virology lab Flashcards
what is the process of diagnositics involving virology *
history
physical examination
lab test -non-specific/virological
make list of ddx
determine which tests to ask for
what can be detected in the virology lab *
infectious virus - isolation and electron microscopy - rare
protein components - antigens of virus eg p24 antigen in HIV, surface antigen in HBV
genetic components of virus - RNA or DNA - quant/qual - PCR
host response - eg Ab or cell responses
what diagnostic methods are used in virology *
cell culture and electron microscopy - rare
Ab detection - serology, enzyme immunoassay
Ag detection - immunoflurescene, enzyme immunoassay
genome detection - PCR - most common
quantification of Ab/Ag
serotyping
viral load - essential for monitoring HIV, HBV, HCV, CMV adn EBV
genome sequenxing - genotyping and antiviral resistance testing
what are the limitations of lab tests *
all assays give fasle +Ve/-ve
sensitivity - ability to correctly identify positive samples - high = few false -ve
specificity - ability to correctly identify -ve samples = few false +ve
what are the typical samples used *
throat swab, sanopharyngeal aspirate (NPA), bronchoalveolar lavage (BAL), ET secretions - for detection of resp muscles by immunofluresence but mainly PCR
stools - for rotavirus, adenovirus and norovirus antigen detection - PCR
urine - for BK virus and adenovirus - PCR
CSF - herped and enterovirus - PCR
blood clotted - serology - Ab detection
blood - EDTA - PCR/viral load testing
saliva - measles - serology/PCR (when difficult to take blood film)
what is serology used to detect *
HIV - Ab and p24 Ag
hep a - IgM/G
HBV surface - Ag/Ab, eAg/eAb, core Ab, core IgM
HCV serology - Ab +/- core antigen
CMV and EBV - IgM/G
VZV - IgG
MMR - IgM/IgG
parvovirus B19 - IgM and IgG
why do you look at IgG and IgM *
both in acute phase of disease
can date infection -duration: M 3 months, G lifelong
IgM - sensitive but not specific - false +ve so do avidity testing
describe avidity testing *
measure of strength of Ab binding
low avidity Ab mixed with denaturising agent eg urea - Ab washed away - not much binding
high avidity - Ab bind even with urea
avidity matures over time - ie longer exposed = more avidity
describe HIV serology *
4th generation EIA - look for Ab and p24 Ag
high sensitivity - pick up antignes early, even if havent made Ab yet
all samples undergo confirmatory testing in 2nd assay to exclude non-specific reactivity - fasle +ves
confirmed +ves undergo typing - 1 or 2
repeat blood sample and EDTA blood for HIV viral load required for all new +ves - also genotype and baseline resistance testing
describe point of care testing *
blood on immunoabsorbant stick
not as good as in lab
suspiscion about false -ve
take to lab to confirm
describe use fo viral isolation in culture *
rare
slow
time consuming - expensive
can quantitate the amoiunt of virus in a sample - 1 dot signifies a single virus
used for phenotypic antiviral suseptibility testing
poor sensitivity and specificity
describe the use of electron microscopy in lab *
sample types - stool/vesicle fluids
rare
describe use of immunoflurescence *
still occaisionally used for direct detection of viral agents in clinical samples - eg resp viruses
rapid and inexpensive
subjective - dependant on skill of technicial and qual of sample
describe the process of immunoflurescene *
incubate Ab with labelled dye
excite dye with right wavelength under microscope
look for flurescence
describe the use of PCR *
look for multiple pathogens in the same sample
amplify DNA
look by electrophoresis
what does teh virus that you look for depend on *
history
what resp tract viruses could be looked for by PCR *
influenza
parainfluenza
RSV
rhinovirus
human metaneumovirus (HMPV)
adenovirus
bocavirus
coronavirus
what is the process of PCR *
denature DNA - 95 degrees
bind primers
TAQ pol - extend primers
amplify DNA
melt data analysis - PCR products melt at different temperatures
