midterm 1 Flashcards
functions of clinical laboratory
diagnosis or confirm diagnosis of infectious disease
guidance of treatment
outbreak detection
support for infection control
collect and collate data/info: trends in resistance, antimicrobial susceptibility summaries
epidemiological studies
what do clinical microbiologists provide advice on?
appropriate specimen collection and transport
interpret test results
patient management - recommendations on antimicrobials
why is it important to understand how the clinical lab works?
need to give the lab the appropriate specimens
decrease frustration
cost effective use - choose test wisely
want physicians to provide clinically relevant information such as allergies or the type of wound/body part/symptoms the sample was collected from
general workflow of lab
collection transport/storage accessioning processing interpretation/identification susceptibility testing/ molecular testing reporting/ documentation
requisition form
must have physicians name and contact info
patients name, birthday, unique identifier such as health card number or passport number
specimen type
method of collection - how it is collected determines how it is processed
time/date of collection
required analysis
relevant clinical info - allergies
things to consider for specimen collection
- will the specimen provide useful information - if you’re not prepared to change the patients management based on the results then don’t do it
- choice of the actual specimen to take
- instructions for collection by patient if required
- transport time to lab - may need transport media
- quality of the specimen
- risk of false positives/negatives
- specimens that required being cultured are never put in formalin
Accessioning of the sample
process of identifying the specimen
is the specimen properly labeled? does the requisition info match the specimen label?
was it properly collected
time/date of collection
is it being processed or rejected
is it STAT/life threatening
received specimens must be coded and entered into the LIS system
criteria for specimen rejection
missing information
info on specimen does not match requisition
specimen is too old- improper transport
swab or media is expired
inappropriate specimen
poor quality specimen
specimen is leaking
duplicate specimens
cant be from physician or their family member
things to consider for transport of specimens
transport time to the lab
type of specimen (swab, urine, blood, stool, fluids, scrapings)
transport media: maintain viability but inhibit growth, Cary Blair Transport Media, SAF (parasites)
incubation/refrigeration/storage
processing of specimens
types of specimen: swab, fluid, stool, blood
what tests are required: culture, molecular point of care (POC), serology, microscope (gram stain, AFB stain)
additional processing such ad decontamination or centrifugation if required
what media
incubation conditions: CO2. low O2 high O2, 37 degrees, 42, etc.
why are molecular diagnostics not great for bacterial identification?
good or viral diagnosis but huge cost for bacterial diagnosis and not super effective because the culture is often more sensitive than the actual PCR itself
molecular diagnostics also does not provide susceptibility information
sputum/specimen grading
Q0- very poor quality: oropharyngeal contamination determined via microscope
Q1- poor quality: oropharyngeal contamination but specimen is still processed - results to be interpreted with caution
Q2- good quality
Q3- very good quality
interpretation/identification and turn around times
microscopy: 30mins-same day
Point of Care (POC): rapid streptococcal antigen test in an hour or less
direct MALDI-TOF: same day
Culture: 24hr to 3 weeks
Serological: same day/ week
molecular: same next day for diagnosis but same/next week for epidemiological studies
susceptibility testing: 24-72 hours or longer for mycobacterium
antimicrobial chemotherapy
use of drugs to combat infectious agents including antivirals, antibiotics, antifungals, and antiparasitic
most are derived from naturally occurring compounds some may be semi-synthetic or synthetic
differential toxicity
drug is more toxic to the infecting organism than to the host
spectrum of activity
broad vs narrow
broad kills a lot of different organisms
narrow kills a select group - try to use these if possible
minimum inhibitory concentration (MIC)
minimum concentration of the antibiotic required to inhibit the growth of the organism
minimum bactericidal concentration (MBC)
minimum concentration required to kill the organism
bacteriostatic vs bactericidal drugs
bacteriostatic drugs inhibit the organism - MBC is higher than MIC
bactericidal drugs - kill, MIC and MBC are the same
time dependent killing vs concentration dependent killing
time dependent killing: goal is to maximize exposure of the drug to the bacteria - dont care how high the concentration is just want to maintain the MIC for as long as possible - dosed more frequently bc want to keep it stable
Concentration dependent killing: goal is to maximize the concentration of the drug - only need to dose one or two times per day
prophylaxis
antimicrobial agents are given prevent an infection - do this before a surgery for example
treatment
antimicrobial agents are administered to treat an existing infection
therapeutic index
+ examples of drugs with low therapeutic index
therapeutic dose/ effective dose
drugs with a low therapeutic dose may require therapeutic drug monitoring to ensure drug levels are both effective at treating the infection and not killing the patient
examples:
aminoglyosides
vancomycin
the ideal antibiotic
no/low toxicity to the host
low probability of having resistance mechanisms
does not induce hypersensitivities in the host eg penicillin
rapid and extensive distribution to the tissues
relatively long half life but not too long
free of interactions with other drugs
convient for administration
cheap
empiric therapy
some infectious disease require immediate treatment - need to prescribe a drug before getting test results back
make a prescription based on:
- epidemiology (most probable diseases and etiologies)
- severity of the disease
- local rates of resistance
once the organism is identified the treatment should be adjusted - narrowed is possible or a new drug given if the first guess was wrong
advantages and disadvantages of combination therapy
advantages:
- treating polymicrobial infections
- initial empiric treatment
- synergy - 1 +1 = 4
- may prevent the emergence of resistance (this is the case with TB)
disadvantages:
- may be antagonistic - 2+2=1
- cost
- increased risk of side effects and drug-drug interactions
- usually not required for maximum efficacy
what influences you choice of antibiotics
activity against isolated or suspected organism
acute vs chronic disease
antibiotic history of the patient - can’t give the same one more than once in 3 months
site of infection
mode of administration/toxicity/cost
metabolism and excretion
duration of treatment / frequency of dose
local rates of resistance
concomitant medications that may react with antibiotic
