Systems For Detection Of Pathogenesis 2

Question 1

What is molecular gene targeting?

Answer 1

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

Nucleic acid amplification techniques (NAAT)

Polymerase chain reaction (PCR)

Question 2

What is quantitative PCR?

Answer 2

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

Question 3

Which genes are suitable targets?

Answer 3

Constitutive

Virulence

Antibiotic resistance

Pathogenic phenotype Repetitive

Question 4

What makes a good molecular gene test?

Answer 4

Specificity

Reliability - non essential? Transmissible?

Sensitivity - how many organisms does it take to suggest disease

Accuracy - do we need to detect liver organisms?

Rapidly

Question 5

What are molecular signatures?

Answer 5

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

• single gene target
• Mass spectrometry

Question 6

How can mass spectrometry be used for bio-signature profiling?

Answer 6

Isolate organism

Lyse with crystallising matrix

Ionise and detect time of flight for each

Calculate Mwt for each protein produced

Compare against archival databases

Question 7

What are the advantages for MALDI TOF profiling?

Answer 7

Rapid

Specific identification

Question 8

What are the disadvantages for MALDI TOF profiling?

Answer 8

Requires pure culture

Requires rigorous calibration and protocol standardisation

Will only identify known profiles

Question 9

What are biomarkers of virulence?

Answer 9

Looking for selected genes or gene products that drive the disease process

Question 10

What are examples of cell wall antigens that are predictive of virulence?

Answer 10

Gram negative cell wall

Question 11

Describe how shiva toxin is detected in E. coli O157

Answer 11

1) Enterohaemolysis

2) Agglutination with anti-toxin antibodies

3) PCR for the presence of the gene

Question 12

What are the advantages of biomarkers of virulence?

Answer 12

Good specificity

Good sensitivity

Easily automated

Question 13

What are the disadvantages of biomarkers of virulence?

Answer 13

Serological response is not rapid - therefore not useful in acute infections

Single sera results are meaningless - due to possible previous exposure

Some antibodies are cross reactive

Virulence is only inferred by the presence of a biomarkers
- ONLY in vivo testing of cultured pathogen
- infected into an animal model can prove virulence

Question 14

What is rapid next generation sequencing?

Answer 14

Look at the whole product instead of the assembled one

Question 15

What are the advantages of using rapid next generation sequencing?

Answer 15

Can show differences between single bases in strains or resistance mutations to antibiotics

Question 16

What are the disadvantages of rapid next generation sequencing?

Answer 16

Not all of the possible mutations are involved in virulence

Silent mutations can be interagency (between genes) or synonymous (not altering coding)

Question 17

What are the advantages of molecular detection methods?

Answer 17

Rapid - Faster detection of pathogens than traditional techniques

Allows appropriate, timely anti microbial therapy and infection control intervals

Increased sensitivity over culture and microscopy based techniques in posative samples

Can be automated and has potential for point of care testing

Question 18

What are the disadvantages of molecular detection methods?

Answer 18

Expensive

Does not screen for unknowns

Requires expertise to interpret

Still requires intensive technical labour

Possibility of contamination

Require complex and efficient methods for extractions of nucleic acid

Negative samples STILL may need a gold standard culture

In culture-negative patients, PCR tests do not significantly decrease time to tuberculosis exclusion.

Question 19

What does the future look like for bio signalling profiling?

Answer 19

Urine after malaria infection using nuclear magnetic resonance for excreted metabolites

Breath urine after tuberculosis infection using gas chromatography for volatile organic compounds

Rapid point of care testing is being developed
- microfluidics
- nanotechnology
- lab on chip

Question 20

How can lab on chip become a reality?

Answer 20

Be able to take a sample and get results through phone

Still being developed