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clinical microbiology > midterm 1 > Flashcards

midterm 1 Flashcards

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1
Q

functions of clinical laboratory

A

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

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2
Q

what do clinical microbiologists provide advice on?

A

appropriate specimen collection and transport
interpret test results
patient management - recommendations on antimicrobials

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3
Q

why is it important to understand how the clinical lab works?

A

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

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4
Q

general workflow of lab

A 
collection
transport/storage
accessioning
processing 
interpretation/identification
susceptibility testing/ molecular testing 
reporting/ documentation
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5
Q

requisition form

A

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

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6
Q

things to consider for specimen collection

A
  • 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
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7
Q

Accessioning of the sample

A

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

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8
Q

criteria for specimen rejection

A

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

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9
Q

things to consider for transport of specimens

A

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

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10
Q

processing of specimens

A

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.

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11
Q

why are molecular diagnostics not great for bacterial identification?

A

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

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12
Q

sputum/specimen grading

A

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

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13
Q

interpretation/identification and turn around times

A

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

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14
Q

antimicrobial chemotherapy

A

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

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15
Q

differential toxicity

A

drug is more toxic to the infecting organism than to the host

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16
Q

spectrum of activity

A

broad vs narrow

broad kills a lot of different organisms

narrow kills a select group - try to use these if possible

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17
Q

minimum inhibitory concentration (MIC)

A

minimum concentration of the antibiotic required to inhibit the growth of the organism

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18
Q

minimum bactericidal concentration (MBC)

A

minimum concentration required to kill the organism

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19
Q

bacteriostatic vs bactericidal drugs

A

bacteriostatic drugs inhibit the organism - MBC is higher than MIC

bactericidal drugs - kill, MIC and MBC are the same

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20
Q

time dependent killing vs concentration dependent killing

A

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

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21
Q

prophylaxis

A

antimicrobial agents are given prevent an infection - do this before a surgery for example

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22
Q

treatment

A

antimicrobial agents are administered to treat an existing infection

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23
Q

therapeutic index

+ examples of drugs with low therapeutic index

A

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

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24
Q

the ideal antibiotic

A

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

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25
Q

empiric therapy

A

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

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26
Q

advantages and disadvantages of combination therapy

A

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
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27
Q

what influences you choice of antibiotics

A

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

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28
Q

re-infection vs reoccurrence

A

reinfection = same infection by different organism

reoccurrence = same infection by same organism - could indicate drug resistance

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29
Q

antimicrobial resistance

A

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

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30
Q

antimicrobial selection of resistance

A

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

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31
Q

mechanisms of resistance gene transfer

A

transduction
transformation
conjugation

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32
Q

causes for the spread of antimicrobial resistance pathogens

A

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

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33
Q

factors that accelerate the development of resistance

A

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

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34
Q

inappropriate use of antibiotics

A

63% of adults with upper respiratory tract infections received antibiotics even though most of them just had a cold

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35
Q

how you use antibiotics in your own health care system

A

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

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36
Q

what happened when fluroquinolones were used more than once in 3 months

A

the chances of being infected with resistant S. pneumoniae increased dramatically compared to the chances when using other drugs

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37
Q

implications of resistance

A

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

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38
Q

impact of lacking drug development and antibiotic resistance

A

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

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39
Q

the threat of antimicrobial resistance

A

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

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40
Q

antimicrobial stewardship

A

limiting the use of antibiotics to those patients who absolutely require them:

