1. Why was PulseNet developed?
In the beginning of the 1990’s, an increasing number of outbreaks of foodborne infections was detected and investigated in the United States. Such an increase placed a strain on the resources available at the Central Reference Laboratory at the Foodborne and Diarrheal Diseases Branch, the Centers for Disease Control and Prevention (CDC) for subtyping of outbreak-related isolates. To cope with this situation, PulseNet was developed as a result of the geographic decentralization of molecular surveillance of foodborne infections.in the USA (see PulseNet USA history). This decentralization led to the transfer of subtyping by Pulsed-Field gel electrophoresis (PFGE) from the Central Reference Laboratory at CDC to the local Public Health Laboratories in the states. Subtyping data were analyzed locally and forwarded electronically to CDC for further analysis. Besides shifting some of the subtyping workload away from the CDC, this change benefited foodborne disease surveillance in others ways: isolates were subtyped more rapidly with no delay that would be incurred by transporting them to CDC and the results were simultaneously available for analysis both locally and at CDC. Today, most states perform subtyping for their own isolates, however, the CDC still acts as a national reference laboratory that offers its services to states requesting assistance.
2. What is the Role of PulseNet?
- Detect foodborne disease case clusters by PFGE, thereby facilitating early identification of common source outbreaks
- Separate outbreak-associated cases from sporadic cases (case definition)
- Assist in identifying and confirming the source of the outbreak (culture confirmation)
- Act as an efficient means of communcatiom between public health laboratories
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3. Why is PulseNet important to public health?
PulseNet plays a vital role in the surveillance and investigation of foodborne illness outbreaks that were previously difficult to detect. By identifying bacterial isolates with indistinguishable subtype through PulseNet, scientists may link cases associated with the same outbreak even if the affected individuals are geographically distant. Outbreaks, along with their sources, can now be identified in a matter of days rather than weeks.
4. How does PulseNet work?
Pulsed-Field Gel Elecrophoresis (PFGE) Fingerprint
- PulseNet participants perform DNA "fingerprinting" by pulsed-field gel electrophoresis (PFGE, see next section) on disease-causing bacteria isolated from humans and from suspected food using standardized equipment and methods.
- Once the PFGE patterns are generated, the laboratory enters them into an electronic database and analyzes the results at the local level. The analyzed patterns may also be uploaded to the regional PulseNet database. If a cluster with indistinguishable patterns is identified indicating an outbreak may be occurring, the laboratory consults with local epidemiologists and initiates an outbreak investigation. If the outbreak is suspected to extend beyond the local level an alert is posted on the regional PulseNet listserv and the PulseNet international forum.
- Database curators at the regional level will also perform regular cluster searches. If a cluster that has not been reported by a local laboratory is identified and an outbreak is suspected, an investigation is initiated. An alert is posted on the regional PulseNet listserv and the PulseNet international forum.(external link)
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5. What is the PFGE process?
PFGE Process showing bacterial suspension, mix with aragose, plug mold, chemical lysis and washing, dna in plugs, restriction enzyme, electrophoresis, documentation capture gel image, data analysis
PFGE is a subtyping method that detects polymorphism in restriction fragments of genomic DNA (macrorestriction). Genomic DNA is restricted by enzymes that recognize short DNA sequences and cut the DNA whenever such a sequence occurs. In this way genomic DNA is cut into a small number (usually 10-20) of restriction fragments in a very specific and reproducible manner.
These DNA fragments are usually too large to separate by conventional agarose gel electrophoresis. In stead, they can be effectively resolved according to their size by a process termed pulsed-field gel electrophoresis (PFGE), developed in the early 80’s.
PFGE facilitates the differential migration of large DNA fragments through agarose gels by constantly changing the direction of the electrical field during electrophoresis. The contour-clamped homogeneous electric field (CHEF) gel electrophoresis method has become the method of choice for resolving DNA macrorestriction fragments of bacterial genomic DNA.
6. Advantages of using PFGE
PFGE is a universal method that may be used for subtyping of bacteria with small modifications dependent on the bacterial species investigated. Usually, only the choice of the restriction enzyme and conditions for electrophoresis need to be optimized for each species.
DNA restriction patterns generated by PFGE are stable and reproducible at the intra- and inter-laboratory levels when the method is highly standardized (see section 8 below). Due to its relative simplicity, high discriminatory power and reproducibility, PFGE has become the gold standard for bacterial subtyping.
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7. Limitations of PFGE
- Time consuming
- Requires a high-level of skills
- Does not work for everything (i.e. clonal patterns)
- The DNA fragments are separated by their size, not by their sequence
- Fragments with the same size may represent different parts of the genome
- Observed differences in PFGE patterns may be difficult to interpret
- Some strains are untypable by PFGE
8. What makes interlaboratory comparison of DNA patterns possible?
For PulseNet, the quality and uniformity of the data is ensured by the implementation of a quality assurance and quality control (QA/QC) program. The components of the QA/QC program that allow for the comparison of DNA patterns across all laboratories participating in PulseNet are:
- Standardised protocols(external link)
- Same molecular size standard ( S. Braenderup H2812, ATCC BAA-664)
- Standardized software used by all participants
- Standardized nomenclature of PulseNet patterns
- Training workshops (lab & software): most participating labs have attended a week of combined laboratory and analysis software training
- Certification: all individuals who submit data must be certified by stringent PulseNet standards
- Proficiency testing: all certified individuals must participate and pass annual proficiency testing in order to maintain certification
- Annual update meetings(external link): provide a forum for the live exchange of information
9. Future objectives of PulseNet
To do what we do but do it better:
- Maintain and strengthen collaboration during international outbreak investigation
- Increase the number of PulseNet regional networks and the number of participants in each network
- Achieve real-time subtyping and real-time communication in all PulseNet regional networks
- Increase the level of communication between laboratorians and epidemiologists at the national and international levels
- Strengthen collaborations and establish partnerships/affiliations with other stake holders for food safety, e.g. academia and the food industry, as well as non-profit national and international organizations worldwide
- Improve the subtyping methods used by PulseNet
- Develop PFGE protocols for more organisms such as Yersinia enterocolitica,and Enterobacter sakazakii
- Develop new faster and more simple subtyping methods
- Multiple-locus variable-number tandem-repeats analysis ( MLVA)
- Single nucleotide polymorphism (SNP) analysis
- Use PulseNet data to study the geographic distribution of major clones of foodborne bacteria
- Use PulseNet data to assist in studies attributing the sources of sporadic foodborne infections
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