virology lab Flashcards by Emily Southall

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

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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

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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

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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

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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)

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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

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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

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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

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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

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describe point of care testing *

blood on immunoabsorbant stick

not as good as in lab

suspiscion about false -ve

take to lab to confirm

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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

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describe the use of electron microscopy in lab *

sample types - stool/vesicle fluids

rare

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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

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describe the process of immunoflurescene *

incubate Ab with labelled dye

excite dye with right wavelength under microscope

look for flurescence

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describe the use of PCR *

look for multiple pathogens in the same sample

amplify DNA

look by electrophoresis

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what does teh virus that you look for depend on *

history

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what resp tract viruses could be looked for by PCR *

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influenza

parainfluenza

RSV

rhinovirus

human metaneumovirus (HMPV)

adenovirus

bocavirus

coronavirus

what is the process of PCR *

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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 *(

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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

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HHV-6 and parechovirus

what would you look for in the immuncomp - cns

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CMV

EBV
JC

what would you look for in someone who travelled to endemic region

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Japanese Encephalitis

West Nile virus,

equine encephalitides,

tick borne encephalitis…

what would you look for in context of an outbreak - cns

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mumps

what would you look for in someone with SSPE (subacute sclerosing panencephalitis)

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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

virology lab Flashcards

(41 cards)