32 Diagnosis of infection - NNUH Flashcards
Which virus tests/ viral serology is available at NNUH?
Adenovirus Antigen
Adenovirus DNA PCR
CMV DNA
(viral load)
Congenital CMV (cCMV) Screening
CMV Serology
EBV DNA (viral load) EBV Serology Enterovirus RNA PCR Enterovirus Serology
Hepatitis A Serology
Hepatitis B anti-HBc (total) Hepatitis B anti-HBc IgM Hepatitis B anti-HBe Hepatitis B anti-HBs (post vaccination) Hepatitis B DNA (viral load) Hepatitis B e Antigen Hepatitis B Surface Antigen (HBsAg) (including confirmation)
Hepatitis C RNA
(viral load)
Hepatitis C Serology
(including confirmation)
Hepatitis E Serology
HIV-1 RNA
(viral load)
HIV-1&2 Antibody/Antigen
HSV-1 & 2 DNA PCR
Measles Serology
Norovirus PCR
Parechovirus RNA PCR
Parvovirus B19 Serology
Respiratory viruses by PCR
Respiratory Serology
Rotavirus Antigen
Rubella serology
VZV DNA PCR
VZV IgG Immunity
Which bacterial/ parasite antigen/serology testing is available at NNUH?
Bartonella serology
Borrelia burgdorferi IgG Serology
Brucella serology
Chlamydia trachomatis NAAT
Helicobacter pylori Antigen
Legionella Antigen
Leptospira Serology
Mycoplasma IF
Mycoplasma pneumonia Serology
Neisseria gonorrhoea NAAT
Pneumocystis jiroveci
Pneumococcal Antigen
Streptococcal Serology
Syphilis Serology
Toxoplasma Serology
Trichomonas vaginalis NAAT
Which viral tests/ serology require to be sent away?
Adenovirus DNA PCR
BK virus PCR
Chikungunya Serology*
Dengue Serology*
Hepatitis D Serology
Herpes simplex Serology
HIV-1 Proviral DNA
HIV-1 Resistance Testing
HIV-2 viral load
HTLV 1&2 Serology
JC virus PCR
Measles IgM
Mumps Serology
Viral Haemorrhagic Fever
West Nile Virus Serology*
*go to PHE rare and imported pathogens lab
Which bacterial/parasite/fungal tests require to be sent away?
Amoebic Serology (IFAT)
Aspergillus antigen (Galactomannan)
Beta-glucan serology
Bordetella pertussis Serology
Campylobacter Serology
Candida Serology
Clostridium botulinum toxin
Cryptococcal antibodies/antigen
E coli 0157 Serology
Filarial Serology
Histoplasma Serology
Hydatid Serology
Leishmania Serology
Listeria PCR
Malarial antibodies
Meningococcal Serology / PCR
Neisseria gonorrhoeae supplementary NAAT
Rabies exposure
Rickettsia Serology*
Schistosoma Serology
Strongyloides Serology
Toxocara Serology
Trichinella Serology
Trypanosome antibodies
*go to PHE rare and imported pathogens lab
Which requests might we receive, which should be re-directed to immunology department?
Aspergillus Serology
Avian Serology
functional Ab - diphtheria/ Hib/ pneumococcal
Farmers Lung Serology
Haemophilus influenzae b Serology (Hib antibodies)
Tetanus immunity
HIV1/2 diagnosis
What is general principle behind Architect EIA?
HIV Ag/Ab combo - p24 and HIV1/2 antibodies
HIV 1 has groups M/ N/O, with many sub-groups. HIV2 is structurally similar, although less pathogenic.
Common feature is transmembrane protein (gp41), so aim to detect antibodies to this. p24 will be detectable sooner, prior to sero-conversion occurring
HIV1/2 diagnosis
What are steps required in Architect EIA?
Add sample, wash buffer, assay diluent, paramagnetic microparticles together
If p24 Ag present will bind to mouse monoclonal Ab
If HIV Ab present, will bind to HIV antigen
Wash sample
Add acridinium labelled-conjugates, which bind to Ag-Ab complexes
Acridinium is chemiluminescent, and amount of luminescence can be used to determine level of antibodies.
Compare to signal to cutoff (S/CO), and if >1.0 then positive test
Must then perform confirmatory test, and HIV serotype
HIV1/2 diagnosis
After initial testing on Architect, result must be confirmed on VIDAS. (VIDAS can in some cases be used as screening test,
What is principle behind VIDAS?
