieved using each of the three methodologies, with discriminatory indices between 89.5% and 91.9% with overlapping 95% confidence intervals. There was also a high level of concordance of groupings made using each method. MLST/SCCmec typing distinguished 10 groups containing at least two isolates, and these correspond to the majority of nosocomial MRSA clones described in the literature. PFGE and spa typing resolved 34 and 31 subtypes, respectively, within these 10 MRSA clones, with each subtype differing only slightly from the most common pattern using each method. The HARMONY group has found that the methods used in this study differ in their availability and affordability to European centers involved in MRSA surveillance. Here, we demonstrate that the integration of such technologies is achievable, although common protocols (such as we have developed for PFGE) may also be important, as is the use of centralized Internet sites to facilitate data analysis. PFGE and spa-typing data from analysis of MRSA isolates from the many centers that have access to the relevant equipment can be compared to reference patterns/sequences, and clonal designations can be made. In the majority of cases, these will correspond to those made by the (more expensive) method of choice - MLST/SCCmec typing - and these alternative methods can therefore be used as frontline typing systems for multicenter surveillance of MRSA. Copyright © 2007, American Society for Microbiology. All Rights Reserved