re-infection vs reoccurrence
reinfection = same infection by different organism
reoccurrence = same infection by same organism - could indicate drug resistance
antimicrobial resistance
resistance = the inability to kill or inhibit the organism with clinically achievable drug concentrations
resistance can be innate such as gram negatives are resistant to vancomycin because it only works on gram positives - don’t really care about this stuff
resistance may be acquired through: mutation and acquisition of DNA
this results in: up/down regulation of things such as efflux pumps or OMPs, expanded spectrum of enzymatic activity (beta lactamases), and target site modification
antimicrobial selection of resistance
the use of antibiotics doesn’t create resistance it selects for it
have a colony of bacteria (billions of mutations happening in the genome) and one of them mutates to modify something in the cell that will resist the effect of antibiotics
the treatment with the antibiotics selects for that mutation
then have a resistant population develop
mechanisms of resistance gene transfer
transduction
transformation
conjugation
causes for the spread of antimicrobial resistance pathogens
global travel - can travel the world in 12 hours
don’t know you’re infected until you get there and then arrive with a new bacteria
factors that accelerate the development of resistance
main reason: overuse and misuse of antibiotics
other reasons:
- inadequate levels of antibiotics at the site of infection
- duration of treatment too short/long
- overwhelming numbers or organisms
- poor quality counterfeits
- over the counter/ no prescription needed
- animal husbandry
- frequent exposure to the same class
inappropriate use of antibiotics
63% of adults with upper respiratory tract infections received antibiotics even though most of them just had a cold
how you use antibiotics in your own health care system
france over presrcibed antibiotics and resulted in more resistant bacteria strains in the country
germany did not over prescribe and resulted in much less resistance
what happened when fluroquinolones were used more than once in 3 months
the chances of being infected with resistant S. pneumoniae increased dramatically compared to the chances when using other drugs
implications of resistance
treatment failure - patient death mortality rate of 42% with resistant strain infection
forced to use more expensive or more toxic alternatives
longer hospital stays = increased health care cost
possibility of not alternative agents - much less drugs are being made
impact of lacking drug development and antibiotic resistance
too expensive to make antibiotics now because there is not a wide enough market to make the money back suing the patent given the time it takes to go through the trials
the threat of antimicrobial resistance
carbapenemase resistance enterobacteriaceae (CRE)
kills up to half of the people it infects - resulted from the colistan resistance gene being passed on through china feeding their animals antibiotics
antimicrobial stewardship
limiting the use of antibiotics to those patients who absolutely require them:
- right patient
- right drug
- right time
- right dose
- right route
cellular targets of antibiotics
cell wall synthesis
nucleic acid synthesis
protein synthesis
cell membrane
general mechanism of resistance
altered permeability - OMP alteration in gram negatives inactivation/destruction of antibiotic novel binding sites efflux mechanisms bypass of metabolic pathways
cell wall synthesis inhibitors
beta lactams
glycopeptides
fosfomycin
beta lactam antibiotics
penicillins cephalosporins: as you go up generations you get more gram negative activity - 1st gen - 2nd gen - 3rd gen - 4th gen - 5th gen carbapenem
structure/mechanism of beta lactam drugs
have a beta-lactam ring that is a substrate analogue of D-Ala-D-Ala
therefore the target of beta-lactams are transpeptidases (penicillin binding proteins) that are involved in the crosslinking of the PG
beta lactams work through competitive inhibition
beta lactam resistance
beta lactamases (most common method) - inactivate the drug by opening up the beta lactam ring
altered PBP (S. pneumoniae)
novel PBP (MRSA)
altered permeability
beta lactam/beta lactamase inhibitors
inhibits the beta lactamase which prevents the destruction of the drug
examples of these include:
- piperacillin-tazobactam
- amoxicillin - clavulanic acid
- ceftolozane - tazobactam
allergies to beta lactams
only about one in every 25 000-40 000 patients
the allergy is caused by beta lactam R groups
rash occurs in 5% of patients develop a rash but this is not an allergic reaction
cell wall active agents and how they work: glycopeptides
glycopeptides such as vancomycin and teicoplanin
these are gram positive agents only
mechanism of action: they bind to the terminal D-Ala of the cell wall peptide, preventing crosslinking to make PG
glycopeptide (Vancomycin) resistance
this primarily happens/is concerning for enterococcus and staph. aureus
the D-Ala-D-Ala target is altered - subsitutes D-lac instead
prevents vancomycin from being able to bind
vancomycin specturm of activity
gram positive cocci:
- MRSA, coagulase positive staph
- pen resistant S.pnemoniae and enterocuccos
gram positive rods:
- C. jeikeium - multidrug resistant
- C. difficile (vancomycin must be given orally bc it needs to be in the gut)
fosfomycin
cell wall synthesis inhibitor
good drug bc you dont have to worry about renal function problems - good for old people
one 3 gram satchel (like a tea bag)
in Canada mainly used for uncomplicated cystitis (multidrug resistant UTI) caused by E.coli or E. faecalis
fosfomycin mechanism of action
inhibits the synthesis of cell wall building blocks in the cytoplasm
does this by inactivating the enzyme enol-pyruvyl transferase
this blocks the condenesation of UDP-NAG with p-enolpyruvate
evolution of fluoroquinolones
1st gen: nalidixic acid
2nd gen: ciprofloxicin - broader spectrum and anti psuedomonal (can be given orally or through IV)
levofloxacin
3rd gen: moxifloxacin - enhanced gram positive and (+/-) anaerobic activity
fluoroquinolones
dna synthesis inhibitors
concentration dependent and highly bactericidal
very good oral bioavailablilty (don’t need an IV)
fluoroquinolones mechanism of action
binds DNA gyrase and DNA complex - allows gyrase to cut and unwind the DNA but does not allow it to re-anneal = toxic to the cell
also does this with topoisomerase 4
development of fluoroquinolones resistance
spontaneous mutations in gyrA and parC (most common method)
- this results in amino acid substitution causing reduced affinity
- requires both mutations to occur
over expression or up-regulation of efflux pumps
- pumps drugs out - when combined with the other mutations it is really bad
- pmrA (Gm+)/norA (Gm-)
- can also have down regulation of porin channels in gram negatives
also have acquired resistance but not as common
adverse events of fluoroquinolones
hyper and hypoglycemia
can have cartilage toxicity - causes tendons to rupture - restrict paediatric usage
what are fluroquinolones used to treat?
