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SEM 4: Medical Microbiology > Diagnosis of Viral Infection > Flashcards

Diagnosis of Viral Infection Flashcards

1
Q

Why is it not always possible to diagnose an infection clinically?

A
  • Most viral infections present with very non-specific symptoms such as fever and coughing
  • This is why it often requires a lab diagnostic test
  • To aid the diagnosis, history, examination and special investigations are needed.
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2
Q

Why is rapid diagnosis of viral infections important?

A

To reduce need for unnecessary tests and inappropriate use of antibiotics.

  • Could leave to antibiotic resistance
  • Key to prevent spread of infection due to viral infections being highly infectious.
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3
Q

What does significance of test results depend on?

A
  • Depend heavily on prevalence in population e.g. HIV.
  • Sometimes false positives and false negatives and it all depends on how common the infection is in the population.
  • The specificity of a test changes with the prevalence of a disease in a population
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4
Q

Why is it important to know the natural history of the pathogen in the type of patient being tested?

A

This will affect test selection and interpretation

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5
Q

Give an example of a test where a consent must be obtained

A

HIV test

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6
Q

What are the 3 different ways of using viral tests?

A
  • Diagnostic: checking if the pt has the virus or not
  • Monitoring: see if the pt needs treatment or not
  • Screening: used when the patient is perfectly fine but what to check if they have any underlying viruses such as in pregnant women
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7
Q

Possible test types

A
  • Electron microscopy
  • Virus Isolation (cell culture)
  • Antigen detection
  • Antibody detection by serology
  • Nucleic acid amplification tests (NAATs and PCR)
  • Sequencing for genotype and detection of antiviral resistance
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8
Q

Why do you need to use a electron microscopy to see a virus?

A

Viruses are a lot smaller compared to other pathogens which can be seen with a light microscopy or even sometimes with the naked eye. Need a magnification of x20000

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9
Q

What techniques have replaced electron microscopy?

A

Molecular techniques

  • Possibly still in useful for faeces and vesicle specimens
  • Also useful in characterising emerging pathogens
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10
Q

Describe the specimen preparation

A
  1. Specimens are dried on a metal grid.
  2. The speciman is then stained with heavy metal such as uranyl acetate.
  3. The specimen can then be concentrated by adding antibodies which will bind to the virus and bring them all together.
  4. Then use a immune electron microscopy to concentrate the virus.
  5. The beams of electrons are used to produce an image.
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11
Q

Why is an electron microscopy better than a light microscopy?

A

The wavelength of electron beams is much shorter than light resulting in much higher resolution than light microscopy and hence why using electron microscopy is better at detecting viruses than light microscopy.

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12
Q

Advantages of electron microscopy

A
  • Rapid
  • Detects viruses that cannot be grown in culture
  • Can visualise many different viruses
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13
Q

Disadvantages of electron microscopy

A
  • Low sensitivity therefore, you need lots of the virus to be there
  • Requires manitenance
  • Requires skilled operators
  • Cannot differentiate between viruses of the same virus family
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14
Q

Viruses that are easy to detect via electron microscopy

A
  • Rotavirus
  • Adenovirus
  • Coronavirus
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15
Q

Rotavirus under electron microscopy

A
  • Causes gastroenteritis
  • Can be seen in faeces
  • Has a wheel like structure
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16
Q

Adenovirus under electron microscopy

A
  • Causes gastroenteritis

- Has an icosahedral shape

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17
Q

Coronavirus under electron microscopy

A
  • Causes gastroenteritis

- Has a crown structure around a dumbbell like structure

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18
Q

Viruses that are harder to detect via electron microscopy

A
  • Astrovirus

- Norovirus

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19
Q

Astrovirus under a microscopy

A
  • Star like structure
  • Star of David
  • Causes gastroenteritis
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20
Q

