diagnosis of viral infections

Question 1

what is electron microscopy

Answer 1

viruses visualised with electron microscope
replaced by molecular techniques
still useful for faeces and vesicle specimens
useful in characterising emerging pathogens

Question 2

procedure of electron microscopy

Answer 2

1. specimens dried on grid. can be stained with heavy metal
2. can be concentrated with antibody
3. beams of electrons used to produce image
4. wavelength of electron beam = shorter than light = higher resolution than light

Question 3

advantages and limitations of electron microscopy

Answer 3

rapid, detects viruses that cannot be grown in culture, can visualise many diff viruses

low sensitivity, requires maintenance, requires skilled operators, cannot differentiate between viruses of same virus family

Question 4

herpes viruses that cause vesicles

Answer 4

herpes simplex and varicella zoster virus

EM cannot differentiate these diff viruses so depends on clinical context, site of vesicle and symptom

Question 5

virus isolation in cell culture

Answer 5

viruses require host cells to replicate and can cause cytopathic effect of cells when patient sample containing virus is incubated within a cell layer

old technique
led to discovery of hMPV
use diff cell lines in test tubes or plates, selection of cell types important
slow but occasionally useful

Question 6

antigen detection

Answer 6

direct direction of viral antigens
viral antigens = proteins. capsid or secreted proteins. detected in cells or free in blood

these techniques being replaced by nucleic acid detection methods due to improved test performance
variety of methods avl incl
direct immunofluorescence = cell associated antigens

enzyme immunoassay = free soluble antigens or whole virus
immunochromatographic method

Question 7

immunochromatographic methods

Answer 7

flavivirus
arthopod vector
common infection in returning travellers
useful as a near patient test or point of care test

Question 8

ELISA for antigen detection

Answer 8

enzyme linked immunosorbent assay = component of reaction adhered to solid surface

can be indirect, direct or sandwhich

1. plate coated with capture antibody
2. sample added and any antigen present binds to capture antibody
3. enzyme conjugated primary antibody added, binds to detecting antibody
4. chromogenic substrate added, converted by enzyme to detectable form = colour change

Question 9

antibody detection by serology

Answer 9

indirect detection of pathogen
diagnostic method of choice for organism which are refractory to culture

used to:
detect antibody response in asymptomatic
determine if vaccination has been successful
directly look for antigen produced by pathogens
serological tests not limited to blood and serum- saliva and semen too

Question 10

detection by antibody detection

Answer 10

when infected with virus, humoral immune response occurs occurs resulting in production of immunoglobulins

IgM antibodies specific to virus are produced first
IgM present for a variable period 1-3 months
as IgM declines, igG produced
diagnosis made by detection of IgM or demonstration of seroconversion = igG then presence of igG

Question 11

molecular diagnostic tests

Answer 11

nucleic acid amplification = NAAT
pcr
can detect RNA/DNA
ability to multiplex using fluorescence probes
qualititative or quantitative
require nucleic acid extraction prior to amplificaton

Question 12

stages of NAAT

Answer 12

1. specimen collection
2. extraction of nucleic acid
3. dna transcription for rna viruses
4. cycles of amplification of DNA target
5. detection of amplicons - too many amplicons = contamination

Question 13

multiplex PCR

Answer 13

term used when more than one pair of primers is used in PCR

enables amplification of multiple DNA targets in one tube