Quiz 4 Content Flashcards
What does phenotypic subtyping of pathogens include? [3]
- Serotyping
- Phage typing
- Antimicrobial resistance profiling
Phenotypic - can be observed with the eyes
These have some utility for characterizing bacteria but are not adequate for outbreak detection principally due to low diversity. Plus, some require a high amount of specialization and the reagents are not available to all public health labs.
Why is the methodology of phenotypic subtyping of pathogens not adequate for outbreak detection?
Principally due to low diversity, but also because some require a high amount of specialization and the reagents are not available to all public health labs.
Phenotypic subtyping methods are useful in terms of initial characterization, but they are not very specific.
What are the criteria for effective subtyping methods? [4]
- Markers must be (1) stable, (2) consistent, and (3) present in all isolates
- Method must have (1) a high discriminatory power with (2) the ability to assign a designation for all isolates
- Must be reproducible over time
- Convenience criteria: (1) Be flexible and (2) be technically uncomplicated
What do molecular subtyping methods generally rely on?
Detecting differences in the nucleic acid sequence of organisms
Name two molecular subtyping methods.
- Fragment-based genotyping; older approach (e.g., PFGE)
- Nucleic acid sequencing-based genotyping; newer approach (e.g., MLST; WGS)
What does fragment-based genotyping rely on?
- Comparison of one or many DNA fragments produced from an isolate
e.g., pulsed-field gel electrophoresis (PFGE)
Give two examples of nucleic acid sequencing-based genotyping.
- Multiple-locus sequence typing (MLST)
- Whole genome sequencing (WGS)
Describe pulsed-field gel electrophoresis.
- Gold standard for bacterial subtyping
- Produces a genetic ‘fingerprint’ of a bacterial isolate
- Achieved through breaking of chromosomal DNA into segments by restriction enzymes
- Creates and visualizes segments of DNA from a bacterial sample to be compared with other samples
- Advanced method of gel electrophoresis
Describe bacterial chromosomal DNA.
- Circular DNA molecule within bacterial cell
- Carries all ‘normal’ genes employed for growth
- Sequence specific for each species and even each strain
How does PFGE work?
- Uses molecular scissors, called restriction enzymes, to cut bacterial DNA at the restriction sites
- These molecular scissors are selected to generate a small number of DNA pieces that can be separated based on size
- Usually these DNA pieces, or restriction fragments, are large and need to be specially treated and separated to generate a DNA fingerprint
How does PFGE differ from regular gel electrophoresis?
- Regular gel electrophoresis cannot separate large size DNA molecules that are more than 50 KB - genome of foodborne bacterial pathogens is usually 3-5 MB
Describe the PFGE process [5].
- Bacterial cells are taken from an agar plate
- Cells are mixed with melted agarose and poured into plug mold
- Cells are broken open with biochemicals, or lysed, so that the DNA is free in the agarose plugs
- DNA gelatin plug is loaded into a gel, and an electric field is applied that separates the DNA fragments according to their size
- The gel is stained so that the DNA can be seen under UV light; a digital camera takes a photo and stores it on a computer.
How are gels visualized in PFGE?
- Similar to PCR product visualization, following electrophoresis, PFGE gels are:
- Stained with ethidium bromide
- Visualized through exposure to UV light
Describe the analysis of PFGE patterns.
- Comparison of patterns using software
- Statistical measure of similarity
- Can produce dendograms
What is PulseNet?
A global network of PFGE results
What are the advantages of PFGE? [3]
- Easily applied to different species
- Generally yields a high amount of pattern diversity that provides good discriminatory power and other measures of subtyping validity
- DNA restriction patterns are stable and reproducible
What are the limitations of PFGE? [8]
- Time consuming
- Requires trained and skilled technician
- Does not discriminate between all unrelated isolates
- Pattern results vary slightly between technicians
- Don’t really know if bands of the same size are the same pieces of DNA
- Change in one restriction site can mean more than one band change
- “Relatedness” should be used as a guide, not true phylogenic measure
- Some strains cannot be typed by PFGE
What are the advantages of multiple-locus sequence typing (MLST)? [4]
- Uses sequence data and can therefore detect changes at the DNA level
- A generic technique that can be readily reproduced and does not require access to specialized reagents or training
- Based on PCR, do not require direct access to live bacterial isolates or high-quality genomic DNA
- The data generated are fully portable among laboratories and can be shared throughout the world
What are the limitations of multiple-locus sequence typing? [2]
- Uses only seven loci (for the housekeeping genes), which limits the ability to detect some switches
- Requires performing PCR and sequencing of the PCR products using an automated sequencer, which is not always available
Briefly introduce whole-genome sequencing.
