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1-Scientific & lab basis of microbiology > How tests work > Flashcards

How tests work Flashcards

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Biology 101 flashcards
1
Q

What are the pros vs.cons of pyrosequencing?

A

Pros:

  • Cheap
  • Sometimes all you need to ID different organisms within a narrow taxon (e.g. NTMs)

Cons:

  • Very short reads (~50-70 bases)
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2
Q

What are the pros/cons of Sanger sequencing?

A

Pros: Long reads (700-1000 bases)

Cons: Expensive

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3
Q

What is the optimal volume of a Blood Culture Set, for adults?

A

40 mL

10 mL/vial

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4
Q

How does the GeneXpert MTB-RIF system work?

A
    1. Sputum is liquified + inactivated with the sample reagent (provided).
  2. 2mL sputum transferred into test cartridge.
  3. Cartridge inserted into the test platform/machine.
    Automatically:
  4. Sample filtered, caught in filter, + washed.
  5. Sample lysed through ultrasonication –>releases DNA.
  6. DNA mixed with PCR reagents (“dissolves reaction beads”).
  7. Semi-nested real-time amplification (PCR) + detection in reaction tube. Detects PCR amplification + adherence of probes to rif.
  8. Result printed out.
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5
Q

Which species will come up as false +ve’s in the QFT test?

A
  1. M.kansasi
  2. M.szulgai
  3. M.marinum

All 3 also have the proteins that QFT is testing for: ESAT-6, CFP-10, TB7.7

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6
Q

HIV confirmatory tests

A

Geenius HIV-1/HIV-2 differentiation test. If Geenius result = indeterminate: RT-PCR HIV-1 NAT (for viral load)

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7
Q

HBV serology result interpretation: + anti-HBsAg

A
  • Acute infection
  • Recent HBV immunization
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8
Q

HBV serology result interpretation:

+ Total anti-HBc

+ IgM anti-HBc

A
  • Acute infection
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9
Q

What markers are used in Hepatitis B virus serology,

& what do each of them signify?

A
  1. HBsAg: acute hep B infection.
  2. Anti-HBs: immune to HBV (usually).
  3. HBcAg: {}
  4. Anti-HBc IgM: acute infection
  5. Anti-HBc IgG: prior exposure (or chronic infection.
  6. HBeAg: active replication.
  7. Anti-HBe: transmissibility
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10
Q

How does the Respiratory Pathogen Panel (RPP) work?

A
  1. Cells received on swab of URT/LRT.
  2. Extract NA
    (URT = Hamilton automated;
    LRT = EasyMAG manual.)
  3. 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:

  1. Pulls beads down to a monolayer.
  2. Red LED -> Classifies the hybridized, tagged beads.
  3. Sorts them on the ‘bead map’ according to their classification.
  4. Green LED -> Quantifies the analyte in each bead region (excited reporter fluorescence)
  5. Signals in each rxn well are analyzed for each of the 22 targets + internal controls
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11
Q

Which viruses are on the BBFE serology panel?

(3)

A
  • HBV (sAg, sAb)
  • HCV
  • HIV
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12
Q

Which virology NATs are done daily (M-F)?

A
  • HIV-1
  • HBV
  • HCV
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13
Q

What is the modified Kinyoun stain used for?

A

To detect coccidian parasites

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14
Q

Which pathogens are on the prenatal serology panel?

(5)

A
  1. HIV
  2. HBV sAg
  3. Syphilis
  4. Rubella IgG
  5. VZV IgG
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15
Q

Which protozoa do we culture at PLNA?

A
  1. Leishmania
  2. Trichomonas
  3. Acanthamoeba

​(Subsequent stains are:

  • Giemsa,
  • modified Kinyoun,
  • Trichrome)
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16
Q

Which tests are best for detecting B.pertussis at different times after infection:

  • 1-2wk
  • 2-3wk
  • 4wk
A
  • 1-2 weeks: PCR = cx
  • >2wk: PCR >cx
    (cx reliability dramatically decreases)
  • >4w: PCR reliability decreases, do serology
17
Q

Which HIV test should be used if you suspect maternal +ve and tmx to newborn

A

HIV proviral DNA test

18
Q

A clinician thinks their patient has Nocardia bacteremia.

How should you process these samples differently than for another bacterium?

