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

Gastrointestinal Tract Infections: Laboratory Detection Flashcards

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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
Q

Talk about the EntericBio for enterics

A

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
Q

What platform is used along side the entericbio

A

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
Q

What platform is used along side the entericbio

A

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
Q

What are the four panels on the entericbio, what is the benefit of these?

A

GP1
GP2
Clostridium difficile
Norovirus

Seperate panels allow for chopping and changing as needed, to suit each hospitals needs

29
Q

How does GP1 differ from GP2?

A

GP2 includes cryptosporidium and giardia

30
Q

What is the enteric bio gene for campylobacter?

A

ssrA gene

31
Q

What is the enteric bio gene for salmonella?

A

invA gene

32
Q

What is the enteric bio gene for shigella?

A

ipa gene

33
Q

What is the enteric bio gene for VTEC?

A

stx 1 and stx 2

34
Q

What is the enteric bio gene for c. diff?

A

tcdB

35
Q

What is the enteric bio gene for c. diff?

A

tcdB

36
Q

How are results read on the entericbio PCR?

A

You have access to the ct value and amplification curve
Typical sigmoidal amplification curve

37
Q

What is the main problem with finding information on the entericbio

A

EntericBio is widely used platform worldwide but there are very few papers wrote on its used, some with very limited sampling

38
Q

How does entericbio compare to routine culture

A

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
Q

What does the entericbio struggle with

A

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
Q

In general what should be done with entericbio positives?

A

they should be confirmed by culture -> think of the slopes we put up for salmonella etc

41
Q

What are the limitations of the entericbio?

A

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
Q

What can entericbio not differentiate between

A

Shiga toxin stx1 or stx2
cryptosporidium species
Campylobacter species

IpaH gene of shigella vs enteroinvasive E. coli

43
Q

Talk bout the geneXpert for enteics

A

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
Q

What is the gene xpert epi: Cepheid

A

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
Q

Talk about the pros and cons of the cepheid genexpert

A

Much more sensiive then EIAs for c diff but your overcalling a lot of them positive

46
Q

Talk about multiplex GI platforms

A

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
Q

What is end point PCR array

A

A form of multpilex PCR
Hybridisation to array
e.g. xTAG Gastrointestinal pathogen panel

48
Q

Talk about th xTAG, what is it?

A

Detection of a broad range of virus, bacteria and parasites in a single test
Internal control = bacteriophage MS2

49
Q

Talk about Xtag, how does it work

A

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
Q

What are the pros and cons of Luminex xTAG

A

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
Q

What is the transport medium used for faeces, pros and cons?

A

Cary-Blair

Great for shigella isolation
Impossible to tell if sample is liquid or not

52
Q

Talk about xTAG compared to culture

A

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
Q

Talk about the Biofire for enterics

A

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
Q

Compare the biofire to culture

A

5 false negatives and 5 false positives

55
Q

What is the main question with pcr positives but culture negatives

A

Is it really a significant infection if your not able to culture it

56
Q

What is the main question with pcr positives but culture negatives

A

Is it really a significant infection if your not able to culture it

57
Q

How do multiplex panels affect the patient, compare to culture

A

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
Q

How can multiplex PCR be used in infection control

A

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
Q

What are the five pros of the film array

A

Greater sensitivity than culture
Rapid TAT
Target therapy
Reduced time to treatment
Value of the NPV

60
Q

What are the main limitations/cons of the film array?

A

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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