  • right patient
  • right drug
  • right time
  • right dose
  • right route
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41
Q

cellular targets of antibiotics

A

cell wall synthesis
nucleic acid synthesis
protein synthesis
cell membrane

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42
Q

general mechanism of resistance

A 
altered permeability - OMP alteration in gram negatives
inactivation/destruction of antibiotic 
novel binding sites
efflux mechanisms 
bypass of metabolic pathways
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43
Q

cell wall synthesis inhibitors

A

beta lactams
glycopeptides
fosfomycin

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44
Q

beta lactam antibiotics

A 
penicillins
cephalosporins: as you go up generations you get more gram negative activity 
- 1st gen 
- 2nd gen
- 3rd gen 
- 4th gen 
- 5th gen 
carbapenem
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45
Q

structure/mechanism of beta lactam drugs

A

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

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46
Q

beta lactam resistance

A

beta lactamases (most common method) - inactivate the drug by opening up the beta lactam ring

altered PBP (S. pneumoniae)

novel PBP (MRSA)

altered permeability

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47
Q

beta lactam/beta lactamase inhibitors

A

inhibits the beta lactamase which prevents the destruction of the drug

examples of these include:

  • piperacillin-tazobactam
  • amoxicillin - clavulanic acid
  • ceftolozane - tazobactam
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48
Q

allergies to beta lactams

A

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

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49
Q

cell wall active agents and how they work: glycopeptides

A

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

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50
Q

glycopeptide (Vancomycin) resistance

A

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

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51
Q

vancomycin specturm of activity

A

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)
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52
Q

fosfomycin

A

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

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53
Q

fosfomycin mechanism of action

A

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

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54
Q

evolution of fluoroquinolones

A

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

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55
Q

fluoroquinolones

A

dna synthesis inhibitors

concentration dependent and highly bactericidal

very good oral bioavailablilty (don’t need an IV)

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56
Q

fluoroquinolones mechanism of action

A

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

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57
Q

development of fluoroquinolones resistance

A

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

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58
Q

adverse events of fluoroquinolones

A

hyper and hypoglycemia

can have cartilage toxicity - causes tendons to rupture - restrict paediatric usage

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59
Q

what are fluroquinolones used to treat?

A

gram negative/atypical infections

  • oral step down from serious infections
  • pseudomonas (cipro/levofloxacin only)
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60
Q

inhibitors of protein synthesis

A

macrolides, lincosamides, streptogramins (MLS)
tetracyclines
aminoglycosides
linezolid

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61
Q

the macrolides

A

examples: erythromycin (not nice), clarithromycin, azithromycin

binds to the 50S subunit of bacterial ribosomes - inhibiting protein synthesis

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62
Q

macrolides mechanism of resistance

A

M phenotype: efflux pump - only effects macrolides

MLS phenotype: target site modification - effects macrolides, lincosamides, streptogramin

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63
Q

adverse events of macrolides

A

GI upset
infusion related phlebitis
ventricular arrhythmia
cyto P-450 interactions

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64
Q

clindamycin

A

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

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65
Q

tetracyclines

A

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

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66
Q

adverse events of tetracyclines

A

discolouration of teeth (only tetracycline not doxycycline), photosensitivity, depression of skeletal growth, esophageal ulceration

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67
Q

mechanisms of resistance against tetracyclines

A

energy dependent efflux
enzymatic inactivation
ribosomal protection

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68
Q

aminoglycosides

A

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

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69
Q

how aminoglycosides work

A

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)

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70
Q

aminoglycosides mechanism of resistance

A

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

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71
Q

adverse events of aminoglycosides

A

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

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72
Q

aminoglycosides: monitoring therapy

A

once daily - trough levels only

six hours before the next dose should be less than 1mg/L

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73
Q

inhibitors of metabolic pathways

A

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

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74
Q

TMP-SMX mechanism of action

A

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

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75
Q

TMP-SMX mechanisms of resistance

A

chromosomal (less common):
metabolic bypass
overexpression of DHFR (dihydrofolate reducatse)

Plasmid (most common):
drug resistant variants of DHFR/DHPS (synthetase enzyme)

