Download Review of D+D Antimicrobials - the University of Colorado School of

Preamble
Sunday, January 3rd, 2010
What follows is the original preamble (more or less the same as those from M2M, etc). A
few notes on ID:
1) The title is a joke. The title is a joke. The title is a joke.
2) The title is a joke. No, really. ID is the arch-example of step one material that you
need to learn, use, and forget—it’s dense, full of more specifics than you thought
possible, and nearly impossible to cram. If you go to class at all (and, for my
money, you should, otherwise you’re kind of wiping your ass with your tuition
money), go to ID. The lectures aren’t always great but you need repetition to learn
this material.
3) The lectures aren’t always great. Don’t expect them to be. Microbiologists and
pharmacists are not, Dr. French excepted, the most scintillating lecturers. The
point of this class is mainly to listen and read and write all this array of
information, again and again, until it sticks. The fact that this class comes right
before step one is a huge boon to you—you can rote-memorize data without
having to worry about losing it by game day. So rote-memorize.
4) The lab sessions can be fun or awful depending on who your instructor is. That
said, it’s actually good information (as opposed to the D+D labs) and can help
solidify some of your information.
5) First Aid is extremely helpful as an adjunct to figure out where the gaps in
information that you need to close on your own are. It’s also extremely helpful to
know which lists of information you should write down (again and again) until
you have them. Learn the stupid selective-growth-media information, it’s tedium
on top of tedium but it does show up.
6) Clinical Microbiology Made Ridiculously Simple is also very helpful to
understand the material when the lecture wasn’t so hot and First Aid is
particularly opaque. I occasionally cite sections from the book in these LOs.
7) More than any other category of medicine, I found ID to be the part of step one
where raw knowledge counted for more than being able to integrate different
areas of knowledge. If you don’t know that X bacterium is Gram-negative but
strictly aerobic, you’re hosed, because you’re not going to be able to use subtle
test-taking strategy to infer it from other info in the stem. So learn it.
8) Don’t kill yourself worrying about drug side effects (like most other categories of
drugs except for cardiac and neuro), but DO know contraindications (eg.
fluoroquinolones in kids can cause tendon rupture) and know which ones you
can’t use in pregnant women.
9) Miller Time Guy makes his reappearance for the fungi lectures. You have my
blessing to miss those, but be warned the notes are pretty useless. I’d just read
CMBMRS and skim his Powerpoints.
10) For anyone who’s been using these things all the way through the first two years,
god help you—good to have you along for the ride. Hope they helped.
–jcr
PS- if you find these useful and can go without a latte for a couple of days, I’d like you to
give another $5 to charity.
Hey, everybody. These are my compiled learning objectives for Life Cycle when I
took it in the spring of 2009. I hope you find them useful. A few notes:
1. These aren't to be taken as everything-you-need-to-know material, or
anything close to it. They can be, however, extremely useful, if only to
look at the material a second time in a different format.
2. Learning objectives change. Granted, in our vaunted institution, they
often don't change a lot. But it's worth figuring out where these
overlap with what you're studying and where they don't to avoid any
unnecessary learning (God forbid).
3. They can be incorrect. I hope this is infrequent, but I'm sure there are
things in here that aren't accurate. I've tried to curate them
reasonably well; I hope one of your classmates will do likewise. If you
find an error, kindly let him or her know.
4. They are nothing more or less than my personal take on what we
happened to be learning on a given day. Sometimes they're very
detailed, sometimes they're uncomprehending, frequently they're
irreverent. I occasionally call babies vampires and things like that
(dude, they are). Internet lesson: free trumps tasteful. In any case
you are free to disagree with me.
5. To anyone who's wondering: I honored this block and all the rest in
my first two years. That's not supposed to impress you, but it is
supposed to give you some kind of confidence that I have a reasonably
good handle on what's going on.
6. To the many of you who are thinking, "How can I repay this wonderful,
wonderful man?" I would reply that I never turn down free beer if I
can help it. The problem with that is that I suspect I will never meet
most of your class, and beer-buying in absentia is a cold and heartless
thing. So if you find these useful and would like to do something for
me, I would prefer it if you donated $5 to the charity of your choice; if
you're stumped, I suggest browsing www.charitynavigator.com for
some good options. Kindly do not donate money to armed
insurrectionist groups.
i.
Addendum on donating to charity: always always ALWAYS have
an email account that you set aside purely to sign up for or
donate to things (thus ducking all the spam associated
therewith). I think I have 1,200 emails in mine, mostly from a
donation I made to the SPCA a couple of years ago. Gmail and
Hotmail work well. I also recommend using a false street
address to avoid direct-mail campaigns.
ii.
"That seems like a lot of trouble to go through to donate five
bucks"-- yeah, well, welcome to the world, sonny Jim. Doing
things for other people frequently is a pain in the ass. Doesn't
make it less worth doing.
Review of D+D Antimicrobials
Tuesday, January 06, 2009
8:02 AM
Review of D+D Antimicrobials, 1/5/08:
(The LOs say, essentially, "know drugs." General notes follow.)
Note that the Powerpoint for this lecture has a copy of the drug list with various
features of antibiotics highlighted. Might be a good idea to check that out for where
to put emphasis when studying.
Stuff she expects us to know from D+D: penicillins, cephalosporins, carbapenems,
monobactams, vancomycin, aminoglycosides, macrolides, tetracyclines,
chloramphenicol, clindamycin, streptogramins, linezolid.
Imagine the bacteria vs. the immune system as a foot race, tortoise v. hare style.
The immune system is the tortoise-- the bacteria can usually grow faster than it can
kill them, at least at first. You can either tie an anvil around the hare's ankle,
allowing the immune system to catch up (bacteriostatic drugs), or you can just drop
the anvil on the hare and be done with it (bacteriocidal drugs). Cheating? How did
you think the tortoise REALLY won, anyway?
Penicillins (beta-lactams): time-dependent killing (the amount of time in which the
concentration of the drug is above the minimal bacteriocidal concentration, or MIC,
determines the rate of bacterial killing).
Aminoglycosides: concentration-dependent killing (the concentration of drug
determines the rate of bacterial killing).
 Note that this has to do with why penicillins are dosed three times a
day while aminoglycosides are dosed once: you're trying to keep the
penicillins at a steady, relatively low concentration (no gain in efficacy
above the MIC), while you're trying to reach a quite high dose of
antibiotic with the aminoglycosides (and there winds up being less
toxicity associated with one big dose anyway).
"Post-antibiotic effect:" most antibiotics have some effect for a while even after their
concentration dips below the MIC/MBC.
Note narrow-spectrum antibiotics have their place-- they're much better at not killing
off a bunch of commensal flora than the broad-spectrum ones.
Beta-lactam antibiotics:
 Target peptidoglycan wall (inhibit its polymerization); vancomycin also
acts there, but acts at a different step.
 They're all bacteriocidal.
 All penicillins are renally excreted (note oxacillin is also hepatically
metabolized).
 Extended-spectrum penicillins are better at getting through the outer
membrane of Gram-negative organisms (thus the extended spectrum).
Note vancomycin doesn't get into the bacterium itself at all (only good
vs. Gram-positives).
Cephalosporins:
 As generation advances, more Gram-negative activity and less Grampositive activity.



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As generation advances, more ability to cross into the CSF.
Less allergenic than penicillins.
1st generation: like ampicillin (extended-spectrum penicillin, targets E.
coli and Klebsiella proteus); doesn't cross BBB. 1st-generation
cephalosporins can be used in place of penicillins in the face of mild
allergic reactions; often used for surgical prophylaxis.
 Note if you have a strong (type I) allergic reaction to penicillin,
you want to use a macrolide instead (no structural resemblance
at all).
2nd generation: more Gram-negative (also targets H. influenzae,
Enterobacter); mostly doesn't cross BBB. Often used for otitis media
and enteric infections.
3rd generation: yet more Gram-negative (some anti-Pseudomonas),
but poor effect vs. Gram-positive; crosses BBB into the CSF. Used for
sepsis and meningitis.
Monobactams are narrow-spectrum (Gram-negative); carbapenems are broadspectrum.
Aminoglycosides, macrolides, chloramphenicol, tetracyclines, clindamycin:
 All act on bacterial ribosomes.
 Aminoglycosides and tetracyclines: 30S subunit.
 Macrolides, chloramphenicol, clindamycin: 50S subunit.
 Note that aminoglycosides are bacteriocidal, but the others are mostly
bacteriostatic.
 Aminoglycosides cause incorrect translation, which is what
causes cell death.
 Aminoglycosides:
 Not well absorbed orally (the others all are); mainly acts on
Gram negative aerobes (taken up with oxygen).
Streptogramins is a newer, effective broad-spectrum antibiotic whose use is limited
to severe cases. Linezolid is used for multiply-resistant Gram-positives.
Streptococci
Monday, January 05, 2009
1:21 PM
Streptococci, 1/6/08:
[General notes, largely from CMBMRS:]
 5 types of strep:
 Group A: pyogenes (beta-hemolytic)
 Group B: agalactiae (beta-hemolytic)
 Group D: Enterococcus species, bovis, equinus (mostly nonhemolytic)
 pneumoniae (alpha-hemolytic)
 viridans (alpha-hemolytic)
 (beta-hemolytic = clears RBCs; alpha-hemolytic = leaves green
biliverdin residue)



 Note all have lipoteichoic acids (LTAs) in their cell envelope.
Group A (pyogenes):
 M protein--inhibits complement, prevents phagocytosis;
targeted by antibodies.
 Streptolysin O (destroys red and white blood cells).
 Streptokinase (breaks down clots by activating plasminogen).
 Pyrogenic exotoxin (produces scarlet fever).
 Note that the exotoxin gene is carried from one
bacterium to another on a transmissible bacteriophage
("lysogenic conversion").
 Note that the lipoteichoic acids in pyogenes are attached to the
M protein and inverted so that the lipophilic end is sticking
outwards. This promotes adherence to epithelial cell
membranes.
 Pyogenes strains without M protein are avirulent; antibodies
against the particular flavor of M protein present are
immunoprotective.
 Pyogenes has an anti-phagocytic hyaluronate capsule in
addition to the M proteins, etc, that are in its cell wall. Note
that the capsule is non-immunogenic.
 Group A diseases:
 Pharyngitis (direct invasion)
 Skin infections, particularly erysipelas (direct invasion)
 Necrotizing fasciitis (direct invasion)
 Scarlet fever (exotoxin)
 Toxic shock syndrome (exotoxin)
 (delayed) Rheumatic fever and post-streptococcal
glomerulonephritis (cross-reaction of anti-pyogenes
antibodies)
 Recall that acute rheumatic fever occurs in a
month or two after infection, but the valvular
damage usually only become apparent decades
later.
 Note that PSGN is immune complex mediated
(type III), while RF is direct antibody mediated
(type II).
 Note pyogenes forms chains (vs. staph's clusters).
 Note that there's a rapid antigen detection test (results in
minutes) for pyogenes-mediated pharyngitis, using a throat
swab.
 Note that pyogenes is the only beta-hemolytic streptococcus
that's sensitive to bacitracin.
Group B (agalactiae):
 Looks just like pyogenes on stain; however, has an
immunogenic capsule and no protein M or streptolysin O.
 Lives in the vagina and rectum.
 Group B diseases: tend to have to do with pregnancy or birth:
 Neonatal meningitis (most common cause under 6
months) and sepsis.
 Can also infect pregnant mothers, causing bacteremia,
sepsis, and stillbirth or spontaneous abortions.
Group D:



Enterococci: now their own genus, these are the only
classified streptococci that are widely resistant to penicillin.
Main strains are E. faecalis and E. faecium.
 Random fact for the boards: bovis is often associated with
colon cancer. (eg.: "blood is drawn from a patient with
malignancy of the large intestine. Gram-positive organisms are
cultured. What is the likely pattern of hemolysis when this
organism is grown on blood agar?" -- answer: gamma or nonhemolysis.)
Viridans:
 Bunch of alpha-hemolytic species. Significant members:
mutans, which causes dental caries, and sanguis, which
causes endocarditis.
 They're optochin resistant (to differentiate them from:)
Pneumoniae, aka pneumococci:
 Optochin-sensitive, alpha-hemolytic.
 Immunogenic, widely polygenic capsules (84+ different
variations).
 Causes bacterial meningitis in adults.
 Most common cause of pneumonia in adults.
 Frequent cause of otitis media in kids.
 Increasing resistance to penicillins.
1. Describe the classification system of streptococci based on visible hemolysis on
sheep blood agar.
 As described above: three patterns: clears blood entirely (betahemolytic), leaves heme remnants like biliverdin to leave a green
residue (alpha-hemolytic), or has no hemolytic activity at all (nonhemolytic or gamma-hemolytic).
2. Describe the hemolytic pattern of common streptococci species.
 Alpha: pneumoniae, viridans.
 Beta: pyogenes, agalactiae.
 Non/Gamma: group D (bovis, enterococci).
3. Describe Lancefield classification of streptococci and the antigens involved in
classification
 Has to do with immunologic features of the streptococci; mainly uses
cell wall carbohydrates for differentiation.
4. Assign the common streptococci species to the correct Lancefield groups.
 Group A: pyogenes.
 Group B: agalactiae.
 Group D: bovis, enterococcus.
 Non-assigned: pneumoniae, viridans.
5. Describe the structure of streptococci and the role in virulence of capsule, cell wall
and cell wall components, teichoic acids, and enzymes and toxins.
 Largely as mentioned; have anti-phagocytic capsules. Note that the
capsule of pyogenes isn't immunogenic (as opposed to pneumoniae);
pyogenes's M-protein is its primary antigen.
 Note M protein is anti-phagocytic. Note also there are lots and lots of
M protein serotypes (not to be confused with the lots and lots of
pneumoniae capsule serotypes).
 Recall: pyogenes has streptolysins, pyrogenic exotoxins, and
streptokinase. Also has pro-motility factors (hyaluronidase, DNAase,
proteinase)-- the pus of strep infections is thin and watery for this
reason.
 Note streptolysin O is cardiotoxic. Recall that anti-streptolysin O
antibodies are used to determine recent strep infections.
 [Scarlet fever: red rash, strawberry tongue, spares the perioral area.]
 Erysipelas: well-delineated, deep red, indurated cellulitis. Usually
pyogenes.
 Strep spreads very rapidly-- lots of pro-motility factors.
6. Describe the mechanisms of the non-suppurative complications of group A
streptococci infections: acute rheumatic fever and acute post-streptococci
glomerulonephritis
 Acute rheumatic fever: as mentioned, not an acute manifestation of
the strep (no strep present)-- type II cross-reaction. Rheumatic fever
generally follows pharyngeal strep infections only (not a sequel of
cellulitis).
 ARF can be prevented by treatment of the acute infection-which is why you want to treat strep throat when you see it.
 PSGN: as mentioned, also not an acute manifestation of strep. Type III
immunopath. Note that PSGN can arise after either pharyngitis or skin
infections.
7. Describe the pathogenesis of group B streptococci infections, particularly the role
of the capsule.
 Unlike group A, group B strep's capsule is its virulence factor (and is
immunogenic). Antibodies against the capsule (6 serotypes) are
immunoprotective.
 Group B is a frequent cause of newborn infections (sepsis, meningitis);
~25% of women of childbearing potential are colonized in the
vagina/rectum with group B, which is where the baby picks it up.
 Group B was originally identified in cows that, when infected, wouldn't
give milk-- thus "agalactiae."
8. Differentiate enterococci and group D streptococci.
 Enterococci: penicillin-resistant. Can grow in 6.5% NaCl.
 Bovis and equinus: aren't penicillin-resistant. Can't grow in 6.5% NaCl.
 Note they can both hydrolyze esculin.
 Enterococci are another of those species of normal flora with
high levels of resistance to more or less everything
(cephalosporins, penicillins, aminoglycosides, sulfonamides,
clindamycin, some vancomycin). They're generally only a
problem in the hospital with radical changes in the underlying
bacterial flora.
9. Describe common infections due to viridans streptococci.
 (1) Dental caries (mutans)
 (2) Endocarditis (sanguis)
 Note that, as per our small group cases, mutans can get into the
bloodstream and cause endocarditis as well, mainly after dental work
or with poor dental hygiene.
10. Describe the metabolism of streptococci and the common tests used for
classification, including catalase, bacitracin sensitivity, CAMP test, bile esculin
hydrolysis and growth in NaCl.
 Metabolism: aerotolerant, ferment carbohydrates to form lactic acid.
Grow much better with blood.
 The only streptococcus that's bacitracin-sensitive is pyogenes.

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The only alpha-hemolytic streptococcus that's optochin-sensitive is
pneumoniae.
The only streptococci that is widely resistant to penicillin are the
enterococci.
No streptococci are catalase-positive (no bubbles when you apply
H2O2) (used to distinguish them from staphylococci).
Group D strep species are able to hydrolyze esculin.
CAMP test: B group strep enhances the hemolysis of staph aureus.
Staphylococci
Monday, January 05, 2009
2:55 PM
Staphylococci, 1/6/08:
[General notes, largely from CMBMRS:]
 Note that all staphylococci are catalase-positive (can break down H2O2
to water and O2); this helps differentiate them from streptococci,
which are catalase-negative.
 Staph aureus:
 Beta-hemolytic, but leaves behind a gold pigment (thus
'aureus').
 Catalase-positive, as mentioned (defends against reactive
oxygen species).
 Coagulase-positive (can convert fibrinogen to fibrin).
 Note that this has the effect of coating the bacterium in
fibrin-- this inhibits phagocytosis.
 Defense proteins:
 Protein A: binds the complement-fixing region of IgG,
preventing fixation and lysis. Not to be confused with
IgA protease, which is secreted by the encapsulated,
meningitis-causing bacteria of the next lecture.
 Hemolysins: lyse RBCs.
 Leukocidins: lyse white blood cells (highly associated
with virulent, invasive MRSA).
 Penicillinase: breaks open the beta-lactam ring of
penicillin, conferring penicillin resistance.
 Can also have methicillin resistance,
cephalosporin resistance, sulfonamide resistance,
etc.
 Most recently, we're getting vancomycinintermediate (due to multiple peptidoglycan
layers) and vancomycin-resistant (due to a
resistance gene it picked up from Enterococcus)
staph aureus.
 Note you can also get erythromycin-induced
clindamycin resistance when the two are coadministered. Who knows why?
 Invasion/motility proteins:
 Hyaluronidase (helps spread through connective tissue)
Staphylokinase (plasminogen activator, helps get
through clots)
 Lipase, proteases (help get into skin and through tissue)
Exotoxins (3 different types):
 Exfoliatin (causes skin to slough off in scalded skin
syndrome in kids)
 This causes desmosomal separation (granular cell
layer is sloughed off).
 Enterotoxins (cause food poisoning when pre-formed
toxin is ingested)
 Toxic shock syndrome toxin (TSST):
 Superantigen: causes massive IL-1, IL-2, TNFalpha release.
 Sort of does it all: diarrhea, fever, peeling
skin.
 Note this is not pre-formed toxin, but is produced
in the course of the infection.
 Note also that most people have TSS toxin
antibody from sub-clinical exposures.
Diseases caused by staph aureus:
 Direct-invasion:
 Pneumonia (usually in hospitalized patients)
 Meningitis/brain abscesses
 Osteomyelitis (most common cause of bone
abscesses in boys)
 Endocarditis (rapid onset; vegetations on valves
can flip septic emboli as per group case).
 Note First Aid mnemonic on bacterial
endocarditis signs and symptoms (p. 282
of 2008, under CV), bacteria FROM JANE:
 Fever
 Roth's spots (white spots on retina)
 Osler's nodes (raised lesions on
toes/fingers)
 Murmurs
 Janeway lesions (nonraised lesions
on palms/soles)
 Anemia
 Nail-bed hemorrhage
 Emboli
 Septic arthritis (invasion of joint space)
 Skin infections (impetigo/"honey-colored crust",
cellulitis, sutures, etc)
 Catheter infections
 Toxin-mediated:
 Scalded-skin syndrome
 Toxic shock syndrome
 Food poisoning
 [Note distinction between blisters from scalded-skin
syndrome and those from, say, impetigo-- impetigo is a
direct infection and the blisters will culture staph, while
SSS is toxin-mediated and the blisters won't culture
staph.]

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
Note that most staph species are penicillin-resistant (secrete
penicillinase).
 Note also that staph aureus has a capsule.
Staph epidermidis:
 Catalase-positive
 Coagulase-negative
 Grows on skin; frequent contaminant of blood cultures and
frequent colonizer of indwelling catheters (like Foleys).
 Secretes a biofilm (glycocalyx, or polysaccharide web) to bind
itself tightly to its environment (which is why it sticks to
catheters so well).
Staph saprophyticus:
 Catalase-positive
 Coagulase-negative
 Second leading cause (behind E. coli) of UTIs in young women.
[Recall that the primary human site of colonization for staph aureus is the anterior
nares.]
[Staph mainly undergoes genetic shift/drift through bacteriophage-mediated
transduction.]
[Phagocytosis by PMNs is main defense against staph-- which is why you see
recurrent staph infections in chronic granulomatous disease (PMNs can't make
oxidative burst to kill engulfed staph) and Job's disease (ineffective macrophage
activation, thus inefficient IL-8 secretion, thus not much neutrophil chemotaxis).]
1. Describe how Staphylococci are distinguished from other gram positive cocci like
streptococci.
 All species of staph are catalase-positive; in addition, aureus is
coagulase-positive.
 Staph aureus also forms characteristic clumps (as opposed to strep's
chains).
 Note that aureus likes to cause abscesses (likes to clump), as opposed
to pyogenes, which like to spread quickly. That said, it can still cause
necrotizing fasciitis if it gets deep enough.
 Generally: drain abscesses.
 General pattern: local colonization in respiratory or cutaneous tissue,
with following bacteremia.
2. List important Staph. aureus virulence factors and describe how they contribute to
the symptoms of infections.
 Capsule, teichoic acids (project from surface and mediate attachment),
protein A, clumping factor (causes aggregation of bacteria), exotoxins
as listed above, etc (complete list on slides).
 Capsule: inhibits phagocytosis, enhances attachment.
 Lipoteichoic acid: attaches to fibronectin residues on host cells.
 Protein A: inactivates attached complement.
 (Kind of amazing these little bastards haven't already gotten
us.)
3. Explain how protein A interacts with antibodies.
 As mentioned, binds to Fc region of IgG heavy chain and prevents
complement binding.
 Note that this means you have a bunch of relatively inactivated IgGs
sticking off the bacterium-- which further inhibits phagocytosis.
4. What is coagulase? How would it contribute to disease?

As mentioned, cleaves fibrinogen to fibrin; the fibrin forms a mini-clot
around the bacterium and prevents phagocytosis. Not the be confused
with clumping factor, which aggregates cocci.
5. How would Staph. aureus infections be identified in the clinical laboratory?
 McConkey inhibits staph (can't grow staph on McConkey)
 Hemolysis (beta)
 Catalase test (positive)
 Coagulase test (positive)
 Can also use labeled clumping factor and coagulase
Encapsulated Pathogens
Monday, January 05, 2009
3:23 PM
Encapsulated Pathogens, 1/6/08:
[Here we're talking about Hemophilus influenzae, Neisseria meningitidis, and
Streptococcus pneumoniae, three organisms whose polysaccharide capsules largely
determine the extent of invasion (this often, but not always, also correlates with the
extent of the immune response against them). The capsule is thus the "critical
virulence factor." Note that, as with Strep pyogenes's association with M protein, if
an 'encapsulated organism' doesn't produce a capsule, it's avirulent or only weakly
virulent (it can still produce localized infections like otitis media in the case of S.
pneumoniae). Note also that the relevant antibodies against these organisms are
mainly directed at the capsule.]
[Note that these aren't the only encapsulated organisms with immunogenic capsules- Group B strep would be another example. These are presented as representative
samples only.]
1. Compare and contrast H. influenzae, N. meningitidis, and S. pneumoniae in terms
of (i) basic bacteriological features (e.g., morphology etc.). (ii) methods and
potential problems for laboratory diagnosis. (iii) major virulence factors (e.g.
capsules).
 H. influenzae:
 Small, Gram-negative, often coccoid. Note it really, no really,
does not cause the flu. Note it also doesn't require blood to
grow (though it does need heme, ie. factor X, and NAD, ie.
factor V).
 Note influenzae likes to grow near staph aureus (staph
produces NAD).
 6 capsule serotypes. The one that most commonly causes
disease ("b") has a very effective vaccine now. Type "a" is
hence now more common.
 Note that the capsule of "b" contains teichoic acid, which
is also found in all Gram-positive bacteria (so anti-"b" =
anti-Gram positive).
 N. meningitidis:
 Gram-negative diplococcus, shaped somewhat like a kidney
bean.

