Diagnostic Virology Flashcards
Summarise the diagnostic virology tools
History
Physical examination
Laboratory tests (non-specific and virological)
Make a list of possible causes
Determine which tests to request
Essentially, the tests are non-specific- so use history and examination to request possible tests
Describe a maculopapular rash
Some areas are raised and some areas are flat- measles
What is a dermatomal rash indicative of
Shingles- VZV
What can we detect in virology
Infectious virus
(virus isolation & EM)- culture and look on microscope- replaced by PCR now
Protein components (antigens) of the virus
(p24 antigen in HIV, surface antigen in HBV, etc)- virus in serum, plas, CSF- goof for blood borne
Genetic components of the virus (DNA or RNA) - quantitative or qualitative tests are available
The host response (e.g. antibody or cell responses)
How can we detect the different components used in virology
Antibody detection (serology - EIA) Antigen detection: (Immunofluorescence - IF; enzyme immunoassay - EIA) Genome detection (polymerase chain reaction - PCR)
Culture and microscopy- replaced by PCR
What are the key limitations of laboratory tests
All assays give rise to false negative and false positive results
Sensitivity: the test’s ability to correctly identify positive samples (few false negatives)
Specificity: the test’s ability to correctly identify negative samples (few false positives).
What are the key features of an ideal test
High specificity i.e. have a low level of cross reactivity.
Sensitive- detect the virus or the antibody at very low levels.
Rapid – results should be available in a timely fashion.
Non-invasive. This reduces the risks of the procedure and makes then easier to repeat if necessary.
Cost effective. Most virology tests only cost a few pounds each but some of the molecular tests are significantly more expensive, so use them wisely.
What should a good history include
When talking a history it is important to include vaccination history, travel (especially in the previous 3 weeks), contact with animals/pets, contact with infected persons and occupation. This information may give you some important clues.
Essentially, what does diagnosis depend on
Diagnosis depends on the clinical findings, the detection of specific antibodies and/or the detection of a virus in the appropriate clinical sample.
Summarise the different viral detection methods
Quantification of antibody or antigens e.g. HBsAg in hepatitis B infection or RSV antigen in respiratory sampl
Serotyping (eg HIV)
Quantification of genomes –
“viral load”
(essential for diagnosis and monitoring of HIV, HBV and HCV, and also for CMV and EBV in the immunocompromised)
Genome sequencing- e.g. using PCR to detect viral DNA or RNA
Genotyping
Antiviral resistance testing
Describe the sample used for respiratory viruses
Throat swab, Nasopharyngeal aspirate (NPA), bronchoalveolar lavage (BAL), ET secretions – for detection of respiratory viruses by (IF or) PCR
Describe the tests done with stool samples
Stools - for rotavirus, adenovirus & norovirus antigen detection (EIA) or PCR- useful for gastroenteritis or viruses that cause diarrhoea
Describe the tests done with urine samples
Urine – for BK virus & adenovirus PCR also CMV (KEEP IN STERILE CONTAINER).
Describe the tests done with CSF
CSF - for herpes viruses and enteroviruses PCR
(in sterile container, VTM (viur transport medium) not required) – for the diagnosis of viruses causing meningitis or encephalitis such as HSV, VZV, enteroviruses, mumps, etc.
What tests are done with clotted blood
Blood (clotted) - for serology (antibody detection)
What tests are done with blood (EDTA)
Blood (EDTA) - for PCR / viral load testing
Used for detection and quantification of HIV, HBV and HCV.
What tests are done with saliva
Saliva – for serology &/or PCR (eg measles)
In patients where you want to do serology- but difficult to take blood
When are throat swabs useful
Throat swab – for virus isolation (in virus transport medium, VTM) – useful in the diagnosis of enteroviruses and respiratory viruses.
When are nasopharyngeal aspirates useful
Nasopharyngeal aspirate (NPA) – for respiratory viruses using Immunofluoresence (IF) or PCR, such as RSV, influenza A&B, adenovirus, parainfluenza viruses, SARS etc.
Describe the different viruses that serology will be used to detect
HIV serology (antibody + p24 antigen) Hepatitis A IgM and IgG HBV surface Ag/Ab, eAg/eAb, core Ab, core IgM HCV serology (antibody +/- core antigen) CMV and EBV IgM & IgG VZV IgG Measles, mumps, rubella IgM & IgG Parvovirus B19 IgM & IgG
Compare IgM to IgG
Both involved in the acute phase of disease
IgM- lasts for around 3 months (pentameric)
IgG- lifelong
IgM is a marker of RECENT infection
IgG rises later on
So positive IgG and absent IgM- indicates immunisation or past infection.
Describe a typical immune response to HAV
Virus infects patient
Virus in faces after about one week
Symptoms after about 2 weeks
Detectable IgM after 3 weeks- will peak 4 weeks later then come down
Detectable Igg after 4 weeks- will continue to rise and plateau
Can detect these antibodies to date the infection.
What is a key issue with serology
The tests are sensitive (i.e good when antibody conc is low)- but they aren’t specific- so won’t distinguish between IgG and IgM well- issue in dating the infection- use antibody avidity testing instead.
Describe antibody avidity testing
Add anti-human IgG HRP conjugate to detect bound IgG
Add urea
Acutely- avidity maturation will be low- thus urea will degrade most of IgG- low level
Chronically- avidity maturation taken place- IgG will bind strongly to antigen- even in presence of urea- infection can’t have been less than 3 month ago- had it around 6 months ago.
