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4.1 Medical Microbiology > Gastrointestinal Tract Infections: Laboratory Detection > Flashcards

Gastrointestinal Tract Infections: Laboratory Detection Flashcards

1
Q

How do we detect VTEC/EHEC, what sample type is used, how does it work?

A

Real Time PCR on the EntericBio
Faeces sample, no pre-enrichment needed
DNA extraction amplification and detection
Rapid results within 3 hours on day 1

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2
Q

What can be detected on the EntericBio panel, list the bacteria, parasites and viruses?

A

Bacteria:
- C. difficile
- Salmonella
- Shigella
- Campylobacter
- Shiga toxin 1 and 2 (stx1/2)
- Yersinia Vibrio

Parasites
- Cryptosporidium/Giardia
- Entamoeba

Viruses:
- Norovirus
- Rotavirus
- Adenovirs

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3
Q

Where is the reference lab for VTEC, why is this important to know?

A

Cherry Orchard
Any VTEC positives are sent out to cherry orchard e.g. in the Mater where nothing was done once detected -> ID’d molecularly and then sent straight out to reference

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4
Q

What is the main issue with the molecular detection of VTEC/EHEC on the Enteric bio?

A

The EntericBio only detects if the Stx1 or 2 genes are present
It cant distinguish between these two genes
ie. will only tell you shiga toxin gene detected

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5
Q

How did we used to culture VTEC, why do we no longer use this?

A

Used to use CTSMAC agar
Commensal E. Coli and E. Coli 0157 will grow on CTSMAC -> agar designed for O157
Typical E. Coli = sorbitol fermenter = pink colonies
O157 = non-sorbitol fermenter = colourless
This agar was too selective/too harsh for many other serotypes of shiga-toxin producing E. coli other than O157

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6
Q

What is the preffered agar for STEC now?

A

STEC Chromagar:
- wil grow most of the common STECs as well as oher enterobacteriacae

-STECs = mauve
- Enterobacteriacae = colourless, blue or inhibited

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7
Q

What is the conventional micro way of detecting salmonella?

A

Faeces sample
Selective enrichment in selenite broth
XLD or DCA agar
Biochemical ID
Serotyping

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8
Q

What is the contemporary micro way of IDin salmonella

A

Direct detection using Real time pcr on the entric bio

Or syndromic testing using the biofire GI panel

Detection of the InvA gene

Reference lab in NUIG

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9
Q

What is the selective enrichment used for Salmonella, how does this work, pros and cons?

A

Selenite broth
Works by surpressing gut flora while simultaenously enriching for Salmonella
Can enrich from as little as a single bacterial cell
Only downside is that it takes days

NB: this is the gold standard for Salmonella
-> we did this in the Mater before plating

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10
Q

For what salmonella strains do you need a cat 3

A

Salmonella typhi and salmonella paratyphi

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11
Q

In Salmonella enteric fever, bacteria often spreads to other locations other than the gut, what other specimens might be seen with this disease and how do we isolate from them?

A

In enteric fever Salmonella typhi might be found in blood cultures, faeces, urine or even bone marrow aspirates

Since these are usually sterile sites there is less of an isse culturing from these

Salmonella will normally grow from these on normal plates in normal incubation etc

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12
Q

Talk about XLD agar for Salmonella

A

Xylose lysine deoxycolate

Lactose fermenters/commensals = yellow
Shigella = xylose lysiene = red
Salmonella = red with black centres due to H2S+ production

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13
Q

Talk about DCA for Salmonella

A

Lactose fermenters/Commensals grow pink
NLFs are colourless
Shigella = NLF => pale
Salmonella NLF = pale as well except H2S production => black colonies

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14
Q

Talk about the conventional micro method of detecting shigella

A

Faeces sample
Selective culture on day 2
XLD or DCA agar
Biochemical ID on Day 3
Serotyping on Day3

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15
Q

Talk about the contemporary micro method of IDing shigella

A

Direct detection using real time PCR on the Enteric Bio
Or syndromic testing on the biofire GI panel
Detection of the Ipa gene
DNA can be detected as early as day 1

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16
Q

What is the gene target for salmonella on the enteric bio

A

InvA gene

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17
Q

What is the gene target for Shigella on the Enteric Bio?