gram negative/atypical infections
- oral step down from serious infections
- pseudomonas (cipro/levofloxacin only)
inhibitors of protein synthesis
macrolides, lincosamides, streptogramins (MLS)
tetracyclines
aminoglycosides
linezolid
the macrolides
examples: erythromycin (not nice), clarithromycin, azithromycin
binds to the 50S subunit of bacterial ribosomes - inhibiting protein synthesis
macrolides mechanism of resistance
M phenotype: efflux pump - only effects macrolides
MLS phenotype: target site modification - effects macrolides, lincosamides, streptogramin
adverse events of macrolides
GI upset
infusion related phlebitis
ventricular arrhythmia
cyto P-450 interactions
clindamycin
this antibiotic is most commonly associated with C. difficile colitis
a lincosamide drug
also treats anaerobic infections (usually with a gram negative agent)
gram positive infections such as necrotizing fasciitis and staph infections
also used to treat C. perfringens in penicillin allergy cases
tetracyclines
examples: tetracycline, doxycycline, minocycline
bind reversibly to the 30S ribosomal subunit
excellent for treating atypical bacteria such as RTI and chlamydia
good activity against most gram positives, many enterobacteriaceae and community acquired MRSA
also treat animal born pathogens such as yersinia pestis, burcella, B. burgdoferi, rickettsiae
adverse events of tetracyclines
discolouration of teeth (only tetracycline not doxycycline), photosensitivity, depression of skeletal growth, esophageal ulceration
mechanisms of resistance against tetracyclines
energy dependent efflux
enzymatic inactivation
ribosomal protection
aminoglycosides
natural or semi-synthetic antibiotics - streptomycin (1944)
excellent gram negative activity including pseudomonas
good gram positive activity
bactericidal and concentraatoin dependent
examples = gentamicin, tobramicin, amikacin
how aminoglycosides work
enter through the inner membrane via an energy dependent transport system
- this step is rate limiting and blocked by divalent cations and anaerobiosis
the aminoglycosides irreversibly bind to the 30S ribosomal subunit
do not work in anaerobic environments because they require the ETC to enter the cell (this requires O2 as final e acceptor)
aminoglycosides mechanism of resistance
enzymatic modification is the most common
more than 70 enzymes
different substrate species
plasmid mediated
less common methods:
- altered ribosomal binding sites (only for streptomycin) and reduced uptake or decreased permeability
adverse events of aminoglycosides
have a narrow therapuetic index - therefore long term use required therapuetic drug monitoring
at too high of a concentration they can interfere with mammalian protein synthesis
ototoxicity - causes deafness due to cochlear and vestibular damage
nephrotoxicity - proximal tubule damage
aminoglycosides: monitoring therapy
once daily - trough levels only
six hours before the next dose should be less than 1mg/L
inhibitors of metabolic pathways
trimethoprim/sulfamethoxazole (septra, TMP/SMX)
good gram negative activity and some gram positive
blocks folic acid synthesis at two different points
TMP and SMX act additively
TMP-SMX mechanism of action
sulfonamides block tetrahydropteroic acid synthetase which converts PABA to dihydrofolic acid
- first step in converting PABA to purines
trimethoprfim block dihydrofolate reductase which converts dihydrofolic acid to tetrahydrofolic acid
- second step in converting PABA to purines
TMP-SMX mechanisms of resistance
chromosomal (less common):
metabolic bypass
overexpression of DHFR (dihydrofolate reducatse)
Plasmid (most common):
drug resistant variants of DHFR/DHPS (synthetase enzyme)
antibiotics that target bacterial membranes
colistin -gram negative agent (some gram positive activity)
daptomycin - gram postive agents
colistin
displaces divalent cations from phosphate groups of membrane lipids - disrpting the outter membrane
useful for treating multidrug resistant gram negative infections
can cause renal or neurotoxicity
daptomycin
cant be used to treat RTI because lung surfactant inhibits the drug
works by inserting into the cell membrane - causes rapid depolarization and K+ ion flux
bactericidal and concentration dependent
useful for treating MRSA
metronidazole
go to for anaerobic infections
90% bioavailability
very little resistance
cheap
metronidazole mechanism of action
little/no activity against aerobic bacteria
produces short lived toxic intermediates or free radicals under anaerobic (reducing ) conditions
-this inhibits nucleic acid synthesis by damaging or disrupting DNA
metronidazole mechanism of resistance
reduced drug activation
reduced permeability/efflux
altered DNA repair
adverse effects of metronidazole
GI intolerance antabuse effect - alcohol consumption will cause power puking peripheral neurotoxicity metallic tase black/brown discolouration of urine
why do we do susceptibility testing
- as a guide for treatment
- as an epidemiological tool
- can track the emergence of resistant strains
- real time tracking of local susceptibility - can recommend the optimal empiric treatment
why do we need to keep doing susceptibility testing
susceptibility pattern of many pathogens is not always predictable
susceptibility patterns of some pathogens evolve over time
components of susceptibility testing
- identification of the organism
- site of infection
- selection of anitbiotics
- selection of appropriate test method
- interpretation: requires indepth knowledge of resistance mechanisms, some organisms behave differently in vitro vs in vivo
- selective reporting
- quality control
susceptibility testing: identification of the organism- things to consider
is it predictably susceptible?
beta haemolytic strep are all suscetible to beta lactams
does it have intrinsic resistance?
gram negative bacteria to vancomycin
enterococcus to cephalosporins
does it have inducible resistance?
when you put the patient on these drugs (look susceptible in lab) their resistance is activated
hetero-resistance phenotypes
only about 3-4 cells are resistant but we need to find these cells
- eg oxacillin and MRSA
why is the site of infection is important for susceptibility testing?
CNS/Brain infections- need a drug that will cross the blood-brain barrier
UTI- need a drug that will be excreted by the kidneys into the urine
RTIs - daptomycin is inactivated by lung surfactant
do we report all antibiotics when we do susceptibility testing?
no
don’t report:
predictably susceptible organisms
organisms with inducible resistance mechanisms
inappropriate bug-drug combinations
known contraindications
don’t report 2nd or 3rd line drugs if 1st line works
quality control in susceptibility testing
reference strains (ATCC): MIC ranges/zone diameters
media: must be standardized to ensure the diffusion zone will be comparable to reference strains
antibiotics: shelf life and potency
incubation: temp, time, atmosphere, amount of inoculum
test methods
kirby bauer - disk diffusion
broth/agar microdilution
E-test
automated systems (vitek/microscan)
screening methods: agar based and nitrocephin discs
establishment of antibiotic breakpoints for susceptibility testing
interpretive criteria that establish the categories of susceptible, intermediate, or resistant:
MIC distributions
pharmacokinetics - absorption, distribution, accumulation, elimination (all in vivo measurements)
pharmacodynamics: %time/MIC, AUC/MIC(Cmax/MIC)
clinical/ bacteriological response
nitrocephin disks
quick screen for beta lactamase production
drop disk (with cephlosporins in it) on plate- if there is beta lactamase the disk changes colour
disk diffusion
also known as kirby bauer method
filter disc impregnated with antibiotic is place on a plate with a lawn of bacteria
measure the zone of inhibition around the disc
qualitative test
compare the diameters with standard tables - each drug-bug combination has a specific value for susceptible, resistant, intermediate
factors that affect size of zone inhibition
inoculum density - inoculum too light = larger zones
timing of disc application - kept on plate too long = small zones
temperature of incubation - temp less that 35 = larger zones
incubation time - should be 16-20 hours
depth of the medium - too thin = excessive zone size
potency of antibiotic disc - deterioration = reduced size
composition of medium
acidic or alkaline pH of medium
reading o zones - subjective errors
how to determine macrolide resistance phenotypes
do this via double diffusion disc testing - one disk contains clindamycin and the other contains macrolide - placement of these disks at a specific distance is ESSENTIAL
the M phenotype will show a zone of inhibition around the clindamycin disc but not the macrolide disk - therefore can only be efflux mechanism
if there are no zones of inhibition around the disks then the bacteria have the MLS phenotype
if the there is not zone of inhibition around the macrolide disk + there is a skewed zone around the clindamycin disc then the bacteria have an inducible MLS phenotype
- this zone will be smaller on the side closest to the macrolide disk
E-test
strip has a gradient of antibiotics
have a plate with a lawn of bacteria on it
place strip on plate
the MIC is equal to the point on the plate where the zones intersect
dilution method
broth or agar methods
give quantitative results:
- indicated the amount of drug needed to inhibit or kill a bacteria being tested
the MIC is the lowest concentration of the drug that inhibits the growth or multiplication of bacteria
the MBC is the lowest concentration that leaves less than 0.1% of the inoculum population alive
broth dilution method
90% of this testing is done using mueller-hinton broth
prepared in doubling dilutions (0.5,1,2,4,8, etc)
inoculation of bacteria is incubated overnight
- controls + no antibiotic and no bacteria
turbidity visualization = MIC
subculture non-turbid tubes overnight
growth (bacterial count) - MBC
typically if the MIC is determined to be 16 mg/L then the MBC will be 32mg/L - need to go one dilution step further to ensure that there are no bacteria still alive
agar dilution method
agar plates with doubling dilutions of antibiotics
one concentration per plate but can have multiple different strains on one plate bc you can use a replicator
automated systems (vitek)
based on broth dilution
limited break points
useful for most common pathogens
not reliable fo fastidious pathogens (hard to grow)
what must bacterial identification begin with in the clinical setting?
