Systems for Detection of Pathogens II

Question 1

What is the purpose of molecular gene targeting in the detection of pathogens?

Answer 1

Aims to detect a gene or gene products that are pathogen specific via nucleic acid amplification techniques (NAAT) such as PCR

Question 2

Using PCR to detect pathogens

Answer 2

• PCR will detect RNA/DNA in a sample
• amplifies genome by using primers
• primer is developed against a specific gene in an organism and fluorescently tagged → if organism present, fluorescence shows up under UV

Question 3

Pathogens detected by PCRs include…

Answer 3

Influenza
Norovirus
MRSA
HIV
Hepatitis B
Hepatitis C
Mycobacterium tuberculosis
CMV
EBV

Question 4

What is real-time PCR?

Answer 4

Along with PCR reagents an organism specific fluorescent DNA probe is also added which can be detected in real time PCR machine simultaneously during the amplification process

Question 5

What does real-time PCR measure?

Answer 5

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

• more fluorescence= more amplicon product
• less fluorescence= less amplicon product

Question 6

What is a molecular gene targeting technique used for N.gonorrhoea and C.trachomatis?

Answer 6

Strand Displacement Amplification (SDA)

• alternative to PCR (very similar)
• also uses primers which anneal to target and fluoresce

Question 7

What do we need to consider when targeting a gene to detect the presence of a pathogen?

Answer 7

SENSITIVITY

• look for a gene which has more than one copy inside that particular pathogen
• more copies in an organism= more sensitivity and fewer organisms needed to suggest disease

SPECIFITY
-look for a gene only in that organism (not in host)

RELIABILITY
-look for a gene which is non-essential and not transmissible

ACCURACY
-accuracy of detection system (e.g. is it susceptible to genomic shifts/mutations?)

RAPIDITY

• result generated gives an indication to how serious it is
• instant bedside?
• same day?
• next day?
• next week?

Question 8

What is a microarray and how is it used in detection of a pathogen?

Answer 8

Glass slide with thousands of different kinds of short oligonucleotide probes fixed to it in a tightly spaced array or grid
-labelled DNA/RNA molecules hybridise to probes, detecting specific genes (multiple gene targets)

1) Label control DNA green and pathogenic DNA red
2) Hybridise onto microarray
3) Red signal= more pathogenic DNA
Green signal= more control DNA
Yellow signal= balance

Question 9

What is a tiled microarray?

Answer 9

Like traditional microarrays, they function by hybridising labeled DNA or RNA target molecules to probes fixed onto a solid surface.

However, these are not just used for gene detection, but also for profile gene expression levels.

Question 10

Advantages of tiled arrays

Answer 10

· covers the whole genome
· strand dependent
· can be used for RNA and transcriptomics
· can look for microRNA

Question 11

How do we analyse expression of genes from a tiled microarray?

Answer 11

Expression Analysis

-analyse how certain genes have genes switched on/off

Question 12

Molecular signatures

Answer 12

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

Depending on the size of the target, this can be carried out by:
>single gene target (PCR/qPCR)
>multiple gene target (microarray)
>mass spectrometry (MALDI-TOF)→ looks at what the gene is producing

Question 13

What is biosignature profiling?

Answer 13

A technique used to detect for pathogens not necessarily by looking at DNA, but rather at what the organism is producing e.g. something on its cell surface or a metabolite that is unique to that organism

Question 14

How is biosignature profiling carried out?

Answer 14

Mass Spectroscopy (MALDI-TOF)

1) Take a sample
2) Put into a laser which ionises it
3) When it is ionised, it breaks up and pushes up against the accelerator
4) When it goes to the detector, it takes charge and deflects in one way depending on its charge
5) The detector will produce a peak which will be specific for certain small fragments, making a pattern
6) Pattern produced is compared to reference database to identify the pathogen

Question 15

Advantages of Mass Spectroscopy (MALDI-TOF) Profiling

Answer 15

Rapid

Specific Identification

Question 16

Disadvantages of Mass Spectroscopy (MALDI-TOF) Profiling

Answer 16

• Requires pure culture
• Requires rigorous calibration and protocol standardisation
• Will only identify known profiles (from archival database)

Question 17

What are biomarkers of virulence?

Answer 17

genes or gene products by which pathological disease can be identified

Question 18

How are biomarkers of virulence used to detect pathogens?

