B3.003 Prework 2 Culture Independent Diagnostic Tests

Question 1

advantages of molecular microbiology

Answer 1

increased sensitivity over culture

rapid

Question 2

limitations of molecular microbiology

Answer 2

expensive
targeted
no susceptibility testing
no “test of cure”

Question 3

methods of molecular microbiology

Answer 3

nucleic acid hybridization
nucleic acid amplification test (NAAT)
sequencing
mass-spectrometry

Question 4

nucleic acid hybridization

Answer 4

hybridization of nucleic acid probes to DNA

no amplification step

Question 5

NAAT

Answer 5

polymerase chain reaction (PCR)
-reverse transcriptase PCT (RT-PCR) for RNA
-quantitative PCR (qPCR)
isothermal amplification (LAMP, SDA, TMA)

Question 6

sources of false positives

Answer 6

contamination

Question 7

sources of false negatives

Answer 7

organisms below limit of detection

biological inhibitors

Question 8

PCR steps

Answer 8

extract nucleic acid from sample
heat DNA sample to denature
cool to anneal primers (complementary oligonucleotides)
annealed primers serve as starting point for DNA elongation
repeat cycle, leading to exponential synthesis of DNA (35-40 times)
fluorescent reporter allows for detection and quantification of DNA

Question 9

amplification + detection

Answer 9

single target
-single organism/resistance markers
multiple targets (multiplexes)
-syndromic panels

Question 10

amplification + sequencing

Answer 10

“broad range” PCR
-targets 16S rRNA gene, detects any bacterial pathogen
metagenomics
-sequence all DNA/RNA in a sample, compare to database

Question 11

2 types of serology

Answer 11

antigen detection

antibody detection

Question 12

antigen detection

Answer 12

molecule capable of eliciting an immune response

features: polysaccharides, proteins
method: use lab generated antibodies to detect patient antigen
specimens: urine, serum, body fluid, stool CSF

Question 13

examples of antigen detection

Answer 13

galactomannan, serum, to detect invasive aspergillus infection
cryptococcal antigen, urine or CSF, to detect meningitis caused by Cryptococcus neoformans
legionella antigen, urine, to detect pneumonia caused by legionella pneumophila

Question 14

antibody detection

Answer 14

molecule produced in response to an antigen, capable of binding a specific antigen

features: glycoprotein molecules called immunoglobulins (Ig) (M,G,A,D,E)
specimens: serum

Question 15

examples of antibody detection

Answer 15

lyme disease (borrelia burgdorferi)
syphilis (treponema pallidum)
Q fever (coxiella burnettii)

Question 16

IgM

Answer 16

associated with acute infection
detectable within 7d of infection
wanes after 2-3 months

Question 17

IgG

Answer 17

associated with acute or past infection/vaccination
detectable within 14d
detectable for entire life

Question 18

agglutination

Answer 18

antibody or antigen fixed to latex beads

specimen added to latex Ab or latex Ag leads to visible clumping of beads

Question 19

lateral flow immunoassay

Answer 19

antibodies attached to conjugate pad
antigen in patient sample wicked along conjugate pad
accumulation of antigen-antibody complexes detectable as a visible line

Question 20

enzyme linked immunosorbent assay (ELISA)

Answer 20

antibody or antigen immobilized in well
add patient sample
add reporter antibodies/antigen
add substrate and read colormetric change

Question 21

immunoblot

Answer 21

Western blot
antigens printed or transferred onto membrane
incubated with patient serum
bound patient antibodies detected with secondary antibody + chromogenic detection substrate

Question 22

immunodiffusion

Answer 22

antigen put in center of well of gel matrix
patient samples put in outer wells
samples diffuse outward during incubation
precipitation of Ab-Ag complexes visible in gel

Question 23

when should you choose a serology test?

Answer 23

determine vaccine status
infection with organism that won’t grow in culture
infection with an organism that is cleared quickly
screening

Question 24

sources of serology false positives

Answer 24

heterophile antibodies/ rheumatoid factor
cross reaction to closely related antigens (esp IgM)
maternal transfer (IgG)

Question 25

sources of serology false negatives

Answer 25

immunocompromised

too early/late