understanding of epidemiology, patho-physiology, and infectious disease
general steps on classifying using taxonomy
gram negative or positive?
cocci of bacilli?
anaerobic or aerobic?
classification of aerobic gram positive cocci
need to determine if the arrangement is in clusters or pairs and chains
clusters indicates staph species
pairs and chains indicates streptococcus or enterococcus
general properties of staphylococcus
gram positive
facultative anaerobes
catalase positive except for one exception
commonly found on human skin and mucous membranes
20+ species
4 species associated with disease: S. aueus, S. lugdunensis, S. epidermidis, and S. saprophyticus
there is also S. psuedintermedius which is an animal pathogen that was been seen more in humans now
catalase test
determines if the organism produces catalase
catalase breaks hydrogen peroxide into water and oxygen
- this allows organisms to break down harmful metabolic products that result from aerobic respiration
this test is positive for staph nut negative for strep and enterococcus
epidemiology of S.aureus
10-15% of people carry it around in their nose or other mucous membranes (this number is higher for people in hospital settings)
gram positive
most common type of hospital acquired infection
typically infections are endogenous (spread form person to person)
MRSA is a concern in hospitals and community
slide and tube coagulase postive
slide coagulase test
really testing for clumping factor not coagulase bc it is a latex agglutination test (LAT)
newer versions of LAT also test for protein A
S.aureus and S. lugdenensis are positive
tube coagulase test
looks for free coagulase
test performed in rabbit plasma
this causes the plasma to be solid
S.aureus ad S. pseudointermedius (slide negaitve) are positive for this test
S. aureus virulence factors
coagulase - most important virulence factor: can lead to the formation of fibrin coagulum which produces localized infection - protects the organism from phagocytosis
protein A - covalently linked to PG which inhibits opsonization and phagocytosis and also has anti-complementary activity
coagulase negative staphylococcus
gram positive
everybody has this on their skin
can’t get rid of it even with disinfectant and hand washing
have an affinity for synthetic materials therefore devices that are inserted through the skin such as catheters, prosthetics, hip replacements etc.
main reason for surgical prophylaxis
S. lugdunensis
gram positive
clinically significant coagulase negative staph
considered to be as virulent as S.aureus (similar clinical spectrum)
produces clumping factor (slide coag)
PYR and ornithine decarboxylase +
S. saprophyticus
gram positive
UTI pathogen associated with sexually active young women
clinically significant coagulase negative staph
pathogenesis of coagulase negative staphylococcal infections
slime (a polysaccharide) - not the same as a biofilm
slime allows coagulase-negative staph to adhere to synthetic material
slime is also anti-phagocytic and inhibits chemotaxis
S. pseudintermedius
gram positive
slide coag negative, tube coag positive
an animal pathogen (primarily dogs) that is on the rise in human infections
frequently methicillin resistant
until MALDI-TOF it was frequently misidentified as MRSA
enterococcus classification
gram positive
many different species but most are not human pathogens
all intrinsically resistant to cephalosporins
grow in 6.5% NaCl at pH 9.6
growth between 10-45 degrees
growth in 40% bile
esculin hydrolysis, LAP, and PYR +
common enterococcus clinical isolates
most common:
E. faecalis
e. faecium
these are concerning because if they are vancomycin resistant their VRE gene is transferable
less common:
E. casselifavus
E. gallinarum
VRE is chromosomal so less of a concern - not going to be transferred
streptococcaceae
gram positive cocci in pairs chains or tetrads catalase negative most are facultative : streotococcus leuconocstoc aerococcus pediococcus gamella
anaerobic:
peptococcus
peptostreptococcus
peptoniphillus
classification of streptococci
viridans group streptococci
S. anginosus group
pyogenic streptococci (includes group A, B, C, G)
s. pneumoniae has its own group bc it causes so much disease but technically belongs to viridans
types of haemolysis
beta - complete
alpha - partial (greening of the agar)
gama - no haemolysis
lancefield typing
Groups A,B,C,F ,G = group specific antigens and cell wall polysaccharides
- only for beta haemolyitc strep
Group D and enterococci = LTA moiety and are not always beta haemolytic
beta haemolytic streptococcus groups
group A = s.pyogenes group B (occasionally alpha or not at all) = s. agalactiae group C (occasionally alpha or not at all) = s. equismilis/dysgalactiae group D (occasionally alpha or not at all) = s. bovis and enterococcus group F and G (sometimes A and C) = s.anginosus
PYR test
enterococcus and group A strep +
S. bovis and streptococci -
tests for the presence of PYR enzyme
S. agalactiae (group B strep)
weakly beta haemolytic
colonies are smaller than group A
CAMP test positive
all women should be screened for group B strep colonization late in pregnancy bc it can be a problem for baby
CAMP test
differentiates group B form other strep
synergistic haemolysis observed between S. aureus and group B strep
how the test works: streak of staph aureus down the middle - group B strep intersecting at one end - listeria intersecting at other end - should observes arrow head haemolysis pattern at intersections
streptococcus anginosus group
also known as milleri group
3 species: S. anginosus, S. intermedius, S. constellatus
primarily group F but also C, G,A
colonies are small pin point like and smell like caramel or butterscotch
associated with deep liver/brain infections
streptococcus bovis group
group D antigen
capable of growth in bile - hydrolyzes esculin
if found in blood it may be linked to colon cancer
examples: S. equinus, S. gallolyticus, S infantarius, and S. alactolyticus
how to differentiate between enterococci and S. bovis
both are group D antigen positive, usually non-haemolytic and are bile/esculin +
enterococci is PYR positive and can grow in 6.5% NaCl
alpha haemolytic strep (viridans strep)
not groupable by lancefeild typing
streptococcus pneumoniae - primarily human pathogen
S. sanguis
S.mutis
S. mutans
S. salivarius- opportunistic pathogen, periodontal disease, endocarditis
how to differentiate S. pneumoniae from other viridans group strep?