A

Ipa gene

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18
Q

What is the issue with detection of the Ipa gene on the EntericBio?

A

EntericBio cannot differentiate between Shigella and VTEC/STEC etc

Any positives have to be confirmed with biochemical and serotyping

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19
Q

Talk about DCA agar for Shigella, what makes it selective vs differential

A

Deoxycholate Citrate Agar
Selective for Shigella through sodium deoxycholate and sodium citrate
Differential through lactose and sodium ferric chloride

Lactose fermenters/commensals = pink
NLFs = colourless
Shigella = NLF = pale
Salmonella NLF = pale
H2S pos Salmonella = pale with black centre

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20
Q

Talk about XLD for Shigella flexneri

A

Selective through sodium deoxycholate
Differential through xylose, lysine and H2S

Lactose fermenters/commensals = yellow
Shigella = xylose lysiene red
Salmonella = red with black centres

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21
Q

How has our detection of Salmonella and Shigella changed?

A

Moving away from culture based methods to molecular type screens

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22
Q

For all enteric pathogens, what are the different kinds of PCR available, give examples of each

A

Real Time PCR:
- Enteric Bio C. diff / Enteric Bio Gastro Panel
- GeneXpert C. diff
- BD Max

End Point PCR array:
- Xtag GPP

Real Time PCR Array:
- Multiplex
- Film array

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23
Q

Pros and cons of molecular based methods

A

Improvements in specimen workflow -> streamlines the whole process of enterics
Throughput - EntericBio can run multiple specimens at a time
Patient population
Ability to leverage existing molecular resources in the lab

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24
Q

Three kinds of molecular detection methods for enterics

A

Individual pathogen detection assay
Multiplex molecular enteric panels
Syndromic panels