Norovirus under a microscopy

A
  • Belongs to the calicivirus family

- Most common cause of gastroenteritis

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21
Q

Herpes simplex

A
  • Causes vesicles or blisters
  • It has a ‘fried egg appearance’: when the virulent is inside an envelope. The envelope is formed from the host cell.
  • If the patient has concentrated vesicles just around the lips then it is most likely a cold sore.
  • Hard to differentiate between this and varicella zoster virus under the microscope so depends on the clinical context: site of vesicle and symptoms
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22
Q

Varicella zoster virus

A
  • Causes vesicles or blisters
  • If a patient has vesicles/white rash all over the body and fever, then it is most likely chicken pox
  • Electron microscopes cannot differentiate these different viruses so depends on the clinical context: site of vesicle and symptoms.
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23
Q

Poxvirus

A
  • Often described as looking like a ball of wool
  • There are different types of the poxvirus - smallpox, monkeypox, Orf and cowpox.
  • Using an electron microscope will not be able to determinate the difference between the poxviruses therefore it again depends on clinical context
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24
Q

Why are viruses unable to grow on an agar plate?

A

Virus need host cells for them to be able to replicate and carry on living

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25
Q

What is a cytopathic effect (CPE)?

A

The ability of a virus to cause morphological changes in the host cells. The CPE changes can be linked to which virus causes these changes.

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26
Q

Give an example of how an CPE can be seen

A
  • When a virus is added a CPE effect can be seen, the cells might change shape.
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27
Q

How can you find out which virus is causing the CPE?

A
  • Use a cell culture which supports the growth of only one virus
  • Cell cultures can also be used to see if an antiviral drug works or not by looking for inhibition of CPE
  • Do a neutralisation effect which is when you use antibodies against a specific virus antigen and if they stop the CPE then that virus is present.
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28
Q

When is the CPE method used and what has replaced it?

A
  • It has now been replaced by molecular techniques.
  • Sometimes still used for reasearch or for rare viruses’ detection e.g. discovery of hMPV and the Nipha virus
  • This technique is slow but occasionally useful in anti-viral sensitivity testing
29
Q

Why is it important to use different cell lines in test tubes when carrying out cell cultures?

A
  • This is because different viruses may have different affinities for different cells.
  • Some cultures may support the growth of only one particular type of virus. Therefore, selection of cell types is important.
30
Q

How is antigen detection used for direct detection of the virus?

A
  • Viral antigens which are usually either capsid or secreted proteins can be detected.
  • Infected cells may display viral antigens on their surfaces or sometimes those proteins may be found floating free in the blood or the urine of a patient.
31
Q

Examples of where swabs/samples can be obtained

A
  • Nasopharyngeal aspirates (NPA) - viral culture for pts with suspected respiratory tract infection e.g. RSV and influenza
  • Blood (serum and plasma) e.g. Hepatitis B and Dengue
  • Vesicle fluid e.g. Herpes simplex and varicella zoster
  • Faeces e.g. rotavirus and adenovirus
32
Q

What technique has replaced antigen detection?

A

Nucleic acid detection methods due to improved test performance

33
Q

What are the most common methods for antigen detection?

A
  • Direct immunofluorescene
  • Enzyme immunoassay (ELISA)
  • Immunochromatographic methods
  • Often used as a point of care for rapid diagnosis
34
Q

Describe the direct immunofluorescence method

A
  1. Antigen from infected host cells become bound to the slide
  2. The specific antibody (poly- or monoclonal) that is complementary to the antigen is tagged with a fluorochrome and mixed with the sample on the slide.
  3. This is then viewed using a microscope equipped to provide ultraviolet illumination.
35
Q

Advantage and disadvantage of direct immunofluoresence

A

Adv:
- Quick, only takes a couple of hours

Dis:
- Not sensitive, therefore not able to detect the virus in a lot of infected cells

36
Q

Describe the immunochromatographic method

A

Quick, similar to pregnancy test
1. Get the pt blood e.g. pt with dengue fever

  1. Add the blood to the small circular hole
  2. A line will appear if an antigen in the pt blood binds to an antibody in the kit that is specific to Dengue.
37
Q

When is Dengue common?

