System for detection of pathogens I

Question 1

How can we define a pathogen?

Answer 1

1. Commensal Non pathogen (in host)
   PRESENT but NOT CAPABLE of causing disease in the host
2. Zoonotic Non pathogen (in carrier)
   PRESENT but only CAPABLE of causing disease in ANOTHER host
3. Commensal Opportunist (in host)
   PRESENT and CAPABLE of causing disease in the host but only in certain circumstances

→ Not all positive samples are diagnostic of active disease

Question 2

What are some advantages and disadvantages of microscopy?

Answer 2

Advantages:
→ Easy to perform
→ Rapid screening
→ Some parasites have SPECIFIC morphology
eg. Schistosoma mansonii
→ Specific Immunoflourescence staining possible

Disadvantages:
→ Not Sensitive
eg. Mycobacterium tuberculosis
→ Screening sputum smears requires
→ At least 10,000 orgs per ml to be visualised
→ General stains are not specific
→ Labour intensive (expensive)
→ Requires specialist interpretive expertise (more expensive)

Question 3

What types of media are there (for growing colonies etc)?

Answer 3

Media:

→ Non Selective Media
eg. Blood Agar

→ Semi Selective Media
eg. MacConkey Agar, DCA, CLED

→ Selective growth temperatures
eg. Campylobacter specie

Question 4

What are some advantages and disadvantages of Classical culture and identification?

Answer 4

Advantages:
→ Cheap simple, reliable reagents
Sensitive
eg. Single organisms can be grown and identified

→ Validated specificity
eg. ‘Gold Standards’ with multiple parameters

→ Direct in vivo measurement of effectiveness of therapy
eg Antibiotic sensitivity

→ Easily archived
eg. Epidemiology

Disadvantages
→ Some pathogens cannot be grown eg. Mycobacterium leprae
→ Some pathogens cannot be well differentiated by biochemistry alone
→ Slow: culture requires at least overnight incubation:
→ Viral = 3-10 days
→ Mycobacterial = 6-12 weeks
→ Some pathogens grow too slowly to aid rapid diagnosis
eg. Mycobacterium tuberculosis
→ Labour intensive (expensive)
→ Requires specialist interpretive expertise (more expensive

Question 5

What does quantitative PCR (qPCR) measure?

Answer 5

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

Question 6

How does Multiple gene targeting (Microarrays) work?

What are some advantages?

Answer 6

Don’t use so much anymore as technology has progressed but:

Ordered short oligonucleotide probes (40-70mer) attached to slides in defined
spots.
Each spot represents a single gene
Comparative Genomic Hybridisation (CGH) used mostly for DNA

Depends on if genes stick to a spot, produce a different colour (ie red/yellow/green)

Advantages
→ Covers the whole genome
→ Strand dependant
→ Can be used for RNA and Transcriptomics (can use microarrays to tell if an organism is just resting/fighting off toxins etc)
→ Can look for microRNA

Question 7

How does Mass Spectrometry using MALDI-TOF (Matrix Assisted Laser Desorption Ionization-Time-of-Flight) work? What are some advantages and disadvantages?

Answer 7

(Looking at proteins within organisms)

Isolate organism
Lyse with crystalizing matrix
Ionise and detect time of flight for each particle
Calculate Mwt (Daltons) for each protein produced

proteins are broken up and travel down towards a detector and can be measured according to size, when particles hit the detector, this creates a peak. the pattern you get from the peaks depends on which proteins you have. Then you match up profiles using a database.

Advantages:
Rapid
Specific Identification

Disadvantages:
Requires pure culture
Requires rigorous calibration and protocol standardisation
Will only identify known profiles (requires a huge database beforehand)

Question 8

What is the latex agglutination test? (biomarker of virulence)
What are some advantages and disadvantages of using biomarkers of virulence?

Answer 8

You have some dots and add an antibody on one of the dots, this will cause agglutination on just that dot to confirm that it contains a specific antibody.

Latex agglutination test uses particles coated with specific antibody to cell wall antigens eg CSF Direct Agglutination test

not super sensitive but very specific

Specific cell wall antigens are predictive of invasiveness and virulence

we can also serotype with ELISA once agglutinated to identify specific organisms

we can also look for what toxins the organism makes- using toxin detection

Advantages:
Good Specificity
Good Sensitivity
Easily automated

Disadvantages:
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 biomarker
ONLY in vivo testing of cultured pathogen
infected into an animal model can prove virulence

Question 9

What are some overall advantages and disadvantages for molecular detection methods?

Answer 9

ADVANTAGES:
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

DISADVANTAGES:
Expensive
Does not screen for UNKNOWNS
Requires expertise
Labour intensive
Possibility of contamination
Require complex and efficient methods for extraction of nucleic acid
NEGATIVE samples may STILL need Gold Standard culture
Hospitalization costs accounted for 95% of health-system costs
among patients suspected of tuberculosis. In culture-negative
patients, PCR tests do not significantly decrease time to tuberculosis
exclusion.

Question 10

Summary

Answer 10

Any new systems for detecting pathogens MUST be:

→ Reliable, Sensitive, Specific & preferably Rapid
→ Applied to the correct specimen
→ Must derive from a large reference database
→ Constantly updated with new species or variants
→ Must be as good or better than the gold standard
(direct culture