S. pneumoniae is bile soluble and susceptible to optochin
other viridins strep are not bile soluble and are resistant to optochin
nutritionally variant strep
examples include:
-adiotrophia adjacens and A. defectiva
- granulicatella adjacens and G elegans
these make up approximately 5% of infective endocarditis
biochemically similar to viridans group as they are optochin resistant, bile insoluble and alpha haemolytic
require pyridoxal (vitamin B6) for growth PYR positive
how can nutrotionally variant strep be grow on culutre plates?
could add vitamin B6 to the blood agar plates
could also use a regular blood agar plate and streak S. aureus onto it - this lyses RBCs causing them to spill there contents (includes vitamin B6) - therefore the nutrient variant strep can grow in these places
listeria monocytogenes
gram positive bacilli catalase postive tumbling motility at 25 degrees umbrella motility in semi-soft agar beta hemolytic CAMP test positive intrinsically resistant to cephalosporins
which bacteria are intrinsically resistant to cephalosporins?
enterococcus species
listeria monocytogenes
corynebacterium species
gram positive bacilli commonly found on skin look like Chinese characters under the microscope catalase positive contaminant / flora vs pathogen
pathogenic species:
C. diptheriae
C. pseudodiptheriticum
C urealyticum
families in the enterobacterales order
enterobacteriaceae erwiniaceae pectobacteriaceae yersinaceae hafinaceae morganellaceae budviciaceae
common characteristics of enterobacterales
all facultative aneraobes
all ferment glucose
all gram negative rods oxidase negative (except plesimonas)
all reduce nitrate to nitrite
enterobacterales that ferment glucose AND lactose
citrobacter spp.
enterobacter spp.
escherichia coli
klebsiella pneumoniae
enterobacterales that ferment glucose but NOT lactose
proteus spp salmonella enteriditis salmonella typhi shigella spp serratia marcescens yersinia enterocolitica yersensia pestis
oxidase test
all enterobacterales are oxidase negative with one exception
vibrio, psuedomonas, and aeromonas are all oxidase positive
use a drop of culture onto a dry slide
cytochrome oxidase is an enzyme involved in the reduction of oxygen at the end of the ETC
MacConkey Agar
is selective for gram negatives because it contains bile salts and crystal violet (inhibits gram positive)
is differential for lactose fermenting bacteria - lactose is primary carbon source - colonies turn pink when they are lactose fermenters
the nitrate test
detects if the bacteria has nitrate reductase - therefore able to use nitrate as an electron acceptor (nitrate gets reduced)
how it works:
add reagent - if it turns red then this is positive
if it does not change add zinc
no change with zinc means positive result - zinc reacts with nitrate
change with zinc = negative result because nitrate is still present and therefore zinc will react with it, reducing it to nitrite (no nitrate reductase)
Triple sugar iron (TSI) slants contents
1% glucose, 10% lactose, 10% sucrose (glucose is limiting)
sodium thiosulfate for H2S production
FeSO4 for H2S detection
phenol red indicator
how to interpret TSI slants
if the bacterium can ferment glucose it will turn the slant yellow bc it lowers the pH
glucose is depleted first so the bacterium must ferment another sugar source
if slant remains yellow it means the bacterium has metabolized sucrose or lactose
if slant turns back to red this indicates the organism can only ferment glucose - start to metabolize amino acids for glucose - creates alkaline environment = red
- this rection occurs in slant area only bc it requires O2
could also get bubble at bottom of tube if they are gas producers
if bacteria can utilize sodium thiosulfate to produce H2S then there is black precipitation in the tube
general characteristics of vibrio and aeromonas
classified together as vibrionaceae:
- primarily found in water sources
- may cause GI disease
- not closely related by molecular methods
how are vibrio and aeromonas similar and different compared to enterobacterales
similarities:
- gram negative
- facultative anaerobes
- fermentative (glucose)
differences:
- vibrio and aeromonas are oxidase positive
key tests for vibrio
salt tolerance = 6% NaCl oxidase they are susceptible to O/129 while most other bacteria are resistant - resistance in vibrio has been shown - O/129 is a vibriostatic drug
TCBS agar
highly selective for vibrio spp
oxgall added inhibit gram positive organisms
sodium thiosulfate / ferric chloride can detect H2S
sucrose CHO source
vibrio cholerae = yellow
haemophilus influenzae
gram negative bacilli (cocco-bacilli)
oxidase positive
facultative anaerobic (CO2 enhance growth)
fastidious
does not grow on MacConkey also does not grow on blood agar bc it requires RBCs to be lysed
primairly associated with RTI
what factors does haemophilus influenzae require for growth
X and V factors
X = protoporphyrin IX (heme)
V = NAD (nicotinamide adenine dinucleotide)
chocolate agar
blood heated until RBCs lyse
allows haemophilus influenzae to grow bc they have access to internal contents of slide
porphyrin test
determines an isolates X factor requirement
also called ALA test
haemophilus spp that require X factor cannot synthesize it from precursor ALA
How the test works:
-heavy suspension of organism in aminolevuinic acid, incubated 4hr and then illuminated with UV light - then examined for red fluorescence
- fluorescence indicates enzymatic conversion of aminolevulinic acid to porphyrins and therefore X factor independence
where do you find afermenters?
typically found in nature as inhabitants of soil, water and normal flora of animal and human mucous membranes
afermenters
some are truly pathogens while others are opportunistic
comprise only a small percentage of clinical isolates
require more effort for identification
by definition do not ferment glucose
most are obligate anaerobes
some require 48-72 hours for growth
most grow at 35 degrees but some grow at RT
classification of afermenters
no family
includes many genera - names are constantly changing
afermenter morphology and cultural characteristics
typically gram negative cocci or cocco-bacilli
some grow on MacConkey and others dont
when grown on SBA plates - haemolysis, morphology, size, and pigmentation can provide useful info for ID
most are non-fastidious and can be isolated using the same methods as enterobacterales
initial cues that suggest an organism is an afermenter
lack of glucose fermentation
often oxidase positive
may not grow on MacConkey agar
may have unusual antibiotic resistance
commonly isolated afermenters
stenotrophomonas maltophilia acinetobacter elizabethkingia meningosepticum burkholderia psuedomonas - most common and most important
stenotrophomonas maltophilia
gram negative
2nd most frequently isolated
transient normal flora of patients (tyoically in ICU) causes hospital acquired infections
actinebacter
gram negative
this is everywhere - soil, water and skin
common colonizer
less common to cause hospital acquired infections but they tend to be multidrug resistant
elizabethkingia meningosepticum
gram negative
occassionally assocaited with meningitis or speticemia
susceptible to vancomycin in-vitro - this is a major indicator of the species in lab
burkholderia
gram negative three species that are true pathogens: B. cepacia - see this in CF patients B. Mallei - glanders in horses B. pseudomallei - see this in chronic respiratory infection in south east asia
pseudomonas aeruginosa
aerobic gram negative rod oxidase positive able to grow at 42 degrees metallic sheen in colonies greenish colour (most common strain) - can also be black brown or yellow nitrate positive associated with multi-drug resistance
your technologist shows you an SBA plate that has satellite colonies beside a staph streak.
what one test could help you narrow down the identification of the organism?
gram stain
gram postive = nutritionally variant strep
gram negative = haemophilus influenzae
your technologist shows you a gram stain of what appears to be gram positive cocci in clusters. they want to know if it is S. aureus OR a coagulase negative strep. all you have is the gram stain. What do you tell you technologist? what would be the next step if it was S. aureus?
perform both tube and slide coagulase tests.