9 capsule serotypes, of which A, B, and C are the most
important clinically (A is responsible for epidemics). Note that
"B" type is non-immunogenic in humans (no response) due to
its homology with normal ECM substances.
 S. pneumoniae:
 Gram-positive diplococcus with pointy ends ("lancet-shaped").
 Alpha-hemolytic, but largely indistinguishable from viridans
without an optochin sensitivity test (recall viridans are resistant
but pneumoniae isn't). You can also use straight-up antipneumoniae sera.
 90+ (classically "84") capsule serotypes, only 12 of which
usually cause disease.
 Note pneumoniae with capsules causes meningitis; pneumoniae
without capsules causes otitis media.
 Common factors: you can use culture and anti-capsule antisera to
detect them. However, antisera can be misleading, because
encapsulated organisms can shed their capsules in various bodily fluids
and go on their merry way.
 All of these (and N. gonorrhoeae) secrete IgA protease (cleave IgA
antibody).
 The endotoxin (lipopolysaccharide or LPS, or here called
lipooligosaccharide or LOS) of the two Gram-negative encapsulateds
(meningitidis and influenzae) is particularly immunogenic and is
implicated in DIC, etc.
 S. pneumoniae, when it lyses, releases a toxin called pneumolysin
(lyses surrounding cells).
2. What is the age-related incidence of meningitis for these three organisms and
what are the reasons for this age related incidence?
 Influenzae: Under 7 years.
 Meningitidis: 6 months to 6 years (maternal IgG protects infant for
first 6 months).
 Pneumoniae: 6 months to the grave, pretty much.
 Note theme: kids past 6 months but before 6 years are particularly
vulnerable to encapsulated bugs because the T-independent
mechanism of antibody production aren't mature. This is why all three
of the top meningitis-producing bugs in this age group are the
encapsulated organisms described here.
3. What is the relationship between certain capsular serotypes or serogroups and
invasive disease (e.g. meningitis) caused by these organisms?
 As mentioned above; only certain serotypes cause invasive disease.
4. What are the differences in the immunogenicity of the different capsules of H.
influenzae and N. meningitidis and why is this issue medically important?
 Recall that influenzae type b has teichoic acid, as do all Gram-positive
organisms (like pneumoniae). This means anti-b can be anti-Gram+
and anti-Gram+ can be anti-b.
 Recall that the meningitidis type B capsule is non-immunogenic; thus
there is no vaccine for meningitidis B. (B capsule is also identical to a
capsule of E. coli.)
5. How are antibodies to the capsules used to identify the organisms and to rapidly
diagnose disease without culturing the organism? What precautions need to be
recognized when using antiserum for diagnosis? Why is it important to identify the
capsular types of these organisms?

Mostly discussed above; there are antibodies to pretty much every
serotype of these organisms for diagnostic purposes. Watch out for
pre-shed capsules.
 Capsules serve three diagnostic/treatment purposes: ID organism,
discover whether it's virulent, and target appropriate antibody.
6. What is the basis for vaccines against H. influenzae, N. meningitidis, and S.
pneumoniae diseases? That is, what is their composition? How safe and how effective
are they? To what age groups are they given? Why are they not effective in all age
groups? Why are they effective against only some capsular serotypes or serogroups?
 Vaccines: generally composed of capsule polysaccharides. H.
influenzae vaccines are conjugated with a protein-based toxoid (see
next point).
 As stated here, they are generally safe and effective (except possibly
Pneumovax, see next point)-- note the mercury has been removed
from Hib (H. influenzae b) vaccine.
 Age groups:
 vaccine vs. H. influenzae: as young as 2 months (conjugate
vaccine: Tetramune).
 vaccine vs. N. meningitidis: 11-55 years (Menactra) or 2-10
years (Menomune). Note no vaccine is available for the B
serotype (non-immunogenic).
 vaccine vs. S. pneumoniae: elderly/at-risk (Pneumovax) or
infants/toddlers (Prevnar).
 Note also that the situations that tend to lead to bacterial meningitis
outbreaks are day care, military camps, and college dormitories-constant close exposure. Good target groups for vaccination.
 Vaccines are composed of capsule polysaccharides from a variety of
strains; however, it doesn't cover them all.
7. Why are capsular polysaccharides conjugated to proteins more effective as
vaccines than the polysaccharides alone?
 Okay, I'm going to take you back lo these many months to Blood and
Lymph. Remember "T-cell-independent immune responses?" That's
when a polysaccharide-based antigen prompts an immune response by
B-cell stimulation alone (recall that normally you need a T-helper cell
response as well to co-stimulate the B cell).
 These work okay in kids past a few years old, but infants can't do this
yet (they can't do it until about 2 years old). So what you do is, you
tie the polysaccharide antigen to a protein-based antigen (like tetanus
toxoid). This gets the T-helper cells involved and starts co-stimulating
the B cells to make a good response against not only the toxoid, but
the polysaccharide as well.
 This is why the incidence rate of H. influenzae meningitis in kinds and
infants in the US is currently dropping like a brick-- the conjugate
vaccine can be used as young as 2 months.
 Note, of course, that it's also expensive, which means kids in thirdworld countries are still dying from bacterial meningitis at a decent (if
the word can be applied) clip. Dr. Cohen's got a good story about how
a WHO guy did a legal end-run around this to make a $130 vaccine
into a $0.50 vaccine, for all you aspiring do-gooders.
 Note that you have a similar vaccine against most types of
meningitidis, recommended for all kids around 11-12 years old. Note
also that you have no vaccine against serotype B meningitidis, as it
ain't immunogenic.

Note on Pneumovax (vaccine vs. 23 strains of pneumoniae): its
efficacy in preventing pneumococcal pneumonia is being seriously
called into question-- pneumonia doesn't have to go through the
blood, so vaccination doesn't seem to do a lot of good except in
preventing bacteremia.
 Note on Prevnar (vaccine vs. meningitis-causing pneumoniae
strains): works extremely well; conjugated vaccine and thus
efficacious for all infants and toddlers. Notice that it shows some
benefit in preventing otitis media as well.
 Problems: expensive, only covers 7 strains of pneumoniae
(other strains thus on the rise), and can give rise to superresistant strains of pneumoniae.
8. In what kind of situations are prophylactic antibiotics administered for the
diseases caused by these organisms?
 In bacterial meningitis cases, anyone exposed to the patient can be
prophylactically given antibiotics.
 When a person is an asymptomatic carrier they can also be given
prophylactic antibiotics.
[CMBMRS additional notes:]
 H. influenzae b also causes acute epiglottitis (can't swallow, develops
stridor) and septic arthritis in infants (most common cause, vs. N.
gonorrhoeae in young adults). Use third-generation cephalosporins;
treat close contacts with rifampin.
 Neisseria is the only pathogenic Gram-negative coccus. N. meningitidis
can cause petechiae and bilateral adrenal hemorrhage
(Waterhouse-Friderichsen syndrome; can be rapidly fatal). Highrisk groups include infants past 6 months (no more maternal
antibodies) and military recruits. Neisseria is the only thing that grows
on Thayer-Martin VCN agar. Use third-generation cephalosporins or
penicillin G.
 Pneumoniae: as mentioned, it's developing resistance to penicillins.
Cephalosporins and high-dose penicillins are still effective.

Most common causes of meningitis in newborns (to 6 months):
 Group B streptococci (agalactiae)
 E. coli
 Listeria monocytogenes
 (note these are all frequent colonizers of the vagina/rectum.)

Most common causes of meningitis in infants/kids 6 months - 6 years:
 S. pneumoniae
 N. meningitidis
 H. influenzae
 (note these are all encapsulated)
 [First Aid also lists enteroviruses.]
Intracellular Bacteria
Tuesday, January 06, 2009
12:18 PM
Intracellular Bacteria, 1/7/08:
[Some terms: obligate intracellular bacteria can only reproduce inside other cells;
facultative intracellular bacteria can reproduce either inside or outside other cells.]
 Obligate intracellular bacteria include, among others, Chlamydia,
Mycobacterium leprae, and Rickettsia. Note that these depend on the
host cell's proteins (the size of their functional genome is reduced)-eg. they lack genes for citrate synthase, lactate dehydrogenase, etc
(they use the host's).
 Facultative intracellular bacteria include, among others, Listeria,
Legionella, Mycobacterium tuberculosis, Salmonella, and Shigella.
1. Explain the advantages and disadvantages of an intracellular lifestyle inside
phagocytic and non-phagocytic host cells.
 Generally speaking it's good to live inside cells- you're protected from
most of the immune system, it's a great place to wait around while
you're waiting for your host to become immunocompromised, and you
don't have to compete with anything else for space or nutrients (which
are abundant in the cytosol).
 If you're in epithelial cells (as in Listeria or various GI anaerobes), you
can also get from one cell into the next without being exposed to the
extracellular milieu.
 One nice thing about living inside phagocytes, in particular, is that
they're quite mobile-- so if you get inside some, you can spread
yourself to distal parts of the body (Salmonella does this with dendritic
cells in the intestine).
 The problem with living inside phagocytes is that they're designed to
kill things living inside them-- sort of like living inside a garbage
disposal. Specifically, bacteria living inside phagocytes have to avoid
the following:
 NADPH oxidase system (oxidative burst) in the phagolysosome
 Particularly low pH inside the phagolysosome
 NO in the cytosol
 Antimicrobial peptides (lysozymes, defensins, etc)
 Iron sequestration
2. Describe the strategies used by different intracellular pathogens to avoid the
antimicrobial defenses encountered during the maturation of phagosomes into
phagolysosomes.
 One of the easiest ways to avoid the phagolysosome's challenges is to
get out of it. Listeria monocytogenes does this by lysing it open with
"listeriolysin O" (related to streptolysin O).
 Listeria also has an interesting mechanism of escape from the
cell: it causes polymerization of the host cell's intracellular actin
at one of its poles, forming "rockets" of actin that shoot it out
into neighboring cells (avoiding extracellular immune defenses).
 In addition, preventing the fusion of the phagosome with the lysosome
is also useful.
 Finally, a few bugs just deal with all the stuff in the lysosome.
3. Describe how different intracellular lifestyles may impact on antibiotic usage and
susceptibility.

Depending on where the organism is within the host cell, it can be
easier or harder to target different antibiotics to that location.
 Aminoglycosides in particular seem to be poor at combating
intracellular bacteria unless they're in the lysosomes (AGs are
concentrated there).
 Penicillins are also ineffectual (don't get inside the cell).
 By contrast, tetracycline diffuses through the cell membrane and gets
into the cytosol pretty good.
 Note that all obligate intracellular bacteria listed are susceptible to
tetracycline and rifampin (tetracycline is bolded).
4. List intracellular pathogens that escape into the cytosol, or remain in vacuoles that
are fusogenic or non-fusogenic.
 As mentioned, Listeria breaks open the phagosome to escape.
 Fusogenic organisms are those that don't bother preventing the
fusion of the phagosome with the lysosome. There's only one of these
here: Coxiella burnetti (obligate intracellular bacteria). For whatever
reason the lysosome doesn't bother it and it divides like crazy.
 Non-fusogenic:
 Legionella prevents fusion and directs its vacuole instead to the
ER, where it starts making more protein.
 Chlamydia prevents fusion and directs its vacuole instead to the
Golgi.
 (note there are lots of signaling molecules around to
direct vesicles to both of those locations-- probably
pretty easy to fake.)
 Mycobacterium tuberculosis/leprae don't direct their
phagosomes in any particular direction, but just prevent fusion.
 [Note also that certain species avoid the entire question of
phagosome-lysosomes by infecting red cells (which don't have much of
a defense system, lacking both nuclei and ribosomes).]
5. Define the genetic basis for the classification of obligate or facultative intracellular
pathogens.
 As mentioned, obligate intracellular bacteria lack the genes to make
certain essential substances; facultative intracellular bacteria still have
them.
6. Compare and contrast the means by which zipper and trigger mechanisms
contribute to invasion and dissemination of intracellular pathogens.
 Zipper mechanism: this involves activating, from outside the cell, the
cell's phagocytosis mechanisms-- the bacterium has ligands that bind
to particular receptors on the cell membrane. This requires close
contact with the membrane and causes the bug to be 'tugged' into the
cell. It can occur even on cells that aren't normally phagocytic (eg.
epithelial cells).
 Trigger mechanism: This is more of a brute-force approach; the
bacteria injects substances through a protein 'syringe' directly into the
target cell's cytoplasm. These substances cause the membrane to
reach out and engulf nearby structures, including the bacteria
("macropinocytosis").
[A few notes on Listeria monocytogenes:]
 It only affects immune-compromised hosts (including pregnant
women, newborns and the elderly), largely because hosts with intact




cellular immunity can activate macrophages with IFN-gamma to
destroy their intracellular hangers-on.
It's thus one of the leading causes of meningitis in infants under 6
months of age and in the elderly.
In pregnant women it does not cause meningitis but does kill the fetus
(causing miscarriage).
Its pathogenicity is related to the amount of "internalin" it has, a
surface protein that facilitates the zipper mechanism of entry.
Treat with either ampicillin or SMX/TMP.
More Antimicrobials
Tuesday, January 06, 2009
1:56 PM
More Antimicrobials, 1/7/08:
[LOs, condensed: Know everything about the bolded drugs on the list. Know what
drug class the unbolded drugs belong to. Know anything else I tell you about them.
To be fair, this is reasonable when you're talking about antibiotics.]
[General notes:]
Sulfonamides/Trimethoprim:
 Sulfonamides and trimethoprim act on the folic acid/THF pathway
(kind of like methotrexate, remember?). Because bacteria can
synthesize their folate but humans can't, the synthesis pathway can be
interrupted.
 Specifically, folate is synthesized from a compound called PABA. Sulfa
drugs like sulfamethoxazole (SMX) act as PABA analogs that inhibit
the PABA-incorporation enzyme (dihydropteroate synthase).
Trimethoprim (TMP) is NOT (repeat, as in the notes, NOT) a sulfa
drug; it acts downstream after the folate has been synthesized to
prevent dihydrofolate from being turned into tetrahydrofolate.
Specifically, it acts on the bacterial form of the enzyme dihydrofolate
reductase (whose human counterpart is inhibited, recall, by the
chemo drug methotrexate).
 Note that TMP is much more potent than SMX, since
dihydrofolate reductase is the rate-limiting step of this reaction.
 SMX and TMP are nearly always used together, as in Bactrim. By
themselves, each is bacteriostatic, but together they are bacteriocidal
(big synergistic effect, since they target the same pathway at multiple
sites). They have a broad spectrum of action, including some
intracellular organisms, and are absorbed well through the oral route.
Note that there's widespread resistance to sulfa antibiotics.
 Note SMX has a delayed onset of action, since it's interrupting the
pathway at a non-rate-limiting step. TMX, by contrast, acts rapidly
(inhibits the RLS).
 They are well-absorbed through the oral route and cross the BBB (can
be used for meningitis, as in Listeria meningitis).
 Again: TMP is not a sulfa drug.




The metabolism of sulfonamides is weird-- they're N-acetylated, which
makes them less soluble rather than more. This predisposes them to
form urine stones (they're renally excreted). Alkalinizing the urine
renders them more soluble.
Adverse effects of sulfonamides:
 Note sulfonamides are oxidative stressors (cause hemolytic
anemia in G6PD deficiency).
 Sulfonamides can cause Stevens-Johnson syndrome (begins as
a rash, winds up a potentially fatal dermal-epidermal
separation).
 Sulfonamides displace various things - both drugs and bilirubin
- from their binding sites on albumin. This means it can mess
with concentration-sensitive drugs like warfarin and can also
cause an increase in plasma indirect bilirubin concentrations
(which is why it causes kernicterus in babies).
 Note that SMX/TMP cross the placental barrier.
 There's a fairly high rate of hypersensitivity reactions to
sulfonamides.
Mechanisms of resistance:
 Alteration of target enzymes
 Note that bugs without folate can still pick up the end products
they need folate to make (eg. purines, thymine, methionine,
etc) from surrounding tissue. Pus, in particular, is rich in all
these things, which is one reason it's important to drain
abscesses when treating.
SMX/TMP are mainly used vs UTIs, as well as in toxoplasmosis and vs.
Nocardia and Chlamydia. As mentioned in DEMS, sulfasalazine is used
by itself for ulcerative colitis. Sometimes it's used in sinusitis. Of
current interest, most strains of MRSA are susceptible to SMX/TMP.
Quinolones:
 Primarily act on bacterial DNA gyrase and topoisomerase IV, which
are required for replication and transcription (it 'nicks' DNA and then
re-seals it to relieve coiled tension in the DNA).
 Quinolones are bacteriocidal, though exactly how isn't quite
understood, and are broad-spectrum. Note that they don't have much
effect on anaerobes.
 Main mechanisms of resistance: mutations in DNA gyrase, increased
efflux of drug.
 Fluoroquinolones are well-absorbed through the oral route and widely
distributed. However, they can't be taken with antacids or milk (they
bind to divalent cations like Mg or Ca).
 Toxicities:
 Contraindicated for pregnant women and children under 12
years of age (impairs cartilage development).
 Can cause tendonitis and tendon rupture in adults and leg
cramps and myalgias in kids (as per First Aid).
 Some quinolones inhibit theophylline metabolism (recall
theophylline has a very narrow therapeutic index).
 Specific drugs:
 Ciprofloxacin: 2nd generation
 Levofloxacin: 3rd generation
 Monifloxacin: 4th generation


What the 'generation' means: like cephalosporins, signifies their
spectrum of use.
 1st gen: excellent Gram-negative, no Gram-positive, no
antipseudomonal, some use vs. atypicals (no cell walls), no
anaerobic.
 2nd gen: excellent Gram-negative, good Gram-positive, good
antipseudomonal, good atypicals, no anaerobic.
 3rd gen: excellent Gram-negative, excellent Gram-positive, no
antipseudomonal, good atypicals, some anaerobic.
 4th gen: excellent Gram-negative, excellent Gram-positive,
good antipseudomonal, good atypicals, excellents anaerobic.
 [Obviously a pretty wide spectrum here.]
Fluoroquinolones are usually used for UTIs and GI infections from
multiply-resistant bugs (often nosocomial). Infrequently used for
respiratory infections, sometimes for prolonged treatment of soft
tissue or bone infections. Sometimes used in prostatitis and STDs.
Others:

Daptomycin is limited-use, mainly vs. vancomycin-resistant Grampositives. Bacteriocidal.

Polymyxins act as bacterial detergents to lyse the cell membranes.
Bacteriocidal; acts mainly against Gram-negatives. Main use is for
Pseudomonas meningitis.

Nitroimidazoles: metronidazole (for anaerobes and protozoa),
nitrofurantoin (for UTIs). Damage bacterial DNA.
 Metronidazole: used in pseudomembranous colitis due to C.
difficile.
 Metronidazole has a disulfiram-like effect on alcohol
metabolism-- so patients should avoid alcohol.
 Recall that metronidazole is part of 'triple therapy'
against H. pylori in peptic ulcers.
 Nitrofurantoin is rapidly excreted, but accumulates in the renal
tubule-- a "UTI antiseptic." Used to get around
allergies/resistance to sulfa drugs.
Rifampin: inhibits DNA-dependent RNA polymerase. 1st line for antimycobacteria (anti-TB and leprosy). Recall that it induces CYP450 enzymes.
Enteric Bacteria I + II
Tuesday, January 13, 2009
8:01 AM
Enteric Bacteria I + II, 1/13/08:
Types of E. coli:




ETEC: enterotoxogenic E. coli (similar to V. cholerae)
EHEC: enterohemorrhagic E. coli (toxin destroys epithelial cells)
 Note EHEC can cause HUS (hemolytic uremic syndrome)
EIEC: enteroinvasive E. coli (invades epithelial cells)
EPEC: enteropathogenic E. coli (attaches to and directly damages
epithelial cells)
Diarrhea:

Watery/secretory: does not involve bacterial invasion (toxin effects
only)
 Causes: ETEC, V. cholerae
 No white cells in stool, no fever.
 Dysentery: involves bacterial invasion of the intestinal epithelium
(toxin + invasion)
 Shigella, EIEC, Salmonella enteritidis
 PMNs in stool, fever
 Bloody water diarrhea: involves systemic bacterial invasion (toxin +
invasion)
 Salmonella typhi, Yersiniae, Campylobacter jejeuni
 Leukocytes in stool, severe fever
 [Hemorrhagic: does not involve bacterial invasions (toxin effects only)]
 EHEC
Enterotoxic bacteria outbreaks tend to occur during the hot months - temperature
increases the number of bacteria present, which increases the likelihood of ingesting
enough bacteria to cause disease.
 Note Shigella is extremely pathogenic-- only need 10-100 bacteria to
cause disease (vs. 10^8 for Vibrio cholerae) due to its acid resistance
(survives the stomach).
1. Relate morphology, metabolism and genetics of enteric bacteria to pathogenesis.
 They're all Gram-negative-- inner and outer membranes that surround
a layer of peptidoglycan.
 Genetics:
 Can transmit DNA through plasmids (eg. antibiotic resistance),
bacteriophages (Shiga/Cholera toxins), or 'pathogenicity
islands' (both the notes and Wiki are somewhat confusing on
this topic).
 Morphology (also see next LO):
 Need to adhere to the intestine. Generally this is accomplished
by pilli-- little arms that come off the bacteria to attach to
specific sites (this specificity is why certain bugs live only in the
small intestine and others live only in the colon).
2. Understand the diagnostic value of major antigenic structures of enteric bacteria.
 3 general regions where you look for different features to structurally
differentiate different genera and species of enterobacteria:
 Some bacteria have flagella, some don't. Flagellae are classified
as "H" antigens; bacteria that have them are called motile.
 Some bacteria have capsules, some don't. Capsules are
classified as "K" antigens.
 In the outer membrane there's a common feature of most
Gram-negative bacteria, the lipopolysaccharide.
 The "O" antigens are repeating oligosaccharide units in
the LPS.
The "lipid A" portion of the LPS is retained inside the
outer membrane of the bacteria. When the bacteria is
lysed, it's released, which is bad news because it's the
main toxigenic region of the LPS and is largely
responsible for septic shock.
3. Compare and contrast mechanisms of pathogenicity of invasive and non-invasive
enteropathogenic bacteria.
 Non-invasive:
 Note that the treatment of non-invasive enteropathic bacteria is
generally supportive care without antibiotics.
 V. cholerae: comma-shaped rod with a single flagellum (it's
motile).
 Lives largely in shellfish; transmitted fecal-oral.
 Has acquired 2 bacteriophages: one that transmitted the
ability to colonize the intestinal wall, another that
created the cholera toxin.
 Let's talk about the cholera toxin for a moment:
 Like a lot of toxins, there's two subunits: the A
subunit, which is the active or effector part, and
the B subunit, which is a delivery system and
binds to the target on enterocytes.
 The A subunit forms an enzymatic complex
makes adenylate cyclase constitutively active,
leading to a massive increase in cAMP inside the
enterocyte. This activates chloride pumps in the
membrane, leading to a following efflux of water
and cations (diarrheal output is enormous, up to
a liter per hour).
 Enterotoxigenic E. coli: aka traveler's diarrhea:
 Two toxins:
 Heat-labile toxin (LT): more or less identical to
cholera toxin-- just targets different receptors
(which results in a slightly different clinical
picture with less drastic diarrhea).
 Heat-stable toxin (ST): similar, but increases
cGMP instead of cAMP.
 Generally self-limiting (not as bad as cholera).
Treatment is still supportive.
 Enteropathogenic E. coli:
 Not associated with toxin-coding bacteriophages but
with pathogenicity islands in the bacterial chromosome.
 The bacterium attaches to enterocytes and directly
destroys their microvilli.
 Again, not associated with toxins; causes malabsorption
instead (resulting in osmotic diarrhea).
 Enterohemorrhagic E. coli:
 Caused by certain strains of O157:H7 (= the O antigen
type).
 Attaches and effaces like enteropathogenic, but has
toxins as well.
 This toxin is Shiga-like: they inhibit protein synthesis by
binding the 70S ribosomal subunit in the intestinal cells.
This kills the intestinal cells and causes efflux of blood in



the stool. Note that the bacterium itself doesn't seem to
invade the cells.
As mentioned before, it's associated with hemolytic
uremic syndrome (toxin is absorbed into the
bloodstream and gets stuck/activated in the kidney).
Treatment is still supportive only.

Invasive:
 [Note the distinction between invasive organisms that invade
and reside in intestinal cells and invasive organisms that get
into systemic circulation and go to various organs or
lymphatics.]
 Note that the diarrhea due to invasive bacteria is lower-volume
and thicker.
 Shigella:
 A variety of serogroupings depending on O antigen. The
most severe is Shigella dysenteriae; the most common
in the US is S. sonnei, which is pretty tame (of the
watery-diarrhea variety). Humans are the definitive host
for Shigella (not zoonotic).
 Pathogenesis:
 Shigella invades colonic M cells (remember that
these sample colonic contents to present to the
immune system) and enterocytes. Specifically, it
its phagocytosed and then lyses open the
phagosome (like Listeria; it's thus a facultative
intracellular bacterium).
 Note it also spreads from cell to cell in a similar
manner to Listeria (shoots from one to the next).
 Shigella causes apoptosis of macrophages; also
causes a massive influx of neutrophils due to IL-8
and IL-1 secretion (from the macrophages). This
causes lots of tissue damage.
 Note that Shigella has no pilli and no flagellae. That is,
they're nonmotile and they have no attachment factors.
They also have no glycocalyx, capsule, or mucus. This
limits them a bit, but it also greatly reduces the ability of
the immune system to target them.
 Note also that Shigella doesn't spread beyond the
intestinal epithelial cells (gets taken out by PMNs), as
opposed to S. typhi, etc (see below); its invasion is
limited to the intestinal epithelium.
 Causes intestinal cramps.
 Definitely do treat Shigella infections with antibiotics.
 Salmonella:
 A bunch of variants of this. They have different
pathogenicities:
 Typhi
 Enteriditis
 Cholerae-suis
 Ok, technically it's not S. typhi, it's S. enterica,
subspecies enterica, serovariant Typhi. Whatever.
 Pathogenicity:

Can attach and invade enterocytes (induce their
own phagocytosis).
 They can survive inside macrophages and other
professional phagocytes by preventing the fusion
of the phagosome with the lysosome (nonfusogenic).
 S. enteritidis: most common, zoonotic; causes watery
diarrhea (no antibiotic treatment).
 S. cholerae-suis: zoonotic; causes sepsis due to
invasion into the bloodstream, causing high fever but
without diarrhea.
 S. typhi: human-adapted (not zoonotic); invades
intestine; invades macrophages and travels through the
blood. Tends to accumulate in the gall bladder and
produces biofilms there to stick around. The invasion of
the intestinal wall can be so severe that it perforates the
bowel. Clinically, see jaundice, RUQ tenderness, and
hepatosplenomegaly.
 Use antibiotics for septicemia and cholerae-sius.
 There's a (moderately effective) vaccine vs. S. typhi.
 Camplyobacter:
 Spiral ("gull wing")-shaped (not to be confused with
spirochetes).
 The main problem with Camplyobacter is C. jejeuni (not
C. coli).
 C. jejeuni is the most frequent cause of gastroenteritis
in the US.
 Note that it's microaerophilic (can't deal with high
oxygen tensions, as opposed to most of the rest of
these, which are facultative anaerobes).
 Zoonotic; prevalent in commercial poultry (cook your
chicken thoroughly).
 Invasive, motile, carries a toxin, induces an
inflammatory/bloody diarrhea. We don't actually know a
whole hell of a lot about it for such a widely-distributed
pathogen.
 Generally supportive care; can use antibiotics if it gets
really bad.
 [Helicobacter pylori:]
 (most common bacterial pathogen in the world)
 Not exactly invasive-- invades mucosa of the stomach, not the
cells. Localizes to the gastric pits (recall that underneath the
mucus the pH is much closer to normal).
 Looks a lot like Campylobacter (spiral-like rod); has multiple
flagellae on one end; is microaerophilic like Campylobacter.
 Produces urease, which breaks urea down to ammonia (which
neutralizes the acidity of the stomach). Damages cells due to a
toxin which causes big vacuole formation. Pro-inflammatory,
causes ulcers (both gastric and duodenal) and predisposes to
gastric adenocarcinomas.
4. Explain how acidity of the stomach and bacterial virulence factors contribute to
the pathogenicity of gastrointestinal pathogens.

