Pathogen Detection & Diagnosis

Question 1

List which viral infections may be diagnosed by electron microscopy

Answer 1

• useful for faecal and vesicle samples
• SarsCoV 2
• herpes simplex
  1. specimen dried on grid
  2.urynal acetate staining (heavy metal)
  3. antibody application = concentration of sample
  4. electron beams produce images (3D, monochrome, higher resolution)

📈: visualise virus quickly, rapid, detect viruses that cannot be grown in culture
📉: cannot differentiate between viruses from same family (low specificity) high maintenance and skilled worker required, low sensitivity

Question 2

Describe the principles of serologic diagnosis of viral infections

Answer 2

• detection in symptomatic treatments or successful vaccination treatment
  (antibody detection)
  IgM used for mostly non-specific (false positives)
  igG⬆️ as IgM ⬇️
• ELISA [hepaptitis A]
• abbot architect (serology machine)
• micropartical immuno-chemiluminescence
• may not grow in every cell line
• can analyse cytopathic effects (changing morphology of host cell)
• used in combination with EM

Question 3

Describe the principles of antigen detection and PCR for diagnosis of viral infections

Answer 3

• individual capsomeres (proteins made during viral replication)
• be identified in different specimens
• influenza (RSV)
• vesicles (herpes simplex)
• faeces (rotavirus, adenovirus)
• nucleic acid detection test
• direct cell immunofluorescence [flourochrome tagging]
• immunoassays
• lateral flow [rapid / point of care]
• ELISA (immobilising antigens w/ antibody)
• sandwich (hepatitis B)

Question 4

Describe the methods employed for molecular diagnostic tests

Answer 4

• PCR / NAAT
• nucleic amplification
• strand displacement amplification
• detect RNA/DNA
• multiplex [real time PCR/ looking for multiple virus with primers from one sample]
• automated / point of care/ high sensitivity = quicker diagnosis
• cytomegalovirus (chronic= reactivation) immunocompromised can be detected
• useful to monitor treatment (what quantity of viral nucleic acid is present before/during/after treatment?

📉 = amplicons contamination = potential false positives
- you have to know what you’re looking for [primers must bind to target]
- sudden target mutations

Question 5

list some methods of detecting and diagnosing viruses

Answer 5

• electron microscopy
• cell culture virus isolation
• antigen detection
• serology => antibody detection
• genotype sequencing (detecting antiviral resistance)

Question 6

why can’t some viruses be detected in cell culture?

Answer 6

specific viruses grow with specific cell lines which might not be available in cull culture

Question 7

Describe the methods employed for detection of antiviral resistance

Answer 7

eg: HSV, CMV, hepatits-C
- genome sequencing can detect antiviral resistance expression [looking for known mutation which ⬆️ resistance]