A

Common infection in returning travellers

38
Q

ELISA

A

Enzyme linked immunoassay (ELISA) used for antigen detection
- Common technique

39
Q

What are the 3 formats of ELISA? Which one is primarily used for antigen detection?

A
  • Indirect
  • Direct (primarily antigen detection)
  • Sandwich
40
Q

Mechanism of antigen detection by ELISA

A
  1. Well/plate is coated with an antibody that is specific to antigen on the virus
  2. Sample is added to the well and any antigen present will bind to the capture antibody.
  3. Add another antibody that is conjugated to an enzyme. This enzyme-conjugated primary antibody will bind to the captured antigen. This forms a sandwich. This enzyme will therefore only be present if that target antigen is present.
  4. Add a chromogenic substrate, which the enzyme is able to change the colour of. Therefore, the substrate will only change colour if the enzyme conjugated antibody is present and the enzyme conjugated antibody will only be present if the antigen is also present.
41
Q

What is another use of ELISA other than detecting antigens?

A

Used to detect antibodies instead

42
Q

Describe the production of antibodies during an infection

A
  1. When infected with a virus, the humoral immune response takes place resulting in production of immunoglobulins (antibodies).
  2. When an individual is initially infected, the body produces IgM. These are present for a variable period of time between 1 to 3 months.
  3. Later on, the body then produces IgG. As the levels of IgG antibody increases, the levels of IgM antibodies decrease.
43
Q

What can detection of these antibodies tell us?

A

Can tell us at what stage of the infection the individual is.

44
Q

Define Serology

A

The indirect detection of the pathogen

45
Q

When is serology the chosen method of detection?

A

Diagnostic mode of choice for organisms which are refractory to culture

46
Q

What is serology used for?

A
  • Detect an antibody response in symptomatic patients
  • Determine if vaccination has been successful
  • Directly look for antigen produced by pathogens
47
Q

Where are serology tests usually done?

A

Normally done on blood and serum however can be performed on other bodily fluids as semen and saliva.
This is good when testing on children because it is harder to obtain blood samples from children so saliva would be a lot easier.

48
Q

What is serum?

A

The clear bit of blood once it has been centrifuged containing proteins, antigens, antibodies, drugs and electrolytes.

49
Q

How to detect meales antibodies in patient serum?

A
  1. Prepare the well by adding measles antigens to the bottom of the well.
  2. Add the patient serum and if the patient has antibodies, they will bind to the antigen.
  3. Then add an enzyme conjugated antibody which will bind to the patient antibody.
  4. Between these stages wash the well so only the antibodies that are bound to the antigen stuck on the bottom of the well are present.
  5. This ensures that any floating antibodies are not present and that the enzyme conjugated antibody is only bound to the antigen bound antibody.
  6. Finally, add the substrate which will change colour and lead to the different colours seen in the wells above.
  7. Any purple colour seen means a positive result, a dark purple means a strong positive result and a light purple means a weak positive result.
50
Q

What do the levels of antibodies in serology tell you about the stage of infection?

A
  • Positive for IgM and IgG means that the infection must be recent or acute which relates to the earlier graph
  • If a patient is positive for IgG and negative for IgM then it’s a resolved infection and the patient will have lifelong IgG or a patient who is immunised. This is an example of an infection only once.
51
Q

What happens if someone is infected with the same virus more than once?

A
  • The level and type of antibody varied with the pathogen, exposure and time.
  • Someone might get infected with a virus and make IgG antibodies then.
  • Many years later, they might get infected again and because they made memory cells then, they get a very high IgG response.
52
Q

What is the modern lab detection method of antibodies and antigens in blood?

A

Abbott Architect

53
Q

What is the Abbot Architect?

A

A machine used that:

  • Detects either antibodies or antigens
  • Usually by enzyme immunoassays such as ELISA or related technology such as microparticle immune-chemiluminescence
54
Q

Why is the detection of both antibodies and antigens used?