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76
Q

antibiotics that target bacterial membranes

A

colistin -gram negative agent (some gram positive activity)

daptomycin - gram postive agents

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77
Q

colistin

A

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

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78
Q

daptomycin

A

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

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79
Q

metronidazole

A

go to for anaerobic infections

90% bioavailability

very little resistance

cheap

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80
Q

metronidazole mechanism of action

A

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

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81
Q

metronidazole mechanism of resistance

A

reduced drug activation
reduced permeability/efflux
altered DNA repair

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82
Q

adverse effects of metronidazole

A 
GI intolerance 
antabuse effect - alcohol consumption will cause power puking
peripheral neurotoxicity 
metallic tase 
black/brown discolouration of urine
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83
Q

why do we do susceptibility testing

A
  1. as a guide for treatment
  2. as an epidemiological tool
    - can track the emergence of resistant strains
    - real time tracking of local susceptibility - can recommend the optimal empiric treatment
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84
Q

why do we need to keep doing susceptibility testing

A

susceptibility pattern of many pathogens is not always predictable

susceptibility patterns of some pathogens evolve over time

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85
Q

components of susceptibility testing

A
  • 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
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86
Q

susceptibility testing: identification of the organism- things to consider

A

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

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87
Q

why is the site of infection is important for susceptibility testing?

A

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

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88
Q

do we report all antibiotics when we do susceptibility testing?

A

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

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89
Q

quality control in susceptibility testing

A

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

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90
Q

test methods

A

kirby bauer - disk diffusion
broth/agar microdilution
E-test
automated systems (vitek/microscan)

screening methods: agar based and nitrocephin discs

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91
Q

establishment of antibiotic breakpoints for susceptibility testing

A

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

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92
Q

nitrocephin disks

A

quick screen for beta lactamase production

drop disk (with cephlosporins in it) on plate- if there is beta lactamase the disk changes colour

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93
Q

disk diffusion

A

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

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94
Q

factors that affect size of zone inhibition

A

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

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95
Q

how to determine macrolide resistance phenotypes

A

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

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96
Q

E-test

A

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

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97
Q

dilution method

A

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

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98
Q

broth dilution method

A

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

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99
Q

agar dilution method

A

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

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100
Q

automated systems (vitek)

A

based on broth dilution
limited break points
useful for most common pathogens
not reliable fo fastidious pathogens (hard to grow)

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101
Q

what must bacterial identification begin with in the clinical setting?

A

understanding of epidemiology, patho-physiology, and infectious disease

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102
Q

general steps on classifying using taxonomy

A

gram negative or positive?
cocci of bacilli?
anaerobic or aerobic?

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103
Q

classification of aerobic gram positive cocci

A

need to determine if the arrangement is in clusters or pairs and chains

clusters indicates staph species

pairs and chains indicates streptococcus or enterococcus

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104
Q

general properties of staphylococcus

A

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

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105
Q

catalase test

A

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

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106
Q

epidemiology of S.aureus

A

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

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107
Q

slide coagulase test

A

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

108
Q

tube coagulase test

A

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

109
Q

S. aureus virulence factors

A

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

110
Q

coagulase negative staphylococcus

A

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

111
Q

S. lugdunensis

A

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 +

112
Q

S. saprophyticus

A

gram positive
UTI pathogen associated with sexually active young women

clinically significant coagulase negative staph

113
Q

pathogenesis of coagulase negative staphylococcal infections

A

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

114
Q

S. pseudintermedius

A

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

115
Q

enterococcus classification

A

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 +

116
Q

common enterococcus clinical isolates

A

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

117
Q

streptococcaceae

A 
gram positive cocci in pairs chains or tetrads 
catalase negative 
most are facultative :
streotococcus
leuconocstoc
aerococcus
pediococcus
gamella