Describe the role of EIA in viral serology
4th generation EIA: Ab + p24 Ag detection
Good- sensitive
Use antigen detection to pick up early stages- where the patient may not have made antibodies yet.
Describe the importance of confirmatory testing in HIV
Detection of specific antibody is an indirect method of detecting infection.
Non-specific reactions can be a problem, therefore it is important to use multiple formats (generally use 3 different assays).
Interpretation of results must take the clinical circumstances into account.
All reactive samples undergo confirmatory testing in a second assay to exclude non-specific reactivity (false positives)
Repeat blood sample + EDTA blood for HIV viral load required from all new +ves (also genotyping and baseline resistance testing)- to ensure that the blood came from the correct patient
What else should be done in HIV confirmatory testing
Confirmed positives undergo typing (HIV 1 vs 2)
Summarise point of care testing
PCR for flu patients- within 15 mins
HIV-strip with patient blood on to detect Abs
RSV
Not as accurate as lab methods- so confirmation with lab essential.
Describe viral isolation in cell culture
Now rarely used - Reference lab only
Slow, time consuming (hence expensive)
Still useful for phenotypic antiviral susceptibility testing (HSV)- add antiviral- see how much growth reduces with different concentrations
Low specificity and sensitivity
Describe the use of EM to detect viruses
Viruses are too small to be seen by light microscopy
They can be visualised using an electron microscope
Sample types: stool and vesicle fluids
Now rarely used
Describe the use of immunofluorescence to detect viruses
Still occasionally used for the direct detection of viral antigens in clinical samples (DIF)
(e.g. respiratory viruses)
Rapid and inexpensive but subjective and very dependent on the skill of the technician and the quality of the sample
Can be used for typing and culture confirmation
How does immunoflourescence work
Put sample on glass
Add antibody with fluorescence attached and put in spectrometer at wavelength of fluorescence. - low for colour.
Commonly used for RSV.
Describe the respiratory viruses that should undergo PCR
(Influenza, parainfluenza, RSV, rhinovirus, human metapneumovirus [HMPV], adenovirus, bocavirus, +/- coronavirus…)
“multiplex PCR”
Describe PCR
Target amplification to allow detection and quantification over very large dynamic ranges (> 5-8 logs)
Can be very sensitive (as low as 1 genome copy)
Can subtype viruses from PCR products
Problems with contamination. This can be overcome using “Real Time” PCR.
Can multiplex- screen for multiple viruses - § Multiplex PCR – rather than use a single test tube for each virus, use several viruses in one tube – only do 3 or 4 at once.
What is sampled when detecting CNS diseases (such as meningitis and encephalitis)
CSF for PCR (HSV, VZV, enterovirus…)
Stools and throat swab for enterovirus detection (ie by PCR)
Blood for serology and/or PCR for West Nile and/or Japanese Encephalitis virus infection and other arboviruses
Summarise typical clinical histories for CNS diseases
Meningitis or encephalitis: HSV, VZV + enterovirus
Young child with febrile fits: Add HHV-6 + parechovirus
Immunocompromised (eg HIV): Add CMV, EBV & JC virus
Recent travel to endemic region: Consider Japanese Encephalitis, West Nile virus, equine encephalitides, tick borne encephalitis…
In context of an outbreak: eg mumps
SSPE (subacute sclerosing panencephalitiis) : measles antibody index
What tests can be done with diarrhoea and vomiting
Stool (preferred)
Vomit – lower yield …
PCR or antigen detection assays (EIA)
(norovirus, rotavirus, adenovirus, sapovirus, astrovirus)
Norovirus, rotavirus- community
The others- immunocompromised
What is important to remember about enteroviruses and enteric viruses
Enteroviruses are a type of enteric virus
What is PCR
Polymerase Chain Reaction
Method for amplifying specific RNA (RT-PCR) or DNA sequences
Cycle ( x 30):
What must happen first before PCR is performed to identify the genome of an RNA virus?
The RNA must be reverse transcribed by reverse transcriptase to dsDNA
dsDNA is the start point of PCR
Outline the process of PCR
Denature dsDNA (95 degrees)
Cool to 50 and anneal primers
Heat to 72 and add Taq polymerase and nucleotides
Repeat for 30 cycles
Put products in lanes of electrophoresis- match bp to that of virus
What is real time melt PCR analysis
Different viruses have genes that melt at different temperatures- can use these peaks at different temperatures to detect the virus
dF/dT- more fluorescence
Quantification during linear phase gives better reproducibility, precision and dynamic range.
Readily adapted to multi-plexing i.e. detect multiple viruses in the samples simultaneously.
Closed tube monitoring eliminates contamination.
Describe the applications of PCR
Genotyping
Antiviral resistance testing
Phylogenetic analysis - for tracing- if closely mathced- shows transmission between individuals.
Summarise viral gene detection and quantification
Polymerase chain reaction PCR (a target amplification system)
bDNA (signal amplification system)
Both assays used to measure “viral load” in HIV, HCV and HBV infection.
Summarise enzyme immunoassays (serology to detect antibodies and antigens)
Can be adapted to detect specific antibody classes e.g. IgM IgG or IgA
Sensitive and can quantify amounts of antibody (e.g. anti-HBs antibody)
Adaptable to antibody or antigen detection
