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Medical Microbiology > Systems of Pathogen Detection II > Flashcards

Systems of Pathogen Detection II Flashcards

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USMLE® Step 1 flashcards Biology 101 flashcards
1
Q

What is the aim of molecular gene targeting?

A

Aim to detect a gene or gene products that are pathogen specific

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2
Q

What are the 2 molecular gene targeting techniques

A
  • Nucleic acid amplification techniques (NAAT)

- Polymerase Chain Reaction (PCR)

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3
Q

What is PCR?

A

PCR is amplifying DNA specifically using primers / oligonucleotides in a cyclical process viewed using banding on gel electrophoresis or fluorescence

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4
Q

How is product amplified in PCR?

A

Two DNA primers (18-20bp) specific for opposite DNA strands, used to amplify DNA region

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5
Q

How is the product visualised in PCR?

A

Product is visualised by fluorescent tags or staining in gels for an amplicon of an exact size

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6
Q

Which pathogens are detecting using NAAT and PCR?

A
  • Influenza/H1N1
  • Norovirus
  • MRSA
  • HIV
  • Hepatitis B
  • Hepatitis C
  • Mycobacterium tuberculosis
  • CMV
  • EBV
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7
Q

What is the role of Quantitative PCR?

A

Measures the speed at which a PCR amplicon product accumulates by the amount of fluorescence released

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8
Q

What is the advantage of using PCR?

A

PCR provides specificity and a good approximation of quantitation

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9
Q

What is SDA?

A

Strand Displacement Application (SDA) is similar to PCR; uses primers and fluorescence as well

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10
Q

Which pathogens are identified using SDA?

A

Used to identify N. gonorrhoea and C. trachomatis

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11
Q

Which genes are suitable targets for pathogen detection?

A
  • Constitutive
  • Virulence
  • Antibiotic resistance
  • Pathogenic phenotype
  • Repetitive
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12
Q

How is specificty of a test determined?

A

Is the test unique to the Genus
Species
Type

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13
Q

How do we determine if the test is reliable?

A

If the test tells us if the target is:

  • non-essential
  • transmissible
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14
Q

How do we know how sensitive a molecular test is?

A

By the no. of organisms it takes to suggest disease

  • for every sample type
  • for every host type
  • for every epidemiological niche
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15
Q

What factors determine the accuracy of a molecular test?

A
  • Detecting live organisms

- Susceptibility of detection system to genomic shifts/mutations

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16
Q

Why is the rapidity of a molecular test significant?

A

Is result generated going to be beneficial to patient?
Instant Bedside? - Diagnosis of paediatric meningitis
Same Day? - Transmission/Quarantine
Next Day? - Antibiotic resistance
Next Week? - Chronic persistent infections

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17
Q

How is multiple gene targeting conducted?

A

Microarrays

Comparative Genomic Hybridisation (CGH) used mostly for DNA

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18
Q

Outline how microarrays are used to detect multiple genes

A

Ordered short oligonucleotide probes (40-70mer) attached to slides in defined spots.
Each spot represents a single gene

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19
Q

What info does a microarray provide?

A

Can look for genes and their expression

Some genes may be present but not expressed

20
Q

What are the advantages of using microarrays?

A
  • Covers whole genome
  • Strand dependant
  • Can be used for RNA and Transcriptomics
  • Can look for microRNA
21
Q

What is the use of Microarray expression analysis?

A

Can identify genes over time and how certain expressions change over time
- Some genes may be present to start off with but lost later on

22
Q

What is the purpose of molecular signatures?

A

Aim to detect a gene or gene products that are pathogen specific

23
Q

Outline single gene target signatures

A

Single gene target (PCR)

  • PCR
  • qPCR
24
Q

Outline the multiple gene target signatures

A

Microarray

25
Q

What is the use of Mass Spec?

A

Identifies different components of the organism (not just DNA)

26
Q

What is Mass Spec MALDI-TOF?

A

Mass Spectrometry MALDI-TOF (Matrix Assisted Laser Desorption Ionization-Time-of-Flight)

27
Q

Outline how mass spec MALDI-TOF is conducted

A
  1. Isolate organism
  2. Lyse with crystalizing matrix
  3. Ionise
  4. Detect time of flight for each particle
  5. Calculate Mwt for each protein produced
  6. Compare against database
28
Q

What does each peak represent in mass spec?

A

Each peak is specific for small peptide fragments

29
Q

How does mass spec identify a pathogen?

A

Different organisms produce specific patterns that are compared with known pathogen patterns to identify which it is

30
Q

Outline the advantages of using MALDI-TOF

A
  • Rapid
  • Specific Identification
  • Automated
31
Q

What are the disadvantgaes of using MALDI-TOF mass spec?

A
  • Requires pure culture
  • Requires rigorous calibration and protocol standardisation
  • Will only identify known profiles
32
Q

Define biomarkers of virulence

A

Selected genes or gene products that drive the disease process

33
Q

Outline biomarkers of virulence used

A

We use biomarkers of bacterial cell components e.g. O-antigens, peptidoglycan etc.

34
Q

How does latex agglutination test detect the virus?

A

Latex agglutination test uses particles coated with specific antibody to cell wall antigens

CSF Direct Agglutination test

35
Q

Which viruses are detected using an agglutination test?

A

Neisseria meningitidis Group B Haemophilus influenzae type b Streptoccoccus pneumoniae type 3

36
Q

What do cell wall antigens tell us about a virus?

A

Specific cell wall antigens are predictive of invasiveness and virulence

37
Q

How is the Shiga toxin of E.coli 0157 detected?

A

1) Enterohaemolysis
2) Agglutination with anti-toxin antibodies
3) PCR for the presence of the gene

38
Q

What are the advantages of the 0157 Stx detection method?

A
  • Good Specificity
  • Good Sensitivity
  • Easily automated
39
Q

What are the disadvantages of the E.coli Type O157 detection approach?

A
  • Serological response is slow; not useful in acute infections
  • Single sera results are meaningless as possible previous exposure
  • Some antibodies cross-reactive
  • Virulence only INFERRED by presence of a biomarker
    ONLY in vivo testing of cultured pathogen infected into an animal model can prove virulence
40
Q

How is rapid sequencing conducted?

A

Rather than looking at the assembled product we look at ALL the pieces individually

41
Q

What is the benefit of using direct sequencing to identify pathogens?

A

Sequencing can show differences between SINGLE bases in strains Or resistance mutations to antibiotics

42
Q

What are the 2 types of possible silent mutations?

A
  • Intragenic (between genes)

- Synonymous (not altering coding)

43
Q

Outline the advantages of molecular detection methods

A
  • Rapid
  • Faster detection than traditional techniques
  • Allows appropriate, timely antimicrobial therapy and infection control interventions
  • Increased sensitivity over culture + microscopy based techniques in +ve samples
  • Automated and has potential for Point of Care testing
44
Q

Outline disadvantages of molecular detection methods

A
  • Expensive
    Doesn’t screen UNKNOWNS
  • Requires expertise
  • Labour intensive
  • Contamination
  • Require complex + efficient methods for nucleic acid extraction
  • NEGATIVE samples may STILL need Gold Standard culture
45
Q

What are the future techniques of pathogen detection?

A
  • Bio-signature profiling
  • Metabolic profiling
  • Rapid Intrinsic Fluorescence
  • Rapid Point of Care Testing
46
Q

What does a biosignature profile show us?

A

Following the host transcriptomic profile with microarrays

During active disease can identify which genes are turned ON to create profile

Medical Microbiology (15 decks)

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