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diagnositics > virology lab > Flashcards

virology lab Flashcards

1
Q

what is the process of diagnositics involving virology *

A

history

physical examination

lab test -non-specific/virological

make list of ddx

determine which tests to ask for

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2
Q

what can be detected in the virology lab *

A

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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3
Q

what diagnostic methods are used in virology *

A

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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4
Q

what are the limitations of lab tests *

A

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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5
Q

what are the typical samples used *

A

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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6
Q

what is serology used to detect *

A

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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7
Q

why do you look at IgG and IgM *

A

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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8
Q

describe avidity testing *

A

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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9
Q

describe HIV serology *

A

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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10
Q

describe point of care testing *

A

blood on immunoabsorbant stick

not as good as in lab

suspiscion about false -ve

take to lab to confirm

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11
Q

describe use fo viral isolation in culture *

A

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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12
Q

describe the use of electron microscopy in lab *

A

sample types - stool/vesicle fluids

rare

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13
Q

describe use of immunoflurescence *

A

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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14
Q

describe the process of immunoflurescene *

A

incubate Ab with labelled dye

excite dye with right wavelength under microscope

look for flurescence

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15
Q

describe the use of PCR *

A

look for multiple pathogens in the same sample

amplify DNA

look by electrophoresis

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16
Q

what does teh virus that you look for depend on *

A

history

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17
Q

what resp tract viruses could be looked for by PCR *

A

influenza

parainfluenza

RSV

rhinovirus

human metaneumovirus (HMPV)

adenovirus

bocavirus

coronavirus

18
Q

what is the process of PCR *

A

denature DNA - 95 degrees

bind primers

TAQ pol - extend primers

amplify DNA

melt data analysis - PCR products melt at different temperatures

19
Q

what would you look for with CNS disease *(

A

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

20
Q

what would you look for in a young child with febrile fits - CNS

A

HHV-6 and parechovirus

21
Q

what would you look for in the immuncomp - cns

A

CMV

EBV
JC

22
Q

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

A

Japanese Encephalitis

West Nile virus,

equine encephalitides,

tick borne encephalitis…

23
Q

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

A

mumps

24
Q

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

A

measles Ab index

25
Q

how would you look for virus in someone with diarrhoea or vom

and what would you look for *

A

PCF or antigen detection assays

norovirus, rotavirus, adenovirus, sapovirus, astrovirus

26
Q

what sample would you wnat with someone with diarrhoea/vom *

A

stool preferred

vomit - lower yeild

27
Q

why would you sequence patient samples *

A

to genotype them

see if resistant - determine treatment

look at phylogenetic analysis - if SNPs - might have transmission between patients

28
Q

different virology specimen types *

A

blood sample - serology - rust top - 5ml SST tubes

blood sample - PCR - 6ml EDTA (pink top)

virology swabs - flocked swab, viral transport medium (VTM)

29
Q

why would you want clotteed blood and what tube would you use *

A

to get serum for serolgy

rust coloured top

30
Q

why would you want to use EDTA tube - purple top tube *

A

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

31
Q

timeline for diagnosis of HBV *

A

surface antigen from 2wks post infectiom

clinical presentation - 6wks to 6months

32
Q

timeline for diagnosis for HCV *

A

PCR from 1-2 weeks post infection

Ab can take up to 9 months post infection - 7-8wks

clinical presentation 3wks to 3months

33
Q

timeline for HIV diagnosis *

A

PCR 2-3wks

seroconversion 3-4 wks post infection

34
Q

what are the markers for Hep B *

A

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

35
Q

what do you look for on hepatitis screens *

A

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

36
Q

what are the surfaec Ab titres *

A

< 10mIU/ml : Non-responder - ? cAb+

10–100mIU/ml : Weak response (single booster!)

> 100mIU/ml : Strong response

37
Q

what do you test for fo vesicular rash *

A

vesicle swab/fluid

HSV - not IgM

VZV - not IgM

enterovirus

38
Q

what would you test for for a maculopapular rash *

A

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

39
Q

how do you interpret EBV markers *

A

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

40
Q

what is PCR used to investigate with CSF *U

A

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

41
Q

basics of PCR *

A

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

diagnositics (5 decks)

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