A
  • Useful for some infections such as Hepatitis B, HIV and Hepatitis C
  • This is because it allows you to establish whether the infection is acute or chronic.
  • This may have therapeutic implications.
55
Q

Give an example of a molecular diagnostic test

A

Nucleic acid amplification (NAAT)

56
Q

Describe the NAAT technique

A
  1. Denaturation; heat briefly to separate DNA strands
  2. Annealing: Cool to allow primers to form hydrogen bond with ends of target sequences
  3. Extension: DNA polymerase adds nucleotides to the 3’ end of each primer.
    Cycle 1: yields 2 molecules
    Cycle 2: yields 4 molecules
    Cycle 3: Yields 6 molecules; 2 molecules match target sequence
57
Q

Purpose of NAAT

A
  • Can detect DNA or RNA depending on the virus
  • Ability to multiplex using fluorescence probes, for example, can look for several targets in one sample
  • This can be qualitative or quantitative
  • This requires nucleic acid extraction prior to the amplification
58
Q

Advantages of using NAATs

A
  • May be automated
  • Highly sensitive and specifc, generates huge numbers of amplicons
  • It is rapid
  • Useful for detecting viruses to make a diagnosis
    • > At first time of infection, such as measles and influenza
    • > During reactivation, such as cytomegalovirus
  • Useful for monitoring treatment response
    • > Quantitative such as HIV, HBV, HCV and CMV viral loads
59
Q

Limitations of using NAATs

A
  • May detect other viruses which are not causing infection
  • Exquisitely sensitive and so may generate large numbers of amplicons
    • > This may cause contamination
  • Need to know what viruses you are looking for because you need primers and probes that are specific for the target
60
Q

What is real time PCR?

A

Amplification and detection occur in real time. For example, simultaneously by the release of fluorescence

61
Q

Advantages of RT-PCR

A
  • Avoids the use of gel electrophoresis or line hybridisation
  • Allows the use of multiplexing
62
Q

What is multiplexing?

A
  • A term used when there is more than one pair of primers used in a PCR.
  • It enables the amplification of multiple DNA targets in one tube
  • For example, detection of multiple viruses in one CSF specimen such as HSV1, HSV2, VZV, enterovirus and mumps virus
63
Q

Describe the process of RT-PCR

A
  1. Taqman probe complimentary to the region of interest binds primers.
  2. Oligonucleotide probe with a fluorescent reporter at the 5’ end a quencher at the 3’ end.
  3. The quencher prevents the reporter fluorescing when excited if in close proximity.
  4. When the Taq polymerase extends from the 3’ end of primer as normal.
  5. The Taq possesses 5’-3’ nuclease activity and hydrolyses the probe.
  6. The reporter is removed rfom the quencher and fluorescence can be detected.
  7. For any given cycle within the exponential phase, the amount of product and hence fluorescence signal is directly proportional to the initial copy number.
64
Q

What is Ct?

A
  • Cycle threshold (CT) is all real time PCR formats that detect fluorescence in real time and use the Ct value to perform quantitation and presence/absence amplicon detection.
  • Ct is the number of times you need to repeat the cycle to detect the virus
65
Q

How is relative fluorescence plotted and what is it used for?

A

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.

66
Q

Which substances inhibit PCR?

A

Haem and bile salts

- Assays should always include an internal positive control as results could incorrectly be reported as negative.

67
Q

What can genome sequence give us?

A

A whole genome of a virus to look at it’s systems and how it works.

68
Q

How are the methods for virus detection combined?

A
  • Can initially use antibody and antigen detection for initial diagnosis: screening test and confirmation test
  • Can use NAAT to find out viral load and to monitor treatment: Quantification of virus in the blood
  • Resistance testing (sequencing); This is to look at the genome of the virus and how it works but also look for mutations to see if the virus has become resistant or not.
69
Q

Why is testing done?

A
  • Testing for specific infections on risk groups: Testing for HIV, HBV and HCV.
  • This is done because it may have an implication for others such as antenatal.
  • In these situations, the patients are asymptomatic.
  • The screening test needs to be sensitive.
  • May have some false positives so need a specific confirmatory test

SEM 4: Medical Microbiology (15 decks)

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