Generally the acidity of the stomach kills off most pathogens you
ingest (note exception of Shigella with its acid resistance). Conditions
of low acid content or acid resistance tend towards higher
pathogenicity of ingested bacteria. I think his slide also said that
pathogenicity goes up when you ingest food with bacteria, though I'm
not sure why.
5. Identify virulence factors and host cell targets of enterotoxigenic bacteria.
 Not directly covered; presumably it's more or less covered already.
6. Identify mechanisms by which type III secretion systems contribute to
pathogenesis.
 Type III secretion systems: effectively, little molecular needles.
 They're kind of shot out of the bacteria's membrane into the cytoplasm
of the host cells; this allows the bacteria to shoot up its host's cells
with effector molecules that accomplish a variety of ends (eg. inject
toxin, invade the cell by causing phagocytosis of the bacteria, etc).
7. Classify enteric bacteria according to environmental and host range distribution.
 As noted?
Bacterial STDs I + II
Tuesday, January 20, 2009
7:56 AM
Bacterial STDs I + II, 1/20/09:
[I refer you to Bob's excellent handout on this lecture and the two that immediately
follow.]
[General notes:]
 65 million in US with an incurable STD; incidence of all STDs is 18
million per year.
 He took us, just for kicks, through the definitions of specificity and
sensitivity.
 Note that the rates of infection of men and women distinguish these
STDs:
 They're roughly equal for N. gonorrhea.
 More women than men get Chlamydia.
 More men than women get Treponema (spyhilis).
1. What are the major characteristics of Neisseria and Moraxella species?
 As you know, Neisseria are Gram-negative diplococci; they're roughly
0.8 micrometers in diameter.
 Moraxella is standard respiratory flora, related to Neisseria. It's the
third major cause of otitis media behind S. pneumoniae and H.
influenzae and can worsen COPD (catarrh being an exudate of blood
and white cells). Note that it also shows up as Gram-negative
diplococci. It wasn't mentioned in the lecture (this is from CMBMRS).
2. What are the standard procedures for diagnosis of gonorrhea? Are some more
effective for diagnosis in men than in women? Why?

Rapid diagnosis: Gram stain of an exudate (eg. sterile swab of
urethra). What you're looking for, for a positive test, is the Gramnegative diplococci inside neutrophils.
 The rapid test has good specificity and sensitivity for men, but
the sensitivity is poor in women (~50%).
 Culture is the gold standard. Note that Neisseria is strictly aerobic and
needs to be put in a high-CO2 environment while it cultures.
 Note that his notes say culture really isn't the gold standard-use molecular testing (nucleotide amplification, eg. on selfcollected specimens) instead.
 Neisseria is picky and fragile. The boards seems to like one particular
media you use for culturing Neisseria: Thayer-Martin VCN media.
This is a chocolate agar (heated sheep's blood) implanted with
different antibiotics: Vancomycin (kills all Gram-positives), Colistin
(polymyxin, kills all Gram-negatives aside from Neisseria), and
Nystatin (kills all fungi). The antibiotics are there for isolation
(Neisseria doesn't grow as well as most of the others).
 Dr. Vasil suggests plating on both antibiotic-treated and nonantibiotic-treated chocolate agar (some Gonorrhoeae are
sensitive to vancomycin).
 Other tests:
 Neisseria sp. are oxidase-positive (colonies turn black on
certain media).
 Neisseria Meningitidis ferments Maltose. Gonorrhoeae doesn't.
3. Are men or women more likely to have an asymptomatic infection with N.
gonorrhoeae? What is the epidemiological significance of asymptomatic carrier? Why
is an asymptomatic infection of any concern if there are no symptoms?
 Women are more likely to have asymptomatic gonorrhea infection (5080%).
 No symptoms = no treatment; no treatment can lead to further
invasion and pelvic inflammatory disease (infection of uterus,
fallopian tubes, and/or ovaries), leading to possible sterility, ectopic
pregnancy, abscesses, peritonitis, etc.
 The epidemiological consequences of someone who's infectious but
doesn't know it should be fairly obvious.
4. What are the clinical manifestations of gonorrhea and what organs, besides the
ones in the urogenital tract, can N. gonorrhoeae infect?
 Men: discharge of pus, pain during urination.
 Women: sometime vaginal discharge, but often asymptomatic as
noted.
 Gonorrhea spreads from urethra into prostate, uterus, etc; if it
spreads, can cause endocarditis, arthritis (most common cause of
septic arthritis in young, sexually active adults), meningitis, rash, etc.
 Note babies delivered to a gonorrhea-infected mother frequently get
ophthalmia neonatorum (eye infection). Can treat with silver nitrate
drops.
 Note that Chlamydia also infects the eyes of newborns. Give
erythromycin drops at birth to cover both species. Note also
that Chlamydia infection requires follow-up treatment with
systemic antibiotics.
 Sexual abuse of prepubertal girls by gonorrheic men causes a
particularly virulent form of gonorrhea in the girls-- the attachment
points for the bacteria are more exposed in the younger girls'
epithelia.
5. What is the importance of antigenic heterogeneity in the pathogenesis of
gonorrhea? Which bacterial surface structures undergo antigenic variation and/or
phase variation? How does this antigenic heterogeneity relate to the ability of a
single person to be infected multiple times with this organism?
 Virulence factors of N. gonorrhoeae:
 Main adherence and antigenic factor is their pilus (attachment
factor). This swaps around genes a lot (antigenic variation) so
that it's tough to make antibodies to all the varieties out there.
What this means, functionally: you don't develop immunity to
gonorrhea.
 Antigenic variation: change the amino acid sequence
of the protein (recombination variance, like your
antibodies). Note that this can occur during the course
of a single infection.
 Note colonies seem to be able to 'turn the pili off' (ie.
phase variation) to allow themselves to detach from a
surface and infect a new host. Due to accidental
insertion of a guanine during replication.
 Note that N. gonorrhoeae seems to actually have
several types of pilus, any one of which can be
turned on or off and each of which go through
antigenic variation-- for added difficulty in
making antibodies against it.
 Recall that Neisseria (both species) has IgA protease.
 Like most Gram-negative bacteria, Neisseria sheds some
endotoxin (oligosaccharide and Lipid A) to produce an
inflammatory response.
 "Opa" proteins (ie. "opaque") also allow extremely close
adherence and invasion of host tissue. "Porins" seem to allow
systemic invasion.
6. What are the recommended guidelines for treatment of gonorrhea? How do these
relate to the treatment of other common STD’s?
 N. gonorrhoeae is penicillin-, tetracycline-, and quinolone-resistant.
 Current treatment is ceftriaxone (third-gen cephalosporin). Might add
tetracycline or azithromycin to cover concomitant Chlamydia
trachomatis infection.
 As mentioned, newborns should get silver nitrate (covers N.
gonorrhea) or erythromycin (covers N. g. and also Chlamydia) drops.
Chlamydia infections require systemic antibiotics.
7. Define the major characteristics of spirochetes? How are they different from other
bacteria?
 Gram-negative but lack LPS; spiral-shaped and motile. Can't generally
be seen on light microscopy because they're so thin.
 Can't be well cultured in vitro (T. pallidum will consent to survive
about 3-6 days under very particular conditions). Can barely grow
some species in tissue culture cells (specifically rabbit testis cells, if
you were wondering).
 Course goes through episodes of active disease punctuated by long
periods of latency.
 Note that spirochetes have no identified virulence factors (hard to
culture them to find out) and thus no vaccines.

There are four clinically important species of Treponema pallidum:
 T. pallidum pallidum: syphilis.
 T. pallidum pertenue: yaws (see below).
 T. pallidum endemicum: bejel (see below).
 T. pallidum carateum: pinta (see below).
8. What are the various stages of syphilis? Describe the major aspects of the
pathogenesis and clinical manifestations of each stage?
 How syphilis progresses:
 Infection occurs. No, not from a toilet seat.
 There's an incubation period of 3 weeks during which the virus
is multiplying at the local site of infection.
 You get primary syphilis (chancre) for 2-6 weeks (localized to
the site of entry.. so to speak). Then it resolves.
 About 25% of people spontaneously heal at this point; the
other 75% go on.
 After this you have an asymptomatic but bacteremic phase for
2-24 weeks (organisms get into blood vessels and spread
hematogenously to other tissues).
 Secondary stage symptoms (eg. rash) come up for 2-6 weeks.
 Then those go away and you go into latent syphilis for
anywhere from 3-30 years. The organisms are present, but
asymptomatic.
 Eventually you show up with tertiary symptoms.
 Primary stage: painless chancre. An ulceration, often on the genitals
but sometimes on the skin, tongue, or anus.
 Note the distinction between a chancre (syphilitic) and a
chancroid (looks like chancre but ain't, caused by Hemophilus
ducreyi)-- the latter is minimally invasive and is painful,
whereas the chancres of syphilis are painless. See 1/23 small
groups for more on chancres, chancroids, and
treatment/diagnosis.
 Secondary stage: acne-like vesicular. Classically, the rash is on the
palms and soles of the feet and its fluid is quite infectious. Also see
lymphadenopathy.
 Tertiary stage: eye, skin, nerve, cardiac, and fertility involvement.
Note that tertiary symptoms usually only come up after many years of
latent infection.
 Cardiac involvement: aortic arch aneurysm and root dilatation.
[eg. "an immigrant from a former Soviet republic with an aortic
regurg murmur;" caused by obliteration of the vasa vasorum
vessels feeding the wall of the aorta]
 CNS infection: tabes dorsalis (posterior column disease), plus
you go nuts.
 Skin/mucosal epithelia: "gummas:" localized granulomatous
lesions.
 Fertility: can cause stillbirth in syphilitic women.
 Syphilis is transmissible in the primary and secondary stages only. The
tertiary stage is largely due to type 4 hypersensitivity reactions in the
host, and where it's not, it's usually the spirochetes invading the
central nervous system, which doesn't get a lot of exposure. Infection
occurs through contact with the chancres or rash.

Note that it is a fatal disease; about 30% of tertiary syphilis patients
die of it. The rate is higher and more immediate in children born with
syphilis.
9. How is syphilis diagnosed? What are the best tests for each stage of the disease?
Why are serological test not very suitable for the diagnosis of the primary stage of
syphilis? How infectious are the various stages of syphilis?
 If it's available, take a sample from the chancre; can't see Treponema
with normal light microscopy (too thin) but can be diagnosed with
darkfield microscopy or immunofluorescence.
 In general, serology is the best way to diagnose:
 Rapid (non-treponemal) test (aka RPR or VDRL): you're looking
for antibodies against certain lipids that are released secondary
to spirochete invasion. Note you don't actually look for
antibodies against treponemal antigens.
 Fluorescent test (aka FTA): apply patient's serum to killed
treponema, look for antibodies.
 What tests are good for which stages:
 Darkfield microscopy is good for primary syphilis (person may
not have antibodies yet and thus serology is unhelpful) but not
as good as the others for the secondary and tertiary stages.
You can also use direct, non-host-derived fluorescent antibodies
to pick up primary syphilis.
 Rapid test is great for secondary stage, but not so good for
primary stage or tertiary stage; fluorescent test is good for
both secondary and tertiary stages. Neither is good for
detection of primary syphilis because the body hasn't had a
chance to make antibodies (though our small groups seem to
say that if you've got a patient with a painless chancre and a
negative serological test, wait a week and test them again).
10. What is the epidemiology of the various diseases caused by Treponema sp.?
 T. pallidum: causes syphilis. Worldwide distribution, can affect the
entire age range of sexually active adults plus infants through vertical
transmission.
 Note that infantile syphilis is often fatal.
 The incidence of syphilis is, as of recently, higher in men that
women (particularly in gay men).
 T. pertenue: causes yaws; yaws can look like syphilis but isn't an
STD. Can cause facial disfigurement. Mainly in the tropics; not sexually
transmitted; affects all ages.
 T. endemicum: causes bejel; also can look like syphilis but isn't an
STD. Mainly in desert regions; not sexually transmitted; affects all
ages.
 T. carateum: causes pinta: hyperpigmented (then later, depigmented)
lesions on the skin. Affects mainly darker-skinned people. Mainly in the
tropics.
 [Immune response to syphilis:]
 Humoral response is diagnostic, but not particularly protective.
 Cell-mediated response targets the organism but also causes damage
to the host's tissues (mainly in tertiary syph).
11. How is syphilis treated? Has antibiotic resistance developed in Treponema
pallidum as it has in other bacteria? How can syphilis be prevented? What is the
behavior that is most associated with small epidemics of syphilis?

Nearly all species of syphilis can be treated with penicillin; if there's a
penicillin allergy, you can also use tetracyclines.
 See some penicillin resistance, but able to be overcome. See growing
macrolide resistance, particularly in the strains more common in the
gay community.
 Jarisch-Herxheimer reaction: 2-24 hours after penicillin treatment;
fever, chills, headache, nausea, etc. May be due to lots of spirochete
lysis at once.
12. Describe the pathogenesis and clinical manifestations of leptospirosis?
 [Not discussed in class]
 Caused by Leptospira interrogans (evidently so named because the
bacterium looks like a question mark).
 Causes bacteremia and fever; mainly targets the liver and kidney and
causes tissue damage (jaundice, hemorrhage, nitrogen retention). Can
also cause meningitis after IgM titers rise.
13. What is the epidemiology of leptospirosis?
 [Not discussed in class]
 Generally from ingestion of contaminated water/food (kidney infection
sheds the bacterium into the urine). Incubation period is 1-2 weeks.
 Leptospirosis is zoonotic.
14. What are the major characteristics that differentiate the Chlamydia from other
bacteria?
 Chlamydia is an obligate intracellular bacterium. It has no way to
generate its own ATP.
 As you might expect, they're tiny-- ~0.3 micrometers in diameter,
about the size of the larger viruses. They have a tiny genome (1 Mb or
less).
 Their cell walls have no muramic acid and are hence resistant to
lysozymes.
15. Describe the life cycle of Chlamydia. How does this unusual life cycle affect
treatment of chlamydial infections?
 2 stages of Chlamydia: the metabolically inert but infectious particle
(elementary body) and the mature, metabolically active form inside
cells (reticulate body).
 The elementary body is taken up by a cell's endosome (EBs use type
III secretions to prompt their own uptake).
 The EB changes to a reticulate body and, since Chlamydia is
nonfusogenic, it prevents the fusion of the phagosome/endosome with
the lysosome. Once this is done, it multiplies inside the endosome.
 Eventually the host cell lyses and releases a bunch more elementary
bodies. Sort of like viruses, actually.
 Since it largely lives inside the endosome inside cells, you need to
target antibiotics to that location.
16. Describe the major diseases caused by the different species of Chlamydia.
 Chlamydia trachomatis: causes trachoma, inclusion conjunctivitis,
urethritis, and lymphogranuloma venereum.
 Trachoma: chronic conjunctivitis; scars the conjunctiva,
eventually leading to corneal scarring (due to abrasion) and
blindness. Leading cause of preventable blindness worldwide.
 Inclusion conjunctivitis: acute conjunctivitis, most common in
neonates directly after birth.
 Recall that inclusion conjunctivitis in infants has to be
treated systemically, unlike gonorrheal conjunctivitis.

Urethritis: similar to gonorrhea; symptoms are often less
severe. Much more frequently asymptomatic.
 Lymphogranuloma venereum: Chlamydial infection into the
lymph nodes. One of the few chlamydial infections that can
disseminate (infects peritoneum, intestine).
 Chlamydia psitacci: causes "psittacosis" (acute pulmonary infection).
Zoonotic (from birds).
 Chlamydia pneumoniae: causes, no kidding, mild pneumonia (another
atypical pneumonia, like Mycoplasma pneumoniae). Quite common
(moreso than trachomatis, actually).
17. What are the potential complications of sexually transmitted chlamydial
infections for women?
 Pelvic inflammatory disease, as mentioned above under gonorrhea.
 High incidence of scarring off of the fallopian tubes (causes
infertility).
 Infected women have a 3-5-fold increased risk for contracting HIV.
 [for boards: post-chlamydia you see a high incidence of Reiter's
syndrome.]
20. Describe the epidemiological characteristics of the different chlamydial infections.
 (LOs #18 and 19 were duplicates of 17)
 trachomatis:
 It's the most common STD.
 It doesn't live long outside the host-- generally need direct
person-to-person contact.
 It is a reportable disease (have to report it).
 Higher incidence in the southern US.
 Unlike gonorrhea, the incidence of Chlamydia in women is much
higher than the incidence in men, at least with current
numbers.
 Note the overall incidence of Chlamydia in both genders
is on the rise.
 75% of women and 50% of men with chlamydial urogenital
infections are asymptomatic.
 psitacci and pneumoniae: as above (zoonotic and aerosol-transmitted
respectively).
21. What diagnostic tests are used to identify Chlamydia sp.?
 Giemsa stain, immunofluorescence (look inside other cells). Gram
stain is not used.
 Hard to culture (have to grow inside other cells), but culture works
well. Most tests (rapid tests, etc) are very specific but not very
sensitive; serology is sensitive but not specific. In general, it's tough
to pin down a Chlamydia diagnosis. His notes suggest nucleotide
amplification testing as the test of choice.
 Upon pus testing, you won't see the Chlamydia inside PMNs as you will
with gonorrhea.
 In infants, you look for basophilic inclusion bodies in the conjunctiva
(congenital trachoma in infants is also caused "inclusion
conjunctivitis").
22. Which chlamydial infection is predominant in developing countries? Why? What
are the consequences of the infection? What preventive measures are being taken to
decrease either the incidence of disease or the complications arising from chronic
infection?


Trachoma has a very high prevalence in developing countries, for
whatever reason. It leads to permanent blindness.
Prevention: what you'd think (safe sex promotion).
Papillomaviruses, warts, and cervical cancer
Tuesday, January 20, 2009
9:59 AM
Papillomaviruses, Warts, and Cervical Cancer, 1/20/09:
[Tumor viruses: HPV, hep B/C, EBV, HHV-8 (Kaposi's-associated).]
1. Define unique features of HPV structure and life cycle.
 Double-stranded, nonenveloped DNA virus.
 Pretty tiny (50 nm). Infects only epithelial cells; associated with warts,
papillomas, and carcinomas.
 Responsible for 100% of cervical cancers; also causes 25% of head
and neck cancers in men (generally squamous cell cancers of the
mouth and throat).
 The viral particle is composed of only two proteins, L1 (major) and L2
(minor).
 L1 is most of the capsid; L2 connects L1-composed structures.
 Note L1 is the basis for all currently-existing vaccines.
 There's one copy of the viral genome inside the capsule; it's a very
small genome, with maybe 8-10 genes. Like most viruses, it expresses
replication, transcription, and transformation genes early in the
infection, and capsid proteins later on.
 HPV expresses oncogenes (in that early phase) mainly with E6 and
E7 proteins. E7 also mediates escape from host immune defenses.
 HPV infects only epithelia. Specifically, HPV infects only
undifferentiated basal proliferating cells in the epithelium. Makes
sense; the oncogenes aren't going to have much effect on fully
differentiated and nondividing cells.
 Note that although the viral particles infect dividing cells, they're
released from the dead differentiated squamous cells that are at the
surface (virus matures and is packaged only as the epithelial cells are
rising toward the surface).
 The majority of people in the US are infected at one point or another;
however, most infections are cleared within a year.
 About 9.2 million young, sexually active people are infected.
 Cervical cancer is the second leading cause of female cancer-related
deaths.
 It's a sexually-transmitted disease (but note it can infect skin and
mouth).
 However, it can also be transmitted, rarely, from mother to
infant and from fomites.
 Note condom use is not fully protective.
 Risk factors: intercourse before 20 (less safe sex), multiple partners,
smoking, long history of oral contraceptives. Regular Pap smears are
important.
2. Identify high and low risk HPVs and their associated diseases.

There are lots of different genotypes of HPV (200+). The persistance
of high-risk types HPV types is necessary for cancer development.
 High risk mucus-infecting: types 16, 18, 31, 33, 45, 56.
 Note types 6 and 11 cause genital warts (not cervical
cancer, but included in the Gardasil vaccine) while types
16 and 18 cause cervical cancer.
 Intermediate/low risk mucus-infecting: lots of others.
 There are also types that infect the skin (cutanotropic).
 Associated diseases:
 Common warts: small, rough tumor, usually on hands and feet.
(2, 7)
 Plantar warts: on sole or toes. (1,2, 4)
 Condyloma accumulata: genital warts. Highly contagious. (6,
11)
 Respiratory warts. (6, 11)
 Epidermodysplasia verruciformis: genetic disorder with
massively increased susceptibility to HPV (pretty dramatic).
 What you're mainly worried about is cervical squamous cancer. HPV
16 and/or 18 are found in about 70% of all cervical cancers.
 As mentioned in Life Cycle, 99% of cervical cancers arise inside the
transitional zone between the columnar and the squamous regions of
the cervix.
 Head and neck cancer: generally involved with the squamous cells of
the mouth and throat, particularly the oropharynx. Common (4th most
common cancer worldwide) and dangerous (less than 50% survival
rate). Definitely don't think it's just warts and cervical cancer (eg.
most frequent cause of rectoanal carcinoma in HIV+ men).
3. Describe the HPV oncoproteins and their important cellular targets.
 Oncoproteins: E6 and E7.
 Inhibit p53 and Rb, respectively; when E6 and E7 are blocked,
the cancer regresses.
 E6 phosphorylates p53 and targets it for degradation, while E7
deactivates Rb. Both of these allow the cell cycle to progress.
 Over a long course of infection, the viral chromosome integrates with
the host genome in such a way that the normal block of E6 and E7
production (E2) is disabled-- you get lots of E6/E7, leading to
increased oncogene effect in host cells.
 One reason high-risk strains are high risk is that their E6 and E7
proteins are particularly good at their jobs (bind Rb and p52 tightly).
4. Explain how HPV-associated lesions could be diagnosed.
 (1) Pap smear from cervical/anal scraping: uses cytological analysis.
About 70% sensitivity.
 (2) HPV test: uses PCR. About 70% sensitivity.
 Note that using Pap and HPV tests together has a pretty good
overall sensitivity.
 (3) Colposcopy (follows positive Pap): microscopic examination of the
cervical surface; generally also take biopsies.
5. Explain how the current HPV vaccines are made and what their limitations are.
 [Note that generally surgery is used as therapy: either freeze lesions
or burn them off.]
 The Gardasil vaccine targets L1 proteins found in types 6, 11, 16, and
18 (6 and 11 are for warts, 16 and 18 are for cervical cancer).