S. auerus would be positive for both
S. lugdenensis wold only be positive for slide
S. pseudointermedius only positive for tube
next step is to determine if it is MRSA - if it is then no beta lactams
- could use vancomycin if this were the case
anaerobiosis
these bacteria will not grow in the presence of oxygen
possible mechanisms:
- no cytochrome systems for the metabolism of O2
- little/no superoxide dismutase
- little/no catalase
what are most anaerobic clinical isolastes?
moderatley obligate anaerobes
have a small amount of catalase and dismutase therefore can tolerate small amounts of oxygen
types of bacteria based on their tolerance of oxygen
obligate aerobic bacteria
obligate anaerobic bacteria
facultative bacteria - dont care either way
microaerophiles - require oxygen but at a lower concentration
why do anaerobic bacteria die when exposed to oxygen
oxygen metabolism has toxic by products
anaerobic do not have enzymes such as dismutase and catalase to detoxify so they die
methods for excluding oxygen during culture of anaerobes
- liquid media containing fresh animal tissue of 0.1% agar containing a reducing agent, thioglycollate
- anaerobic jar - gas packs or gas replacements where O2 is reacted away and replaced with nitrate or any inert gas
- anaerobic chamber - no one really uses these anymore bc most anaerobes don’t cause disease
classification of clinically important anaerobes
gram positive cocci:
peptostreptococcus
peptoniphilus
cutibacterium acnes
gram positive bacilli:
- C. perfringins, tetani, botulinum
- C. difficile
- Propionibacterium spp
- actinomyces
- lactobacillus
- mobiluncus
gram negative cocci
-veillonella spp
gram negative bacilli
- bacteroides
- fusobacterium
- prevotella
- porphyromonas
epidemiology of anaerobes
mainly come from endogenous flora - medications and some surgeries/cancers can favour the growth of anaerobes
skin - propionibacterium (acne), cutibacterium, peptostreptococcus
mouth - actinomyces, fusobacterium
upper respiratory tract - propionibacterium
GI tract - bacteroides, fusobacterium
female GU tract - lactobacillus
roles of anaerobic bacteria in the body
prevents colonization and infection by pathogens - interfere with adhesion and also secrete toxic metabolites
contributes to host physiology through the production of useful cofactors like vitamin K
clinical features of anaerobic infections
typically poly-microbial
source of the infection is endogenous flora
alterations of tissues that provide proper conditions for anaerobic infection
abscess formation
exotoxin involvement
what factors predispose patients to anaerobic infection?
trauma to mucosal membrane or skin
interruption of blood flow - leads to tissue necrosis - which leads to low redox potential in tissues
prior antibiotic therapy and person still isn’t getting better
immuno-suppression
anaerobic virulence factors
necrotizing toxins adherence factors polysaccharide capsule toxins hylauronidase lipases enzymes like proteases and phospholipases
what are indications that an infection might be caused by anaerobic bacteria?
usually purulent (produce pus)
close proximity to mucosal surface
infection does not get better with antibiotic treatment
distinct morphological features in gram stain
presence of gas - bubbling or crackling when pressed on
foul odour
brick red or black fluorescence
methods of collection of anaerobic specimens
Swabs - discouraged
- it is possible they may contain non-infectious anaerobes and they must be transported with little O2 exposure
aspirates - favoured
excess air is expressed from syringe and the syringe and needle is used to take a sample from site of the infection
- transport this in the syringe itself or in an oxygen-free transport tube
specimens that are acceptable for anaerobic culture
tissue
- placed in an oxygen free transport tube or bag
- should be ground up for best analysis
blood
- want to collect blood in anaerobic and aerobic blood culture bottles
specimens that are unacceptable for anaerobic culture
swabs from: throat, nose, nasopharynx, gingival cavities, stool, vaginal, cervical, urethral, decubitis ulcers, feet and other exposed wounds
sputum
voided catheterized urine
don’t want any of these because the patient would already be growing normal anaerobes in this area but we need to know if they’re actually pathogenic or not so this does not help
culturing and interpretation of anaerobic bacteria
typically incubated for 48hrs before examination
no growth - reincubate for up to 5 days and then discard
depending on infection site - only mono-microbial etiologies are frequently worked up
poly-microbal anaerobic infections are typically reported as mixed anaerobic growth
anaerobic culture workup: when do you do it and how?
if there is growth on the plate and it looks like the species could be clostridium or something bad=work up
how to do it:
step 1= asses for aero-tolerance
- subculture aerobically and anaerobically
- after 24 hours determine if the organism is facultative or obligate
step 2= gram stain suspicious colonies
- look for presence of spores (all clostridium and bacillus)
- note the shape and the gram reaction of the bacteria
- look to see if it is filamentous
how do you know you’ve cultured an anaerobe?
foul odour
fluorescence
failure to grow aerobically
double zone of haemolysis - likely clostridium perfringens
antimicrobial susceptibility testing for anaerobic bacteria
typically not performed on poly-microbial isolates sometimes beta lactamase testing drugs of choice include: metronidazole- go to for anaerobes amoxicillin-clavulanate piperacillin-tazobactam meropenem
treatment protocols for anaerobic bacteria
surgical intervention: source control (e.g. draining abscesses), removing necrotic tissue, eliminating obstructions
antibiotics
antitoxins (tetanus and botulism)
vitek microbial ID system
still used for susceptibility testing
requires 16-24 hours of incubation time
the ID cards used are 5-8$
what is MALDI-TOF
matrix assisted laser desorption ionization - time of flight
most significant advance in clinical micro in 30 years
rapidly identifies bacteria and at a much lower cost than vitek
can do this directly from isolated colonies and positive culture bottles using protein biomarkers such as ribosomeal proteins
requires really experienced microtechs
MALDI-TOF mechanism
sample is mixed with the matrix and dried on the plate
- the matrix kills the organsim and lyses it
- matrix is also a source of protons which allows for ionization
laser ionizes the matrix molecules - the sample molecules are ionized by protein transfer from matrix
produces a spectrogram
this is compared to standards and matched = diagnosis
MALDI-TOF vs Vitek
MALDI-TOF - instrument is 220K service/year = 20k cost/test = 50 cents time/test = 20 minutes (16 samples)
Vitek: -reagent rental service/year is built into contract cost/test = 8$ time/test = 16-24 hours
MALDI-TOF can pay for itself in 2.5 years and is far more beneficial for physicians and patients
latest rapid ID systems
BioFire- 1hr ID + 200$/test
- gives youID for most common pathogens
Accelerate Pheno
1hr ID, 6hr susceptibility, 250-300$/test
- gives most common pathogens
very easy to operate systesm
bacteremia: what is it + what causes it
it is bacteria in the blood - can lead to septicemia (septic shock)
caused by:
- drainage from infection site into lymph and then into vascular system
direct entry from contaminated needles/intravascular devices, graft material, trauma
three patterns of bacteremia
transient: happens in minutes to hours
- often occurs following manipulation of infected tissues or instrumentation of contaminated mucosal surfaces
- happens when you brush your teeth
- also the onset of actue infections such as pneumonia, meningitis, and septic arthritis
intermittent: most common with undrained abscesses
continuous: typically indicates an endovascular infection because the bacteria is being continuously added to the blood stream
example: endocarditis
most common infections that cause bacteremia
intravascular device
respiratory tract
urinary tract
intra-abdominal