Tests p24 antigen, HIV antibody
Solid phase receptable (SPR) serves as pipetting devie, as well as solid phase -
- upper part coated in monoclonal p24 Ab, for detection p24 Ag
- lower part coated in HIV gp160 protein, and other HIV-1 group O, and HIV-2 specific peptides
Sample added, and cells lysed Released p24 antigen binds to anti-p24 HIV Ab bind to gp160 or other peptides Wash sample Add conjugate enzyme which provides fluorescence. fluorescnce is proportional to the presence of HIV Ab and p24 antigen
HIV 1/2 diagnosis
After diagnosing on Architect, and confirmed on VIDAS, we must check if HIV1/2 on Geenius.
What is principle behind this test?
Add sample and buffer. further buffer facilitates lateral flow across strip.
As sample flows across strip, HIV Ab are captured by antigens on solid phase, and causes a colour change (pink/purple).
Atingens present on solid phase:
HIV 1 - p24, gp31, gp41, gp160
HIV 2 - gp36, gp140
This Control line serves to demonstrate that sample and reagents have been properly applied and
have migrated through the device.
HIV 1/2 diagnosis
What antigens are present on each band of Geenius HIV1/2 confirmatory test?
Band 1: gp36 HIV-2 ENV
Band 2: gp140 HIV-2 ENV
Band 3: p31 HIV-1 POL
Band 4: gp160 HIV-1 ENV
Band 5: p24 HIV-1 GAG
Band 6: gp41 (Group M and O) HIV-1 ENV
CTRL band: Protein A
HIV 1/2 diagnosis.
Once confirmed, we need to check viral RNA load (only of HIV1, HIV2 is send away).
Extraction firstly performed on Qiagen Symphony.
PCR then performed on Qiagen Artus performed on Rotor-gene.
How is it performed?
HIV1 Master mix A + B contain reagents and enzymes for reverse transcription and amplification of 93 bp region of HIV-1 genome, and for the direct detection of amplicon fluourescence in Rotor-Gene Q
Validated for use with HIV1 A-H subtypes
HIV1 viral load of Qiagen Artus via Rotor-gene
What is viral load reference range?
Not detected
<34 copies/ ml - result is outside the determined test range by assay. LOQ - limit of quantitation
> 34 copies/ml - HIV RNA detected - LOD - limit of detection
What is meant by -
Limit of detection
Limit of quantitation
LOD - is the actual concentration of an analyte in a specimen that can be consistently detected ≥ 95% of the time
LOQ - is the lowest concentration at which the analyte can not only be reliably detected but at which some predefined goals for bias and imprecision are met. This can be same as LOD.
Both used to identify analytical sensitivity
Diagnosis of SARS-Covid19
Can be split into PCR testing, and antibody testing.
What are methods of antibody detection?
Architect
DiaSorin - Liaison
Panther etc are for PCR detection
Diagnosis of SARS-CoV-2
How does Architect assay work?
Combine sample, SARS-CoV-2 Ag coated microparticles, and assay diluent
If Ab present, bind to Ag coated microparticles
Wash sample
Anti-human IgG acridinium-labeled conjugate is added
This triggers chemiluminescent reaction, measured in relative light units (RLU). Direct relationship between amount of IgG antibodies to covid in sample, and RLU detected by system optics
Diagnosis of SARS-CoV-2
How does DiaSorin Liaison assay work for serology testing?
Reagents -
- Add sample
- Recombinant S1/S2 antigens coated magnetic microparticles
Plasma/ serum sample binds to S1/S2 antigens
Wash away unbound material
Add mouse monoclonal Ab bind to Ab-Ag complex, trigger chemiluminescent reaction.
Chemiluminescent reaction produces light signal, which is proportional to antibody present. So quantitative assay. Measures IgG to S1 and S2 antigens
What are pitfalls of SARS-CoV-2 serology testing?
Specificity 97%, but sensitivity approx 64%. Sensitivity increases to 71% if performed 21 days after infection
If not automatic testing, risk of errors/ contamination
Cross-reacting antibodies
Ab tests should always be interpreted in clinical context, cannot be used to inform of infection status
Can have delayed antibody production
Immunocompromised may not produce antibodies
Do not know if antibodies provide immunity
Other coronavirus strains have not been evaluated with common assay
Rheumatoid factor in human serum can react with immunoglobulins, interfering with assay
Diagnosis of SARS-Covid19
Can be split into PCR testing, and antibody testing.