Essentially you grow L1, and L1 only, in yeast, then administer that as
a vaccine.
Problems: no therapeutic use (virus doesn't live outside cells, so
making antibodies against it doesn't do much good if it's already
there), doesn't cover the other high-risk groups (causing ~30% of
cervical cancers), expensive ($300).
HIV
Tuesday, January 20, 2009
11:01 AM
HIV, 1/20/09:
[He switched around the LOs. The following are the 'real' ones, from his slides; they
don't always match up well with what he lectured on, so sometimes the material
doesn't quite fit the LO.]
[I refer you to the notes on HIV from B+L ("Immunology of AIDS," 2/22/08) for
additional info. Also note that CMBMRS and First Aid have a lot more molecular detail
on HIV than this; some of it's at the end of these notes.]
1. Understand the origins of the AIDS endemic.
 HIV-1 epidemic began as a zoonotic infection from chimpanzees,
possibly in the 1900-1925 period, somewhere in southeast Cameroon.
 1981: CDC first describes AIDS; HIV is isolated.
 Currently about 33 million adults are infected with HIV worldwide. For
a while there it was the leading cause of death in our age group,
though now with combined therapy it's dropped way down.
 (HIV-2 is a distinct, much more regional virus acquired from a distinct
species of primate.)
2. Be familiar with the clinical manifestations of acute and chronic HIV-1 infection.
 Acute: signs/symptoms of primary HIV infection:
 Often looks like a febrile illness (fever, fatigue, headaches,
muscle aches, lymphadenopathy, rash); can also resemble
mononucleosis or aseptic meningitis. Can get mucosal
ulceration of mucosal tissues as well. You get some kind of
symptoms in about 50% of patients but the rest are
asymptomatic.
 Rash is erythematous and can be either macular (flat) or
papular (raised).
 Usually occurs 2-3 weeks after exposure. High levels of
circulating virus in the blood and lymphatic systems.
 Chronic: no symptoms during latent period, though CD4 cell levels (T
helper cells) are constantly declining. Once the AIDS stage is reached,
various infections tend to occur at specific CD4 counts:
 [CD4 count > 1000 is normal.]
 < 300: more superficial infections: Candida infections (thrush),
shingles, etc.
 < 200: more serious infections: pneumocystis pneumonia
(PCP), toxoplasmosis, cryptococcus meningitis, etc.
Note that AIDS is defined by a CD4 count < 200 (plus
evidence of HIV) or certain characteristic "AIDS
infections" (eg. PCP).
 < 100: bad mojo: CMV retinitis, mycobacteria intracellulare, etc
3. Understand how HIV-1 infection leads to AIDS.
 HIV-1 attaches to CD4 proteins (and co-receptor surface proteins,
see below) on T-helper cells in order to gain entry into them.
 Once inside, the viral genome is reverse-transcribed into cDNA by
reverse transcriptase and integrated into the host genome, usually in
transcriptionally-active areas.
 The virus can then make structural proteins that assemble at the cell
membrane and bud off as virions; the virions only fully mature after
they have left the cell (without maturation, the virions aren't
infectious, a point that's important for drug targeting). The entire
replication cycle from entry to release is completed in 1.5-2 days.
 Note that infected T cells seem to be able to fuse with noninfected T cells, creating multinucleated giant cells.
 The virus is effectively cleared from the bloodstream by antibodymediated mechanisms; however, there's usually enough time for some
to get into T helper cells (which, recall, are the main cell type with CD4
receptors). Since HIV is an extremely prolific virus and there's lots of
replication going on - about 10^10 virions produced per day, each of
which can in turn infect a T cell - there's lots of T cell death.
 Generally the T cells infected are active; these T cells generally
die after 1 cycle of viral replication.
 If the T cells infected are resting or not actively transcribing
(eg. memory cells), the HIV virus can stay in the genome of the
T cell indefinitely, in a latent stage.
 When these T cells become activated again, the virus can
emerge. [You may recall from B+L that expression of HIV
genes seems to be promoted by the same things that promote
IL-2, which is secreted by Th1 cells when they're activated.]
 CD4 lymphocyte count: good clinical tool to figure out how much
damage has been done to the immune system. CD4 counts correlate
with disease progression. Note that most HIV replication is going on in
the lymphatic system, not the blood, but blood CD4 counts are a
pretty good estimate anyway.
 Can also measure the plasma HIV RNA levels (drops to undetectable
levels with successful treatment). This seems to indicate the rate of
progression (whereas the CD4 level indicates the current stage of the
disease).
 Virus load increases rapidly with initial infection; the host response
generally resolves the acute infection into chronic infection (generally
no symptoms), with low viral loads.
 Without treatment, the CD4 count declines over time, which affects
both the patient's immunity to other disease and the ability to fight the
HIV infection itself-- thus the viral load begins to rise again. At this
point symptoms of immunodeficiency start to crop up and, usually,
treatment is sought and diagnosis is made.
 On average, it takes about 10 years to get from infection to AIDS. The
patient is fully infectious during all of that time.
 Evolution of HIV: HIV evolves rapidly due to a 'sloppy' reverse
transcriptase enzyme-- no proofreading mechanism, thus lots of

wiggle room in what actually gets reverse-transcribed into the
genome. 1/10,000 mutation rate in nucleotides. Fast replication rate
and selective pressures from treatment or immune response also drive
fast evolution.
 A variety of genetically distinct viral variants ("quasispecies") infect
any given individual after a while.
 This is one reason why antibody response tends to be
ineffectual.
 This is also the reason why you want to absolutely stop all viral
replication at a given point in time with treatment-- if you only
partially suppress it, you allow new resistant strains to come up. That's
why you give at least 3 anti-retrovirals at the same time-- a
quasispecies isn't (we hope) going to simultaneously develop
resistance to all 3 at once, but resistance to all 3 could, in principle,
develop if they were given in series.
4. Be familiar with the mechanism of actions of the 5 major classes of antiretroviral
drugs and how these drugs prevent and/or reverse the clinical manifestations of HIV1.
 Classes of drugs used:
 (1) Entry inhibitors: prevent entry of virus into the cell.
 What the virus needs to get into the cell: CD4 (main
receptor), CCR5 (co-receptor with CD4), or CXCR4
(another co-receptor).
 gp41 binds to CD4; gp120 binds to the co-receptor.
 Most HIV uses CCR5 (viruses only attach to a specific
co-receptor).
 Note that people homozygous for a deletion in
the CCR5 receptor gene are immune to most HIV
infection (CCR5 isn't expressed, virus can't
enter).
 Heterozygosity seems to provide some
protection.
 Heterozygosity is pretty common among white
people (10%) but is irritatingly rare in Africa.
 So entry inhibitor drugs can bind CCR5 or prevent fusion
to prevent infection.
 Reverse transcriptase inhibitors: prevent, no kidding, reverse
transcription.
 (2) Nucleoside inhibitors: these have to be activated
once inside the cell; they're analogs of naturallyoccurring nucleosides, but they need to be
phosphorylated to nucleotides to be incorporated into
viral cDNA (once they're incorporated, the cDNA stops
being synthesized).
 (3) Non-nucleoside inhibitors: non-competitive
inhibition: irreversibly binds reverse transcriptase.
 (4) Integrase inhibitors:
 Prevent integration of viral cDNA into human DNA.
 (5) Protease inhibitors:
 Inhibit maturation step in released virus. Competitive
inhibitors of HIV protease; the newly-made virions don't
mature and can't infect other cells. Low bioavailability
(large molecules).
[A few molecular notes from CMBMRS:]
 HIV: single-stranded linear RNA genome, with an envelope. It has only
a few genes that we really care about: gag, env, and pol are structural
and functional, while tat, rev, and nef are regulatory.
 Major capsid protein: p24 (derived from gag gene).
 Surface glycoproteins for attachment: gp41, gp120 (derived from env
gene).
 Reverse transcriptase and integrase (RT makes cDNA, integrase sticks
the cDNA into the host genome): derived from pol gene.
 The entire viral genome has long terminal repeats on each end to
facilitate its insertion into the host genome (it looks like a transposon).
 tat is the transcription activator for the genome.
 rev promotes the production of env proteins.
[You may be wondering how the virus gets into the brain (helper T cells aren't
generally allowed in). Evidently the virus also infects monocytes but doesn't destroy
them; they carry HIV into the brain, where AIDS dementia eventually results.]
[Common malignancies that crop up without T cells: B-cell lymphomas secondary to
EBV infection; Kaposi's sarcoma secondary to herpes virus HHV-8 infection.]
[Recall that B cells don't work so well without T-helper cells around; HIV seems to
cause them to dysfunction further (get hypergammaglobulinemia but can't produce
new antibodies very well).]
[Note oral hairy leukoplakia (caused by EBV) can be distinguished from Candida by
its inability to be rubbed off the tongue with a depressor. OHL is also mainly found
on the sides of the tongue and on the buccal mucosa.]
[Finally, note that HIV is non-transforming-- it expresses no oncogenes, unlike
certain other retroviruses (eg. Rous sarcoma virus).]
Herpesviruses: Diseases I
Wednesday, January 21, 2009
7:28 AM
Herpesviruses: Diseases I, 1/21/09:
[Note that the last two posted LOs for this lecture were taken off but a bunch more
material was covered by Dr. Levin at the end-- we don't, last I checked, have LOs for
this latter material, but I presume it's still fair game for the exam.]
1. Define the shared characteristic properties of all herpesviruses.
 Double-stranded DNA genome within an icosahedral capsid within an
envelope.
 Tegument: a bunch of viral and cellular proteins and viral/cellular
RNA that lies in the space outside the viral capsid but inside the
envelope.
 The glycoproteins coming off the envelope determines the sites the
virus can bind to.
 These are really big viruses-- 2nd biggest known after poxviruses.
Have from 70-200 genes. But note that they're fragile-- envelopes are
sensitive to detergents, heat, etc, and can't live outside the host for
long.
2. Describe the general lytic replication cycle of herpesviruses.




DNA of genome serves as the direct template for transcription.
Herpesviruses tend to induce cytopathic changes in infected cells
(syncytia, cytomegaly, inclusions, etc). In general the cell is eventually
lysed (but see next point).
They also are generally latent: that is, they use certain types of cells
to reside in but not actively replicate in or lyse, though a small subset
of genes are expressed.
Lytic cycle:
 Attachment/Entry:
 The glycoproteins stuck to the outside of the envelope
serve two functions: one, to nonspecifically stick to stuff
to slow the virion down (like neutrophil 'rolling'), and
two, to specifically bind to certain receptors on specific
cell surfaces to induce conformational changes that
cause a fusion of the viral envelope with the cell
membrane (emptying the contents of the envelope into
the cell).
 This releases both the capsid and the tegument into the
cell.
 The tegument contains fully active proteins which allow
the virus to immediately overwhelm any cellular
defenses and get set for immediate replication.
 Uncoating/Gene expression/Replication:
 Capsid goes to the nucleus and 'docks' at a nuclear pore.
 The viral genome is unspooled out into the nucleus
itself.
 The genome circularizes in the nucleus (it's linear in the
virion); transcription begins, using host RNA polymerase
II.
 Immediate early genes: expressed almost
immediately; not dependent on any other protein
expression. Code proteins important for the regulation of
viral and host gene expression and immune evasion.
 Early genes: expressed next; generally entail
polymerases, accessory factors, and other proteins
necessary to replicate viral DNA.
 The viral genome is replicating itself as it goes
here; evidently there are a lot of replicated
copies of its genome all bound together into a big
long double-stranded string, called concatameric
DNA.
 Capsid assembly/Envelopment/Egress:
 Late genes: expressed last and from concatameric DNA;
encode mainly the structural factors of the capsid and
the machinery that assembles them into mature virions.
 The concatameric DNA is stuffed into the capsids
thus produced.
 Envelopment: Capsids bud through the nuclear
membrane, then through the ER (where they acquire
tegument proteins to surround them), then through the
Golgi, where they pick up two more membrane layers;
they keeps the first and use the second to merge with
the cell membrane to be released.

Latency:
 Latency: maintenance of viral genomes in a cellular
reservoir in the absence of the production of infectious
viral particles.
 Has to be able to be stimulated to produce virions again
(reactivation) in response to some kind of stimuli about
the host.
3. Explain the basis for the division of the herpesviruses into three families.
 Alpha, beta, and gamma: based on both biological and molecular
properties.
 Alpha herpesviruses:
 Include herpes simplex viruses (HSV-1 and HSV-2) and
varicella zoster (VZV).
 Grow rapidly (12-24 hours), lyse their host cells, cause mainly
acute illness.
 Establish latent infections in the peripheral nervous system and
sensory nerve ganglia.
 Beta herpesviruses:
 Include CMV, HHV6, and HHV7.
 Grow very slowly (80-120 hours)
 Infect a wide variety of cell types.
 Not only lyse cells but also cause chronic low-level infections.
 Establish latency in myeloid and epithelial cells.
 Gamma herpesviruses:
 Include EBV and Kaposi's-associated herpesvirus (HHV8).
 Replicate in lymphoid cells.
 EBV establishes latency in B cells (can transform these into
malignant cells).
[Dr. Levin's notes on alpha and some beta herpesviruses:]
 Herpes simplex viruses:
 Clinical hallmark of all herpes simplex viruses are skin
vesicles.
 HSV invades through breaks in the epithelia; it lyses cells,
which cause inflammatory markers to be released that cause
fluid release from nearby capillaries (creating the vesicle); the
resultant white cell invasion causes the vesicle to develop into a
pustule, which eventually scars over.
 Latency: HSVs get into the peripheral nerves (which terminate
in the dermal-epidermal junction where HSV likes to live) and
ascend up those nerves to the sensory ganglia.
 On histology, HSV forms synctial giant cells in the dermis.
 HSV type 1: "above the belt," causes mostly oral disease.
 First infection generally causes vesicle outbreak on the
lips and face.
 Mostly it's so minor as to be unapparent; in about 20%
of infections you get systemic symptoms.
 Some kids show up with herpetic gingivostomatitis
(severe inflammation of gums, lips, etc), which resolves
well if treated promptly (use antivirals, eg. acyclovir). In
sexually active young adults it can also cause severe
pharyngitis.
Reactivation of latent infections usually come back down
the same nerve it came in through-- generally much
smaller effects (cold sores) in a well-localized area. The
symptoms are lessened due to preexisting immunity.
 First symptoms of reactivations are tingling (virus
traveling down the sensory nerve). Almost never
cause systemic symptoms.
 Don't bother giving antivirals-- they're for
primary HSV infection, not reactivation (it's too
brief and ineffectual).
 Note that even if you're latent, you're still
shedding virus with some frequency (and thus
contagious).
 Complications:
 If the original infection was in the eye, repeated
bouts of reactivation can cause corneal scarring
and blindness.
 Occasionally it can get into the brain, by traveling
retrograde back up the trigeminal nerve, and
cause herpes encephalitis (very rare but
serious/fatal). You see focal neural deficits and
fever/headache. CSF analysis containing
mononuclear cells and herpes DNA confirms it;
treat with antivirals.
 HSV type 2: "below the belt," causes mostly genital disease.
 Vesicles on the genitals.
 First episode of HSV-2 is pretty similar to first episode of
HSV-1 (albeit on the genitals); mostly unnoticed, but if
it's more involved it can be treated with antivirals.
 Complications: urethritis, meningitis, yeast infections.
 Viral shedding is fairly constant, as with HSV-1-- the
absence of lesions does not mean you're not infectious.
 Reactivation of latency, again, is generally short,
minimally symptomatic, and not treated with antivirals
(although you can use antivirals prophylactically in HSV2-infected mothers).
 Spread by close contact (the virus is fragile).
 In immunocompromised hosts, you're going to have a problem
with herpes simplex viruses; get severe local disease.
Newborns, in particular, pick up herpes from vertical
transmission and can easily die from herpes simplex viruses if
their immune systems don't work.
 Diagnosis: PCR is the gold standard. Can use cytology or
cultures as well.
Varicella zoster virus:
 Transmitted by respiratory droplets; establishes itself in the
back of the throat and infects T cells.
 Varicella (chicken pox): the T cells go to the dermal-epidermal
junction of the skin (same place the herpes simplex virus
goes); after about 10-12 days of incubation, the vesicles begin
to erupt; the virus travels back up the peripheral nerves, as in
HSV.




Clinically: "dew drops on a rose petal" vesicles (wow. give me
the food metaphors again!)
 Primary complication is bacterial superinfections, often with
Staph and Strep. These can be superficial (eg. cellulitis) or
deeper (eg. necrotizing fasciitis).
 Adults who get VSV for the first time can get very sick (can
progress to pneumonia). Note that we have a vaccine against
VZV now-- so if you've never gotten it, go get vaccinated (as I
recall, we all had to do this prior to starting med school).
 Reactivation: causes shingles (zoster): a painful vesicular
eruption strictly limited to one dermatome (the virus has
reactivated and traveled down that sensory nerve). Note that
VSV is present in many sensory ganglia (as opposed to HSV,
which is generally limited to one or two); therefore it can show
up in a lot of different dermatomes at various times.
 The problem with shingles, other than the immediate
painful rash, is that nearby nerve and nerve connections
are damaged by the travel of the virus down the sensory
nerve, causing lasting and prolonged pain in that area-that is, postherpetic neuralgia. It can last weeks to
years.
 Reactivation is a particularly bad problem in
immunosuppression, or just advancing age (decreased
cell-mediated immunity).
 The VZV vaccine also works against the reactivation process
(boosts T-cell immune response); works pretty well and is
indicated in older patients.
Cytomegalovirus:
 A beta herpesvirus-- long replication cycle, and thus tends
towards chronic infection.
 Infects hematopoietic cells. Most common resultant infection is
a mononucleosis-like syndrome (see below and in next lecture
under EBV).
 Maybe 50-60% of adults have had CMV (pretty prevalent).
[CMBMRS says it's closer to 80%.]
 Perinatal infections: acquired from maternal shedding in birth
canal, milk, etc. Generally results in a mild illness; the maternal
antibodies are still protective. If the mother has no antibodies,
that's a bigger problem and can result in hearing loss and
microencephaly.
 Toddler infections: generally a mild illness (virus acquired from
playmates). Occasionally it can be more severe (jaundice,
thrombocytopenia, etc).
 Adolescent infections: CMV is shed in saliva/genital secretions,
so sexual transmission is common.
 Here's where you start getting characteristic syndromes:
looks like mononucleosis (but caused by CMV, not EBV-more on this in the next lecture).
 Diagnosis: look for IgM anti-CMV antibody (acute infection).
Can also use spin amplification and PCR.
 Post-transfusion CMV infection:
 CMV is often latent in blood lymphocytes.
This can cause a febrile illness 3-6 weeks after
transfusion, particularly in individuals who haven't
gotten CMV.
Immunocompromised individuals have a lot of trouble with
CMV:
 CMV is one of the big opportunistic viral infections.
 Lung, liver, GI tract, and retina involvement (classically,
CMV causes retinitis in AIDS patients).
 Post-transplant patients can have trouble with this as
well (classically, causes pneumonia or "transplant
lung").
Treat CMV with ganciclovir, not acyclovir.



[Note a few trends:]
 (1) the older you are when you first get exposed to a herpesvirus, the
more severe the infection tends to be;
 (2) the reactivation symptoms are generally lighter than the original
infection due to pre-existing immunity (possible exception of shingles);
and
 (3) alpha herpesviruses are all susceptible to acyclovir.
Herpesviruses: Diseases II
Wednesday, January 21, 2009
7:30 AM
Herpesviruses: Diseases II, 1/21/09: (beta and gamma)
[General notes:]
 Alphas' latency is in nerve cells; betas'/gammas' are in myeloid and
lymphoid cells respectively.
 Herpesviruses have a bunch of 'fine-tuning' genes to let them infect us
much more efficiently.
 2 other beta herpesviruses (other than CMV, covered last lecture):
HHV-6 and HHV-7. Very little is known about them (recent discovery in
'86)-- they are extremely common and associated with roseola
(subclinical rash of infancy). Essentially the only clinical consequences
are reactivation effects in immunocompromised individuals.
 Gamma herpesviruses: EBV, Kaposi's (HHV8).
 Infect mainly lymphoid cells, nearly all B cells, dendritic cells,
and macrophages. Associated with lymphoid malignancies
(Burkitt's lymphoma with EBV, Kaposi's sarcoma in HIV with
HHV-8).
 Epstein-Barr virus is nearly ubiquitous (infects 90-95% of
population).
 EBV binds to complement- and MHC class II-receptors to get entry to
its host cell-- this means it only lives in professional antigenpresenting cells.



Note that EBV immortalizes B cells-- this allows them to be latent in B
cells (which would otherwise turn over). This can be handy for lab
work.
Treatment of EBV: limited role of antivirals; mononucleosis is selflimiting.
HHV-8: much less prevalent than EBV; causes Kaposi's sarcoma.
1. List the distinguishing features of lytic and latent infection by herpesviruses.
 Lytic infection: all the viral genes are expressed. Lots of new virus
production. Also, y'know, there's lysis. In lytic infection you see lots of
linear DNA (concatameric-- it'll be stuffed into capsids to make
virions).
 Latent infection: limited viral gene transcription (the minimum
necessary to keep it viable for re-emergence). No new virus production
and no lysis. In latent infection you see no linear DNA (circular only).
 Important latent protein: Epstein-Barr nuclear antigen 1. It
tethers the viral chromosome to the host chromosome, to
mimic host genetic material; it's thus replicated by host
enzymes and divides equally just like the chromosomes of the
host cell. It's the only viral protein that's absolutely required for
latency.
2. Describe the contributions of virus infection and host response to mononucleosis.
 The point she's getting at is that in mononucleosis, only a minority of
the immune-cell proliferation is actually made up of infected cells; the
rest is a normal response directed against those infected cells.
3. Compare and contrast EBV and CMV mononucleosis.
 EBV-caused mononucleosis: Inflamed, purulent tonsils, swollen lymph
nodes, and a rash. Causes fever and fatigue (the latter is usually the
major complaint).
 Causes a massive proliferation of B cells (and T cells to control
them).
 See atypical T cells ("Downey cells").
 Self-resolving.
 How to distinguish EBV from CMV infections (important for boards):
 Symptoms are generally similar. However, patients with EBV
mono usually test positive for heterophil antibodies-- get
strong antibody response to sheep red blood cells (the
Monospot test).
 CMV mono isn't positive for heterophil antibodies.
 EBV: transmitted mainly in saliva (mono used to be called "the kissing
disease"); shed for life.
4. Describe the replicative and latent infection of gammaherpesviruses, and the
points at which symptoms and/or diseases occur.
 Not sure what she's getting at here. As mentioned, the viruses go
through replicative (ie. lytic), then latent stages. The main acute
(replicative) disease caused by a gammaherpesvirus is mononucleosis
(by EBV, usually in adolescents or young adults infected for the first
time). The latent diseases include Burkitt's lymphoma (EBV), Kaposi's
(HHV-8), oral hairy leukoplakia (EBV), and a host of other diseases
associated with immunosuppression.
5. List diseases associated with these viruses in immunocompromised hosts.

EBV: mainly, Burkitt's lymphoma and infectious mononucleosis; also
Hodgkin's lymphoma (Reed-Sternberg cells, etc) and oral hairy
leukoplakia. There's a complete list on p. 135.
 Burkitt's: associated with c-myc oncogene (8:14 chromosomal
translocation).
 Note Reed-Sternberg cells are also present in mononucleosis.
 HHV-8: Kaposi's sarcoma.
6. Describe the major gammaherpesvirus associated malignancies and their
distinguishing hallmarks.
 EBV: normally the immune system keeps the proliferation of EBVinfected B cells under control; without a working immune system,
lymphomas result. Note that you generally get Burkitt's not from
reactivated EBV infections but from the slow cranking of latent
infections.
 Kaposi's: classic for AIDS diagnosis; mixed-tissue, highly vascularized
tumor, usually appearing first on the skin and disseminating to a
variety of other tissues.
Human Retroviruses
Monday, January 26, 2009
5:11 PM
Human Retroviruses, 1/27/09:
1. Understand the structure of a retrovirus virion, including the enzymes packaged
with the genome and the nature and packaging of the genomic RNA.
 All retroviruses are enveloped (and hence relatively fragile). They
contain two linear copies of their RNA (positive-strands).
 3 genes:
 gag gene (for capsid)
 pol gene (for reverse transcriptase)
 env gene (for envelope proteins)
 In addition, oncoretroviruses have oncogenes (which aren't required
for infectivity but cause uncontrolled cell proliferation).
 Rous sarcoma virus has the src gene; specifically, it's called vsrc (to differentiate it from the cellular c-src, which isn't an
oncogene in itself). As this implies, all viral oncogenes have
cellular homologs (v-myc has c-myc, etc) that regulate the cell
cycle.
 Note that some oncogenic retroviridae have oncogenes at the
expense of their gag genes-- they are 'helper viruses' that coinfect with other viruses and make use of their gag genes.
2. Understand the features of the retrovirus infectious cycle, including reverse
transcription, provirus integration, and the processes that lead to the formation of
infectious virus.
 Virus de-envelopes and spools out its two RNA strands into the
nucleus.
 Reverse transcriptase does two things: one, makes RNA template into
DNA, and two, synthesizes the complementary DNA strand to the one
just made. It also has integrase and RNAse activity.

Once the two strands of DNA are made (the provirus), it's
time to start trying to integrate it into the host chromosome.
 Note that long terminal repeat regions (LTRs) are inserted on
each end in the transition from viral RNA to viral DNA. This is to
help insert the viral genome into host chromosomes.
 Remember that virions have to undergo modifications after they are
released from the cell.
3. Understand the general similarities and differences between the retrovirus
replicative cycle and the replicative cycles of +stranded RNA viruses and human DNA
viruses that are causative agents of disease (e.g., polio virus and HSV; note that this
aim requires comparison of information from this lecture with information provided in
other lectures).
 Mmm... yeah. I'm going to let this one stand.
4. List the most important retroviruses that infect humans.
 All are lentiviruses ("slow viruses").
 HTLV-1 (human T-cell leukemia)
 Spreads through body fluids, including vertical transmission.
 Gets into T cells, activates genes that cause their proliferation.
 Also infect monocytes/macrophages.
 Long period of latency.
 Can also cause paralysis; associated with a wide variety of
other stuff.
 HTLV-II
 Not much known, doesn't cause T-cell leukemia.
 [HTLVs have two accessory proteins: tax and rex. Tax is like tat, rex is
like rev (see below).]
 HIV-I
 HIV-II
 Some specific notes on HIV (-I):
 [gp160 (env protein) is cleaved in the Golgi into a heterodimer
of gp120 and gp41.]
 gp120 binds to CD4/CCR5 (or CXCR4) on the target cell,
allowing gp41 to insert itself into the host cell membrane and
trigger fusion. Note gp120 mediates attachment and gp41
mediates fusion.
 tat gene: activates viral gene expression (essential).
 rev gene: stimulates export of uncleaved viral RNA out of the
nucleus (essential for export of viral chromosome).
5. Describe the types of human diseases caused by retroviruses.
 Adult T-cell leukemias/lymphomas (rapidly aggressive and can be
fatal), myelopathy/paraparesis, AIDS.
6. Describe attractive retrovirus targets for drug therapy, including why the targets
are attractive.
 Entry inhibitors: blocks uptake of the virus by blocking gp41, etc.
 Reverse transcriptase and integrase inhibitors (there's nearly none of
either in host cells).
 Can also go after tat or rev gene products.
 Protease inhibitors, as previously mentioned, target the maturation
process.
7. Understand the general nature and activity of retrovirus oncogenes.
 As mentioned; they cause uncontrolled cell proliferation.
8. Understand the origin of retrovirus-encoded oncogenes and their differences in
activity from the protooncogenes from which they were derived.

