Diagnostic Microbiology (CH 25) Flashcards
What are the steps to specimen collection?
- sample collection (aseptic, proper transport)
- microscopy & staining (gram stain, AFB, KOH, etc)
- culture & isolation (growth on selective/enriched media)
- biochemical tests (catalase, coagulase, oxidase)
- antigen & toxin tests (ELISA, rapid antigen tests)
- molecular tests (PCR, DNA sequencing)
- antibiotic sensitivity testing (kirby-bauer, MIC, VITEK)
- final diagnosis & treatment decision
Can you use rapid antigen tests before microscopy & collection?
yes, but have to weigh pros/cons rapidness vs false +/-
What samples from human body sites are considered “sterile”?
- blood
- cerebrospinal fluid
- pleural fluid
- synovial fluid
- peritoneal fluid
- any tissues from internal organs
How are blood cultures collected?
- collected by phlebotomy / venal puncture into bottles
- bacterial growth induces fluorescence
- blue top = aerobic
- orange top = anaerobic
Why do we do sample collections?
- to identify sequelae with some bacteiral infections - s/s can overlap
- antimicrobial susceptibility should be known before antibiotics are prescribed
- empirical abx treatment often started before lab testing is completed
- identify abx resistance
- epidemiological surveillance
how are cerebrospinal fluid (CSF) collected?
- collected by lumbar tap / puncture
- for direct microscopy and culture
how are pleural, synovial and peritoneal fluid collected?
collected by thoracentesis, arthrocentesis, and paracentesis punctures
- for direct microscopy and culture
how are skin, abcess, throat, and nasopharyngeal specimens collected?
- skin samples
- deep wound abscesses are sampled via needle aspiration
- special nasopharyngeal swabs used to collect secretions from nasopharynx
how are lung specimens collected?
- sputum comes from wbcs and is the result of inflammation
- bronchoscopy used to collect lung materials if pt has trouble coughing out sputum
- use AFB for mycolic acid (TB)
Urine sample collection
how to collect aseptic collection?
- normal microbiota exist in bladder, are anaerobic & not easily culturable
- skin microbiota may contaminate “clean catch” urine samples
- catheterization ensures aseptic collection of samples directly from bladder
what is specificity used for?
true negatives
what is sensitivity used for?
true positives - testing for who has the disease
disadvantages of POC tests?
- no data about pathogen antibiotic sensitivity
- incr risk of clinician becoming infected
- multiple infections overlooked if initial test is positive
- sensitivity (true positives) may be sacrified for rapid results (false negatives)
Advantages of Point-of-care (POC) tests?
- no culturing required
- clinicians can immediately prescribe right antibiotic
- pts can avoid taking unnecessary antibiotics
- clinicians can quickly determine chain of infection in pts with similar s/s
- clinicians can notify pts while they are still present
- accessible + cheap; can stop spread quicker
how are problem-solving algorithms used to identify bacteria?
culture-based biochemical testing of bacterial isolates individual strains of bac
- differential and selective media
what pathogen identifications use biochemical and protein profiles?
- biochemical algorithms (dichotomous keys)
- culture-based biochemical testing
- biochemical (enzyme reactions)
- protein biochemical composition
What is chocolate agar used for?
contains lysed blood to support growth of fastidious bacteria
- for organisms that cannot get nutrients outside of cells unless it’s already there
what is Hektoen agar used for?
contains lactose, peptone, bile salts, thiosulfate, iron salts, and 2 pH indicators
- if orange colour = lactose (+)
- likely used in anaerobic conditions
- e.g., GI conditions
How are gram-negative enteric bacteria identified?
with biochemical algorithms
put bac in different sites –> see how they interact with the environment and change colour to guide the diagnosis
how are biochemical algorithms used to identify bacteria? and for what?
- dichotomous keys
- automation in clinical laboratories (input into machine, will tell you diagnosis)
- biochemical tests to differentiate between gram -/+
- for identifyinf gram+ pyogenic cocci, gram - enteric bac
- identify acid-fast bacteria
What is used to identify pathogens by pathogen signature? and how is it used?
- matrix-assisted laser desorption ionization-time of flight mass spectrometry or MALDI-TOF MS
- to probe bacterial biochemistry
- each pathogen has specific protein composition that defines its own unique protein signature or fingerprint
- identifies bacteria by measuring/detecting the mass of proteins
What is RT-PCR?
- reverse transcription- polymerase chain reaction
- used when pathogen has RNA, need RT enzyme to make DNA from RNA
what is polymerase chain reaction (PCR)?
- most widely used molecular method in clinical lab
- DNA primers can be made for specific pathogens
- multiple sets of DNA primers can identify individual genes from a pathogen for more specific typing -> takes genomes and makes lots of copies
- gold standard for identifying pathogens
- more specific, knows what makes pathogens different from e/o
- useful for pathogens that are hard/slow to grow
what is qRT-PCR used for?
quantitative reverse transcription-PCR, used for high-throughput diagnosis of viral pathogens, such as WNV