what would you look for with CNS disease *(
things that cause meningitis and encephalitis
CSF - HSF, VZV, enterovirus
stools and throat swab - enterovirus detection by PCR
blood - serology and/or PCR - west nile, japanese encephalitis virus infection and other arboviruses
what would you look for in a young child with febrile fits - CNS
HHV-6 and parechovirus
what would you look for in the immuncomp - cns
CMV
EBV
JC
what would you look for in someone who travelled to endemic region
Japanese Encephalitis
West Nile virus,
equine encephalitides,
tick borne encephalitis…
what would you look for in context of an outbreak - cns
mumps
what would you look for in someone with SSPE (subacute sclerosing panencephalitis)
measles Ab index
how would you look for virus in someone with diarrhoea or vom
and what would you look for *
PCF or antigen detection assays
norovirus, rotavirus, adenovirus, sapovirus, astrovirus
what sample would you wnat with someone with diarrhoea/vom *
stool preferred
vomit - lower yeild
why would you sequence patient samples *
to genotype them
see if resistant - determine treatment
look at phylogenetic analysis - if SNPs - might have transmission between patients
different virology specimen types *
blood sample - serology - rust top - 5ml SST tubes
blood sample - PCR - 6ml EDTA (pink top)
virology swabs - flocked swab, viral transport medium (VTM)
why would you want clotteed blood and what tube would you use *
to get serum for serolgy
rust coloured top
why would you want to use EDTA tube - purple top tube *
whole blood sample eg HHV-8 PCR, HIV proviral DNA PCR, EBV and CMV viral load
plasma - HIV, HBV, HCV viral load, adenovirus, HHV-6 PCR
timeline for diagnosis of HBV *
surface antigen from 2wks post infectiom
clinical presentation - 6wks to 6months
timeline for diagnosis for HCV *
PCR from 1-2 weeks post infection
Ab can take up to 9 months post infection - 7-8wks
clinical presentation 3wks to 3months
timeline for HIV diagnosis *
PCR 2-3wks
seroconversion 3-4 wks post infection
what are the markers for Hep B *
acute infection - surface Ag, core IgM, e AG, HBV DNA +ve - test request - surface Ag
chronic infection - surface Ig, core ab, IgM, e Ag/Ab and HBV DNA +ve - test request - surface Ag
past infection (naturally immune) - core Ab, surface Ab, e Ab, HBV DNA -ve - test request - core Ab
immunised - surface Ab, core Ab -ve
what do you look for on hepatitis screens *
hepitisis (transaminitis) - HAV IgM, BV surface Ag, GCV Ab - alos consider CMV and EBV (tonsillitis, lymphadenopathy, rash), HEV IgM
past hep - HAV IgG, HBV core Ab, HCV Ab
response to vaccine - HAV IgG, HBV surface Ab
what are the surfaec Ab titres *
< 10mIU/ml : Non-responder - ? cAb+
10–100mIU/ml : Weak response (single booster!)
> 100mIU/ml : Strong response
what do you test for fo vesicular rash *
vesicle swab/fluid
HSV - not IgM
VZV - not IgM
enterovirus
what would you test for for a maculopapular rash *
measles IgM or G
rubella IgM or G
parvovirus IgM or G
maybe EBV serology and CMV IgM or G - if lymphaeneopathy and hep
maybe enterovirus PCR in stool/throat swab
how do you interpret EBV markers *
EBV nucear antigen Ab appear 2-6 months post infection
acute - viral capsid antigen IgM positive, VCA G -ve/positive, EBNA IgG negative
past infection - VCA iIgM negative, IgG +ve, Anti-EBNA positive
what is PCR used to investigate with CSF *U
meningitis - HSV and enterovirus and mumps
encephalitis - HSV< VZV, enterovirus
neonates - HSV, enterovirus, adenovirus and CMV
children 1m-3yr - HSV, enterovirus, HHV6
immunocomprimised - encephalitis screen, CMV, positive or negative for JCV and EBV
basics of PCR *
require DNA template - therefore reverse transcription is required for RJNA virus
TAQ pol, primers and nucleotides needed
multiplex PCR >1 primer set per reaction
real time PCR - melt curve, cycle threshold, CT value - numbe of cycles it takes to reach cycle threshold - the more target the lower the CT value