As mentioned, they seem to be derived from host cell cycle regulator
genes. However, the viral genes have been mutated such that their
protein products are constitutively active (thus oncogenic).
Antiretroviral Agents
Tuesday, January 27, 2009
9:00 AM
Antiretroviral Agents, 1/27/09:






Note that non-nucleoside reverse transcriptase inhibitors and fusion inhibitors
only target HIV-1; all other HIV drugs target HIV-2 as well.
Entry/Uptake Inhibitors:
o Enfuvirtide.
o Looks like a sequence in gp41 (HR1). Effectively blocks viral fusion and
entry by inhibiting gp41 activity.
o It's the only approved antiretroviral drug that has to be injected.
Nucleoside Reverse Transcriptase Inhibitors:
o Zidovudine (AZT, a thymidine analog), lamivudine, abacavir.
o Prodrug form taken up by cells and phosphorylated by host kinases;
the triphosphorylated forms have high affinity for the reverse
transcriptase enzyme but little to no affinity for many host DNA
polymerases.
 However, DNA polymerases in the mitochondria can be
affected, resulting in a variety of problems (largely
myopathies).
o Once incorporated into the growing cDNA chain, these drugs terminate
the chain.
o Note this only works well in rapidly proliferating cells.
o Mechanism of resistance: generally, mutations in reverse
transcriptase.
o Zidovudine and abacavir are metabolized in the liver; lamivudine isn't
metabolized at all.
o Lamivudine (cytosine analog) is one of the least toxic agents used.
o Abacavir (purine analog) can cause severe hypersensitivity reactions.
o Note all of these are always used in combination with other agents.
Nucleotide Reverse Transcriptase Inhibitors:
o Tenofovir.
o Like nucleoside RTIs, but has one phosphate group already.
o Same mechanism of action; easier to activate, but less selective for
viral DNA.
o Has poor oral bioavailability.
Non-nucleoside Reverse Transcriptase Inhibitors:
o Nevirapine.
o Don't look like nucleosides or nucleotides; directly bind the viral
reverse transcriptase (and not at its binding site-- irreversible
inhibition by conformational change).
o Used to prevent mother-to-child transmission of HIV.
o CYP inducer (also metabolized by CYP).
o Always used in combination with other agents.
Integrase Inhibitors:
Raltegravir.
Inhibits integrase, no kidding.
Has no effect on human DNA polymerases.
Can result in hepatotoxicity, often preceded by an immune allergic
reaction.
Protease Inhibitors:
o Ritonavir, nelfinavir, atazanavir.
o Selectively target HIV proteases (very little cross-reactivity with
human enzymes).
o CYP inhibitors.
o They bind reversibly to the active site of the proteases.
o Mechanism of resistance: mutations in proteases.
o Ritonavir:
 Metabolized extensively by CYP-- sort of a decoy for CYP
enzymes to allow other drugs to get to a higher effective
concentration. This seems to be its main clinical use.
 Side effects: cause asymmetric loss of body fat (lipodystrophy).
o Nelfinavir and Atazanavir are always administered with meals.
o Always used in combination with other agents.
CCR5 receptor antagonist:
o Maraviroc.
o Inhibits CCR5 receptor (a cytokine receptor).
o Note that not all HIV strains use CCR5 ("R5 tropic"); some use CXCR4
("X4 tropic"). The latter will not be affected by this drug at all.
o Metabolized by CYP enzymes, can cause hepatotoxicity.
o
o
o
o
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Opportunistic Infections
Wednesday, January 28, 2009
7:57 AM
Opportunistic Infections, 1/28/09:
1. Define the terms Opportunistic, Nosocomial and Iatrogenic infections.
 Opportunistic infections: infections caused by organisms that do not
normally cause disease in healthy or immunocompetent individuals.
 Nosocomial infections: infections that occur in an institutional
healthcare setting.
 Iatrogenic infections: infections originating directly from something
that health care workers do.
2. Describe the nature of a biofilm and identify one or more clinical conditions where
biofilms play a role in a specific disease.
 We've more or less learned about biofilms (it wasn't extensively
discussed in class).
 Catheters, in particular, seem to attract things that like to secrete
biofilms (eg. Staph epidermidis).
 Pseudomonas biofilms like to stick to mucus in the lungs of CF
patients, catheters, drains of hot tubs, etc.
 Pseudomonas secretes alginate to combine with the mucus in
CF lungs to form a kind of jelly. The PMNs can't get to the bugs
through the film.
3. Describe some conditions of the host (i.e., the patient) that can contribute to
opportunistic infections and how some of these conditions are more likely to lead to
certain types of infections (e.g., a defect in the production of antibody).
 Granulocytopenia (leads to skin and respiratory infections through
Gram-negatives and Staph species)
 T-cell dysfunction (AIDS, etc; get intracellular pathogens)
 Humoral immunity dysfunction (as due to splenectomy; get
encapsulated pathogens)
 Presence of a foreign body (as in a catheter; see Gram-negatives and
Staph)
 Surgery (see Staph, E. coli, Pseudomonas)
 [There are some others in his notes, as well; these are the ones he
covered in class.]
 He really, really loathes endoscopes. With a passion. Ten-plus minutes
on endoscopes and the infections they can carry.
4. What are the predominant gram-negative organisms associated with opportunistic
infections? Which one is most frequently found and which one is associated with the
highest mortality?
 E. coli, Pseudomonas were mentioned.
 E. coli is probably most frequently found.
 Pseudomonas is associated with the highest mortality rate (on
average, 40-50%).
 Recall that P. aeruginosa infection, particularly in the lungs, is
strongly associated with cystic fibrosis.
 Also associated with burn infections, puncture wounds, hot
tubs, etc.
 Note that P. aeruginosa lives in moist soil; so puncture
wounds from stepping on nails, etc in the dirt are often
associated with it.
5. Discuss the evidence that endotoxin contributes to the pathogenesis of
extraintestinal infections by gram negative bacteria.
 Endotoxin (LPS, in the cell walls of Gram-negative bacteria) contains
Lipid A, which is the pathogenic portion of the toxin and which is
released when the bacterium is lysed (but note many bacteria shed
LPS, including Lipid A, constantly anyway).
 Lipid A activates macrophages, which release IL-1 and TNF-alpha (a
pyrogen and a vasodilator, respectively); this mediates fever and
septic shock (though note that septic shock also requires lots of other
factors, many of which are also activated by endotoxin: clotting
factors, bradykinins, etc).
 Note that endotoxin can also cause DIC, largely through the same procoagulant mechanisms.
6. If Pseudomonas aeruginosa produces a toxin, which has the same mechanism of
action intracellularly as diphtheria toxin does, then why don't patients with
Pseudomonas aeruginosa infections have the same symptoms as a patient with
diphtheria?
 The delivery mechanism interacts with different receptors (think ETEC
and Cholera: same toxin effect, different targeting, which is why
Cholera can kill you and ETEC will just make you unhappy).
 Also, Cornyebacterium diphtheriae only invades superficial tissue
(respiratory tract and skin); by contrast, Pseudomonas gets into the
blood and deep tissues.
7. What are the other virulence factors of Pseudomonas aeruginosa that might
contribute to its pathogenesis? Where (what organ) and under what conditions (kind
of infection) may some of these virulence factors be more significant than others?
 Tough to treat: high innate resistance through lots of efflux pumps to
get rid of antibiotics, and tend to form biofilms (the biofilm likes to
attach to mucus in lungs, which is one reason why it's almost
impossible to get rid of in cystic fibrosis patients).
 Other virulence factors:
 Endotoxin/Lipid A, as mentioned.
 Phospholipases, proteases.
8. What mechanisms does the host have to limit free iron that might be available to
an invading microbe? What mechanisms have microorganisms developed to
overcome the limiting amount of free iron in a host? What factors could upset the
balance of limiting nutrients in a host leading to disease by opportunistic pathogens?
 Nearly all bacteria require iron for growth (except lactobacilli and
Treponema).
 Generally, iron in the plasma is bound to transferrin or lactoferrin,
both to deny free iron to microorganisms and to avoid creating free
radicals from the hyperferremia.
 Upon invasion by microorganisms, iron begins to get shunted into
storage rather than floating around in the plasma. You decrease
absorption from the gut as well.
 The microorganisms, in response, have proteins with really absurdly
high affinity for iron (siderophores), to wrest it away from the binding
proteins. They can also either synthesize products to take iron out of
transferrin/lactoferrin or just absorb lactoferrin whole and degrade it
apart.
 Note that toxins that destroy cells also release lots of iron.
 So basically: if you give people, particularly kids, lots of iron, they
have a higher incidence of disease (as in constant transfusions in
sickle cell or beta-thalassemia).
Antimycobacterials
Wednesday, January 28, 2009
9:04 AM
Antimycobacterials, 1/28/09:
The LOs here say "Use of systemic antimycobacterials in patients."
 Right. Seeing as how we haven't actually learned about mycobacteria,
she gave us a little background:
 Mycobacteria have, outside their peptidoglycan layer, a couple
unique structure layers. One is lipoarabinomannan; on top of
that you have very long-chain fatty acids that form the mycolic
acid layer.
 Mycobacteria have very slow growth rates (doubling times on
the order of days). This poses a problem for typical antibiotics,
which often target rapidly dividing cells.
 Mycobacterium tuberculosis, in particular, grows in widely
different parts of the body, and with different growth rates



(including latency) in each. Thus you need different drugs to
target each of the different locations.
 There is serious emerging resistance to current drugs. In
addition, TB drugs generally carry serious toxicities, which
decrease patient compliance with their regimens.
 Note that TB is reportable.
You always always always treat TB with multiple drugs. Why:
 Many different strains of M. tb are usually found mixed
together. This means a couple things: one, you probably have a
lot of drug-resistant M. tb mixed in there, and two, it's a great
environment for creating multiply-resistant bugs through one
strain passing its resistance to other strains (which are
resistant to other drugs) around it.
 About 8-10% of cases are resistant to one or more of the firstline TB agents.
First-line TB treatment ("short-course therapy"): four drugs:
 Isoniazid (INH) and Rifampin for 6 (to 9) months.
 Pyrazinamide and Ethambutol for the first 2 months.
 After the first two months, if there are no resistant bugs about,
can discontinue the latter two.
 Note that with AIDS patients you have to use different drugs.
 Note that all of these are orally available. They are all relatively
safe during pregnancy.
 The patients are often observed taking the drugs to enforce
compliance, particularly in the third world.
 Note that of these four, only rifampin can be used to treat other
bugs besides mycobacteria.
Isoniazid:
 Related to nicotine; inhibits mycolic acid synthesis through
inhibition of a mycobacterial enzyme (InhA). As such it has
good selectivity; its adverse affects come about mostly due to
the toxicity of the metabolized form.
 Bacteriocidal for growing cells; bacteriostatic for latent cells.
 Note that isoniazid is a prodrug (has to be modified inside the
cell).
 Resistance can pop up due to mutations in the target enzyme
or the enzyme that activates the prodrug.
 Distributed into every tissue in the body (excellent distribution),
including granulomas; metabolized by N-acetylation
(USMLEWorld seems to love N-acetylation, so let's talk about it
a minute):
 Remember how different people have different rates of
CYP metabolism? There's a similar, but bimodal,
phenomenon with N-acetylation. One set of patients
(Caucasian) tend to be "slow" acetylators and
metabolize isoniazid slowly; another set (Asian/Inuit)
tend to be "fast" acetylators and metabolize it quickly.
 The acetylated isoniazid is hepatotoxic. Thus if you're a
fast acetylator, you can get liver damage quickly with
isoniazid use.
 Isoniazid depletes vitamin B6 (pyroxidine) and can cause
nervous system disturbances.






Note also that isoniazid is a CYP inhibitor (she may have said
"inducer" in class).
Rifampin:
 Inhibits the action of bacterial RNA polymerase. Note the MoA
is wide enough that you can use rifampin for other bugs aside
from mycobacteria.
 Bacteriocidal.
 Also has excellent distribution-- however notice it can turn
those tissues red.
 Rifampin is a CYP inducer.
 Adverse effects: fairly standard, aside from CYP induction. The
high level of CYP induction is why you don't usually use
rifampin for AIDS patients. Note also that birth control pills are
metabolized through pathways induced by rifampin.
Ethambutol:
 Inhibits an enzyme that creates the other (non-mycolic acid)
unique layer of mycobacteria, the lipoarabinomannans. So like
isoniazid, it inhibits cell wall synthesis.
 Like isoniazid, can be either bacteriocidal (growing cells) or
bacteriostatic (latent cells).
 Resistance mechanisms, as with isoniazid, involve mutations in
the target enzyme.
 Side effects: high incidence of optic neuritis; contraindicated in
children. Also decreases clearance of uric acid (can precipitate
gout).
 Again, wide distribution.
Pyrazinamide:
 Newer drug, not well understood.
 Similar in structure to isoniazid but a different, unknown,
mechanism of action. The current theory is that it messes with
mycobacterial membrane potential.
 Administered, like isoniazid, as a prodrug that has to be
activated by mycobacterial enzymes.
 Like isoniazid, has a high hepatic toxicity. Like ethambutol, can
produce gout.
A few notes on second-line drugs: generally they're not orally
available, or have higher toxicities.
Note that for Mycobacteria leprae (causative agent of leprosy) you use
a drug called Dapsone.
Mycobacterial Diseases I + II
Tuesday, February 03, 2009
7:54 AM
Mycobacterial Diseases I + II, 2/3/09:
1. Describe the unique properties of mycobacteria and how they create special
problems for the isolation and identification of these organisms.

Gram-positive rod, extremely slow-growing. The slow-growing part is
irritating for trying to figure out what kind of resistances the strain
you're looking at has got.
 Mycobacteria are acid-fast (red rods which fail to lose their red color
after harsh acid, alcohol, and heat destaining) due to their thick and
intricate cell wall.
 Cell wall substituents:
 Mycolic acids: found only in Mycobacteria and Nocardia. Recall
that this layer is targeted by isoniazid.
 Lipoarabinomannan/phosphatidylinositol mannoside (PIM) are
also present.
 The reason it's classified as Gram-positive is that it doesn't have a
traditional outer membrane. However, the elaboration of the different
layers of cell wall means that it effectively does have a bilayered
membrane.
2. Describe how M. tuberculosis is transmitted and the odds of developing disease.
 TB is transmitted exclusively by aerosol (respiratory droplets).
 Droplets are microscopic-- they evaporate to micron-sized particles,
which are small enough to penetrate all the way to the alveoli (recall
that larger-size particles aren't capable of getting that far).
 Usually, you have to be around someone who's breathing, coughing,
etc TB-infected air to catch it. Of course, that means the closedcirculation ventilation in hospitals or airplanes is a perfect setting.
 Most people exposed to TB don't develop infection (70%).
 Of the other 30%, only about 5% go on to active (primary) infection;
the other 95% get a contained, latent infection.
 Of the people with latent infection, only about 5-10% get reactivation
of TB (generally in the setting of immunocompromise).
 Infection process:
 Macrophages engulf TB. TB has no problem with that and can
live inside macrophages (at least when they're not activated)
just fine. The macrophages bring it to the lymph nodes and
thence to the blood, where it spreads to other organs. Being an
obligate aerobe, it particularly likes the lungs (and in particular
the upper lobes where the V/Q ratio is highest).
 Effective immune-system ways of killing TB: nitric oxide and
cathelicidin (note that you need vitamin D, and hence
sunlight, to make cathelicidin). Activated macrophages can
sometimes kill the TB and sometimes not; usually they just sort
of stay in a stalemate situation.
 TB is highly adapted to humans: it's nonfusogenic (lives
inside the phagosome) and upregulates the uptake of nutrients
in the cell into the phagosome with a compound called PIM
(part of its cell wall).
 Note that TB prevalence worldwide is increasing-- 2.5 million cases in
1980, 5 million in 2005. In the US it's been steadily declining.
 Granulomas, of course, are a big part of TB's pathogenicity.
 How this happens: lots of monocytic immune cells are recruited
in to destroy the infected site; they fail; lots of necrotic debris
accumulates and, in the case of TB, becomes caseous;
macrophages fuse around the periphery of the infection to
make a containment system.

Why TB lets this happen: this gives it an opportunity to
reactivate once the host is immunosuppressed. It's generally
spread and propagated through reactivated disease.
 Note that TB can infect animals, and will kill them, but can't be spread
from them. It's been adapting to humans for a long, long time.
 Potts disease: TB that's spread to the spine.
3. Describe the development of immunity to M. tuberculosis.
 Mainly T-cell mediated; antibodies are largely ineffective.
 Effectively what the T cells are doing is activating the macrophages,
which then can either kill the intracellular TB or slow it down. Recall
that INF-gamma is needed to activate macrophages and that activated
macrophages secrete TNF-alpha; medications that target either of
these cytokines can predispose to reactivation of latent TB.
 This is why HIV plus TB is a bad combination: the T cells that activate
the macrophages to contain the TB are all being killed by HIV.
4. List the immune factors known to control M. tuberculosis.
 As mentioned, NO and cathelicidin.
5. Differentiate between primary, latent, and reactivation tuberculosis.
 [Initial infection: looks flu-like or asymptomatic.]
 Primary TB: after initial contact; may cause an active infection (failure
of containment) or may not (progressing to latency).
 Latent TB: contained infection (non-contagious). Can last for decades.
 Reactivated TB: just that (contagious, usually follows
immunosuppression).
 Reactivated TB occurs almost exclusively in the upper lobes of
the lung. They necrose their way into an airway, from which
they can be spread by coughing.
 Note that most cases of TB occur within a year or two of infection, as
primary TB.
6. Describe how M. tuberculosis survives within a phagosome.
 As mentioned, prevents fusion and upregulates uptake of nutrients
with PIM.
7. Discuss the primary goal of tuberculosis control.
 The point is to interrupt the cycle of infection, latency, reactivation,
and more infection.
 Ideally you use a vaccine. We ain't got one, or at least one that works
reliably. They're in development.
 So:
 (1) You want to find active cases, diagnose them rapidly, and
treat to cure. (2) Then you want to treat their contacts for
latent infection.
 (3) Finally you want to find high-risk people for TB and treat
them.
 Risk factors for latency for recent infection:
 HIV infection or other immunosuppression
 Diabetes
 IV drug users
 Recent immigrants from TB-prevalent countries
 Being a healthcare worker
 Being in close contact with TB-active individual
8. Describe the symptoms of active tuberculosis and two methods for detecting
latent infection.
 Cough > 3 weeks, chest pain, hemoptysis


Fever, chills, night sweats, weight loss.
Chest X-ray is extremely useful.
 Primary TB shows up with some consolidation, a little like
pneumonia.
 Active TB usually looks like upper lobe opacity with cavitations.
 Miliary (blood-borne) TB is disseminated and hence looks like
little nodules widely distributed throughout the lung.
 Sputum smear analysis is also useful (get an acid-fast stain of the
smear). If positive, can treat for TB. But note that negative doesn't
prove much. The smear is relatively specific but not sensitive.
 For latent infection: can use the PPD skin test or newer, ELISA-based
techniques.
 How the PPD works: you present mycobacterial antigens and
look for a predictable memory T cell reaction (delayedhypersensitivity, type IV rxn).
 PPD and symptoms normal: not infected.
 PPD and/or symptoms abnormal but X-ray normal: latent.
 PPD and/or symptoms abnormal and X-ray abnormal: active.
 Note that you read the induration of the PPD, not the erythema.
High-risk people (eg. HIV) have a lower diagnostic threshold (5
mm); no-risk people have a higher diagnostic threshold (15
mm); medium-risk people (eg. healthcare workers) are in the
middle (10 mm).
 Note that the TB skin test does have some flaws: false
negatives (as in HIV, where you can't mount the necessary
immune response), false positives (picks up vaccinated
individuals and other mycobacteria).
 The newer screens tend to be ELISA-based tests; they're much
more sensitive and specific than the PPD and measure the
interferon-gamma response. You also don't have to wait 48
hours (which means you can diagnose same-day) and the
diagnosis is standardized (as opposed to getting out a ruler and
making a judgment call on how big something is).
 Because you're testing an antigen more or less specific to TB,
you can use ELISA-based tests to differentiate BCG-vaccinated
individuals from people with active TB infection.
 To look at active TB:
 Nucleic acid amplification test: 100% positive predictive value
for TB and extremely rapidly (1 day). However, 5-10% false
negative results. Again, good specificity but less good
sensitivity. At that point you come up to a clinical judgment
call.
 Cultures: take freakin' forever (2-8 weeks depending on
technique) but can tell you about resistance. Generally you
want a liquid culture for speed (such as it is: 2 weeks) and a
solid culture for slower confirmation.
 Note that TB is still the #1 killer of HIV patients worldwide.
9. Describe a typical antimicrobial regimen for treatment of M. tuberculosis (drug
sensitive).
 As mentioned in "Antimycobacterial Drugs," for active TB infections,
INH, ethambutol, rifampin, pyrazinamide are first-line. Recall that
ethambutol and pyrazinamide are usually used only for the first 2
months.

Note that, if it's followed for the full 6 months, there's a 98%
cure rate. But shorter regimens would really be helpful.
 Fluoroquinolones are currently very useful as backups. Note you can
use a fair number of standard antibiotics in extremis (azithromycin,
imipenem, augmentin, etc).
 For high-risk latent TB infections, you generally use INH for 9 months
to treat.
 Note that TB acquires resistance mainly through mutation, not by
horizontal transfer from other organisms.
 Note also that previous treatment of TB is the biggest indicator of
multidrug-resistant TB in a patient.
10. Describe the pros and cons of BCG vaccination.
 BCG: live attenuated organism.
 Amazingly, it's the most widely-used vaccine in the world. The reason
that's amazing is that it really doesn't do much against adult
pulmonary TB. Its main function seems to be in minimizing the extent
of miliary or meningeal TB in infants and kids.
 As mentioned, BCG vaccination causes positive results in all
subsequent PPD tests (but not ELISA-based assays).
11. Compare and contrast M. tuberculosis and non-tuberculosis mycobacterial (NTM)
infections.
 There's a lot of mycobacterial bugs (130+) other than TB.
 Generally, unlike TB, they are not transmissible from human to
human. They are infrequently found as pathogenic in the US.
 Note that there generally isn't a latent infection stage of NTM bugs.
 They like to live in water and are often environmental.
 Most common ones that cause lung disease in the US: MAC (see next
point), kansasii, abscessus.
 Some (eg. kansasii) are easy to treat; some (eg. abscessus) are
nearly impossible to treat.
12. Describe disease caused by MAC.
 Mycobacterium avium complex: contains Mycobacterium avium and
Mycobacterium intracellulare. We've seen this as MAI (Mycobacterium
avium-intracellulare) in the DEMS block, where we talked about
differentiating it from Whipple's disease.
 Causes pulmonary disease (similar to TB), AIDS wasting, and
disseminated disease.
13. Describe the definitive treatment for Buruli ulcer in early disease.
 Buruli ulcers: caused by M. ulcerans, which infects the skin and
secretes an extremely nasty toxin that kills everything around it
(including nerves, which means it's painless).
 Generally you use rifampin and streptomycin for 8 weeks, then use
surgery to remove all the necrotic tissue. I presume the surgery is the
definitive part.
14. Describe how M. leprae is transmitted.
 Causes leprosy; only hosts are humans and armadillos (bad luck,
armadillos!).
 Affects peripheral nerves and skin.
 It's spread from person to person through nasal secretions, but the
rate of transmission is very low.
 It causes massive, painless, tissue destruction (nose and digits are
lost).
15. Compare and contrast the two extreme forms of leprosy in terms of their
bacteriological and immunological characteristics.
 Two poles of a spectrum of leprosy: tuberculoid leprosy, in which the
immune system are kicking up granulomas to contain the infection,
and lepromatous leprosy, in which the cell-mediated mechanisms are
failing and humoral immunity is kicked up (to no effect).
 Generally the classic leprous symptoms are only present in
lepromatous leprosy; the bacteria are widely disseminated, and are
found in the nerve sheaths.
 Tuberculoid leprosy often shows up as a small hypopigmented rash
and, sometimes, single enlarged nerves.
Mycoplasma/Legionella
Tuesday, February 03, 2009
10:06 AM
Mycoplasma/Legionella, 2/3/09:
1. Describe the clinical syndrome of atypical pneumonia.
 More gradual onset, less dramatic illness; on chest exam, relatively
minimal physical findings. On X-ray, the findings are more dramatic
than the physical findings. Only rarely causes pleural effusion.
 Specifically, you see fever, malaise, myalgia, headache, sore throat,
and cough (generally non-productive).
 These are reasonably common but certainly not atop the list of
pneumonia-causing organisms (Legionella is 4th, Mycoplasma is 5th)-though notice they're both much more common in inpatient settings.
They're also the most common cause of bacterial bronchitis and
pneumonia among young adults and teenagers.
 Doesn't respond to the same antibiotics that the usual pneumonia
suspects are susceptible to. To treat mycoplasma infections you use
tetracyclines, macrolides, and quinolones.
 Note (as per CMBMRS) that about 7% of Mycoplasma patients get
Stevens-Johnson syndrome (aka erythema multiforme).
2. Describe and compare biological characteristics of Mycoplasma and Ureaplasma.
 Mycoplasma organisms:
 They're the smallest 'free-living' (ie. nonviral) organisms in the
world.
 They have no cell wall (no peptidoglycan) and are therefore
resistant to all antibiotics that target the cell wall (eg.
penicillins) and stain poorly with Gram stain (hard to see with
light microscopy as well).
 Due to the flexible external membrane, they can look like a
wide variety of shapes (pleomorphism).
 Their genomes are small and they lack many enzymes that are
necessary to synthesize essential compounds-- have to pick
them up from their environment. Note, however, that they
aren't invasive or intracellular.
 Tend to make tiny "fried egg" colonies.
 M. pneumoniae is the main pathogenic mycoplasma.

