Micro Lab Diagnosis Flashcards
what part of gram-negative cell walls contribute to shock
lipopolysaccharides, particularly lipid A, which is an endotoxin
stain for mycobacteria
acid-fast, gram staining won’t work because of waxy lipid layer
stain for spirochetes
wright-giemsa, gram staining won’t work, even though they’re gram negative, because they’re too thin to resolve
culture dependent diagnostic tools
microscopy and tissue cultures from a sample off where you think the organism resides
culture independent diagnostic tools
serology and molecular biology (nucleic acid detection, viruses)
what are all the places you can collect specimen from
sputum, urine, feces, tissue lesion (center and border), pus, blood culture, abscess fluid, CSF, vaginal discharge
what are the tools with which you can collect specimen
blood culture and swabs
Normal flora of the upper respiratory tract
staphylococcus, streptococcus pneumonia and viridans, haemophilus, and anaerobes
normal flora of the skin
staphylococcus, coryneform bacteria or diptheroids, and propionibacterium
normal flora of the GI tract
anaerobes, enterococcus, enterobacteriacaea (e coli and klebsiella), streptococcus anginosus, lactobacillus, and candida
normal flora of the GU tract
lactobacillus and streptococcus agalactiae
neutrophils on slides look like
multi-lobed nuclei
epithelial cells in sputum
rejected because heavily contaminated with saliva
selectivity of MacConkey
inhibits growth of gram positive organisms, grows gram negative
differentiation of MacConkey
distinguishes between those with the ability to ferment lactose (pink), and this without (colorless)
the keys to a good blood culture
draw before antibiotics, volume, two bottles of 2 draws for 4 bottles total
alpha hemolytic will appear ___ on blood agar
green
beta hemolytic will appear ___ on blood agar
cleared
gamma hemolytic will appear ___ on blood agar
white/colorless, no clearing
if you see yellow colonies on blood agar, think
staphylococcus aureus
if you see red/pink colonies on blood agar, think
serratia marcescens
if you see blue/green metallic colonies on blood agar, think
pseudomonas aeruginosa
if you see “feet” or projections on a colony, think
yeast infection, or candida albicans
if you see mucus on a colony, think
klebsiella pneumoniae
if you see swarming off the streak link on blood agar, think
proteus vulgaris
know a species is gram positive, what’s the next think you want to know
cocci or bacilli?
know your species is a gram positive cocci, what’s the next question?
clusters or diplococci/chains?
clusters of gram - cocci indicate
staphylococcus
diplococci or chains of gram - cocci indicate
streptococci
know you have a streptococci, what’s the next step?
aerobic or anaerobic
anaerobic streptococci need to know
peptostreptococcus
know you have an aerobic streptococci, what’s the next question?
what type of hemolysis does it undergo
alpha hemolytic streptococci
S. pneumonia, mutans, and viridans
beta hemolytic streptococci
S. pyogenes (group A strep, GAS), and agalactiae (group B strep, GBS)
gamma hemolytic streptococci
enterococcus
know you have a gram negative isolate, whats the next question?
bacillus/coccobacillus or cocci?
gram negative cocci to know
neisseria and moraxella
know you have gram negative bacili/coccobacilli, what’s the next question?
does it grow on macconkey agar
gram negative isolate grows on macconkey agar, what’s the next question?
does it ferment lactose (pink) or not (colorless)
lactose-fermenting macconkey positive gram negative organisms
Escherichia, klebsiella, or enterobacter
non lactose-fermenting maconkey positive gram negative organism
does it ferment glucose
gram negatives that ferment glucose
are also lactose non-fermenters, and grow on maconkey agar, and include salmonella, shigella, proteus, serratia
gram negatives that don’t ferment glucose
also don’t ferment lactose, grow on maconkey agar, and include pseudomonas burkholderia and acinetobacter
if your gram negative isolate doesn’t grow on maconkey
test its oxygen tolerance
oxygen tolerant gram negative isolates
campylobacter, helicobacter, vibrio
oxygen intolerant gram negative isolates
bacteroides, fusobacterium, prevotella, porphyromonas
PCR-based susceptibility testing
detects genes associated with drug resistence
mecA gene
associated with methicillin resistance in staph aureus
vanA/B genes
associated with vancomycin resistance in enterococcus faecium
bla KPC gene
associated with carbapenem resistance (beta lactamase, bla) in Klebsiella pneumonia carbapenemase (KPC)
pros and cons of PCR susceptibility testing
fast, sensitive, specific, but expensive and only single target, when resistance may be caused by a combination of many mutations
MIC
minimal inhibitory concentration, minimum concentration of a drug that will inhibit the growth of an organism
culture-dependent phenotypic susceptibility testing
agar dilution, broth microdilution, disk diffusion, strip test
microbroth dilution
broth and increasing concentration of antibiotic in well plate, lowest concentration without bacterial growth is the MIC
strip testing (Etest)
paper/plastic strip with increasing concentration of a drug, placed in center of a lawn of isolate on a plate, MIC is point of growth inhibition
disk diffusion test