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25
Talk about the EntericBio for enterics
Realtime detection of pathogens directly from faeces No pre-enrichment of samples needed DNA extraction, amplification and detection on day 1 Rapid results taking only 3 hours on day 1
26
What platform is used along side the entericbio
Roche light cycler 480 6 detection channels for single or multiplex Primers and Taqman probes Limited targets available to multiplx (entericbio) 5-6 targets
27
What platform is used along side the entericbio
Roche light cycler 480 6 detection channels for single or multiplex Primers and Taqman probes Limited targets available to multiplx (entericbio) 5-6 targets
28
What are the four panels on the entericbio, what is the benefit of these?
GP1 GP2 Clostridium difficile Norovirus Seperate panels allow for chopping and changing as needed, to suit each hospitals needs
29
How does GP1 differ from GP2?
GP2 includes cryptosporidium and giardia
30
What is the enteric bio gene for campylobacter?
ssrA gene
31
What is the enteric bio gene for salmonella?
invA gene
32
What is the enteric bio gene for shigella?
ipa gene
33
What is the enteric bio gene for VTEC?
stx 1 and stx 2
34
What is the enteric bio gene for c. diff?
tcdB
35
What is the enteric bio gene for c. diff?
tcdB
36
How are results read on the entericbio PCR?
You have access to the ct value and amplification curve Typical sigmoidal amplification curve
37
What is the main problem with finding information on the entericbio
EntericBio is widely used platform worldwide but there are very few papers wrote on its used, some with very limited sampling
38
How does entericbio compare to routine culture
Enteric bio has higher sensitivity - higher deterction rate for campylobacter, shigella, campylobacter and e coli o157 - additional 17 positive samples on entericbio with reduced TATs Highly specific >99% for all pathogens Excellent for Salmonella, VTEC, Giardia and crypto
39
What does the entericbio struggle with
In one study it was seen to miss 32 salmonella (19%) when compared to enrichment broth for salmonella Difficult to confirm if camps detected are true positives as a lot didnt grow Very low numbers of shigella positives were confirmed-> issue considering shigella should be notified to public health A lot of the crypto detected never had a parasite test reuested - what do you do with a positive result with no test reuest
40
In general what should be done with entericbio positives?
they should be confirmed by culture -> think of the slopes we put up for salmonella etc
41
What are the limitations of the entericbio?
Clinical details and other lab results must be used to interpret ressults - colonisation vs infection Qualitative not quantitiative - only tells us if present or not Positive PCR does not indicate presence of viable organism samples submited for test of clearance/cure have to be put up on routine cultre methods Does not differentiate between certain targets Detection of target is dependent upon specimen collection, transport, storage, handling, preparation etc mutation in primer binding/probe bonding site can cause false negatives Strongly positive targets present in sample can interfere with detection of other low concentration targets
42
What can entericbio not differentiate between
Shiga toxin stx1 or stx2 cryptosporidium species Campylobacter species IpaH gene of shigella vs enteroinvasive E. coli
43
Talk bout the geneXpert for enteics
Qualitative real-time polymerase chain reaction Rapid and automated Used for C. diff Cycle threshold Ct value <36 = positiv If sample processing control (SPC) fails the result is invalid
44
What is the gene xpert epi: Cepheid
Targets three genes: - toxin B gene (tcdB) - binary toxin gene (cdt) - tcdC gene deletion at nt 117 It is for the presumptive ID of ribotype 027 -> not used anymore
45
Talk about the pros and cons of the cepheid genexpert
Much more sensiive then EIAs for c diff but your overcalling a lot of them positive
46
Talk about multiplex GI platforms
Syndromic testing Broad coverage of organisms withou need to select specific tests Enhanced ability to detect co-infections Increased sensitivity Higher throughput Reduced sample volume requirements - not really, still need pea sized amount which would be sufficient to inoculate a plate
47
What is end point PCR array
A form of multpilex PCR Hybridisation to array e.g. xTAG Gastrointestinal pathogen panel
48
Talk about th xTAG, what is it?
Detection of a broad range of virus, bacteria and parasites in a single test Internal control = bacteriophage MS2
49
Talk about Xtag, how does it work
Pre-PCR: - Sample pre-treatment - Nucleic acid extraction and purification - Multiplex PR and reverse transcription Post-PCR: - bead hybridisation and detection - data acquisitoin and analysis by Lumminex - detection
50
What are the pros and cons of Luminex xTAG
Pros: - 100ul fresh or frozen stool - stool colleted in transport medium e.g. Cary-Blair - 24 samples can be completed in approximately 5 hours Cons: - open system - separate molecular areas - risk of amplicon contamination
51
What is the transport medium used for faeces, pros and cons?
Cary-Blair Great for shigella isolation Impossible to tell if sample is liquid or not
52
Talk about xTAG compared to culture
High sensitivity for all pathogens >88% for mot Specificity moderately high for most Specificity for salmonella only 60% - overcalls a lo of these positives
53
Talk about the Biofire for enterics
Less hands on time User friendly to perform Reduces the requiremen for highly trained personnel Automated recording and reporting of results they are often less subjective 23 targets but low sample throughput
54
Compare the biofire to culture
5 false negatives and 5 false positives
55
What is the main question with pcr positives but culture negatives
Is it really a significant infection if your not able to culture it
56
What is the main question with pcr positives but culture negatives
Is it really a significant infection if your not able to culture it
57
How do multiplex panels affect the patient, compare to culture
Much more sensitive Much broader range of GI pathogens Reduced turn around time Patients received targetted therapy -> less broad spectrum Stopped antimicobrials in STEC patients etc etce Enhanced detection - lots of false negatives by culture Parasite detection much higher Much less imaging needed for patients - reduction in health service costs
58
How can multiplex PCR be used in infection control
regulating use of isoltion rooms (a major cost on HSE) through the use of negative predictive value -> have to wait for a negative to leave room etc Reduces time needed to de-isolate patient - film array = 1.5 days earlier film array group had much higher antimicrobial use but more targetted therapy - reducing isolation stay etc Overall improved operational capacity of the hospital and flow of patients
59
What are the five pros of the film array
Greater sensitivity than culture Rapid TAT Target therapy Reduced time to treatment Value of the NPV
60
What are the main limitations/cons of the film array?
Clinical relevance of co-infections Differentiation of carrier vs active infection Over prescribing unnecesary therapy Potential for contamination Changes in test performance over time as new strains emerge Cost-effectiveness

4.1 Medical Microbiology (30 decks)

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