anaerobic:
peptococcus
peptostreptococcus
peptoniphillus

118
Q

classification of streptococci

A

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

119
Q

types of haemolysis

A

beta - complete
alpha - partial (greening of the agar)
gama - no haemolysis

120
Q

lancefield typing

A

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

121
Q

beta haemolytic streptococcus groups

A 
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
122
Q

PYR test

A

enterococcus and group A strep +
S. bovis and streptococci -

tests for the presence of PYR enzyme

123
Q

S. agalactiae (group B strep)

A

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

124
Q

CAMP test

A

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
125
Q

streptococcus anginosus group

A

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

126
Q

streptococcus bovis group

A

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

127
Q

how to differentiate between enterococci and S. bovis

A

both are group D antigen positive, usually non-haemolytic and are bile/esculin +

enterococci is PYR positive and can grow in 6.5% NaCl

128
Q

alpha haemolytic strep (viridans strep)

A

not groupable by lancefeild typing

streptococcus pneumoniae - primarily human pathogen
S. sanguis
S.mutis
S. mutans
S. salivarius- opportunistic pathogen, periodontal disease, endocarditis

129
Q

how to differentiate S. pneumoniae from other viridans group strep?

A

S. pneumoniae is bile soluble and susceptible to optochin

other viridins strep are not bile soluble and are resistant to optochin

130
Q

nutritionally variant strep

A

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
131
Q

how can nutrotionally variant strep be grow on culutre plates?

A

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

132
Q

listeria monocytogenes

A 
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
133
Q

which bacteria are intrinsically resistant to cephalosporins?

A

enterococcus species

listeria monocytogenes

134
Q

corynebacterium species

A 
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

135
Q

families in the enterobacterales order

A 
enterobacteriaceae
erwiniaceae
pectobacteriaceae
yersinaceae
hafinaceae
morganellaceae
budviciaceae
136
Q

common characteristics of enterobacterales

A

all facultative aneraobes
all ferment glucose
all gram negative rods oxidase negative (except plesimonas)
all reduce nitrate to nitrite

137
Q

enterobacterales that ferment glucose AND lactose

A

citrobacter spp.
enterobacter spp.
escherichia coli
klebsiella pneumoniae

138
Q

enterobacterales that ferment glucose but NOT lactose

A 
proteus spp
salmonella enteriditis 
salmonella typhi
shigella spp
serratia marcescens 
yersinia enterocolitica 
yersensia pestis
139
Q

oxidase test

A

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

140
Q

MacConkey Agar

A

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

141
Q

the nitrate test

A

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)

142
Q

Triple sugar iron (TSI) slants contents

A

1% glucose, 10% lactose, 10% sucrose (glucose is limiting)
sodium thiosulfate for H2S production
FeSO4 for H2S detection
phenol red indicator

143
Q

how to interpret TSI slants

A

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

144
Q

general characteristics of vibrio and aeromonas

A

classified together as vibrionaceae:

  • primarily found in water sources
  • may cause GI disease
  • not closely related by molecular methods
145
Q

how are vibrio and aeromonas similar and different compared to enterobacterales

A

similarities:

  • gram negative
  • facultative anaerobes
  • fermentative (glucose)

differences:
- vibrio and aeromonas are oxidase positive

146
Q

key tests for vibrio

A 
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
147
Q

TCBS agar

A

highly selective for vibrio spp
oxgall added inhibit gram positive organisms
sodium thiosulfate / ferric chloride can detect H2S
sucrose CHO source
vibrio cholerae = yellow

148
Q

haemophilus influenzae

A

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

149
Q

what factors does haemophilus influenzae require for growth

A

X and V factors
X = protoporphyrin IX (heme)
V = NAD (nicotinamide adenine dinucleotide)

150
Q

chocolate agar

A

blood heated until RBCs lyse

allows haemophilus influenzae to grow bc they have access to internal contents of slide

151
Q

porphyrin test

A

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

152
Q

where do you find afermenters?

