B3.003 Prework 2 Culture Independent Diagnostic Tests Flashcards
advantages of molecular microbiology
increased sensitivity over culture
rapid
limitations of molecular microbiology
expensive
targeted
no susceptibility testing
no “test of cure”
methods of molecular microbiology
nucleic acid hybridization
nucleic acid amplification test (NAAT)
sequencing
mass-spectrometry
nucleic acid hybridization
hybridization of nucleic acid probes to DNA
no amplification step
NAAT
polymerase chain reaction (PCR)
-reverse transcriptase PCT (RT-PCR) for RNA
-quantitative PCR (qPCR)
isothermal amplification (LAMP, SDA, TMA)
sources of false positives
contamination
sources of false negatives
organisms below limit of detection
biological inhibitors
PCR steps
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
amplification + detection
single target
-single organism/resistance markers
multiple targets (multiplexes)
-syndromic panels
amplification + sequencing
“broad range” PCR
-targets 16S rRNA gene, detects any bacterial pathogen
metagenomics
-sequence all DNA/RNA in a sample, compare to database
2 types of serology
antigen detection
antibody detection
antigen detection
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
examples of antigen detection
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
antibody detection
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
examples of antibody detection
lyme disease (borrelia burgdorferi) syphilis (treponema pallidum) Q fever (coxiella burnettii)
IgM
associated with acute infection
detectable within 7d of infection
wanes after 2-3 months
IgG
associated with acute or past infection/vaccination
detectable within 14d
detectable for entire life
agglutination
antibody or antigen fixed to latex beads
specimen added to latex Ab or latex Ag leads to visible clumping of beads
lateral flow immunoassay
antibodies attached to conjugate pad
antigen in patient sample wicked along conjugate pad
accumulation of antigen-antibody complexes detectable as a visible line
enzyme linked immunosorbent assay (ELISA)
antibody or antigen immobilized in well
add patient sample
add reporter antibodies/antigen
add substrate and read colormetric change
immunoblot
Western blot
antigens printed or transferred onto membrane
incubated with patient serum
bound patient antibodies detected with secondary antibody + chromogenic detection substrate
immunodiffusion
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
when should you choose a serology test?
determine vaccine status
infection with organism that won’t grow in culture
infection with an organism that is cleared quickly
screening
sources of serology false positives
heterophile antibodies/ rheumatoid factor
cross reaction to closely related antigens (esp IgM)
maternal transfer (IgG)
sources of serology false negatives
immunocompromised
too early/late