- Bacterial genomes are roughly 1000 times smaller than the human genome (e.g., Listeria monocytogenes genome is ~3 MB)
- WGS platforms involve many different technologies all of which generate large, genome-scale datasets
How does whole genome sequencing work?
- Specimen - selection and cultivation
- Bacterial culture - DNA isolation
- Genomic DNA - quality control and DNA processing
- Sequencing library - sequencing
- Raw sequence data - data processing and analysis
- Analysis results - central data repository; summarize
- Diagnostic report
What are the advantages of whole genome sequencing? [1]
- Higher discriminatory power compared to PFGE (e.g., > 50% S. enteritidis isolates share identical PFGE types)
- The use of WGS-based characterization of foodborne pathogens by both public health and regulatory agencies expands very quickly and may replace PFGE in the future
- Routine use of WGS was initiated by US CDC in 2013
What are the limitations of whole genome sequencing? [2]
- Rapid analysis remains a challenge
- Requires trained bioinformaticians
What is surveillance? [4]
- Collection
- Analysis
- Interpretation
- Dissemination
… that leads to action to prevent and control disease.
How are cases of foodborne illness usually reported?
Someone gets sick and seeks medical care
A specimen is submitted for testing
Lab tests for pathogen
Lab identifies pathogen and reports it to surveillance
These reported cases are important, but are only the tip of the iceberg.
In the spectrum of foodborn disease outbreaks, describe the old scenario. [5]
‘Old’ refers to the fact that people are most familiar with this type of outbreak.
- Large number of cases in one jurisdiction
- Detected by affected group
- Local investigation
- Local food handling error
- Local solution
In the spectrum of foodborne disease outbreaks, describe the new dispersed scenario. [6]
- Small number of cases in many jurisdictions
- Detected by lab-based data
- Multijurisdictional investigation
- Industrial contamination events
- Broad implications
- Makes coordination among multiple organizations even more important
List the steps in an outbreak investigation.
- Detect possible outbreak
- Find cases in outbreak
- Generate hypotheses through interviews
- Test hypotheses through analytic studies and laboratory testing
- Solve point of contamination and original source of outbreak vehicle
- Control outbreak through recalls, facility improvements, and industry collaboration
If cases stop, decide outbreak is over.
If cases continue, continue to generate hypotheses for testing.
What is step 1 of an outbreak investigation?
- A foodborne illness is caused by the consumption of a contaminated food
- When two or more foodborne illnesses were identified from consumption of the same contaminated food, the event is called a foodborne outbreak
Step 1: Determine existence of an outbreak
How does public surveillance help in outbreak detection?
- Reportable diseases via public health surveillance
- Cluster: a larger number of people than expected appear to have the same illness in a given time period and area
How does lab information help with outbreak detection?
- Clusters based on further typing (e.g., PFGE)
- When several strains have the same DNA fingerprints all at the same time, and there are more with that one serotype than is expected, that’s a sign of a possible outbreak
How do clinicians help with outbreak detection?
- Cases of HUS or Salmonellosis in a hospital
- Stool samples submitted to clinical laboratory
How does community help in outbreak detection?
- Community detection - informal reports (e.g., weddings, catered events, etc.)
Why is it important to find more cases in an outbreak? [4]
- The first illnesses recognized are only a small part of the outbreak
- Finding more ill persons is important to help public health officials understand the (1) size, (2) timing, (3) severity, and (4) possible sources of the outbreak
Think of crime scene investigators trying to identify a serial killer; more cases gives detectives more information to identify the perpetrator.
