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.
