32 Diagnosis of infection - NNUH Flashcards

Question

HBV serology How does anti-HBc assay performed?

Answer 1

Add: Sample assay diluent rHBcAg coated parmagnetic microparticles Anti-HBc present in the sample binds to the rHBcAg coated microparticles. The reaction mixture is washed and anti-human acridiniumlabeled conjugate is added. The resulting chemiluminescent reaction is measured as relative light units (RLUs).

Answer 2

False positive anti-HBc Previous infection with spontaneous clearance Current acute infection - anti-HBc bind to HBsAg forming complex, so no free antigen detected Current chronic infection - anti-HBc keeps blood free from antigen Current infection - mutated HBsAg not detectable by current assay As we move from top to bottom, anti-HBc levels increase. So absolute levels can help us determine what the clinical picture is

Answer 3

High risk e.g starting rituximab - start treatment for HBV immediately, and continue until 12 months after completing therapy. Do not vaccinate Moderate risk e.g long term steroids, most DMARDs - check HBV DNA. If positive - treat. If negative - monitor LFTs/ HBV serology. Vaccinate Low risk e.g low dose steroids, methotrexate - HBV DNA does not matter, monitor LFTs/ HBV serology. Vaccinate Moderate/ low risk groups - start treatment if signs of re-activation. Treatment includes tenofovir/ entecavir

Answer 4

solid phase indirect ELISA Polystyrene microwell strips are pre-coated with HEV recombinant During the first incubation step, HEV specific antibodies, if present, will be bound to the solid phase pre-coated HEV antigens. The wells are washed to remove unbound serum proteins and then, anti-human IgG antibodies (anti-IgG) conjugated to horseradish peroxidase (HRP-Conjugate) is added. Chromogen solution addeed, which is hydrolysed by HRP-immunocomplex to produce blue colour. Colour change proportional to amount of antibody in wells.

Answer 5

134 bp region of HBV genome - detect in fluorescence channel Cycling Green Internal control in Cycling Yellow 10.22 IU/ml

Answer 6

Qiagen Artus - NNUH Abbott Realtime Roche - Cobas Taqman Siemens VERSANT HBV assay

Answer 7

240bp region of HCV genome 21 IU/ml

Answer 8

assay covers clinically relevant regions of NS3, NS5A and NS5B and detects genotypes 1, 2, 3, 4, 5, and 6, and subtypes 1a and 1b. Studying the most important regions is quicker/ more convenient than whole genome sequencing, and provides same results about phylogenic information. Gives information on resistance, mutation, epidemiology The limit of detection is 1000 IU/mL for genotypes 1 (including 1a, 1b and others), 2, 3 and 4, The limit of detection is 2000 IU/mL for genotypes 5 and 6.

Answer 9

Detects norovirus I and II Use stool sample - high viral load, so does not require amplification. Isolated RNA transcribed to cDNA by RT. Gene fragments specific for norovirus GI and GII are subsequently amplified by real-time PCR. The amplified targets (ORF1/ORF2 junction region) are detected with hydrolysis probes, which are labelled at one end with a quencher and at the other end with a fluorescent reporter dye (fluorophore). During the extension step the Taq-Polymerase breaks the reporter-quencher proximity. The reporter emits a fluorescent signal which is detected by the optical unit of a real-time PCR instrument.

Answer 10

<50 IU/ml neg 50-100 IU/ml borderline >100 IU/ml pos

Answer 11

IgM <20 IU/ml neg 20-25 IU/ml borderline >25 IU/ml pos IgG <2 IU/ml neg 2-3 IU/ml borderline >3 IU/ml pos

Answer 12

105bp region of CMV genome 42 copies/ml LOD

Answer 13

CMV IgG avidity enables weak avidity antibodies, produced at the early stage of a primary infection, to be differentiated from high avidity antibodies, which are characteristic of a past infection. It may be tested to help exclude or diagnose a recent primary infection. The detection of high avidity antibodies is a strong indication that a primary infection occurred more than 3 months ago, whereas the detection of weak avidity antibodies is a strong indication that a primary infection has occurred within the last 3 months. Avidity indicates the strength of the link between an antibody and an antigen. The addition of urea which disrupts antigen-antibody linkage has little effect on the high avidity antibody link but has a great effect on the weak avidity antibody linkage. The comparison of results obtained with and without urea corresponds to a measure of avidity, the Avidity Index Same VIDAS process as before, use two strips. One strip remove buffer well, and replace with urea.