Describe the list of criteria used in case definition (step 2 of outbreak investigation) [6]
- A list of criteria used to determine if an individual is included as a case in an outbreak investigation
- (1) Features of the illness
- (2) DNA fingerprint (if the pathogen is traced by PulseNet)
- (3) The pathogen or toxin (if known)
- (4) Certain symptoms typical for that pathogen or toxin
- (5) Time range for when the illnesses occurred
- (6) Geographical range, such as residency in a state or region
Step 2: Case definition- then find more cases!
How can more cases be found? [6]
- Reviewing laboratory reports to PulseNet
- Reviewing regular surveillance reports
- Asking local clinical and laboratory professionals to report cases of the particular illness more quickly, as soon as they suspect the diagnosis
- Reviewing emergency room records for similar illnesses
- Surveying groups that may have been exposed
- Asking health officials in surrounding areas to look for illnesses that might be related
Finding more cases is based on the case definition.
How are the effects of recalls tracked in outbreak investigation?
- Investigators use an epidemic curve or epi curve to track the number of illnesses over time.
What is a hypothesis in outbreak investigation?
A suggested explanation for the source of foodborne illness outbreak
How are hypotheses generated? [3]
- Using 3 types of data
(1) Epidemiologic
(2) Traceback
(3) Food & environmental testing
Also remember that hypothesis generating interviews are important too.
What is step 3 of outbreak investigation?
- Pinpoint how the pathogen spread - review details such as:
- The specific pathogen causing illness
- Where sick people live
- How old they are, their sex, and race/ethnicity
- Did they have contact with a sick person
Hypothesis-generating interviews
Describe step 4 in outbreak investigation.
- Case-control studies are the most common type of epidemiologic study conducted during foodborne outbreaks - whether ill persons are more likely than people who did not get sick to have eaten a certain food or to report a particular exposure
Test hypothesis
What is an odds ratio?
A measure of association between an exposure and an outcome
What does OR = 1 mean?
The exposure does not affect odds of outcome
What does OR > 1 mean?
Exposure associated with higher odds of outcome
What does OR < 1 mean?
Exposure associated with lower odds of outcome
How is odds ratio calculated?
Example:
Exposed & Diseased: 7
Exposed & No disease: 10
Non-exposed & Diseased: 6
Non-exposed & no disease: 56
OR = (7/10) / (6/56) = 6.5
or
OR = (7/10) * (56/6) = 6.5
OR > 0, so exposure is associated with higher odds of disease.
Describe step 4 of outbreak investigation.
- Food testing can help to support a hypothesis
- Misleading results due to : food items with short shelf-life; spoiled food; leftover food or food in open containers; no test exists
- Sometimes no link can be found between a specific food and an illness
Analysis using case-control studies is also important during this step.
Step 4: Testing hypotheses
Describe step 5 of outbreak investigation.
- A traceback investigation:
- Starts with the sick people or restaurants and
- works its way back through the chain of production
- to search for a common point between people or places where contamination is likely to have happened
Note: every outbreak is different, and not all steps always apply!
Describe step 6 of outbreak investigation. [5]
- Cleaning and disinfecting food facilities
- Temporariliy closing a restaurant or processing plant
- Recalling food items
- Telling the public how to make food safe (such as cooking to a certain temperature) or to avoid it completely
- Telling consumers to throw away the suspect food from their pantry or refrigerator
Step 6: Controlling an outbreak
Describe step 7 of outbreak investigation.
- An outbreak ends when the number of new reported illnesses drops back to the normal number
- The epi curve shows that illnesses are declining
- Even when illnesses from the outbreak appear to have stopped, public health officials continue surveillance for a few weeks to be sure cases don’t start to increase again
Step 7: Decide an Outbreak is Over
Why is food testing during hypothesis testing not always helpful during outbreak investigation?
- Misleading results due to: (1) food items with short shelf-life; (2) spoiled food; (3) leftover food or (4) food in open containers, or (5) no test exists
- Sometimes no link can be found between a specific food and an illness