A

typically found in nature as inhabitants of soil, water and normal flora of animal and human mucous membranes

153
Q

afermenters

A

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

154
Q

classification of afermenters

A

no family

includes many genera - names are constantly changing

155
Q

afermenter morphology and cultural characteristics

A

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

156
Q

initial cues that suggest an organism is an afermenter

A

lack of glucose fermentation
often oxidase positive
may not grow on MacConkey agar
may have unusual antibiotic resistance

157
Q

commonly isolated afermenters

A 
stenotrophomonas maltophilia 
acinetobacter
elizabethkingia meningosepticum 
burkholderia 
psuedomonas - most common and most important
158
Q

stenotrophomonas maltophilia

A

gram negative
2nd most frequently isolated
transient normal flora of patients (tyoically in ICU) causes hospital acquired infections

159
Q

actinebacter

A

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

160
Q

elizabethkingia meningosepticum

A

gram negative
occassionally assocaited with meningitis or speticemia
susceptible to vancomycin in-vitro - this is a major indicator of the species in lab

161
Q

burkholderia

A 
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
162
Q

pseudomonas aeruginosa

A 
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
163
Q

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?

A

gram stain

gram postive = nutritionally variant strep
gram negative = haemophilus influenzae

164
Q

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?

A

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

165
Q

anaerobiosis

A

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
166
Q

what are most anaerobic clinical isolastes?

A

moderatley obligate anaerobes

have a small amount of catalase and dismutase therefore can tolerate small amounts of oxygen

167
Q

types of bacteria based on their tolerance of oxygen

A

obligate aerobic bacteria
obligate anaerobic bacteria
facultative bacteria - dont care either way
microaerophiles - require oxygen but at a lower concentration

168
Q

why do anaerobic bacteria die when exposed to oxygen

A

oxygen metabolism has toxic by products

anaerobic do not have enzymes such as dismutase and catalase to detoxify so they die

169
Q

methods for excluding oxygen during culture of anaerobes

A
  1. liquid media containing fresh animal tissue of 0.1% agar containing a reducing agent, thioglycollate
  2. anaerobic jar - gas packs or gas replacements where O2 is reacted away and replaced with nitrate or any inert gas
  3. anaerobic chamber - no one really uses these anymore bc most anaerobes don’t cause disease
170
Q

classification of clinically important anaerobes

A

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
171
Q

epidemiology of anaerobes

A

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

172
Q

roles of anaerobic bacteria in the body

A

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

173
Q

clinical features of anaerobic infections

A

typically poly-microbial
source of the infection is endogenous flora
alterations of tissues that provide proper conditions for anaerobic infection
abscess formation
exotoxin involvement

174
Q

what factors predispose patients to anaerobic infection?

A

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

175
Q

anaerobic virulence factors

A 
necrotizing toxins
adherence factors 
polysaccharide capsule
toxins 
hylauronidase
lipases
enzymes like proteases and phospholipases
176
Q

what are indications that an infection might be caused by anaerobic bacteria?

A

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

177
Q

methods of collection of anaerobic specimens

A

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

178
Q

specimens that are acceptable for anaerobic culture

A

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

179
Q

specimens that are unacceptable for anaerobic culture

A

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

180
Q

culturing and interpretation of anaerobic bacteria

A

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

181
Q

anaerobic culture workup: when do you do it and how?

A

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
182
Q

how do you know you’ve cultured an anaerobe?

A

foul odour
fluorescence
failure to grow aerobically
double zone of haemolysis - likely clostridium perfringens

183
Q

antimicrobial susceptibility testing for anaerobic bacteria

A 
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
184
Q

treatment protocols for anaerobic bacteria

A

surgical intervention: source control (e.g. draining abscesses), removing necrotic tissue, eliminating obstructions

antibiotics

antitoxins (tetanus and botulism)

