How tests work Flashcards
(34 cards)
What are the pros vs.cons of pyrosequencing?
Pros:
- Cheap
- Sometimes all you need to ID different organisms within a narrow taxon (e.g. NTMs)
Cons:
- Very short reads (~50-70 bases)
What are the pros/cons of Sanger sequencing?
Pros: Long reads (700-1000 bases)
Cons: Expensive
What is the optimal volume of a Blood Culture Set, for adults?
40 mL
10 mL/vial
How does the GeneXpert MTB-RIF system work?
- Sputum is liquified + inactivated with the sample reagent (provided).
- 2mL sputum transferred into test cartridge.
- Cartridge inserted into the test platform/machine.
Automatically: - Sample filtered, caught in filter, + washed.
- Sample lysed through ultrasonication –>releases DNA.
- DNA mixed with PCR reagents (“dissolves reaction beads”).
- Semi-nested real-time amplification (PCR) + detection in reaction tube. Detects PCR amplification + adherence of probes to rif.
- Result printed out.
Which species will come up as false +ve’s in the QFT test?
- M.kansasi
- M.szulgai
- M.marinum
All 3 also have the proteins that QFT is testing for: ESAT-6, CFP-10, TB7.7
HIV confirmatory tests
Geenius HIV-1/HIV-2 differentiation test. If Geenius result = indeterminate: RT-PCR HIV-1 NAT (for viral load)
HBV serology result interpretation: + anti-HBsAg
- Acute infection
- Recent HBV immunization
HBV serology result interpretation:
+ Total anti-HBc
+ IgM anti-HBc
- Acute infection
What markers are used in Hepatitis B virus serology,
& what do each of them signify?
- HBsAg: acute hep B infection.
- Anti-HBs: immune to HBV (usually).
- HBcAg: {}
- Anti-HBc IgM: acute infection
- Anti-HBc IgG: prior exposure (or chronic infection.
- HBeAg: active replication.
- Anti-HBe: transmissibility
How does the Respiratory Pathogen Panel (RPP) work?
- Cells received on swab of URT/LRT.
- Extract NA
(URT = Hamilton automated;
LRT = EasyMAG manual.) - Add extracted NA into foil-sealed wells in 96wp with lyophilized magnetic beads + reagents.
@ thermocycler:
4. Multiplex RT-PCR + bead hybridization.
Read results on the MAGPIX:
- Pulls beads down to a monolayer.
- Red LED -> Classifies the hybridized, tagged beads.
- Sorts them on the ‘bead map’ according to their classification.
- Green LED -> Quantifies the analyte in each bead region (excited reporter fluorescence)
- Signals in each rxn well are analyzed for each of the 22 targets + internal controls
Which viruses are on the BBFE serology panel?
(3)
- HBV (sAg, sAb)
- HCV
- HIV
Which virology NATs are done daily (M-F)?
- HIV-1
- HBV
- HCV
What is the modified Kinyoun stain used for?
To detect coccidian parasites
Which pathogens are on the prenatal serology panel?
(5)
- HIV
- HBV sAg
- Syphilis
- Rubella IgG
- VZV IgG
Which protozoa do we culture at PLNA?
- Leishmania
- Trichomonas
- Acanthamoeba
(Subsequent stains are:
- Giemsa,
- modified Kinyoun,
- Trichrome)
Which tests are best for detecting B.pertussis at different times after infection:
- 1-2wk
- 2-3wk
- 4wk
- 1-2 weeks: PCR = cx
- >2wk: PCR >cx
(cx reliability dramatically decreases) - >4w: PCR reliability decreases, do serology
Which HIV test should be used if you suspect maternal +ve and tmx to newborn
HIV proviral DNA test
A clinician thinks their patient has Nocardia bacteremia.
How should you process these samples differently than for another bacterium?
- Process like a fungal culture (e.g. fungal BL cx).
- Add a modified acid-fast smear step.
- Add a ‘Nocardia culture’ process for the spx.
What is an ‘Agglutination reaction test’?
A test to see if the patient’s serum contains Ab cognate for an insoluble Ag.
If it does, it results in visible clumping (= +ve agglutination reaction).
What are the different types of agglutination assays?
-
Direct agglutination: Inactivated whole organism + pt serum. (Tests Ag’s found on naturally occurring particulates, e.g. RBCs, whole bacteria.).
- Done for rare pathogens (e.g. F.tularensis, Brucella spp.).
-
Indirect/passive: Ag coupled to particles/beads composed of an inert material (e.g. latex, colloidal gold, gelatin, silicates).
- Often preferred since +ve reactions are very visible.
- Reverse passive: Ab-coated particles detect soluble Ag in patient serum.
Note: agglutination tests that use RBCs are ‘hemagglutination’ assays.
Explain how Complement Fixation tests work.
Principle: Detects a specific antibody orantigen in a patient’s serum.
- Add pt serum into a tube.
- Heat-inactivate complement (but not Ab’s) in the serum.
- Add in a known quantity of complement proteins.
- Add in RBCs. If the Ab IS in the pt’s serum, the Ag + Ab will bind, and complement will react with these complexes + be depleted from the solution. RBCs will NOT lyse = see a pellet.
If the Ab is NOT in the pt’s serum = Ag + complement remain unbound.
Complement lyses the RBC’s, see red haze.
What are the 3 major types of enzyme immunoassay?
Direct EIAs:
- Ag’s from the sample are attached to a surface.
- A matching Ab (linked to an enzyme) is applied over the surface so it can bind to the antigen.
- The enzyme’s substrate is added, producing a detectable signal, most commonly a color change.
Competitive EIAs:
- Unlabeled Ab bound to solid phase.
- Add pt spx (w Ag of interest) + purified, labeled Ag to well
- = Compete for the adhered Ab bound to the solid phase.
- Wash.
- Add enzyme substrate.
- If pt spx contains target Ag, it will reduce the colour change.
- *Noncompetitive EIAs:**
(a. k.a. capture/sandwich assay). - Rxn wells coated w purified Ag bind to specific Ab’s in the spx, which will be detected with an enzyme-labeled 2’ Ab.
- Mainstay of immunologic testing in the clinical micro lab!
What are the 2 types of immunofluorescence assays?
Direct IFAs:
- Apply spx to slide.
- Add a fluorescently-tagged anti-influenza virus Ab.
- Review with a fluorescence microscope at the appropriate excitation wavelength for the fluorescent tag.
- If the tagged Ab has bound to influenza Ag in the spx, fluor light will be emitted + observed.
Indirect IFAs:
- Unlabeled target AbAb/whole organism is adhered to a microscope slide.
- Pt spx is added –>inc.
- Ab-Ag complexes form.
- Unbound spx washed away.
- Then, a fluor-labeled 2’ Ab is added.
- Slide observed using a fluor microscope.
Explain how an immunodiffusion assay works.
When is it used?
Ag and pt serum are placed near to each other in a semisolid matrix and allowed to diffused towards each other. If a complementary Ab to the test Ag is in a patient’s serum, they will bind and form a precipitate.
This test is used to see if a patient’s serum contains antigens/antibody to a pathogen of interest.