Answer 18

Look for selected genes or gene products that drive the disease process:

• Latex Agglutination test
• Serology by ELISA
• Toxin Detection

Question 19

Latex agglutination test

Answer 19

method which detects certain antigens or antibodies in a variety of bodily fluids such as blood, saliva, urine or cerebrospinal fluid

The sample to be tested is sent to the lab where it mixed with latex beads coated with a specific antigen or antibody

If the suspected substance/organism is present, the latex beads will clump together (agglutinate)

Question 20

Serology by ELISA

Answer 20

e.g. paired sera for influenza virus antibodies to compare the different types of antibodies against different types of antigens

Question 21

Toxin Detection

Answer 21

Looking for the toxin an organism produces via:

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

Question 22

Advantages of using biomarkers of virulence to detect pathogens

Answer 22

Good specificity
Good sensitivity
Easily automated

Question 23

Disadvantages of using biomarkers of virulence to detect pathogens

Answer 23

• Serological response is not rapid (10-14 days to produce antibodies), therefore not useful in acute infections
• Single sera results are meaningless due to possible previous exposure- may have already encountered the disease but doesn’t mean you have the disease now; however, if you titre antibodies and quantify, and if antibody increases in a specific time period you are making antibodies and you have the disease now (paired sera results)
• Some antibodies are cross-effective
• Virulence is only INFERRED by the presence of a biomarker
• ONLY in vivo testing of cultured pathogen infected into an animal can prove virulence

Question 24

Rapid Next Generation Sequencing to detect pathogens

Answer 24

• Rapid next generation sequencing gives a lot of data
• There are many different ways of sequencing all done very quickly; however it produces a lot of data to be analysed
• sequencing can show differences between single bases (e.g. SNPs) in strains or resistance mutations to antibiotics

Question 25

Purpose of sequencing to detect pathogens

Answer 25

We infer from the DNA sequences which will tell us theoretically what is happening. For example, in antibiotic resistance, we may be sequencing to see which organisms are resistant to a particular antibiotic. It gives an idea of what is happening in the body, but it doesn’t give the PHENOTYPE (only gives the genotype). The only way of seeing effect on phenotype is the traditional method of taking the organism and seeing if it kills the antibiotic in culture.

Question 26

Advantages of sequencing (molecular detection methods) to detect pathogens

Answer 26

· Rapid · Faster detection of pathogens than traditional techniques · Allows appropriate, timely antimicrobial therapy and infection control interventions · Increased sensitivity over culture and microscopy-based techniques in POSITIVE samples · Can be automated and has potential for Point of Care Testing

Question 27

Disadvantages of sequencing (molecular detection methods) to detect pathogens

Answer 27

· Expensive= look for multiple bases when you may only be looking for one base · Does not screen for UNKNOWNS= sequence blind- not sure what you are actually looking for · Requires expertise · Labour intensive · Possibility of contamination= important to have pure sample · Require complex and efficient methods of extraction of nucleic acid (e.g. small bacterial DNA in human blood→ most of the sequencing will be human DNA) · NEGATIVE samples may STILL need gold standard culture

Question 28

Future techniques to detect pathogens

Answer 28

Bio-signature Profiling Metabolic Profiling Rapid Point of Care Testing

Question 29

Future bio-signature profiling technique to detect pathogens

Answer 29

- Take an active disease and see which genes are switched on when a patient has this disease - Also, look at what is being made, and biochemical problems that occur dependent on the disease - A profile is then made- this makes it easier to identify a disease based on what has already been observed or tested for - It can also be used to predict which patients are at higher risk

Question 30

Future metabolic profiling technique to detect pathogens

Answer 30

- Looking at how the whole body has reacted to a specific disease (not only looking at gene changes)→ e.g. smell of someone - For example- bees were trained to know which patients had TB. This is because tuberculosis patients had a specific genetic peak for a specific smell that was a volatile fatty acid only made in TB patients

Question 31

Rapid Point of Care Testing

Answer 31

Be able to buy tests to tell what disease you may be showing symptoms of. -lab on a chip -nanotechnology -microfluidics -rapid sequencing of samples direct at the bedside DNA extraction, PCR and sequencing on site However, this may not be the way forward as it should still be in the hands of the trained professionals. Therefore, the debate circling around is: - Who will do the analysis? - Where? - How will the testing be controlled? - Who will interpret the results? - When does the science become medicine?