paper disk with single concentration of antibiotic placed on bacterial lawn on agar, measure zone of inhibition, does not give a MIC
PCR test
extract nucleic acid from sample, denature, anneal primers, elongate, repeat, fluorescently detect DNA
NAAT test
single target, multiplex, or broad range amplification and sequencing/detecting
single target NAAT
uses amplification and detection to identify single organism or resistance marker; most sensitive
multiplex NAAT
uses amplification and detection to identify a pathogen from a panel of common syndromic options i.e. patient has respiratory symptoms, PCR compared to respiratory pathogen pane, less sensitive than single
broad range PCR
targets 16S rRNA on any bacterial pathogen, can only prove that you have an infection, last resort, done when all other tests are negative, least sensitive
MALDI-TOF MS
isolate spotted onto metal plate, coated with protective matrix, laser vaporizes bacteria, travel up to analyzer, mass spec profile compared to database
current use of MALDI TOF MS
identification of cultured bacteria
future use of MADLI TOF MS
susceptibility testing, strain typing, direct from sample detection
Serology: antigen detection
administer a lab generated antibody to detect antigen in specimen, which can be urine, serum, sputum, stool, CSF
Serology: antibody detection
administer lab generated antigen to detect IgG or IgM, or total antigen-specific patient antibodies, only works for blood serum
IgM
present in acute infection, detectable within 7 days of infection, wanes after 2-3 months
IgG
present in chronic disease and immunity, detectable within 14 days, detectable for entire life, indicative of either current, chronic, or past infection
agglutination
antibody or antigen fixed to latex beads, specimen added, visible clumping is a positive result for the antibody/antigen in question
lateral flow immunoassay
e.g. pregnancy test, antibodies on conjugate pad, antigen in sample wicked along pad, accumulation of antigen antibody complex detectable s visible line
ELISA sensitive or specific?
high sensitivity, low specificity, high negative predictive value, so all positive results must be confirmed with a secondary assay, which, if negative, indicates negative result
ELISA method
antibody or antigen immobilized in well, patient sample added, reporter antibodies/antigen added, substrate for colometric change added, read
immunoblot aka western blot
antigens printed/transferred onto membrane, incubated with patient serum, bound antibodies detected with secondary antibody and color detection substrate
immunodiffusion
antigen put in center well of gel matrix patient samples put on periphery, samples diffuse outward during incubation, precipitation complex visible in gel
when to use as serology test
determining vaccine status, infection with culture-independent organisms, infection with organism cleared too quickly, screening for HIV/HCV/HBV
why would you get a false positive with a serology test
heterophile antibodies, rheumatoid factor, closely related antigens cross react (especially with IgM), and maternal transfer aka passive immunity (especially of IgGs)
why would you get a false negative with a serology test
patient is immunocompromised or the test is administered either too soon or too late
factors to consider when ordering a lab
cost-benefit ratio, time to result, do I know how to interpret, will the result change anything
role of bacterial cell wall
structural rigidity, shape, osmoregulation, defense
drugs that target bacterial cell walls
penicillins, monobactams, carbapenems, cephalosporins, vancomydin
how do bacteria develop resistance to cell wall attacking drugs
enzymatic inactivation, modification of target, porin mutations to reduce permeability, and efflux pumps
components of peptidoglycan layer
NAG and NAM, NAM tetrapeptide side chain, peptide inter bridge cross-linker
steps of building the peptidoglycan layer
peptidoglycan polymers exported from cell, transglycosylase attaches them, penicillin binding protein creates transpeptidase crosslinks
how do beta lactam drugs work?
structurally mimic NAM, bind the enzymatic domain of PBP, preventing peptide crosslinking, leading to weak peptidoglycan wall
beta lactamase
which cleaves and degrades the beta lactic ring at the N-C=O bond e.g. penicillinase, secreted extracellularly in gram positives and into periplasmic space in gram negatives
mechanisms of resistance to beta lactams
beta lactamase synthesis, modification of target, porin mutations, efflux pumps
modification of target in staph aureus
via mecA gene that encodes the PBP2a protein, low affinity for penicillin after it’s been modified, develops resistance
ways to check for resistant mega gene
targeted molecular testing for mecA, antigen testing for PBP2a, and phenotypic testing for oxacillin resistance
what are them most likely causes of culture negative endocarditis
recent antimicrobial therapy, fastidious organism
opportunities to narrow down your broad spectrum antibiotics
3 hours after presentation, direct gram stain reported, take rapid AST, de-escalate
1-2 days after presentation, organism growth and ID reported, take another rapid AST, de-escalate antibiotics if differential further narrowed
finally, when AST report comes back after 2-3 days, narrow antibiotics based on organism resistance