Ureaplasma: closely related to mycoplasma bacteria but lives in urine
and produces urease. Normal vaginal flora, but seems to be one of the
things that cause vaginosis and urethritis.
 Note that mycoplasma organisms need exogenous cholesterol in their
cell membranes to function (nearly unique among pathogenic
bacteria).
 Most infections don't cause pneumonia-- can cause runny nose, sore
throat, or a host of other minor symptoms.
 How they cause disease:
 Transmission is through respiratory droplets (Mycoplasma likes
to live in and on the ciliated epithelial cells of the respiratory
tract). Close quarters tend to cause outbreaks, but the
outbreaks are pretty slow-- the incubation period is about 3
weeks.
 They have these unique "terminal attachment structures:"
these are composed of cytoskeletal elements that seem to
facilitate tight attachment to the ciliated cells mentioned above.
 As you might expect, the attachment structure is antigenic; the
main antigen seems to be called P1.
 They cause cilial stasis (thus hindering clearance of the
bacteria).
 They have some cytopathic effects (produce a mild pertussislike toxin and hydrogen peroxide); the majority of the tissue
damage seems to result from the immune response from PMNs
and from cytokine release.
3. Explain how diagnostic tests for infections caused by Mycoplasma pneumoniae and
Ureaplasma urealyticum work, and how they are used in medical practice.
 Mycoplasma infection causes the production of autoantibodies
(similarity between the membranes of Mycoplasma and host cell
membranes); thus you can see cold agglutinins in infected patients'
blood. Very few people use this anymore (poor sensitivity/specificity),
but good for boards.
 Similarly, you can use complement fixation levels for diagnosis (much
better than agglutinins).
 ELISA and PCR seem to be your best bets but they're still not great.
 Sputum culture takes about 2-3 weeks (needs cholesterol-rich media).
4. Describe and compare biological characteristics of Legionella pneumophila and
related species.
 Legionella: Facultatively intracellular, Gram-negative rod (but stains
poorly with Gram stain). Lives inside of macrophages and are nonfusogenic (similar to mycobacteria).
 Directs the phagosome to the ER, which works out well for it.
 Resistant to penicillins (makes beta-lactamases).
 Disease states caused by Legionella:
 (1) Pontiac fever (acute, self-limiting flu-like illness without
pneumonia)
 Note that Pontiac fever is highly contagious but nearly
never fatal.
 (2) Legionnaire's disease (pneumonia, chest pain, headache,
diarrhea, myalgias).
 Legionnaire's disease has a lot more extra-pulmonic
symptoms than other pneumonias.
Legionnaire's is generally not highly contagious but can
be fatal.
 Legionella likes to grow in water (also like mycobacteria). Air
conditioning systems and plumbing can be reservoirs, and often are in
hospitals.
 Note some significant similarities to mycobacteria (facultatively
intracellular, lives in macrophages, nonfusogenic, likes water, goes to
lungs). But note that the treatment of Legionella is identical to that of
Mycoplasma.
5. Explain how diagnostic tests for infections caused by Legionella species work, and
how they are used in medical practice.
 Use charcoal yeast extract to grow it (need cysteine and iron)-requires 3-5 days.
 Use silver stain to see it inside of macrophages. Nonspecific.
 Can use direct fluorescent staining instead-- highly specific but
not highly sensitive.
 Can also use a monoclonal antibody-based urine antigen test. Rapid,
highly sensitive, and highly specific.

Adenoviruses and Viral Gene Therapy Vectors
Thursday, February 05, 2009
9:41 AM
Adenoviruses and Viral Gene Therapy Vectors, 2/5/09:
[I saw that Dr. Schaack was lecturing, followed by Dr. Vasil, and fled in terror. These
are from the notes and from what little CMBMRS says about it, but notice that the
notes seem to be written first in Vulcan and then translated to Czech and then to
English.. so take with a grain of salt.]
1. Understand the general structure of the adenovirus virion.
 Non-enveloped, double-stranded DNA virus, with an icosahedral
capsule.
 Contain no lipids; heat- and detergent-resistant (can thus make it
through the GI tract).
 Attaches to the host cell (generally respiratory epithelium) with fiber
proteins and is endocytosed.
 The primary exposed antigens of adenoviruses are "typespecific reactive sites on hexons." Make of it what you will.
 It carries its own viral DNA polymerase; it uses a viral terminal
protein to prime its 5' DNA for replication.
 Within 8-10 hours after infection, the host cell stops its own DNA
replication and translation. Note that the cells are generally not lysed
quickly.
 49+ serotypes of virus.
2. Understand the types of diseases caused by adenoviruses (but not the groups or
serotypes that cause them).
 Upper respiratory tract infections, particularly common in childhood:
rhinitis, sore throat, fever, conjunctivitis. Generally self-limited with
persistent but type-limited immunity. Can occasionally cause serious
pneumonia. With immunosuppression you can occasionally get
disseminated infection.
 3rd most common cause of respiratory infection in children
(after RSV and parainfluenza; CMBMRS makes it 4th and adds
rhinovirus).
 Adenoviruses cause pink-eye (aka epidemic keratoconjunctivitis).
 These are transmitted through oral and respiratory secretions. Note
that adenovirus infection is usually asymptomatic (only 4-5% of
symptomatic viral respiratory illness is caused by adenovirus) but the
person is still shedding virus.
 Can occasionally cause ARDS in military recruits.
 Certain serotypes cause diarrhea; these are the ones that are
transmitted fecal-oral.
 It causes several other things, evidently including obesity (..); these
are listed on p. 204 of the notes.
 There is no specific treatment for adenovirus; can use ribavirin and
cidofovir, but not shown to be that effective.
 There are some military vaccinations against the ARDS-producing
strains.
 Transforming (immortalize host cells), but not associated with tumors
in humans.
3. Understand the commonly used DNA virus gene therapy vectors.
 Most commonly used: retroviruses, adenoviruses, and adenoassociated viruses.
 Retroviruses:
 Keep the LTRs to facilitate genomic insertion; keep the signal to
transcribe and package into a virion; the rest of the retroviral
sequence can be gene(s) of choice.
 Use "vesicular stomatitis G protein" as an envelope protein to
facilitate the stability of the virion. I have no idea what this
means.
 Size capabilities: 8 kb DNA.
 Problems: virion is fragile (enveloped) and slow to make its
product; also generally can't infect non-mitotic cells
(integration of viral cDNA requires nuclear transport, which
evidently doesn't happen). We're getting around this by some
HIV-based vectors which target DNA to the nucleus.
 Adeno-associated viruses:
 Parvoviruses: single-stranded, small (5 kb) DNA genome.
 Usually has to have adenovirus gene products around to do its
thing; can go solo if there's enough of it. No clinical symptoms.
 Can't infect non-mitotic cells without adenovirus superinfection.
 Inserts itself into the host DNA like retroviruses.
 Size capability: 5 kb (fairly small).
 Problems: small, integration in host DNA, slow.
 Adenoviruses:
 Take out the transforming gene product (E1) so that it doesn't
immortalize the cells.
 Can carry a large amount of DNA (8-36 kb).
 Can transduce non-mitotic cells.
 Stable, non-enveloped virions.
 Problems: induces a major inflammatory response (although
note that the virus can be targeted to tumor cells, which the
inflammation can help destroy). You only limited expression in
mitotic cells, since the DNA isn't integrated into the host's.
4. Understand the advantages and disadvantages of these vectors.
 As above.
Models of Toxin-Mediated Bacterial Disease
Thursday, February 05, 2009
10:44 AM
Models of Toxin-Mediated Bacterial Disease, 2/5/09:
[C. diphtheria/B. pertussis both infect the upper respiratory tract]
[C. diphtheria: Gram-positive rods. B. pertussis: Gram-negative rods.]
1. What is the evidence that diphtheria toxin is the most important virulence
determinant of this organism?
 Non-toxin-producing bacteria don't produce disease.
 Antibodies against the toxin prevent disease.
2. How is the toxin used in the prevention of diphtheria? What is used to treat
diphtheria besides antibiotics? Why does it not always prevent mortality?
 Diphtheria toxin is an A-B toxin (B binds to cell and enters to deliver A,
which is the active toxin). It is produced by the bacteria in the throat
but circulates systemically, particularly targeting the heart, peripheral
nerves, and kidneys. It targets and inactivates elongation factor 2
(by ADP-ribosylating it) in eukaryotic cells, resulting in cell death (and
cardiac failure). Recall that it forms a pseudomembrane (although note
also that the other toxin that forms a pseudomembrane, from C.
difficile, has a completely different mechanism of action).
 Note that ADP ribosylation is a somewhat common motif in
bacterial toxins; the examples he gives are B. pertussis and V.
cholerae, which ADP-ribosylate G proteins to increase adenylyl
cyclase (in that case activating it, rather than inactivating).
Note that adenylyl cyclase activation doesn't kill the cell like
EF2 inactivation.
 Note also that the production of the diphtheria toxin is
regulated by iron-- increases in iron will shut off toxin
production.
 Diagnose diphtheria clinically and treat rapidly (don't wait for lab
results-- they take too long and diphtheria can be fatal). You need
special media (Loeffler's and potassium tellurite agar) to culture it.
 Treat diphtheria with antibiotics (erythromycin or penicillin),
but also antitoxin and vaccination. Note that infection with C.
diphtheria doesn't always provide lasting immunity; want to
vaccinate as part of treatment.
 Antitoxin used to treat diphtheria: horse-derived antibodies
against toxin. Need to treat quickly, since the antitoxin only
targets unbound, circulating toxin (once the toxin is bound it
can't be neutralized). Note that the antitoxin is only carried by
the state department of health.
 Vaccine used to prevent diphtheria: formalin-inactivated toxin.

The only thing I can think of about not preventing mortality is
that if you don't give the treatment promptly, mortality goes
up.
3. How safe and effective are the respective current vaccines against Diphtheria and
Whooping Cough? Would a person 20 yrs old who had received all his/her childhood
DPT vaccinations, and none since, be sufficiently protected against Diphtheria and
Whooping Cough if they came in contact with someone with either of these diseases?
Why or why not for each disease?
 Diphtheria vaccine is extremely efficacious (consists of toxoid alone).
It is extremely safe.
 Pertussis vaccine in the DPT or DPT-Hib combined vaccines is
efficacious, but is a killed, whole-cell vaccine (not just the toxoid) and
has some side effects (not that he says what they are-- CDC website
says anaphylaxis, "intractable crying," encephalopathy, arrhythmias,
Reye syndrome, etc).
 He says to use an acellular pertussis vaccine (aP vaccine, composed of
purified antigens) instead of the whole-cell type (P vaccine). It's not
clear to me if Tetramune (the DPT-Hib vaccine) is acellular or not.
 Regarding the 20 yr old: no, he wouldn't be immune to either. This
age group is the one is which you find the most new cases of
diphtheria these days. However, adult cases of pertussis are generally
much milder than the childhood form. Booster vaccinations should be
given in the 11-18 age range and every 10 years after.
 If you're curious why some vaccines are called "DTaP" and others are
"Tdap": evidently the capitalization indicates higher concentration.
 Re why the immunity fades over time, I suspect it's what it usually is:
gradually lowering concentrations of circulating antibodies against the
toxins. However, note that for pertussis, cell-mediated immune
mechanisms seem to be effective as well.. which maybe is why you
have more extensive lasting pertussis immunity (your memory T cells
are still working even if your humoral immunity is flagging).
4. If it is known, how or why do these vaccines protect against Diphtheria and
Whooping Cough?
 Diphtheria: neutralize toxin.
 Pertussis: neutralize toxin and other antigens.
 Note that you don't use antitoxin to treat pertussis (doesn't
seem to do much good)-- treatment is primarily supportive or,
if caught early enough, can use antibiotics to limit its course.
Once they start the convulsive coughing, antibiotics have no
effect at all.
5. Describe other known virulence factors of C. diphtheriae and B. pertussis besides
Diphtheria toxin and Pertussis Toxin and their possible role in disease, if they are
known.
 C. diphtheria: no other known virulence factors.
 B. pertussis: tracheal cytotoxin that destroys epithelial cells, adenylyl
cyclase toxin to stop macrophage attraction, filamentous
hemagglutinin mediates attachment to epithelial cells.
6. What are some specific problems associated with making a laboratory and clinical
diagnosis of Diphtheria and Whooping Cough? How do they influence initiation of
treatments for these diseases?
 Need specific media (Loeffler/potassium tellurite agar) to culture
diphtheria; you also need to send it to the CDC or a state health lab to
see if it produces toxin. This takes about 4-5 days.

It's hard to get the pertussis organism off the epithelial cells (tight
attachment). To culture pertussis you need a special sticky swab
(apparently you use alginate, like from Pseudomonas) in the nose
while the patient is coughing. Past the first two weeks (catarrhal
stage), you can't get the organism. Even if you culture it, it still take
3-5 days to grow.
 You don't wait for lab confirmation if you suspect either of these
diseases.. I think? Maybe with pertussis. Hard to say from his notes,
unless I'm missing something.
7. Should the physician always wait for a laboratory diagnosis before treating these
diseases? Why or why not?
 No- disease can progress rapidly (waiting 4 days to treat diphtheria
can cause mortality to reach 20%).
 Note that you use toxin tests to test for diphtheria, but not pertussis.
8. What are the environmental factors, if any, that influence the expression of the
virulence factors of C. diphtheriae and B. pertussis?
 As mentioned, diphtheria toxin is inhibited by higher levels of iron.
Note that the toxin, like V. cholera toxin, is carried by a bacteriophage
(so you need bacteriophage infection to produce the toxin).
 Pertussis toxin production is regulated by the vir gene, which is turned
on and off by various things. No iron/bacteriophage dependence.
9. What is the value of antibiotic usage in Diphtheria and Whooping Cough when the
major manifestations of these diseases are due to toxins, against which antibiotics
have no effect?
 (1) eliminate transmission of the infection to others
 (2) reduce toxin-producing bacterial load
 (3) prophylaxis of exposed people
 Note that erythromycin is used for both diphtheria and pertussis, while
penicillin can only be used for diphtheria.
10. Who should not be vaccinated with the whole cell pertussis vaccine? Why? What
is the difference between the split pertussis vaccine and the whole cell pertussis
vaccine?
 According to his notes, you shouldn't use the whole cell vaccine at all.
Harrison's says that in kids 7 years and older the whole-cell vaccine
has particularly bad side effects.
 Split pertussis vaccine: purified antigenic components. Whole cell:
whole, killed, pertussis bacteria.
Rota-/Calicivirus and Viral Diarrhea
Monday, January 12, 2009
8:04 AM
Rota-/Calicivirus and Viral Diarrhea, 2/6/09:
1. List/Name





viruses that can cause gastroenteritis.
Rotavirus
Calicivirus (norovirus and sapovirus)
Astrovirus
Adenovirus
Essentially there are a ton of viruses that cause this. There are a ton
of tons of serotypes of virus that cause this. Immunity would be hard
enough under these conditions without the fact that epithelial (IgA)
immunity is generally more short-lived than serum (IgG) immunity.
 [General stuff about viral diarrhea:]
 Spread oral-fecal (sometimes the infection is asymptomatic but still
infectious).
 Short incubation period. Acute disease only lasts a few days. Local
infection of GI epithelial cells only.
 After it infects intestinal epithelial cells, you see some blunting of the
intestinal villi for the duration of the infection (replaced after the
infection is over).
 Some different ways that viruses can cause diarrhea:
 Malabsorption due to enterocyte damage
 Villus ischemia
 Possibly an enterotoxin (good recent evidence for this)
 Very stable-- can survive in food/water.
2. Explain how diagnosis of gastroenteritis viruses is made even though most of
these viruses cannot be grown in cell culture.
 Use PCR and viral detection assays from feces.
 Can also take EM of stool (wow, really?), immunoprecipitation of
viruses, or "virus discovery chip."
 However, note that virus can be found in the feces of healthy people
as well (asymptomatic but infectious disease).
3. Describe the replication and pathogenesis of Rotaviruses.
 Double-stranded, segmented, nonenveloped RNA virus.
 Only 4 types of rotavirus serotypes in humans.
 Double-protein shell, very stable. Two surface proteins of note: VP4 or
P (spike) and VP7 or G (outer capsid protein).
 The genome of rotavirus, like that of the flu, is segmented (11,
in this case).
 Rotavirus is a much more serious disease and is responsible for
500,000 deaths, mainly children, per year worldwide, largely due to
dehydration.
 It makes an enterotoxin, NSP4-- essentially it causes increased
intracellular calcium through a Gq kind of mechanism, which increases
chloride efflux (sense a theme here?). Varying susceptibility to this
toxin may be why adults don't get nearly as much serious disease
from rotavirus.
 Rotavirus is the classical virus that infects daycare centers.
 Like influenza, reassortment of rotavirus genome segments can result
in epidemics from animal flu-human flu recombination.
4. Describe the replication of Noroviruses.
 Norovirus is a calicivirus.
 Small, single-stranded, positive strand, nonenveloped RNA virus. Very
sturdy. Lots of serotypes.
 Proteins: RNA-dependent RNA polymerase, 2 structural proteins.
 Like most positive-strand RNA viruses, it produces a polypeptide which
needs to be cleaved by a viral protease in order to function. This
protease is a good drug target.
 Stool contains 10 million+ virions per mL (!). Infectious dose is 10100.
 Mainly spread from person-to-person contact (possibly through Not
100% Safe Sexual Behavior); can also be spread through shellfish
(filter nutrients from feces and concentrate virus).

Illness lasts 1-2 days and is frequently incapacitating; it can be lifethreatening in infants, elderly, and immunocompromised. Spreads like
crazy-- attack rate is 40-50%.
 Classically you see norovirus outbreaks on cruise ships, schools, and
long-term care facilities.
 Note that it's really hard to get rid of in a facility (hardy little bastards
and there's gazillions of them). However, note that cooking destroys
the virus.
5. Compare the epidemiology of different gastroenteritis viruses. Which would be
most likely to cause epidemics in a newborn nursery, daycare, school, family,
nursing home? Which would most likely be associated with contaminated shellfish?
Which is most likely to be spread by water?
 Prevent with isolation and clean environment (treat with oral
rehydration solution).
 As mentioned, schools and nursing homes tend to be norovirus haunts.
 Shellfish-borne viral diarrhea is usually due to norovirus as well.
 Daycare and nursery is rotavirus.
 Family: not, I think, mentioned in lecture, but probably norovirus.
6. Describe the type of immunity that is most likely to prevent viral gastroenteritis,
and explain the current problems in eliciting this type of immunity.
 IgA (epithelial). As noted, it's relatively short-lived.
7. Compare the types of vaccines being developed for gastroenteritis viruses and
explain which vaccines will need to contain multiple serotypes of virus.
 For noroviruses, can target the viral protease, or can make more
classical viral capsid antigen vaccine. However, there are lots of
serotypes of noroviruses.
 For rotoviruses, targeting a single serotype has widely variable results;
there was a vaccine targeting many serotypes, but it was associated
with rare but serious intussusception (telescoping of the intestines), so
it was withdrawn.
 2 new rotavirus vaccines approved in 2006: live attenuated
vaccines. One (Rotateq) is a pentavalent bovine virus with
capsid proteins from a variety of strains; the other (Rotarix) is
a monovalent human rotovirus that provides cross-protection
against other serotypes (possibly through immunization against
the toxin?).
 She mentioned that you could, in principle, stick rotavirus
antigens in baby food and build up immunity that way.
Anaerobes
Monday, February 09, 2009
8:51 AM
Anaerobes, 2/9/09:
[General notes:]
 Anaerobes: predominant component of human flora. You've got 'em in
your mouth, colon, female genitals where appropriate, and skin.
 Why oxygen is bacteriocidal to anaerobes (not just bacteriostatic):
oxygen spontaneously produces free radicals (H2O2 and superoxide).
These, unless they're reduced back down to O2 by catalase,
peroxidase, and superoxide dismutase, are toxic to bacteria.
 If you find catalase, etc, in a normally anaerobic organism,
that's a virulence factor, making them aerotolerant-- won't
grow in aerobic environment but can survive for a certain
amount of time in the presence of oxygen. Most pathogenic
anaerobes fall into this category.
 In order to culture them you need to order special anaerobic
conditions.
 Features of anaerobic Gram-negative rods and all anaerobic cocci:
 (1) Generally they're all normal microbial flora. The ones that
cause disease are aerotolerant.
 (2) Usually disease follows autoinfection (normal flora gets
into sites it shouldn't be in) through trauma, aspiration, etc.
 (3) These organisms cause abscesses (fibrin wall around
purulent infection).
 (4) The infections are most often polymicrobial (lots of different
species in there, including aerobes).
 Soft tissue abscesses:
 Acute stage: aerobic and facultative anaerobes predominate,
using up the oxygen in the microenvironment. Clinically:
hypotension and fever.
 Chronic stage: Fibrin encases the oxygen-depleted infection,
further reducing its oxygen exposure; anaerobic
microorganisms dominate.
 Bacterioides fragilis (anaerobic coccus):
 Normal, if minor, component of gut flora, but found in 80% of
abdominal abscesses.
 Virulence factors:
 Relatively aerotolerant (has superoxide dismutase and
catalase).
 Secretes enzymes that damage collagen and cell
membranes.
 Has a pilus.
 Has a capsule.
 Resistant to many antimicrobials (like most normal
flora).
1. Discuss the ramifications of oxygen toxicity for the ability of anaerobes to cause
significant disease in humans.
 See above.
2. Discuss the role of the anaerobic normal flora in the formation of soft tissue
abscesses.
 See above.
3. Identify and describe the pathogenesis of disease(s) caused by the major species
of Clostridium (C. tetani, C. botulinum, C. perfringens, C. difficile). Identify
similarities and differences in terms of acquisition of the organism, virulence factors
associated with disease, treatment, prevention, and public health implications.
 Note C. tetani, perfringens, and difficile (not botulinum) are all normal
flora. C. botulinum, tetani, and perfringens are all found in soil and
water (whereas difficile colonizes fomites).
 Clostridium: all spore-forming, Gram-positive anaerobic rods.
 C. tetani:



Very widespread; common in feces and soil. Spores are
introduced into wounds, infecting minor infection in dead tissue.
 Produces toxin which is transported retrograde up neurons into
the CNS.
 The tetanus toxin blocks the release of inhibitory NTs
(GABA/glycine) onto skeletal muscle-- makes it impossible to
relax the muscles. This seems to involve damage to the axon
terminals.
 Treatment:
 Supportive respiratory therapy
 Debridement of wound (if it can be found-- they're often
small)
 Antimicrobial use to stop toxin production
 Passive immunization (won't have much effect shortterm - the nerve terminals are still damaged - but will
prevent things from getting worse)
 Vaccination-- humans do not acquire natural immunity
to tetanus (it takes more toxin to immunize than it does
to kill).
C. botulinum:
 Spores are, again, widespread and common in soil/water.
 Spores can be destroyed by enough heat and pressure, but
home canning is generally insufficient and the spores survive.
However, notice that acidic foods (eg. canned tomatoes) don't
let spores grow.
 Botulinum toxin is heat-labile (destroyed by cooking).
 Classically you see foodborne botulism affecting adults with
home-canned food (contains pre-formed toxin). Alternatively,
infant botulism is generally seen after ingesting unpasteurized
honey (contains spores).
 Note that the adult foodborne version involves eating
preformed toxin without an active infection (the spores
are killed in the GI tract), while the infant version
involves an active infection (gut isn't acidic enough to
kill the spores) that doesn't contain preformed toxin.
 CMBMRS says the adult form is more dangerous
(respiratory failure vs. floppy-baby).
 There's a rare wound-involved botulism form.
 Inhaled botulinum toxin makes a reasonably good candidate for
bioterrorism (see next LO).
C. perfringens:
 Causes wound and tissue infections, including gas gangrene.
Can also cause food poisoning.
 Grows very fast (doubling time = 8 minutes).
 Perfringens produces real nasty exotoxins. The pivotal one is
alpha toxin:
 Cleaves cell membranes (mimics phospholipase C). In
particular it targets vascular endothelium, lymphocytes,
platelets, and muscle cells.
 Perfringens spreads by killing cells, then moving into the
dead area.
 Generally you get perfringens from traumatic injuries; can also
get it from seeding of normal flora.

Progression of wound infection: cellulitis to fasciitis to
myonecrosis (aka gas gangrene). In the last stage you can feel
gas bubbles under the surface of the skin, due to the large
volumes of CO2 formed during the destruction of host cells.
 Some perfringens species cause food poisoning, often in cooked
food (boiling process deoxygenates the food and activates the
spores). Perfringens, unlike botulinum, has no trouble
colonizing your gut, and causes a watery, generally self-limited
diarrhea for a few days.
 C. difficile:
 Mainly involved in hospital-acquired diarrhea and
pseudomembranous colitis due to broad-spectrum antibiotic use
(allows overgrowth of normal flora).
 A/B toxins: transfers glucose from UDP-glucose onto a variety
of GTP-binding proteins and inactivates them; this screws with
the actin cytoskeleton and causes cell death.
 Treat C. diff patients with metronidazole and isolate them (easy
to transfer difficile due to colonization of fomites).
4. Discuss the likely means by which botulinum toxin would be used as a
"bioweapon".
 Growing botulinum toxin is extremely easy. It's extremely lethal. Bad
combination.
 It can also be aerosolized (toxin, not organisms); it can then be
absorbed across the lung mucosa into the blood. This would be (and
has been) the favored route of weaponization.
 While it wouldn't necessarily kill the targets, it would require intensive
hospitalization.
Respiratory Viruses I
Tuesday, February 10, 2009
7:27 AM
Respiratory Viruses I, 2/10/09:
[Orthomyxovirus: large, enveloped, single (-) strand RNA virus. Like the rotavirus it
has a segmented genome and is capable of genetic reassortment. Each segment
encodes a single protein. It brings its own RNA-dependent RNA polymerase, like
most SS (-) RNA viruses.]
1. Explain how influenza virus, which replicates in the respiratory epithelium, causes
severe systemic symptoms.
 They promote cytokines, chemokines, and interferon release-- cause
aches and fever.
 [Note that you shed flu in respiratory droplets a day before you get
symptoms. Incubation period is about 2 days, very rapid. This is why it
spreads so fast.]
2. Describe the influenza virus hemagglutinin (HA) glycoprotein and its role in
infection and disease. Explain how protease cleavage of HA affects virus virulence,
and how changes in antigenicity of HA occur and affect vaccine strategy.
 Hemagglutinin (HA): trivalent envelope "spike" (makes up 95% of
the spike proteins).