185
Q

vitek microbial ID system

A

still used for susceptibility testing

requires 16-24 hours of incubation time

the ID cards used are 5-8$

186
Q

what is MALDI-TOF

A

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

187
Q

MALDI-TOF mechanism

A

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

188
Q

MALDI-TOF vs Vitek

A 
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

189
Q

latest rapid ID systems

A

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

190
Q

bacteremia: what is it + what causes it

A

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

191
Q

three patterns of bacteremia

A

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

192
Q

most common infections that cause bacteremia

A

intravascular device
respiratory tract
urinary tract
intra-abdominal

in 1/4 of patients the source cannot be determined

193
Q

etiology of bacteremias

A

most common are S.aureus and E.coli

others include:
 coag negative staph
K. pneumoniae
enterococcus
P. aeruginosa 
viridans group strep
194
Q

prognosis of bacteremia

A

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

195
Q

positive blood culture interpretation

A

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

196
Q

blood culture collection

A

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

197
Q

principles of laboratory detection for blood cultures

A

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

198
Q

automated blood culture systems

A

24hr continuous monitoring

fluorescence detection

199
Q

blood culutres

A

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)

200
Q

anaerobic bacteremia

A

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

201
Q

MALDI-TOF direct blood culture bacterial ID

A

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
202
Q

MALDI-TOF smudge plates

A

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

203
Q

conventional ID vs MALDI

A

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

204
Q

origin of viral names

A 
associated diseases
cytopathic effects they cause to host cells 
places or people who discovered them 
biochemical features
appearance
transmission
205
Q

hierarchal virus classification

A

decided by the international committee on taxonomy of viruses (ICTV)

suffix based on classification"
orders=virales
families = viridae
subfamilies = virinae
genera = virus
206
Q

classification of viruses based on common physical properties

A 
size and shape 
envelope or naked
genome organization
replication mechanism 
nucleic acid composition 
antigenic differences
207
Q

subunits of viral structure

A

capsomere: protein subunits of capsid
capsid: capsomeres assemble to form viral capsid - gives virus its shape
nucelocapsif: capsid + nucleic acids

208
Q

virus shapes

A

cube
helical
complex

209
Q

viral surface structures

A

bind to host cell receptors

surface projections - spikes, fibers, knobs, peplomers

surface glycoproteins

210
Q

viral envelopes

A

lipid layer surrounding some viruses

these are derived from the host cell membrane during egress

211
Q

non-enveloped vs enveloped viruses

A

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
212
Q

viral genomes

A

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

213
Q

transmission of viruses

A 
aerosol
droplet
contact
fomites 
sexual
parenteral
vectors
zoonotic
vertical
food/waterborne
fecal/oral
214
Q

stages of virus-host interaction

A 
entry into host 
primary replication
spread
cell/tissue tropism
secondary replication 
cell injury or persistence
host immune response
215
Q

consequences of virus-cell interactions

A 
viral proliferation and cell lysis: cold sores, chicken pox
latent infection: herpes
persistent infection: HIV, HBV
oncogenesis - HPV, EBV
no apparent disease - hep G
216
Q

methods to diagnose viral infections

A

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

217
Q

what antibodies are produced in response to viral infection

A

acute infection = IgM

past exposure = IgG

218
Q

serology: immunologic method

A

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

219
Q

latex agglutination assays

A

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

220
Q

lateral flow assays

A

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

221
Q

enzyme immunoassays (EIA)

A

example = ELISA

antibody or antigen detection
qualitative or quantitative
manual or automated

222
Q

limitations or serology

A

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

223
Q

confirmatory methods for serology

A

if EIA negative - repeat over time (window period)

if EIA positive - confirm with western blot or immunoblot

224
Q

replacement of the HIV-1 WB

A

Geenius HIV-1/2 from Bio-Rad

works like a pregnancy test
done rapidly in about 30 minutes

225
Q

when is molecular diagnostics a good idea

A 
non-cultural agents 
non-viable organisms
slow-growing of difficult to grow
culture confirmation 
agents present in low numbers
226
Q

types of molecular tests

A

nucleic acid amplification techniques - PCR most common
sequencing (microbiology/research applications
molecular epidemiology

