Diagnosis of Viral Infections Flashcards

Question

Diagnosis by antibody detection - how can this be made

Answer 1

Diagnosis can be made by Detection of IgM (can be non specific) ``` Or by demonstration of seroconversion - Negative IgG antibody at first - Then presence of IgG antibody ```

Answer 2

Indirect detection of the pathogen Diagnostic mode of choice for organisms which are refractory to culture

Answer 3

Detect an antibody response in symptomatic patients Determine if vaccination has been successful Directly look for antigen produced by pathogens

Answer 4

Serological tests are not limited to blood & serum - can also be performed on other bodily fluids such as semen and saliva

Answer 5

Produced from processing blood: Blood is coagulated with micronized silica particles Gel used to trap cellular components

Answer 6

Routinely serum tubes are centrifuged for 10 min at 1000xg Supernatant (serum) is removed and stored 4ºC short term -20ºC long term Routinely serum tubes are centrifuged for 10 min at 1000xg

Answer 7

Serum contains proteins, antigens, antibodies, drugs (some) and electrolytes

Answer 8

No past or current infection or immunisation = Negative Acute/recent infection = Positive Resolved infection or immunisation = Negative

Answer 9

No past or current infection or immunisation = Negative Acute/recent infection = Negative/Positive Resolved infection or immunisation = Positive

Answer 10

The level and type of antibody varies | with both the pathogen, exposure and time

Answer 11

Serology: Detection of antibody and or antigens Usually by enzyme immunoassays e.g. ELISA or related technology e.g. microparticle immuno-chemiluminescence

Answer 12

This is useful for some infections such as Hepatitis B HIV Hepatitis C This is because it allows us to establish whether acute or chronic infection This may have therapeutic implications

Answer 13

Combined antigen and antibody 4th Generation HIV tests detect both antibody and antigen in as assay. Results in increased sensitivity and a reduced window period (time between infection and being able to get a positive result).

Answer 14

Nucleic acid amplification (NAAT) e.g. PCR although there are other examples Can detect RNA or DNA Ability to multiplex using fluorescence probes i.e. can look for several targets in one sample May be qualitative or quantitative Requires nucleic acid extraction prior to the amplification

Answer 15

May be automated Highly sensitive and specific, generates huge numbers of amplicons Rapid Useful for detecting viruses to make a diagnosis - At first time of infection e.g. measles, influenza - During reactivation e.g. cytomegalovirus Useful for monitoring treatment response - Quantitative e.g. HIV, HBV, HCV, CMV viral loads

Answer 16

May detect other viruses which are not causing the infection Exquisitely sensitive and so may generate large numbers of amplicons. This may cause contamination. Need to have an idea of what viruses you are looking for as will need primers and probes that are specific for that target.

Answer 17

Different chemistries but all similar Real time as amplification AND detection occur in REAL TIME i.e. simultaneously by the release of fluorescence Avoids the use of gel electrophoresis or line hybridisation Allows the use of multiplexing

Answer 18

Multiplex PCR is the term used when more than one pair of primers is used in a PCR. It enables the amplification of multiple DNA targets in one tube e.g. detection of multiple viruses in one CSF specimen e.g. HSV1, HSV2, VZV, enterovirus, mumps virus

Answer 19

Taqman probe complementary to region of interest, binds between primers. Oligonucleotide probe with a fluorescent reporter at the 5’ and a quencher at the 3’. The quencher prevents the reporter fluorescing when excited if in close proximity

Answer 20

Taqman probe hybridizes to the region of interest This occurs during the annealing phase of PCR Fluorescence is prevented due to the proximity of quencher

Answer 21

Taq polymerase extends from the 3’ end of primer as normal. The Taq possesses 5’-3’ nuclease activity and hydrolyses the probe. The reporter is removed from the quencher and fluorescence can be detected.

Answer 22

For any given cycle within the exponential phase, the amount of product, and hence fluorescence signal, is directly proportional to the initial copy number

Answer 23

Relative fluorescence can be plotted against the number of cycles This can be used to determine relative concentrations of DNA present by construction of standard curve using standards of known concentration.

Answer 24

Some substances inhibit PCR e.g. haem, bile salts. Assays should always include an internal positive control as results could incorrectly be reported as negative. The IC can be anything as long as RNA/DNA respectively depending on nature of target. Include primers specific for the internal control material.

Answer 25

Used to predict response to anti-virals e.g. for HIV in Rx naïve patients, or if clinical suggestion of resistance in drug experienced patients Useful for outbreak investigation by showing identical sequences in suspected source and recipient

Answer 26

Consensus sequence based on clinical observation of resistance or in vitro evidence

Answer 27

Minority species sequencing | May be selected by treatment

Answer 28

``` Antibody and antigen detection for initial diagnosis Screening test (EIA) Confirmatory test (EIA) ``` ``` Viral load(NAAT) at baseline and to monitor treatment response - Quantification of virus in blood ``` Resistance testing (sequencing)

Answer 29

Multiple viral enzyme targets Reverse transcriptase, protease, integrase, viral receptor binding proteins)

Answer 30

Testing for specific infections in at risk groups e.g. HIV, HBV and HCV Testing because it may have an implication for others e.g. antenatal HIV, HBV, rubella In these situations the patients are asymptomatic Needs a sensitive screening test

Answer 31

May have some false positives, so need | A specific confirmatory test

Diagnosis of Viral Infections Flashcards

(55 cards)