What are methods of PCR detection?
Extraction + amplification -
Hologic - Panther
Extraction only -
Altona
MTprep
Symphony
Amplification -
Rotorgene
MTPCR
Diagnosis of SARS-Covid19
What is mechanism of Hologic Panther test PCR detection?
Pre-amplification:
L6 buffer added to samples, which lyses cells, and releases RNA
Oligomers with magnetic microparticles bind to target site on RNA as complementary. Targets ORF1ab region 1/2 sections of RNA
Magnetic force applied to move RNA to side of tube, and supernatant aspirated
Sample washed to remove potential amplification inhibitors
Amplification:
RT-PCR performed converts to DNA. DNA then amplified by polymerase.
Chemiluminescent nucleic acid probes bind to nuclear mateiral. They contain acridinium which is chemiluminescent
Luminescence measured in Relative Light Units (RLU). The internal control has different fluorscence, so can determine difference between
Panthers performs extraction, reverse transcription, and amplification in one unit
AusDiagnostic can be used to perform multiplex PCR, and identify up to 16 respiratory pathogens.
Describe the assay for covid19
Extraction -
- AusDiagnostics MT-Prep performs extraction - lyse cells to release nuclear material. Add protease to remove debris. Add RNAase or DNAase to remove unwanted nuclear material. Wash with alcohol to remove cellular debris - left with pure nuclear material. elute into another tube
Pre-amplification -
- AusDiagnostics MT-Processor
- Two pre-amplification steps where primary amplification involves“target enrichment”using target-specific outer primer sets with a small number of PCR cycles, followed by secondary amplification where inner primers amplify a target region within the product from the primary amplification. ORF1 and ORF8 are gene targets
- uses SYBR green labelled probes
Amplification/ analyser -
- MT-Analyser - PCR performed, and real-time measurement of fluorescence producing melt-curve
AusDiagnostic can be used to perform multiplex PCR. Has 16 wells, which can identify a number of pathogens.
What is included in respiratory panel?
Influenza A Influenza B Influenza A typing H1/H3 Parainfluenza 1, 2, 3, 4 Respiratory Syncytial Virus A & B Adenovirus groups B, C, E, some A, D Rhinovirus Enterovirus Metapneumovirus Coronavirus 229E, HKU-1, NL63 & OC43 Bocavirus
Process same as for covid AusDiagnostics
HBV serology
How does HBsAg/ HBeAg test performed?
Add:
sample
anti-HBs/ anti-HBe coated paramagnetic microparticles
anti-HBs/ anti-HBe acridinium-labelled conjugate
Ag present in the sample binds to the antibody-coated microparticles and to the acridinium‑labeled conjugate.
Sample washed
Causes chemiluminescent reaction measured as relative light units (RLUs), and term if it is above S/CO
Antibody tests performed the same way
HBV serology
After testing HBsAg positive, what other tests are required?
HBsAg confirmation test on Vidas
HBsAg neutralisation test
HBeAg/ HBeAb
HBV serology
As cross-reacting antibodies can occur, neutralisation test must be performed.
How is this done?
In contrast to the goat and mouse antibodies used in the HBsAg assay, the specific antibody used in the confirmatory reagent is derived from horse, this precaution minimises the risk of confirming false positive samples containing anti-species antibodies.
For each test sample two samples cups are used. The Abbott Architect HBsAg assay is run according to the usual procedure except that the sample is incubated with a specific reagent in one sample cup and with a control reagent in the other.
During the first incubation the horse anti-HBs in the Specific Reagent will compete with the mouse anti-HBs immobilised on the microparticles for any HBsAg present in the sample and will reduce the amount of HBsAg binding to the well; in the control well there is no competition and the HBsAg will bind normally. The rest of the assay is completed in the normal way.
HBV serology
How does anti-HBc assay performed?
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).
HBV serology
What is significance of isolated anti-HBc? Termed IAHBc
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
HBV serology.
Isolated raised anti-HBc (IAHBC) in patients who become immunosuppressed/ HIV. This can mean chronic infection, so risk of re-activation.
What are steps for monitoring patient?
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
HEV serology
How is anti-HEV (IgM/IgG) checked?
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.