So the entire virus (envelope and all) is endocytosed by the host cell
after binding to the host's surface sialic acids. This is a little different
from most of the enveloped viruses we've talked about, which merge
their envelopes with the outer cell membrane to gain entry.
 After endocytosis, host proteases inside the endosome cleave the HA
glycoprotein on the surface of the virus envelope. Once the endosomal
pH drops below a certain point, the cleaved HA protein is activated to
fuse the viral membrane with the endosomeal membrane, thus
releasing the capsid into the cytosol.
 Viruses with high pathogenicity have a longer series of basic amino
acid sequences at the HA cleavage site. I think the point of this is that
they cleave especially readily (recall that trypsin, a ubiquitous human
protease, cleaves between the basic residues lysine and arginine) and
are hence more pathogenic.
3. Describe the influenza virus neuraminidase (NA) glycoprotein and its role in
infection and disease. Explain the mechanism of action of the neuraminidase
inhibitors oseltamivir and zanamivir.
 Neuraminidase (NA): tetravalent envelope "spike" (makes up the
other 5% of the spike proteins).
 Binds sialic acid on host cell membranes to facilitate endocytosis; also
allows release of new virions from the infected cell.
 How that part works: the virus sticks to sialic acid residues. NA
cuts off all the sialic acids from the newly-synthesized
membrane proteins of the host cell, effectively denuding the
surface of sialic acid. If it didn't, the virions budding off from
the host cell would just stick immediately back onto the host
cell and re-infect it.
 The -amivir drugs, then, block release of new virions, which limits the
spread and shedding of the virus. Resistance is developing.
4. Describe the unique features in the intracellular replication cycle of influenza
viruses that may be used as targets for drugs. Explain the mechanism of action of
amantadine and rimantadine.
 Amantadine and rimantadine target the M2 protein, an ion pump under
the viral envelope that causes the pH of the endosome to rise (this
seems to prevent the pH from lowering too fast and gives the virus
enough time to prepare for fusion.. maybe?).
 Note that there is widespread resistance to amantadine and
rimantadine and that they're not generally used for flu. Recall,
however, that amantadine has some beneficial effect in
Parkinson's.
 Replication cycle:
 As mentioned, the fusion with the endosome occurs.
 The RNA segments are dumped into the cytosol; they all go
immediately to the nucleus (note this is a big distinction-- most
RNA viruses replicate in the cytosol) and start replicating.
 The reason it's in the nucleus is to make caps on its
mRNAs (it steals them from the host).
 The replicated segments are reassembled and pushed out
through the cell membrane.
5. Explain the epidemiology of influenza including antigenic drift, antigenic shift,
reassortment, interspecies transfer of viral genes, and pandemics. Explain the
significance of global surveillance for influenza strains.

3 types of influenza (A, B, C). The only one that can cause pandemics
is A, and the reason it causes pandemics is that it infects other species
aside from humans (interspecies reassortment)-- B and C are humanhost only.
 Recall that influenza viruses are typed according to their HA and NA
genotypes (thus "H5N1:" 5th type of HA, 1st type of NA).
 Antigenic drift: slow, minor genetic change, generally due to point
mutations and sloppy transcription errors. Causes your regular flu
season epidemic.
 Antigenic shift: rapid, major genetic change, generally due to
swapping of genome segments (generally the ones encoding NA or HA)
between two different types of virus that have infected the same cell
and are undergoing reassortment. The rate-limiting step is the
exposure of the same cell to two different species of virus (which is
why working with chickens when you've got the flu is a bad idea).
Causes big pandemics (big infection rate the first year due to no
immunity, diminishing as time goes on). Tends to be due to
recombination between avian and human influenza.
 The flu can go around the world pretty quickly (1918 being a good
example). If you keep a reasonably close eye on what strains are
coming up, you can figure out what kind of vaccines to make.
6. Describe the current Trivalent, Inactivated influenzavirus Vaccine (TIV) vs. Live
Attenuated Influenza Vaccine (LAIV) for influenza and explain how and why different
strains of virus are selected for inclusion in the influenza vaccine every year.
 TIV: not just a clever name, it really is trivalent and inactivated. Made
out of 3 flu strains that circulated the previous winter (2 A strains, 1 B
strain). Given parenterally.
 LAIV: live and attenuated; viruses are made from recombining NA/HA
genome segments from various strains. LAIV = the best at producing a
strong IgA response. Administered intranasally.
 If your first reaction is "wow, we're really just kind of guessing here
about what to cover, aren't we?" then you're correct.
 Note that influenza vaccines are about 70% effective.
7. Compare the patients most at risk of death from influenza in a normal influenza
epidemic vs. the 1918 influenza pandemic. Describe the basis for concern about a
possible pandemic of H5N1 (bird flu) and how it might be anticipated or prevented.
 Normal influenza epidemic: kills mainly the elderly, people with chronic
cardiac and pulmonary problems, people in nursing homes, people
with renal disease, or diabetes, or immunosuppression. In other words
people with weak immune systems.
 1918 pandemic: killed mainly young adults. As we discussed in Blood
and Lymph, this was due to cytokine storm-- the younger people had a
more extensive intact immune system, hence a more extensive
cytokine response, hence a greater risk of death. The 1918 strains
were particularly effective in triggering cytokine response.
 We're worried about H5N1 because its case fatality is crazy high (>
60%; 1918 flu was 2.5%). Again, it doesn't spread from human to
human as of yet. Look for cyanotic chickens.
Respiratory Viruses II
Tuesday, February 10, 2009
7:45 AM
Respiratory Viruses II, 2/10/09:
[I highly recommend reading CMBMRS and First Aid as adjuvants here.]
1. Compare the viruses that cause primarily respiratory infections. Identify those
that have multiple genotypes and serotypes.
 Rhinoviruses (picornavirus): (+) strand RNA, nonenveloped-- lots of
serotypes.
 Various paramyxoviruses (no relation to orthomyxoviruses): (-) strand
RNA, enveloped
 Pneumovirus (ie. respiratory syncytial virus)
 Metapneumovirus
 Measles (Rubeola)
 Mumps
 Parainfluenza (ie. croup)
 Coronaviruses: (+) strand RNA, enveloped.
 Adenovirus: DS DNA, nonenveloped.
 Respiratory parvoviruses and polyomaviruses.
2. Describe rhinoviruses and their role in common colds, explaining why vaccines are
not practical. Describe the mechanism of action of a drug against rhinoviruses.
 Colds: infection of epithelial cells only; short (1-2 days) incubation
period, like flu. Generally the interferon response is blocked. IgA is
produced, but there are lots of serotypes and the memory IgA
response isn't so great, so reinfections are common.
 The short incubation period is due to the lack of need for a
viremic phase.
 Rhinovirus: acid labile (can't infect intestine), temperature sensitive.
 Like most (+) strand RNA viruses, encode a big polyprotein
which is chopped up by a viral protease to produce a bunch of
proteins. This protease makes a pretty good drug target but
drug resistance pops up quickly.
 All rhinoviruses use only 2 host receptor proteins to bind to
cells.
 Eg.: host cell's ICAM-1 binds to a "canyon" on the
surface of rhinovirus (canyon is too small for
immunoglobulin binding).
 Vaccines aren't practical because there are 100+ serotypes of
rhinovirus.
 Note that certain rhinoviruses can cause more severe lower respiratory
tract infections.
[General notes on paramyxoviruses: enveloped SS, (-) strand nonsegmented RNA
viruses; all cause cell fusion (to get from an infected cell to an uninfected cell
without being exposed to antibodies, etc) with viral fusion (F) protein. Most of them,
except RSV, have a single protein that serves both HA and NA functions (HN
protein). Usually, part a certain point the massively fused, multinucleated giants cells
get too big and die off; however, note that you can sometimes get persistent
infection. See cytoplasmic inclusions of viral proteins and capsids.]
3. Describe parainfluenzaviruses 1, 2, and 3 and their roles in upper and lower
respiratory tract infections. Explain the features needed for a successful vaccine to
prevent serious parainfluenzavirus disease.
 Not much mentioned in lecture, unless I missed it.
 Don't cause systemic infections.
 Type 3: causes pneumonia and bronchiolitis in infants.
 Pretty much everything else she says about them is standard:
stimulate immune responses, clinical syndrome varies by strain, etc.
 For a vaccine, you need to induce IgA-mediated immunity with longterm memory in the respiratory tract. Hasn't yet been done.
 Reinfection is common.
4. Describe the roles of respiratory syncytial virus in respiratory disease in infants,
children, normal adults, and immunosuppressed patients. Explain the early killed
RSV vaccine disaster, and compare new vaccines, immunotherapy and drugs to
prevent or control serious RSV disease.
 Yearly outbreaks every winter (same time as flu). Causes lower RTIs
and bronchiolitis, predominantly in infants, particularly infants with
cardiac or immunological abnormalities.
 Relatively serious-- hard to vaccinate kids that young, mortality rate
can reach 5.6% (35% in more chronically ill kids). Can cause further
respiratory complications down the line. Note that it's acquired
nosocomially (colonizes fomites).
 Early vaccine: killed the virus in formalin, tried it out in kids, and
several older kids (RSV is usually only seen in kids) died-- massive
viral fusion (evidently it wasn't quite killed after all).
 Drugs: aerosolized ribavirin works okay, but it's toxic. Instead can use
intravenous monoclonal antibodies (passive immunization), but this is
absurdly costly and only given to highest-risk infants as a prophylaxis
(IVIg isn't a treatment, it's a prevention).
 New vaccine involving live attenuated strains are in clinical trials.
5. Describe human metapneumovirus and its pathogenesis.
 Newly discovered in 2001.
 Causes interstitial pneumonia (5-7% of resp. infections) with some
persistent respiratory problems.
 No drugs or vaccines.
6. Describe coronaviruses and their replication strategy. Compare the pathogenesis
of coronaviruses in common colds, pneumonia and SARS.
 Coronavirus: binds to host cells (mainly in upper respiratory epithelia),
uncoats into cell, makes a large polyprotein and is cleaved by a viral
protease, like many (+) RNA viruses.
 Encodes lots of overlapping mRNAs ('nested set').
 Coronaviruses cause 15-30% of common colds.
 Coronavirus causes epithelial disruption even in subclinical infections.
 SARS is a coronavirus. Insidious onset, high mortality in the elderly
(but note it was milder in children)-- causes ARDS due to interstitial
pneumonia.
 Note that certain strains of coronavirus can infect lower respiratory
tract as well.
7. Analyze how SARS coronavirus jumped from wildlife to humans and caused a
global pandemic.
 Evidently: bats -> Himalayan palm civets -> humans. Don't eat civets
who eat bat crap, that's the lesson.
8. Describe the discovery of new respiratory viruses such as WU (polyomavirus),
boca (parvovirus), human rhinovirus group C.
 WU: polyoma virus (other notable polyoma virus: JC virus, causes PML
in immunosuppressed). It has an oncogenic potential. Its role in lung
disease is poorly defined.
 Bocavirus: parvovirus. Has been found in up to 5% of children
hospitalized with respiratory disease and 0.5% of patients with
diarrhea. Associated with bronchiolitis, pneumonia, and asthma
exacerbations. Usually a co-pathogen.
 Group C rhinovirus: can cause lower respiratory tract infections.
Mycology I: Superficial Fungal Infections
Tuesday, February 17, 2009
9:44 AM
Mycology I: Superficial Fungal Infections, 2/17/09:
[The Miller Time guy returns. Be warned.]
[CMBMRS is much, much better at explaining most of this. FYI.]
[General notes:]
 Rounded single cells, with or without budding: yeasts.
 If they split off to the side, that's budding. If they split down
the middle, that's fission.
 Can form pseudohyphae (eg. Candida): separate organisms
lining up to look like hyphae but ain't. "Hyphoid," I guess.
 Candida, Cryptococcus.
 Hyphae (filamentous forms): molds.
 Aspergillus, Rhizopus.
 Fungi that are yeasts in tissue but molds in environment: dimorphic.
 Species that cause blastomycosis, histoplasmosis,
sporotrichosis, etc. Complete list is two lectures down
("Systemic Fungal Infections").
 Harrison's: fungal infections from species that aren't normal flora
(endemic mycoses) are generally acquired from inhaled spores.
Infections from normal-flora species (opportunistic mycoses) are
generally spread, like most opportunistic infections, from other sites in
the body.
 Structure: note ergosterol is in the cell membrane (not in the rigid cell
wall). Most antifungals either bind to this (nystatin, amphotericin B) or
disrupt its synthesis (azoles and echinocandins).
 Media:
 Used to culture fungi: Sabouraud's agar.
 Somewhat more specific: Dermatophyte test media (DTM).
 Brain-heart infusion agar: used for fastidious species.
1. What are three pathology stains that are routinely used to identify fungal
organisms in tissue?
 [Not pathology stains but useful:]
 KOH (potassium hydroxide) 10-20%, sometimes with DMSO.
 India ink is used to demonstrate capsules.
 Wood's light used for fluorescent species, but declining in use.
 PAS-D (periodic acid-Schiff with diastase): stains fungi magenta. Most
common. Fungi stain purplish-red.
 Silver stain (GMS): used for all fungal species, though not great for
small yeast. Fungi stain black.
 Mucicarmine: used to identify Cryptococcus. Capsules stain pink.
 Note he mentioned that it would be a good idea to know these pretty
well.
2. What are the three genera of dermatophytes?
 Epidermophyton (least common, only 1 species)
 Microsporum (2nd most common)
 Trichophyton (most common)
 What they all have in common: they all need to eat keratin to survive.
That would be the definition of a dermatophyte.
3. What is the habitat of the dermatophytes and how does this affect the
epidemiology of infection?
 Can live in soil, animals, or humans-- they just need to be somewhere
they can eat dead skin. You can thus get dermatophyte infections from
more or less anything.
 Tinea: clinical syndrome of dermatophyte infection (generally ringlike,
raised, erythematous lesions). The name "tinea" is usually followed by
the Latin word for location, eg. tinea capitis (on the head, ie. scalp).
 Types: capitis, corporis (body; note tinea corporis =
"ringworm"), cruris (crotch), pedis (ie. athlete's foot), unguium
(nails; note tinea unguium = onychomycosis).
 Corporis: look for annular (ringlike) lesions on the body.
 In 10-20% of corporis cases, the organisms fluoresce under a
Wood's light.
 Note that tinea corporis that goes down into follicles is called
Majocchi's granuloma. For some reason he finds this vastly
important.
 Note that you don't want to give steroids for tinea corporis
(makes them grow faster).
 Capitis is almost always in kids. Cruris is almost always in men.
 Note that the scrotum is not involved in tinea cruris.
 Tinea pedis: likes the inter-toe spaces, particularly around the
4th toe. Causes hyperkeratosis. Note it can ascend to instep
and up legs. It can affect one hand but not both-- so two feet
and one hand = tinea pedis.
 Most common organism for tinea capitis: Trichophyton
tonsurans.
 Most common organism for all other tineas: Trichophyton
rubrum.
 Onychomycosis: very common, particularly in older patients-hard to treat (it can get under the nail plate and eats it from
underneath).
 Id reaction: immunogenic eczema. Caused by the immune system
attacking the skin due to result of fungal infection.
 Note that dermatophytes don't invade living tissue unless you're
immunocompromised.
4. What is the preferred food substrate of dermatophytes and how does it affect the
presentation of clinical disease?
 As mentioned, keratin. They're generally only found on the skin.
 Note that tinea syndromes can be immunogenic or nonimmunogenic.
5. What is the preferred food substrate of Candida albicans and how does it affect
the presentation of clinical disease?
 Candida: normal flora, particularly on mucus membranes.
 It doesn't eat keratin, it eats glucose (thus watch out in diabetics). He
said it also eats "serum", which doesn't make a lot of sense to me
unless it's where you find glucose. Anyway, it needs to get deeper
than dermatophytes to feed.
 Technically a dimorphic fungus (produces yeast and pseudohyphae).
 Predisposing factors to candidiasis: antibiotics, immunosuppression,
birth control, etc.
 Classic candidiasis presentation: primary site of infection with
pustular satellite lesions spreading out from there. Miller-Time
approved.
 Can show up between fingers.
 Can show up as a diaper rash (note that unlike tinea, Candida
does affect the scrotum).
 Can show up in oral cavity as thrush.
 If the immune system doesn't recognize Candida, it's bad news
(widespread candidiasis).

Treatment: for superficial infection: nystatin. For systemic infection:
fluconazole.
6. What is the preferred food substrate of Malassezia furfur and how does it affect
the presentation of clinical disease?
 Normal flora; eats sebaceous lipids and thus has to be grown in lipidsupplemented media.
 Causes tinea versicolor: asymptomatic tan-colored slightly scaly
patch, usually on the trunk. Sometimes it can be hypopigmented.
Generally a bigger problem in more humid climates.
 "Spaghetti and meatballs" on KOH stain. He mentioned that we should
know that this is the best way to pick it up (along with clinical
presentation). ("tape prep:" stick some tape on a patient, pull it off,
KOH stain it.)
Mycology II: Localized Fungal Infections
Tuesday, February 17, 2009
11:01 AM
Mycology II: Localized Fungal Infections, 2/17/09:
[General notes:]
 Note that most of these conditions have a history of trauma (to
introduce the organism underneath the skin).
 Sporotrichosis: from Sporothrix schenckii, a dimorphic fungus; found
specifically in peat moss, roses, and cats, but can be in almost any
organic material (wood, hay, etc).
 How it usually shows up: ulcerated primary infection site at site
of trauma with subsequent abscesses following lymphatics.
Boards love this.

Sometimes (20%) you can get an infection without lymphatic
spread.
 Some risk (1%) of dissemination: usually severe alcoholism.
 It's dimorphic and fastidious: at high temperatures it's a yeast,
at lower temperatures it's hyphae.
 On stain: right-angle stalks in hyphae phase. In yeast phase:
it's "cigar-shaped" (elongated). Again, this is good
exam/boards stuff.
 Lots of good treatments for localized disease: standard is
itraconazole. For disseminated disease you have to used
amphotericin B.
 Eumycotic Mycetoma (ie "Madura foot"):
 Generally develops very slowly-- subcutaneous nodule,
following trauma, progressing to fistulas in the skin that drain
purulent, grainy exudates. The "grains" are collections of fungi
and can be one of a variety of colors.
 Species that cause this are generally found in the soil.
 On histology, see huge collections of fungi (the "grainy" in the
exudate) surrounded by neutrophils.
 Hard to manage.
 Chromomycosis:
 Caused by lots of different yeastlike species; they all produce
melanin (thus produce pigmented colonies).
 After trauma, growth of red papule into nodule/verrucous
growth/ulceration.
 Can cause elephantiasis (damage to lymphatics) and death.
 Again, slow-growing and hard to treat.
 On histology, see "copper pennies" or "Medlar bodies".
 Phaeomycosis:
 Not much distinctive-- trauma produces purulent abscess.
Seems to be mainly from puncture wounds?
 On histology, pigmented brown hyphae.
 Usually can take out surgically.
 Lobomycosis:
 Seems to live mainly on dolphins. Don't play with dolphins with
fungal infections.
 Looks like keloids (hard scarred-up overgrowth). Once you
can't surgically excise them, there's no cure.
 Endemic to tropical areas.
 Rhinosporidiosis:
 Causes large polyps, mainly intranasal, studded with white
fungal grains. Cut them out.
 Organisms are big enough to be seen with the naked eye.
 Endemic to tropical areas, generally lives in water.
 Protothecosis:
 Not actually a fungus-- it's a pathogenic algae.
 Prototheca is the genera that causes disease. Generally picked
up by cleaning aquariums.
 Primary ulcer following trauma-- causes ulcerated plaque,
mimics localized fungal diseases. Looks like a blastocyst.
 Can treat with antifungals.
1. What are the ecological habitats of localized cutaneous fungal infections and how
do they typically gain access to the body?

They live in ecological habitats, generally organic. They're nearly all
introduced by trauma.
2. What is meant by a sporotrichoid infection and what is the clinical differential
diagnosis?
 As above; can also be Nocardia, non-TB mycobacteria, or tularemia.
3. What is a dematiaceous fungus and what are three localized fungal infections that
can be produced by a dematiaceous species?
 Dematiaceous: pigmented fungi.
 Eumycotic mycetoma (various colors), chromomycosis (melanin),
phaeomycosis (brown).
4. Name the only localized cutaneous infection produced by an organism that is
classified as an algae and how do people acquire this infection?
 Protothecosis. Cleaning aquariums.
5. What are three clinical diseases that are the result of fungal hypersensitivity
reactions?
 Fungal spores are everywhere. They're frequently inhaled.
 Deposited fungal spores can cause either immunoglobulin-mediated
(types I and II) or T-cell-mediated (type IV) hypersensitivity reaction.
 3 diseases:
 Allergic rhinitis (hay fever)
 Bronchial asthma
 Alveolitis
6. What is a mycotoxicoses? What is an example of human mycotoxicoses and how
does it produce disease in humans?
 Accidental or intentional ingestion of toxic fungal products (eg.
alkaloids).
 St. Anthony's fire: produced by a fungus that infects grain. Produces
ergot alkaloids: these cause massive peripheral vasoconstriction
(notes say it's alpha-adrenergic blockade, which I don't think is right),
necrosis, and gangrene.
Mycology III, Systemic Fungal Infections
Wednesday, February 18, 2009
7:59 AM
Mycology III, Systemic Fungal Infections, 2/18/09:
1. What are dimorphic fungi? What are the classic five fungal infections produced by
dimorphic fungi?
 As mentioned, they live in yeast form at body temperature but hyphae
form at room temperature.
 Five classical dimorphic infections: one localized and four systemic.
 Sporotrichosis (mentioned in the last lecture)
 Blastomycosis
 Coccidioidomycosis
 Paracoccidiomycosis
 Histoplasmosis
 Most of the systemic fungal infection species are dimorphic.
2. Compare the ecological niches of blastomycosis, coccidioidomycosis,
paracoccidioidomycosis and histoplasmosis and how does this affect the distribution
of infection?
 Blastomycosis: Mississippi/Ohio river valleys. Found in the soil; also
in prairie dogs.
 Blastomycosis: very broad, thick-walled, budding yeast (in
yeast phase).
 Generally acquired through inhalation and usually causes
pneumonia.
 When it disseminates, it tends to go to bone, skin, and
prostate.
 Coccidiodomycosis: in the Southwest. Found in the soil.
 Hyphae form grows very rapidly; forms "alternating barrelshaped arthrospores"-- little, joined, articulated barrels. In the
body they form large spherules that are made up of
conglomerated yeast.

Again, acquired through inhalation and usually causes
pneumonia.
 When it disseminates, it tends to go to bone and is frequently
fatal.
 Paracoccidiodomycosis: Confusingly, this seems to be a South
American blastomycosis. Found in soil, particularly everywhere you
grow coffee.
 "Pilot's wheel" or "Mickey Mouse" appearance (see p. 248 of
notes for illustrations) due to narrow-based budding off yeast.
 Again, acquired through inhalation and usually causes
pneumonia.
 When it disseminates, it goes to the adrenal glands and/or
mucosal surfaces.
 Histoplasmosis: Mississippi/Ohio river valleys and caves (bat feces).
Found in the soil, requires a high nitrogen content to grow (which is
why it likes bird droppings).
 Hyphae are very thin and branch frequently. Note that despite
the name (Histoplasma capsulatum) it does not have a capsule.
 Note that on H+E, you find it inside macrophages (this is
prime boards stuff)-- numerous tiny spores inside the
macrophage.
 Once again, acquired through inhalation, tends to cause a flulike illness or pneumonia.
 When it disseminates, it attacks the oral mucosa and the
adrenal glands.
 Note that none of these infections are spread from person-to-person
(unless you stab a syringe into someone).
 Note also that you tend to make these diagnoses on clinical
presentation and KOH.
 [CMBMRS points out that these diseases look a lot like TB: inhaled,
primary infection in the lung, can cause lung
granulomas/cavitations/calcifications, can disseminate through the
blood, and have a skin test that's similar to a PPD.]
3. List four types of patients that are particularly susceptible to disease from
opportunistic fungi and be able to explain why they are susceptible.
 Treated for chemo




[Other fungal

HIV/AIDS
Organ transplant recipient
Diabetic ketoacidosis
(based on his lecture/notes-- they're all pretty self-explanatory.)
organisms that cause systemic disease:]
Cryptococcus neoformans: Yeast, not dimorphic; worldwide
distribution. Found in the soil, particularly in pigeon droppings.
 Acquired through inhalation but doesn't cause pneumonia;
instead it disseminates to cause meningoencephalitis.
 Unlike Histoplasma, it does have a capsule, which means it can
be picked up on India ink stain (how it's identified). Another
good boards piece.
 You get a CSF tap, stain with India ink (won't stain
capsule), and look for big clearings around the yeast.
Can also use mucicarmine (stains capsule pink).
 If it disseminates (generally to CNS), mortality even with
treatment is 15-20%.
 Aspergillus: Mold, not dimorphic; worldwide distribution. Really,
really hard to get rid of.
 Hyphae: acute-angle branching (angles around 45 degrees).
 Acquired through inhalation. Does three potential things:
allergic bronchial reaction, infects pre-formed lung cavities (see
next point), and causes invasive
 Boards like the fact that Aspergillus colonizes lung cavitations
from pre-existing tuberculosis.
 Grows extremely fast and kills immunocompromised patients
very quickly. Likes to infect IV sites.
4. Why are diabetics in ketoacidosis prone to develop mucormycosis?
 Rhizopus and Mucor species: Mold (not dimorphic) but not septate
(no walls in the hyphae).
 Grows extraordinarily rapidly. Large, ribbon-shaped hyphae
without septae; where it does branch it does so at right angles.
 Tends to infect the blood vessels of sinuses and frontal lobe,
but can grow anywhere.
 It really, really likes diabetics, high temperatures and high
concentrations of glucose; it also can grow at low pH and will
eat ketones. You see a diabetic in ketoacidosis with sinus
infections, that's mucormycosis.
5. Describe the pathogenesis of candidiasis and explain how the various symptoms
reflect the habitat of the fungus.
 Candida: as mentioned, a normal-flora yeast that can form
pseudohyphae.
 In normal patients, you can get oral thrush, vaginitis, and diaper rash,
usually only after systemic antibiotic use.
 Tends to only grow out of control in immunocompromised patients,
causing esophagitis, endocarditis, etc (goes to all organs, including the
CNS).
6. Describe the pathogenesis of pneumocystosis and the methods used for its
diagnosis and treatment.
 Pneumocystis: Used to be classified as a bacteria (ie. PCP pneumonia)- turns out to be a fungus without an ergosterol layer and was
renamed Pneumocystis jiroveci.