A
  • 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.
19
Q

What is an ‘Agglutination reaction test’?

A

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).

20
Q

What are the different types of agglutination assays?

A
  1. 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.).
  2. 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.
  3. Reverse passive: Ab-coated particles detect soluble Ag in patient serum.

Note: agglutination tests that use RBCs are ‘hemagglutination’ assays.

21
Q

Explain how Complement Fixation tests work.

A

Principle: Detects a specific antibody orantigen in a patient’s serum.

  1. Add pt serum into a tube.
  2. Heat-inactivate complement (but not Ab’s) in the serum.
  3. Add in a known quantity of complement proteins.
  4. 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.

22
Q

What are the 3 major types of enzyme immunoassay?

A

Direct EIAs:

  1. Ag’s from the sample are attached to a surface.
  2. A matching Ab (linked to an enzyme) is applied over the surface so it can bind to the antigen.
  3. The enzyme’s substrate is added, producing a detectable signal, most commonly a color change.

Competitive EIAs:

  1. Unlabeled Ab bound to solid phase.
  2. Add pt spx (w Ag of interest) + purified, labeled Ag to well
  3. = Compete for the adhered Ab bound to the solid phase.
  4. Wash.
  5. Add enzyme substrate.
  6. 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!
23
Q

What are the 2 types of immunofluorescence assays?

A

Direct IFAs:

  1. Apply spx to slide.
  2. Add a fluorescently-tagged anti-influenza virus Ab.
  3. Review with a fluorescence microscope at the appropriate excitation wavelength for the fluorescent tag.
  4. If the tagged Ab has bound to influenza Ag in the spx, fluor light will be emitted + observed.

Indirect IFAs:

  1. Unlabeled target AbAb/whole organism is adhered to a microscope slide.
  2. Pt spx is added –>inc.
  3. Ab-Ag complexes form.
  4. Unbound spx washed away.
  5. Then, a fluor-labeled 2’ Ab is added.
  6. Slide observed using a fluor microscope.
24
Q

Explain how an immunodiffusion assay works.

When is it used?

A

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.

25
Q

What is the cutoff for a positive QFT test?

A

(TB Ag - Nil) >= 0.35 is a positive LTBI

26
Q

What specimens can the GeneXpert MTB-RIF be used on?

A

Sputum (induced/expectorated)

No others have been examined.

27
Q

What is MPT64?

A

A predominant protein secreted by MTB complex members.

Exception: Some substrains of BCG, and M.bovis.

28
Q

What does the Alere MPT64 Rapid Ag test for?

A

The presence/absence of the MPT64 protein in a culture (liquid/solid) - as a marker of MTBC members.

29
Q

What is our HIV screening test?

What are its targets?

A

ARCHITECT 4th gen HIV immunoassay.

Detects:

  • HIV-1 Ab
  • HIV-2 Ab
  • HIV-1 p24 antigen
30
Q

What are the targets in the QFT assat?

A
  • TB7.7
  • ESAT-6
  • CFP-10
31
Q

What pathogens are detected on the Luminex Respiratory Pathogen Panel (RPP)?

A
  1. Influenza A
  2. A-H1
  3. A-H3
  4. A-H1N1 pdm09
  5. Influenza B
  6. Parainfluenza 1
  7. Parainfluenza 2
  8. Parainfluenza 3
  9. Parainfluenza 4
  10. RSV A
  11. RSV B
  12. Adenovirus
  13. Rhino/Enterovirus
  14. Metapneumovirus
  15. Coronavirus HKU1
  16. Coronavirus NL63
  17. Coronavirus OC43
  18. Coronavirus 229E
  19. Bocavirus*
  20. Chlamydophila pneumoniae
  21. Mycoplasma pneumoniae
  22. Legionella pneumophila*

* Not validated at PLNA

32
Q

How long does the Alere MPT64 Rapid Ag test take for results?

A

15 minutes.

33
Q

How long does the GeneXpert take to run, including processing?

A

1h45’

34
Q

Why are mycobacteria called “acid fast bacilli”? {}

A

Their cell wall contains waxy fatty acids called “mycolic acids”.

This makes them challenging to stain.

However, they do retain stain when you use an acid alcohol decolourizer.

(hence termed “acid-fast”)

1-Scientific & lab basis of microbiology (9 decks)

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