HBV serology
HBV DNA viral load quantified by PCR.
Uses Qiagen Symphony for extraction, then rotor gene
Which region of HBV DNA does it target?
What is Qiagen Artus limit of detection?
134 bp region of HBV genome - detect in fluorescence channel Cycling Green
Internal control in Cycling Yellow
10.22 IU/ml
What assays are available in UK for viral load testing?
Qiagen Artus - NNUH
Abbott Realtime
Roche - Cobas Taqman
Siemens VERSANT HBV assay
HCV serology
HCV RNA viral load quantified by PCR.
Uses Qiagen Symphony for extraction, then rotor gene
Which region of HCV RNA does it target?
What is limit of detection of Qiagen Artus?
240bp region of HCV genome
21 IU/ml
HCV sequencing can be performed used VELA technology, via next generation sequencing.
Being removed from NNUH
What does it detect?
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.
Norovirus has over 7 genogroups, with multiple genotypes.
Pathogenic to humans
Genogroup I - 9 genotypes
Genogroup II - 22 genotypes
Genogroup IV - 2 genotypes
How is RNA test performed on RIDAGENE?
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.
VZV IgG levels.
what are cut-offs for immunity?
<50 IU/ml neg
50-100 IU/ml borderline
> 100 IU/ml pos
Parvovirus B19
What are cut offs for IgM/ IgG?
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
CMV PCR used for viral load. Performed on Qiagen artus, using Rotor-Gene
(PCR for neonatal viral load of urine/ saliva performed on ELITech)
Which region of genome is amplified?
What is LOD?
105bp region of CMV genome
42 copies/ml LOD
CMV serology tests. If IgG positive, can confirm CMV avidity.
What is this, and how is it performed?
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.
CMV avidity
What are normal cut off values?
< 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.
CMV PCR for congenital CMV (cCMV)
Test neonates before age 21 days - usually if fail hearing test. Where a permanent SNHL is identified in children over 1 year of age the saliva test has not been validated.
Performed on ELITech using -
- saliva (at least 1 hour after feeding)
- urine
Why is assessing for cCMV important?
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
cCMV detected - approx 5 cases/ year at NNUH
What are next steps?
Repeat swab to confirm
Inform audiology
Ophthalmology review
CT brain
Virology/ paediatrics to discuss treatment
ELITech for urine/ saliva PCR.
What is limit of detection?
350-450 copies/ml
CMV PCR on ELITech to detect virus.
What is principle of test?
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.
EBV serology performed on VIDAS.
What are the targets?
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
EBV serology.
What is process behind EBV VCA/EA IgG VIDAS test?
Which antigens do antibodies bind to?
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
EBV serology.
What is process behind EBV VCA IgM VIDAS test?
Same as usual VIDAS test
anti-EBV IgM binds to VCA P18 antigens on SPR wall
EBV serology.
What antigens do these antibodies bind to?
anti-VCA IgM/IgG
anti-EBNA IgG
anti-VCA IgM/IgG - p18/ p54
anti-EBNA IgG - p72
CSF testing
Which bacteria/ viruses can be tested at NNUH?
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
What tests are available for syphilis?
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
Syphilis serology.
Initial testing involves EIA on Archiect. What steps are involved?
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
Syphilis serology.
How is TPPA test performed?
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
Syphilis serology.
How is RPR performed?
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
Which tests are performed as complement fixation tests (CFTs)?
Adenovirus Mycoplasma Chlamydia Coxiella Enterovirus Leptospirosis Brucella
How do we test for bartonella?
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.
How is legionella antigen testing performed?
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
How is Streptococcal antigen testing performed?
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.
How is measles antibody detected?
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
group A Streptococci testing involves testing for antibodies against which parts of bacteria?
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.
How to test for GAS? ASO/ADB
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
What is process of testing for rotavirus?
Human serotypes of Group A are major causes of gastroenteritis in young children throughtout world. Faeces samples from children <6 and diarrhoea are tested.
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
What is process of testing for adenovirus?
Adenovirus is among the most common infectious causes of gastroenteritis, mainly in infants and young children. Adenoviral gastroenteritis may result in mortality for populations at risk such as infants, the elderly and immunocompromised patients. The most common Adenovirus serotypes associated with gastrointestinal infection are types 40 and 41.
Faeces samples from children <6 and diarhoea/ vomiting are tested.
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