227
Q

processing specimens for nucleic acid amplification

A

nucleic acid extraction
amplification
detection
interpretation of results

228
Q

nucleic acid extraction

A

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

229
Q

why do we purify nucleic acids

A

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
230
Q

PCR reaction

A

steps = denature, annealing, extension

reagent components: DNA, primers (fwd, rev)
dNTPs
heat stable DNA pol
buffers and MgCl2

231
Q

gel electrophoresis and visualization

A

used to separate amplicons

migration is based on size and charge

dna must be stained to be seen

232
Q

RT-PCR

A

RT converts rna to cDNA

steps = 1st strand synthesis by RT

second strand synthesis by DNA pol

amplification of cDNA by DNA pol

233
Q

mutliplex PCR (mPCR)

A

can detect multiple targets in a single reaction

needs primer pairs for each target

234
Q

qualitative vs quantitative nucleic acid tests

A

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

235
Q

real time PCR

A

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

236
Q

quantitative real time PCR

A

quantitity target is inversely proportional to CT values

more target (DNA or RNA) = smaller Ct values

concept can be exploited for quantitative analyses

237
Q

amplicon contamination

A

the number 1 contaminant of most PCRs is the amplicon form the previous days run

238
Q

prevention of amplicon contamination

A

physical barriers

decontamination

unilateral workflow

239
Q

prevention of amplicon contamination using dUTP and heat labile UNG

A

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
240
Q

how psoralens is used to prevent amplicon contamination of PCR

A

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

241
Q

DNA sanger sequencing

A

only one primer at a time (one strand)

ddNTPs are used - fluorescently labeled chain terminator

242
Q

clinical micro applications of DNA sequencing

A

ID of organisms

predict susceptibility to antimicrobials

molecular epidemiology - outbreak investigations

243
Q

16S rDNA PCR and sequencing

A

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

244
Q

advantages and disadvantages to 16S rDNA sequencing

A

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)

245
Q

molecular epidemiology

A

useful for outbreak investigations

DNA = genetic finger print

two methods are used for this:

  • pulsed feild gel electrophoresis (PFGE)
  • whole genome sequencing (WGS)
246
Q

pulsed field gel electrophoresis

A

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

247
Q

john snow

A

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

248
Q

ignaz semmelweis

A

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

249
Q

epidemiology of infectious disease

A

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

250
Q

ways of looking at infectious diseases

A

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
251
Q

epidemiological triad of disease

A

host

            vector

agent environment

252
Q

infection transmission chain

A 
agent 
reservoir
portal of exit 
mode of transmission
portal of entry
susceptible host
253
Q

agent component of transmission chain

A

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

254
Q

agent characteristics

A

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

255
Q

reservoir component of transmission chain

A

place where the agent persists or lives

agent must be able to exit the reservoir and enter susceptible host via portal of entry

256
Q

5 modes of transmission

A

droplet - lg respiratory droplets over short distance
airborne - small droplets, long distances
contact - direct or indirect
common vehicle contaminated blood products
vector borne - insects

257
Q

susceptible host in transmission chain

A

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

258
Q

basic reproduction number

A

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

259
Q

epidemiology of nosocomial infections

A

nosocomial infection = 48hrs post admission

results in massive healthcare expenses

UTI = 35% (80% assoc. with catheter)

surgical site = 20%

pneumonia = 15%

bacteremia = 15%

260
Q

multidrug resistant organisms

A 
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
261
Q

risk of CRE infections

A

easily transmitted on hands of health care workers

262
Q

evidence for infection control

A

the cost to prevent nosocomial infections and MDR organism infections is significantly less than the cost to treat these infections

263
Q

hand hygiene

A

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

264
Q

precautions principle

A

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

265
Q

infection control activities

A

approp. disinfection and sterilization for device/equipment reprocessing

outbreak management

advice

education

research

clinical microbiology (2 decks)

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