in 1/4 of patients the source cannot be determined
etiology of bacteremias
most common are S.aureus and E.coli
others include: coag negative staph K. pneumoniae enterococcus P. aeruginosa viridans group strep
prognosis of bacteremia
mortality ranges from 20-50%
factors that result in poor prognosis:
- advanced age
- nosocomial infection - harder to treat (more drug resistance)
- enterococcal, gram-neg, fungal etiology
-underlying cirrhosis or malignancy
-primary focus in respiratory tract or skin
surgical wound or abscess
-septic shock
-lack of febrile response to sepsis
positive blood culture interpretation
consider most positives to be a true infection because blood is considered to be sterile
S. aureus, Ecoli, enterobacteriacae, P.aeruginosa, S. pneumoniae and candida ablicans are true infections
corynebacterium spp. bacillus, propionibacterium acnes rarely true infections
problem bacteria are: viridans group strep, enterococci, CNS which are sometimes infectious and true infections and sometimes not
blood culture collection
take two sets of blood
- one set includes two bottles form the same veni-puncture
- each set should be taken from a different veni-puncture site
- one bottle in each set should be anaerobic
more than 95% of bacteremia is detected when more than two bottles are taken
this ensures adequate blood to culture
only 3-5% of blood culutres are typically contaminated
should discard the first few mLs of blood to get rid of skin plug
10mL bottle for adults =40 mL total
principles of laboratory detection for blood cultures
volume of blood:
- fewer than 10cfu/ml in blood
- each mL of blood increases ID by 3%
- best recover occurs when 30-40mL is taken
ratio of blood to broth
- diluting blood with broth greater than 1:5 increases recovery of microbes bc natural microbial growth inhibitors + dilutes any antibiotics
need anticoagulants - don’t want blood to clot
use ion exchange beads to remove antimicrobials
duration of incubation periods
- 5 days for automated blood culture system - maybe more
consider temp for storage - want body temp or at least RT
automated blood culture systems
24hr continuous monitoring
fluorescence detection
blood culutres
instrument flags positive bottle
aliquot is taken
gram stain - call in results to ward right away
blood plated onto several non-selective media (but MacConkey is used if it is gram negative)
anaerobic bacteremia
very uncommon
spectrum of bacteria associated with these infections is very narrow
bacteroides accoutn for 45-75% and of that 75% are typically B.Fragilis
other ones: clostridium, prevotella, fuscobacterium
MALDI-TOF direct blood culture bacterial ID
dont need to wait for bacteria to grow on media
can ID within 1 hour of flaggin positive culture
problems:
- labour intensive
- incorporating into lab workflow
MALDI-TOF smudge plates
not as rapid as direct detection but faster than watiting for overnight culture
after 4-6 hours of incubation most plates have a visible scum
scum can be scraped off and placed on MALDI-TOF matrix
problem:
- because the colonies are not distinct you could get mixed growth and therefore no ID
conventional ID vs MALDI
MALDI can give ID up to 24 hours earlier which could mean the patient lives or dies
also allows for good microbial stewardship because the attending physician can put the patient on a more narrow spectrum of drugs sooner
origin of viral names
associated diseases cytopathic effects they cause to host cells places or people who discovered them biochemical features appearance transmission
hierarchal virus classification
decided by the international committee on taxonomy of viruses (ICTV)
suffix based on classification" orders=virales families = viridae subfamilies = virinae genera = virus
classification of viruses based on common physical properties
size and shape envelope or naked genome organization replication mechanism nucleic acid composition antigenic differences
subunits of viral structure
capsomere: protein subunits of capsid
capsid: capsomeres assemble to form viral capsid - gives virus its shape
nucelocapsif: capsid + nucleic acids
virus shapes
cube
helical
complex
viral surface structures
bind to host cell receptors
surface projections - spikes, fibers, knobs, peplomers
surface glycoproteins
viral envelopes
lipid layer surrounding some viruses
these are derived from the host cell membrane during egress
non-enveloped vs enveloped viruses
non-enveloped:
- stable in the environment bc they are able to resist desiccation, acids, detergents and heat
- they are also transmitted easily via hands and fomites
enveloped:
- not stable in the environment for long bc they will be damaged from drying, acid, detergents, or heat
- they must stay moist to be transmitted - common in body fluids and secretions
viral genomes
composition can be either DNA or RNA
structure can be:
single or double stranded
linear or circular
single or segmented
RNA has polarity: + sense RNA codes directly for protein when in host cytoplasm, negative sense has to be converted to an intermediate first before it can be used to make protein
transmission of viruses
aerosol droplet contact fomites sexual parenteral vectors zoonotic vertical food/waterborne fecal/oral
stages of virus-host interaction
entry into host primary replication spread cell/tissue tropism secondary replication cell injury or persistence host immune response
consequences of virus-cell interactions
viral proliferation and cell lysis: cold sores, chicken pox latent infection: herpes persistent infection: HIV, HBV oncogenesis - HPV, EBV no apparent disease - hep G
methods to diagnose viral infections
guided by clinical presentation or suspicion
lab diagnosis:
electron microscopy - not sensitive
Virus culture + immunoflourescence
detect viral components via: viral antigen testing or molecular diagnostics
histopathology staining to detect the cell damage
serology - test antibodies and immune responses
what antibodies are produced in response to viral infection
acute infection = IgM
past exposure = IgG
serology: immunologic method
principle: detect an antigen or antibody via fluorescence, chemiluminsecence, or colorimetric (this can be quantitative or qualitative)
screening assays: latex agglutination, lateral flow assays, enzyme immunoassays (EIA)
confirmation assays:
western blots, immunoblots
latex agglutination assays
can be antibody or antigen detection
rapid, simple, inexpensive
latex beads are coated with antigen or antibody and added to a patients serum sample
either the patients antibodies will stick to the antigen coated bead or the antibody coated ebad will pick up specific antigen in the blood
lateral flow assays
similar to a pregnancy test
antibodies are attached to a slide and called a test line analyte is added to the machine and capillary flow drags it across - antibodies conjugated to gold nanoparticles produce signal
enzyme immunoassays (EIA)
example = ELISA
antibody or antigen detection
qualitative or quantitative
manual or automated
limitations or serology
competition with other antibodies
cross-reactive IgM antibodies
- connective tissue diseases, pregnancy, vaccination
interfering substances = rheumatoid factor (RF)
- this is an IgM antibody that reacts with the Fc portion of IgG
usually IgM indicates acute infection but there are some cases where they can persist for years - could also be present in viral re-infection and re-exposures
confirmatory methods for serology
if EIA negative - repeat over time (window period)
if EIA positive - confirm with western blot or immunoblot
replacement of the HIV-1 WB
Geenius HIV-1/2 from Bio-Rad
works like a pregnancy test
done rapidly in about 30 minutes
when is molecular diagnostics a good idea
non-cultural agents non-viable organisms slow-growing of difficult to grow culture confirmation agents present in low numbers
types of molecular tests
nucleic acid amplification techniques - PCR most common
sequencing (microbiology/research applications
molecular epidemiology
processing specimens for nucleic acid amplification
nucleic acid extraction
amplification