Answer 14

< 0.40 Low avidity IgG 0.40 ≤ - < 0.65 Borderline avidity ≥ 0.65 High avidity IgG An avidity index greater than or equal to 0.65 is a strong indication of a primary infection dating back more than 3 months. An avidity index lower than 0.40 is a strong indication of a primary infection dating back less than 3 months. For these results, confirmation using another serum collected 3 or 4 weeks later may be justified depending on the clinical context. An avidity index between 0.40 and 0.65 does not enable to distinguish a recent infection from a former infection. For these samples, depending on the context, other markers and/or avidity determination methods should be used and/or a new serum sample (collected 3 or 4 weeks later) should be tested.

Answer 15

Only reversible cause of sensorineural hearing loss Antivirals within 4 weeks can stop hearing loss getting worse If CMV DNA detected on mouth/ urine swab, then test Guthrie card to assess if congenitlaly acquired, or acquired after birth

Answer 16

Repeat swab to confirm Inform audiology Ophthalmology review CT brain Virology/ paediatrics to discuss treatment

Answer 17

350-450 copies/ml

Answer 18

In each well, two amplification reactions are performed starting from DNA extracted from the samples being tested: a specific reaction for the exon 4 region of the CMV MIEA gene (major immediate early antigen, HCMVUL123) and a specific reaction for a region of the human beta Globin gene (Internal Control of inhibition). The CMV specific probe binds, and the internal control probe binds to beta Globin. Both probes are fluorescent. Fluorescent products measured.

Answer 19

EBV VCA/ EA IgG EBV VCA IgM EBV EBNA IgG Diagnosis includes serologu/ monospot test (heterophile antibodies) Different antibodies produced at different time of viral life cycle. During the lytic phase, EBV early antigens (EA) are produced, then viral capsid antigens (VCA) are expressed at the same time as the viral genome. During the latent cycle, Epstein-Barr nuclear antigens (EBNA) are synthesized. When IM occurs, heterophile antibodies appear in 60-80% of cases, anti-EA antibodies in 70-80% of cases, anti-VCA IgM antibodies in 100% of cases and anti-VCA IgG antibodies in nearly 100% of cases. During the convalescent phase, anti-VCA IgG antibodies persist and approximately 95% of patients produce anti-EBNA IgG antibodies Heterophile antibody is antibody produced in response to antigen (non-self). But term is often used to describe specific monospot test for EBV

Answer 20

anti-VCA or EA antibodies will bind to VCA p18 or EA p54 antigens in SPR Wash to remove unbound material Add mouse monoclonal Ab which bind to any human IgG bound to antigen. Substrate added which conjugates the antigen-antibody complex. This catalyses a reaction to produce fluorescent product. fluorescence proportional to antibody level IgG antibodies in Fab' form, conjugated to alkaline phosphatase, are cycled in and out of the SPR and will attach to any human IgG bound to the antigen. During the final detection step, the substrate (4-Methyl-umbelliferyl phosphate) is cycled in and out of the SPR. The conjugate enzyme catalyzes the hydrolysis of this substrate into a fluorescent product (4-Methyl-umbelliferone

Answer 21

Same as usual VIDAS test anti-EBV IgM binds to VCA P18 antigens on SPR wall

Answer 22

anti-VCA IgM/IgG - p18/ p54 anti-EBNA IgG - p72

Answer 23

Neisseria meningitides Strep pneumoniae Haemophilus ``` Adenovirus Enterovirus HSV 1/2 Parechovirus Varicella ``` ``` Send away - E. coli Listeria GBS Fungi Even if get diagnosis at NNUH, may need to send away fro serotyping ``` Remember that EDTA blood can be uses to check N meningitidis, Strep pneumoniae, Haemophilus

Answer 24

Treponemal - - EIA - Architect - TPPA - FTA-Abs Treponemal tests detect antigens or antibodies against specific treponemal antigens and are qualitative, being either positive or negative. disadvantage is results remain positive after infection even with successful treatment. Non-treponemal - - RPR - VDRL - is old test, and been replaced by RPR Non-treponemal tests are not specific for treponemal infections; they detect antibodies that react to a cardiolipin-cholesterol-lecithin antigen in the serum from patients with these infections. There is a 1-2% false positive rate in these tests. Advantage is quantitative and can therefore be used to monitor response to treatment as the tests become negative with successful therapy. Disadvantage is that the level decreases with time; 50% of patients with latent syphilis can actually have a negative RPR 30-40 years after the initial infection, even though they have syphilis. At NNUH - - Screen with EIA on Archiect. - If negative syphilis it is ruled out - 98% predictive value - If positive, proceed to TPPA and RPR