detection
interpretation of results
nucleic acid extraction
lysis-release of nucleic acids: physical/mechanical, chemical, enzymatic
purification: sequential wash steps helps to eliminate contaminants - use silica column to bind to DNA/RNA
recovery
why do we purify nucleic acids
clinical specimens are full of PCR inhibitors that lead to false negative results
urine - urea feces - bile salts, polysaccharides blood - hemoglobin, anticoagulants tissue - melanin, myoglobin processing reagents - formalin, excess salts, detergents, alcohols
PCR reaction
steps = denature, annealing, extension
reagent components: DNA, primers (fwd, rev)
dNTPs
heat stable DNA pol
buffers and MgCl2
gel electrophoresis and visualization
used to separate amplicons
migration is based on size and charge
dna must be stained to be seen
RT-PCR
RT converts rna to cDNA
steps = 1st strand synthesis by RT
second strand synthesis by DNA pol
amplification of cDNA by DNA pol
mutliplex PCR (mPCR)
can detect multiple targets in a single reaction
needs primer pairs for each target
qualitative vs quantitative nucleic acid tests
qualitative:
qualitative PCR for detection of HSV
qualitative RT-PCR for flu
quantitative:
quantitative PCR for CMV post transplant
quantitative RT-PCR for HIV/HCV following treatment
real time PCR
detect amplification using fluorescent chemistries
the fluorescence is proportional to the quantity of DNA produced
CT value: the number of the CR cycle where the fluorescence crosses the threshold
quantitative real time PCR
quantitity target is inversely proportional to CT values
more target (DNA or RNA) = smaller Ct values
concept can be exploited for quantitative analyses
amplicon contamination
the number 1 contaminant of most PCRs is the amplicon form the previous days run
prevention of amplicon contamination
physical barriers
decontamination
unilateral workflow
prevention of amplicon contamination using dUTP and heat labile UNG
dUTP and heat-labile uracil DNA glycosylase (UNG) are added to the pCR reaction prior to amplification
how it works:
- dUTP gets incorporated into DNA (not normal)
- all amplicons generated will have uracil incorporated
- UNG degrades U-DNA if it is present but NOT primers of template DNA (no U present)
- UNG is labile and therefore inactivated after first cycle
how psoralens is used to prevent amplicon contamination of PCR
psoralens is added to pCR prior to amplification
it gets activated by UV light after PCR
psoranlens will the cross link any DNA which prevents it from denaturing and thus the amplicons will not be re-amplified
DNA sanger sequencing
only one primer at a time (one strand)
ddNTPs are used - fluorescently labeled chain terminator
clinical micro applications of DNA sequencing
ID of organisms
predict susceptibility to antimicrobials
molecular epidemiology - outbreak investigations
16S rDNA PCR and sequencing
16S rRNA is a conserved region of prokaryotic ribosomes
16S rDNA is the gene that encodes the rRNA
16S rDNA PCR targets highly conserved regions - if an amplicon is present = positive for bacteria (detection)
DNA sequencing of the amplicon will give the ID - sequencing can be compared to database
advantages and disadvantages to 16S rDNA sequencing
advantages:
can ID most bacteria even if they are not viable
disadvantages:
only good for monomicorbial infections/pure cultures (only applied to normally sterile tissues or bodily fluids)
molecular epidemiology
useful for outbreak investigations
DNA = genetic finger print
two methods are used for this:
- pulsed feild gel electrophoresis (PFGE)
- whole genome sequencing (WGS)
pulsed field gel electrophoresis
isolate DNA - digest it with restriction endonucleases to generate fragments
electrophoresis with system of alternating current angles is used
can look at the national micro library for migration distances - good for outbreak investigation
john snow
pioneer of anaesthesia and epidemiology
famous from determining the source of the broad street pump cholera outbreak in 1854
at the time it was a miasma vs germ theory debate
ignaz semmelweis
determined why women were dying of puerperal fever/childbed fever
noticed that far less women died when midwives birthed their babies compared to being in a hospital
this was bc the medical students were touching cadavers and not washing their hands before birthing
introduced hand washing as a common practice and reduced the rate of death in hospitals down to the midwives rate
epidemiology of infectious disease
only a few organisms are clinically important today but they must be dealt with in a complex manner
variety of clinical manifestations: symptomatic vs asymptomatic
variety of time courses: acute, subacute, chronic
actue to chronic
different routesof transmission
different control methods
ways of looking at infectious diseases
epidemiological: what factors lead to infection/disease
clinical: identifying clinical symptoms and pathophysiology
microbiological: describe characteristics of the microorganisms and susceptibility
all of these lead to an understanding of:
- factors of transmission prevention/control
- diagnosing and treating individual patients
- developing future treatments and vaccines
epidemiological triad of disease
host
vector
agent environment
infection transmission chain
agent reservoir portal of exit mode of transmission portal of entry susceptible host
agent component of transmission chain
first component in chain
environmental characteristics:
- ability to withstand stress
- ability to multiply
- non-human host reservoirs
epidemiologically important if:
- transmitted through environment
-causes infection
produces clinical disease
agent characteristics
infectivity: ability to enter, survive and multiply in host
pathogenicity: extent to which disease is produced in an infected population
virulence: ability to cause serious disease
reservoir component of transmission chain
place where the agent persists or lives
agent must be able to exit the reservoir and enter susceptible host via portal of entry
5 modes of transmission
droplet - lg respiratory droplets over short distance
airborne - small droplets, long distances
contact - direct or indirect
common vehicle contaminated blood products
vector borne - insects
susceptible host in transmission chain
person or animal that allows the agent to live inside
definitive (primary): parasite reaches maturity - sex stage
intermediate (secondary): parasite is in larvae state -asexual, just kind of hangs out
basic reproduction number
R0 = cqd
C= contact rate q = probability of transmission d = duration of infectivity
= the number of people who will get sick from each case of infection
epidemiology of nosocomial infections
nosocomial infection = 48hrs post admission
results in massive healthcare expenses
UTI = 35% (80% assoc. with catheter)
surgical site = 20%
pneumonia = 15%
bacteremia = 15%
multidrug resistant organisms
MRSA VRE CDAD - c.diff associated diarrhea CPE: carbapenemase-producing enterobacteriaciae - this is causing the most concern bc best broad spectrum drug is being destroyed resistant yeast: candida auris
risk of CRE infections
easily transmitted on hands of health care workers
evidence for infection control
the cost to prevent nosocomial infections and MDR organism infections is significantly less than the cost to treat these infections
hand hygiene
proper hand hygiene has resulted in 40-50% reduction in health care associated
method = 4 points of care
- before/after contact with patient/environment
- before aseptic
- after body fluid exposure
- before and after removal of glove
wear gloves
wear gown
wear mask/eye protection
precautions principle
you cannot tell who has what
body substances of all patients are considered potentially infectious
behaviour is determined by risk of encountering body substance not by diagnosis
contact infections = gloves/gowns
droplet infections = masks, eye protection
airborne infections = N95 masks
infection control activities
approp. disinfection and sterilization for device/equipment reprocessing
outbreak management
advice
education
research