Answer 25

Two-step immunoassay Add sample Add microparticles coated with recombinant TP antigens Add assay diluent Antibodies bind to antigens Wash Add acridium-labelled anti-human IgG and IgM Wash Chemiluminescent reaction results, measured in relative light units. Direct relationship between amount of antibodies in sample and relative light units S/CO < 1.0 - negative for T pallidum Ab S/CO >1.0 - <5.0 - weak positive. Centrifuge for 10 mins and repeat test. If negative, then report as neg. If positive, then needs TPPA/ RPR S/CO >5.0 - positive for T pallidum Ab

Answer 26

Particle agglutination assay Have wells will gelatin particles, sensitised with T pallidum. Sensitised particles will agglutinate in presence of Treponema Pallidum Ab Semi-quantitative as can see which dilution still positive

Answer 27

Flocculation test Add serum and reagent Black floculants are formed, and visible macroscopically due to presence of carbon particles Obtain titre by diluting and recording highest dilution at which agglutination visible

Answer 28

``` Adenovirus Mycoplasma Chlamydia Coxiella Enterovirus Leptospirosis Brucella ```

Answer 29

Indirect Immunofluorescent Antibody (IFA) assay Antigen present in wells. Serum diluted with PBS containing 10% goat serum. This acts to block non-specific binding/ background staining. Wash to remove unbound serum antibodies Fluoresceine-labelled antibody binds to Ag-Ab complexes Wash to remove unbound antibody Examine under fluorescent microscopy - apple-green appearance. Semi-quantitative endpoint titers are obtained by testing serial dilutions of positive specimens.

Answer 30

Latex agglutination test Uses antibody sensitised blue latex particles which will agglutinate in presence of specific Legionella cell wall antigens to form visible clumps Tests serogroups 1-14 which are most common causes

Answer 31

Lateral flow immunoassay Urine or CSF Test strip has nitrocellulose membrane, with dried capture antibodies, and conjugate. The conjugate bead is first rehydrated with diluent. Add sample. Antigen will bind to antibody-colloidal gold conjugate. When the sample migrates up the Test Strip to the Test Line, the antigen-conjugate complex is bound to the capture antibody, yielding a pink-red line. When no antigen is present, no complexes are formed and no pink-red line appears at the Test Line.

Answer 32

ELISA Microtitre strip wells are precoated with measles antigens, which bind to serum antibodies. Wash to remove unbound sample material Add HRP labelled anti-human conjugate, which binds to capture measles specific antibodies Add sulphuric acid to stop reaction Note colour change on ELISA microwell plate reader to determine if antibodies present

Answer 33

antistreptolysin-O (ASO) toxin acts as antigen anti-DNAse B (ADB) secreted by bacteria An increase in titre is only observed in 80% of infections, so check two antibodies to increase detection rate. Following acute streptococcal infection, the ASO titre will usually rise after one week, increasing to a maximum level within 3 to 5 weeks and usually returning to the pre infection levels in approximately 6 to 12 months.

Answer 34

ASO - Latex particles coated in recombinant streptolysin-O - Add sample - Ag-Ab complex causes agglutination - ASO levels > 200 IU/ml considered significant ADB - Add DNA substrate, and add ADB, and seurm. - If Ab present to ADB, it will inhibit DNA substrate breakdown, by blocking ADB - If colour change, suggests DNA substrate breakdown by ADB, and absence of antibodies

Answer 35

The ProSpecT Rotavirus test is an immunoassay for the detection of Group A rotaviruses in faecal specimens. The test utilises a polyclonal antibody to detect group specific proteins, including the major inner capsid protein (VP6), present in Group A rotaviruses. Uses direct ELISA to detect antigen Microwells coated with rotavirus polyclonal antibody Faecal suspension added HRP-conjugate added Rotavirus Ag binds to Ab Wash sample Add chromogen which binds to Ag-Ab/ HRP complex, causing colour change

Answer 36

Uses direct ELISA to detect antigen Microwells coated with adenovirus monoclonal antibody Faecal suspension added HRP-conjugate added Rotavirus Ag binds to Ab Wash sample Add chromogen which binds to Ag-Ab/ HRP complex, causing colour change