Usually causes pneumonia, only in immunocompromised
patients.
Generally you look at CXRs and sputum stains.
Histologically, they look round with a "cap."
Treat with Bactrim-- note you can't use the typical antifungals
that target ergosterol.
Antifungals
Wednesday, February 18, 2009
9:02 AM
Antifungals, 2/18/09:
[Essentially "know most things in my handout and everything that's bolded."]
[General notes:]
 Amphotericin B: polyene structure derived from streptomycin.
 MoA: bind to ergosterol, then forms pores in the fungal
membrane. Fungicidal.
 Mechanism of resistance (MoR): thicker membranes/less
ergosterol production. Generally only found in Candida species.
 Pharmacokinetics (PK): has to be given IV. Note it doesn't cross
the BBB (but can be injected intrathecally for CNS infections).
Renally excreted.
 Adverse: renal toxicity, hypotension, arrhythmias, fever,
anemia, phlebitis.
 Note there's a liposomal formulation with no renal
toxicity. Much pricier.
 Generally used only in severe systemic infections: acts very
quickly but has extensive and severe side effects.
 [Note there's a nonabsorbed topical/oral amphotericin
formulation called nystatin used for topical Candida infections
(oral thrush, vaginosis, etc).]
 Imidazoles/Triazoles (azoles):
 MoA: block synthesis of ergosterol (stop conversion of
lanosterol to ergosterol). How they do this is to block the
CYP450 system in fungi (note some cross-reaction with human
CYP system). Fungicidal or fungistatic depending on
concentration.
 MoR: mutations at binding sites.
 PK: Clotrimazole and miconazole (two imidazoles): topical or
oral, no systemic absorption. All others: orally absorbed.
Fluconazole is the only azole that goes to the CSF. Metabolized
by the liver.
 Adverse: mostly GI upset. Can get rare hepatotoxicity and
more common testosterone synthesis inhibition. Note that the
azoles are CYP inhibitors and are one of the drugs that causes
gynecomastia (inhibits CYP breakdown of estrogens). Dr.
Churchill simply says they cause "hormone-related problems."
The azoles are contraindicated in pregnancy (teratogenic).
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Clotrimazole and miconazole are used for topical infections, like
candidiasis and dermatophytes.
 Fluconazole is used for meningeal fungal infections (generally
Cryptococcus); others are used for lots of different stuff.
Terbinafine:
 MoA: blocks synthesis of ergosterol, but at a different point
than azoles (stops conversion of squalene to lanosterol).
Fungicidal.
 PK: Oral or topical.
 Adverse: rare; can get agranulocytosis.
 Used for dermatophyte infections (generally ones resistant to
topical treatment), in particular deep nail infections
(onychomycoses). Works very slowly (3 months of treatment
are necessary).
Echinocandins (caspofungin and micafungin)
 MoA: block synthesis of the beta-1,3 glucans in the fungal cell
wall. Fungicidal.
 (hokey mnemonic: glucans are sugars, which are sweet
like candy. Get it? candy? Because it's echinocandins?
See?)
 Pharmacokinetics: have to be given IV. Metabolized by the
liver.
 Adverse: mild; can cause flushing.
 Used for refractory Candida and Aspergillus infections.
Flucytosine:
 MoA: converted to 5-fluorouracil in fungal cells, which inhibits
fungal DNA/RNA synthesis. Fungicidal.
 PK: oral absorption, gets into the CSF very well.
 Adverse: has some activity in human cells as well (can cause
chemo effects, notably bone marrow suppression).
 Used for cryptococcal meningitis.
Griseofulvin:
 MoA: blocks microtubule synthesis, thus stopping mitosis.
Fungicidal or fungistatic, depending on concentration.
 PK: absorbed orally; absorbed better with fatty foods. Binds to
keratin and goes wherever keratin goes (which is why it's used
for dermatophytes).
 Adverse: headaches.
 Used for dermatophyte infections (generally ones resistant to
topical treatment); however, it works very slowly. Generally it's
been replaced by terbinafine.
[Note that the only antifungals that cross the BBB are fluconazole and flucytosine
("one flu over the BBB?" Ok, I'll stop now.).]
[Note also that the only ones that have to be administered IV are amphotericin and
the echinocandins.]
[Note further the ones that aren't systemically absorbed: nystatin, mitoconazole,
clotrimazole.]
[CMBMRS notes that potassium iodide is used to treat sporotrichosis infections.]
Hepatitis I + II
Monday, February 23, 2009
10:00 AM
Hepatitis I + II, 2/23/09:
1. Describe the presenting symptoms of patients with acute viral hepatitis. Why is
the patient jaundiced?
 Note that acute clinical symptoms are similar regardless of the type of
Hep virus:
 nausea, vomiting
 abdominal pain
 loss of appetite
 fever
 diarrhea
 light-colored stools
 dark urine
 jaundice (elevated serum bilirubin due to its release from
damaged liver cells)
 Notice that hepatitis viruses, by and large, are not cytotoxic. The
damage to the hepatocytes is generally a host-immune-mediated
response (mainly killer T cells).
2. Name the viruses that cause hepatitis, describe their molecular features, and
describe their modes of transmission.
 A and E: oral-fecal (FA: "the vowels go through the bowels."), acuteonly.
 Note that oysters, since they're filter feeders, tend to pick up a
lot of oral-fecal viruses (noroviruses, hep A, etc). Don't eat raw
oysters unless you're prepared to chuck your liver and spend a
week on the toilet, that's my motto.
 B, C, and D: bloodborne, can be chronic.
 D and E are extremely rare in the US.
 Hepatitis D needs hepatitis B co-infection to be itself infectious.
 Hep A:
 Picornavirus: naked (non-enveloped) icosahedral virus with
single-stranded, (+)-strand RNA-genome. Destroyed by
heating.
 Sheds like nuts into the feces.
 Note that acute infections are often clinically inapparent,
particularly in kids.
 Very low mortality associated with Hep A; what there is, is
generally due to fulminant hepatitis (more or less complete
liver destruction) secondary to underlying conditions. Note that
there's no chronic disease associated with Hep A.
 There's only one serotype of the virus-- thus the vaccine is
pretty straightforward. Note that this also means that you can
only get Hep A once.
 Active vaccine: inject killed virus. Use for inducing
immunization before exposure and also for prevention
after exposure.
 Passive vaccine: use human antibodies against HAB. Use
only for prevention after exposure.
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Note Hep A, although it causes acute illness, has a reasonably
long incubation period (30 days). If you catch someone who's
exposed fairly soon afterwards, you can use both active and
passive immunization to protect them.
 To diagnose, look for IgM anti-Hep A, which rises at about the
same time as clinical symptoms. This illustrates the fact that
hepatic damage arises from the host's immune response as
opposed to the viral replication itself (the patient has growing
viremia for several weeks before the IgM and liver enzymes
rise).
Hep E:
 Calicivirus: naked, icosahedral, single-strand, (+)-strand RNA
virus.
 Apparently it's now classified as a "hepavirus" (only
virus in this group).
 Generally associated with water-borne outbreaks, which may
be why it's not a big player in the US (better water
purification).
 No vaccine, no specific therapy. Causes an acute, self-limiting
infection, just like Hep A. However, E is more pathogenic and is
more frequently fatal.
 Hepatitis E is associated with a high frequency of fulminant
hepatitis in pregnant women. No Fooling.
 Again, a long incubation period and increased illness severity
(again increasing with age, like Hep A).
 Note that there's only one serotype of Hep E, so (to paraphrase
our last president) "infect me once, shame on.. .. uh.. well, you
won't infect me again."
 Make diagnosis with IgM anti-Hep E or get PCR of stool. Again,
rise in IgM correlates with the appearance of clinical symptoms
and elevated enzymes.
Hep B:
 Hepadna virus: enveloped, icosahedral, double-strand DNA
virus.
 Actually a little more complicated: it's a partially-singlestranded, partially-double-stranded genome with a
polymerase covalently attached.
 Upon infection and DNA injection, host cells lop off
polymerase and "repair" the weird-looking DNA to be
entirely double-stranded.
 Host RNA pol II starts making mRNAs to be transcribed
(surface, core, polymerase, etc proteins).
 Host RNA pol II also makes a "pregenomic" RNA which
transcribes the entire genome into RNA and sticks it into
capsids along with the lopped-off polymerase.
 It's kind of weird, given that the genome is DNA, that
RNA gets packaged into the virions. However, the
lopped-off polymerase is a reverse transcriptase
which makes viral cDNA from the RNA copy before the
virion buds off from the cell.
 To repeat: Hep B replicates through a process involving
both RNA transcription and reverse transcription. Boards
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likes this. Note that there's some crossover efficacy from
HIV drugs in Hep B infection.
Causes both acute and chronic disease.
Boards just loves all this following stuff, so pay attention.
Neutralizing (protective) antibodies are against the surface
antigen (spike proteins protruding from envelope).
Note that rate of chronic disease tends to vary inversely with
the extent of acute symptoms-- if the patient doesn't mount a
significant immune response to the initial infection, you get less
symptoms (again, immune response is responsible for the
clinical symptoms) but the viral infection remains in the liver
and results in chronic infection (and a chronic low-level immune
response that keeps killing off liver cells). The repeated
destruction and regeneration in the liver leads to hepatic
fibrosis (cirrhosis) and is a risk factor for hepatocellular
carcinoma.
Generally you don't treat immunocompetent adults with acute
Hep B (most of them will clear it by themselves).
You should treat infectious patients (with detectable HBeAg,
see below) with mildly elevated ALT, or noninfectious patients
(without HBeAg) with severely elevated ALT. Also treat when
there's co-infection with HIV.
 There don't seem to be any anti-Hep B drugs that you
can take once and completely clear the infection-- have
to be on them long-term.
 Treat mainly with anti-retrovirals and interferon.
Kids generally don't show severe symptoms for Hep B, just like
for A and E. However, since B can cause chronic disease, that
means that kids who get infected with Hep B are generally
more likely to go on to develop chronic disease.
"Core protein" is responsible for packaging the genome. You
see IgM anti-BcAg (anti-core antigen) as well. See next point.
How to use various serological markers to figure out where in
the course a patient is:
 Anti-core Abs come up early on but are not protective. If
you see anti-core but not anti-surface Abs, the patient is
infected and hasn't been able to fight it off yet. IgM vs
core but no anti-HBsAg = acute infection; IgG vs core
but no anti-HbsAg = chronic infection (the patient has
had it for a while but doesn't have the protective antisurface antibodies which would indicate that she has
immunity).
 Note also that the core antigen isn't part of the vaccine,
so if a patient has anti-HBsAg but no anti-HBcAg,
they've been vaccinated.
 "E" antigen isn't part of the viral particle, nor is it a
surface antigen. However, being positive for "HBeAg" is
indicative of infectivity (again: HBeAg = infectivity).
 Note what chronic infection looks like: HBsAg, no IgG
anti-HBsAg, no IgM anti-HBsAg. Probably have some
IgG anti-HBcAg in there.
 Note that he has a handy slide where he seems to sum
up all the things he wants us to know about this. It's
easier than trying to figure this out from me writing
about it. First Aid has a reasonably good table on this in
its microbiology section, too.
 Again, there's only one serotype, so you can vaccinate against
it pretty good.
 Hep D:
 Single-stranded, circular RNA genome (deltavirus).
 As mentioned, it's defective-- it isn't pathogenic unless you
have a co-infection with hepatitis B. That's because it needs to
steal envelopes and envelope proteins from Hep B ("My name is
Hank Herpes and I sell envelopes and envelope accessories").
 Hep D only encodes one gene, for the delta antigen. Involved in
packaging the genome. Uses all-host machinery to replicate
(uses host DNA polymerase to replicate its RNA, not sure how
that works).
 Packaged in a hepatitis B lipid envelope with hepatitis B surface
antigens.
 Note that vaccination against B thus also confers immunity to
D.
 Note also that the only protective immune response to Hep D is
anti-HBsAg. Thus people who can't clear hep B generally can't
clear hep D either-- so, like B, it can cause a chronic disease.
 Note further that the diagnostic test for HDV is specific for Hep
D-- looking for antibodies to the delta antigen.
 Hep C:
 Flavivirus: enveloped, single-strand (+)-sense RNA virus. 6
genotypes and lots of serotypes.
 Note there is a much higher rate of acute infections being
converted to chronic infections in Hep C than Hep B-- 50-80%
of acute infections will progress to chronic infections, largely
because the acute immune response is so weak (see next
point).
 Anti-HCV antibodies are not protective; thus no vaccine exists.
 Chronic infection frequently (up to 20%) leads to cirrhosis, liver
failure, and/or hepatocellular carcinoma. Generally this is a
long, long time course (20+ years). Note that about a third of
patients with chronic Hep C never have any symptoms.
 Hep C is the most common reason for liver transplant in the US
(the treatment for Hep C that isn't curable by drug therapy, see
below, is a liver transplant).
 Ribavirin: upregulates rate of mutations in the viral RNA
genome. As a monotherapy it has no efficacy.
 Interferon: as a monotherapy it has no efficacy.
 Ribavirin + interferon have synergistic actions and are
effective.
 Different genotypes of Hep C respond differently to
treatment. Anywhere from 40-80% of infections can be
cleared with dual therapy.
 Note this therapy runs about half a year or a full year
with a weekly injection, depending on the genotype of
the virus.
3. Name the hepatitis viruses for which there are vaccines, describe the antigens
used in the vaccines, and explain which populations should be vaccinated.
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A - uses killed virus. There's also a passive (antibody) vaccine.
B - uses HBsAg (surface antigen). There's also a passive (Ab) vaccine.
 Hep B can be transmitted vertically (mother-to-infant) at birth
(perinatally, not in utero). Immediate Hep B vaccination at birth
(both passive and active) prevents infections.
 C - no vaccine.
 D - same as for B.
 E - no vaccine.
4. Explain how active and passive immunization for HAV can be used under different
circumstances and why both types of vaccination are sometimes necessary.
 If they already have symptoms, then they're already making
antibodies to HAV (that's what's causing the symptoms) and, as far as
I can tell from his notes, only supportive treatment is warranted (no
vaccination).
 If they're very recently infected, you want to use both active and
passive immunization, since the passive antibodies will help stop initial
spread and the time to ramp up a neutralizing immune response from
the active vaccine can be shorter than the incubation time of the virus.
 If they're not infected and you just need prophylaxis, you don't need
to use passive immunization, just active.
5. Understand the molecular basis of laboratory tests used to diagnose specific
hepatitis viruses.
 You're looking for various hepatitis antigens and the antibodies to
them. Any questions?
6. Describe circumstances when HBV can cause chronic infection (especially in
neonates), the pathologic consequences of chronic infection, and how HBV vaccine
and HBIG can prevent infection (especially in neonates).
 As mentioned, HBV tends to cause chronic infection when the initial
immune response, and thus clinical symptoms, are mild.
 In neonates, perinatal infection occurs most often when the mother
has active, infectious (HBeAg-positive) HBV.
 Pathologic consequences: cirrhosis and hepatocellular carcinoma.
 The HBV vaccines (active and passive) can prevent disease in
neonates since it's acquired during birth and it has quite a long
incubation period (allowing the kid to ramp up an immune response to
neutralize the virus before it breaks out).
7. Describe HCV infections in patients, the spectrum of disease associated with HCV
infections, and the outcomes of interferon and ribavirin therapy in HCV infected
patients.
 As above.
 Note that Hep C is virtually never transmitted through sexual contact-pretty much blood to blood only. Hep B, on the other hand, is more
frequently an STD.
8. What treatments are available for patients with chronic HBV? What treatments are
available for patients with chronic HCV infections? What patient specific factors (age,
weight, viral load, degree of cirrhrosis, etc.) affect whether to treat (or to monitor
the patient without antiviral therapy) and how to treat the patient?
 For HBV: anti-retrovirals, interferon, some other antivirals.
 For HCV: ribavirin and interferon.
 HBV: as above, treat either mildly elevated ALT with HBeAg or
severely elevated ALT without HBeAg.
Zoonotic Bacterial Diseases
Tuesday, February 24, 2009
7:52 AM
Zoonotic Bacterial Diseases, 2/24/09:
[General notes:]
 Zoonotic infections in the animal host are frequently asymptomatic.
 Diagnosis of these diseases is tricky-- they're rare, their presentation
looks a lot like other diseases, and they can be fatal.
1. Understand societal factors leading to the emergence of zoonotic infections.
 Agricultural and pastoral; with the migration to the city they've
become largely occupational (farmers, vets, slaughterhouse workers,
lab workers, etc) and avocational (pet owners,
hikers/hunters/trappers/fishermen).
 However, note that destruction of animals' habitats causes things that
live there to come to the city.
2. Learn major pathways of transmission of zoonotic infections (e.g. direct contact or
vector borne)
 Cutaneous transmission, including bites
 Arthropod vector
 Inhalation
 Ingestion
3. Recognize the individuals who are at greatest risk for zoonotic infections.
 As above (occupations and avocations).
4. Become familiar with major aspects of the historical origins, microbiology,
epidemiology, clinical features, diagnosis, treatment, and prevention of four
important zoonotic infections:
 Plague:
 Caused by Yersinia pestis (facultatively intracellular Gramnegative rod).
 It's generally accepted as the etiological agent for the Black
Death in Europe.
 There were small pandemics up until about 1920, when the
whole rats-on-ships thing was outlawed.
 Spread by fleas (in CO, watch out for dead prairie dogs).
 Discovered by a guy named Yersin (thus Yersinia).
 Types of plague:
 Bubonic plague: most common form of plague in the
US; lymph gland swelling resulting from the bite of a
flea 2-5 days earlier; 60-90% mortality if untreated.
 Lymph nodes are extremely painful and swollen
(buboes, thus the name); usually inguinal or
axillary.
 Septicemic plague: invasion of almost all organs, no
evidence of prior disease (seems to come after a direct
bloodstream inoculation or consumption of a plaguestricken animal); death occurs in 12-24 hours.
 Presents with ecchymoses, petechiae, and DIC.
Pneumonic plague: least common but nasty
(transmitted human to human through aerosolized
droplets). Primary or secondary (from other 2 types)
lung infection. Highly infectious and 100% fatal if
untreated.
 Note that bubonic plague, left untreated, can get
into the lungs and become pneumonic plague
(which may be what made the Black Death so..
you know, black and deathy).
 Epidemiological terms of plague:
 Enzootic plague: stable rodent-flea infection (not too
much rodent mortality, long-term reservoir).
 Epizootic plague: occurs when plague bacilli are
introduced into rodent/small mammal populations that
are more highly susceptible.
 Zootic plague: transmission from animals to human.
 Demic plague: transmission from human to human
(pneumonic plague only).
 Microscopically, you see bipolar, "safety-pin" type staining of
Gram-negative rods (the ends stain more than the middle, like
the metal density of a safety pin).
 Note animal reservoirs can include cats, rabbits, camels, etc.
Don't get bitten by sick camels, that's my motto.
 Virulence has to do with how well Yersinia gets into nonendocytosing cells. They can kill or shut down macrophages.
VW and F1 surface antigens cause extra virulence (encoded by
plasmids)-- CMBMRS points these out.
 Pretty amazing-- it effectively starves the flea to make it keep
biting people, then causes the flea to regurgitate the blood
(containing Yersinia) back into the host.
 Can be weaponized (pneumonic form is most lethal).
 Prevention: avoidance.
Tularemia:
 Caused by Francisella tularemia (Gram-negative coccobacillus);
carried by rabbits. Generally found in and around Arkansas and
Missouri (big rabbit-hunting country, apparently).
 Note it requires cysteine for growth.
 Most cases are ulceroglandular (direct contact with infected
animals, causes skin ulcers), with some being pulmonic
(inhalation of aerosolized rabbit, no kidding); can also get it
from tick bites or ingestion of contaminated material.
 The skin ulcers are black and well-demarcated with local
swollen lymphadenopathy.
 Extremely virulent (10 organisms cause disease).
 Tularemia, unlike plague, can't be spread person-to-person.
 Can be weaponized (pulmonary form is most lethal).
 Prevention: avoidance.
Brucellosis:
 Not covered in lecture.
 Mostly acquired in the US from the consumption of
unpasteurized goat cheese from Mexico (you Whole Foods
shoppers).
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Doesn't cause primary skin ulcers or buboes; instead you get
systemic relapsing-remitting flu-like symptoms (undulant
fever). Rarely fatal.
Lyme Disease:
 Caused by Borrelia burgdorferi, a spirochete. Very hard to
culture.
 Spread through Ixodes tick bites.
 Causes arthritis, particularly in the knee, fever, and skin rash
(spreading erythema with a pattern of central clearing, ie
erythema migrans). The skin rash is pathognomic (which is a
fancy way of saying it's Miller time).
 Again, the immune response is what causes the
symptoms-- very high levels of IL-1.
 Note it can present in a whole lot of different ways, but
the most common are erythema migrans, heart
palpitations, and arthritis.
 Infection stages:
 Stage I: localized infection (erythema migrans)
 Stage II: disseminated infection (meningitis, carditis,
myositis)
 Stage III: latent infection (chronic arthritis, neuropathy,
etc)
 Note this can go on for decades if untreated.
 Very difficult to diagnose-- the nymph stage of the tick is the
one that most frequently causes the disease, and they're about
the size of a letter on a penny. Once they've fed on you for a
while they're no longer necessary to cause Lyme disease (it's
not a toxin).
 Note that the probability of getting Lyme disease increases
dramatically with the length of the tick's feeding (72 hours
plus).
 Geographically, see cases mainly clustered in the Northeast and
around Minnesota (which is where the other hosts of the
bacteria, white-footed mice and deer, are). Generally see cases
in kids and the middle-aged.
 Note you can't seem to treat chronic Lyme disease with longterm antibiotic use, evidently largely due to the fact that postinfectious symptoms like the chronic arthritis seem to be
autoimmune. Acute Lyme disease can be treated with
doxycycline. There isn't a vaccine on the market.
 Prevention: tick repellant and prompt removal.
 In general PCR is unhelpful for diagnosis.
Rickettsia and Bartonella
Tuesday, February 24, 2009
7:37 AM
Rickettsia and Bartonella, 2/24/09:
[General notes:]
 Nearly all of these are treated with doxycycline.
1. Describe the major biological characteristics of Rickettsiae and related bacteria.
 Rickettsiae: obligate intracellular bacteria; Gram-negative
coccobacillus (but Gram stains poorly).
 Usually have animal reservoirs and are arthropod-borne (louse/tick).
 Humans are generally incidental-only hosts (except louse-borne
typhus).
 Very small genome; uses host metabolites.
2. Compare the intracellular growth cycles of Rickettsiae and related bacteria.
 Replicate in cytoplasm rather slowly (9-12 hours).
 Can't be cultured on artificial media; lose infectivity outside cells.
3. Describe the pathogenesis and clinical presentations of infections caused by
Rickettsiae and related bacteria.
 Most Rickettsia spp. are pathogenic. More or less all of them do the
same thing: invade endothelial cells and spread through the vascular
system.
 Cause increased vascular permeability, leading to edema,
hypovolemia, and ischemia.
 Usually presents with high fever, headache, and a petechial rash.
 If untreated can have severe consequences.
 Three main groups:
 Spotted fever group (transmitted through wood ticks; = Rocky
Mountain spotted fever)
 Typhus group (transmitted mainly through lice; epidemic
typhus, endemic typhus, Brill-Zinsser)
 Scrub typhus group (transmitted through chiggers)
 Clinical differentiation:
 Spotted fever presents with a rash on the extremities spreading
to the trunk (centripetal spread).
 Typhus presents with a rash on the trunk spreading to the
extremities (centrifugal spread).
 Scrub typhus doesn't present with a rash.
4. Identify and describe the etiologic agents, common reservoirs, and modes of
transmission of epidemic typhus, endemic typhus, Brill’s disease, scrub typhus,
Rocky Mountain spotted fever, and Q fever.
 Rocky Mountain spotted fever:
 Caused by Rickettsia rickettsia.
 Most common rickettsial infection in the US.
 Again, presents with a petechial rash which starts on the
extremities and moves to the trunk.
 Spread by tick bites.
 Reservoir is small rodents.
 Incidence is not actually the Rockies but is instead largely
Arkansas and Missouri, like tularemia.
 Usually hits children (60% of patients under 15).
 Epidemic typhus:
 Caused by Rickettsia prowazekii.
 Reservoirs are humans and flying squirrels.
 Transmission is through the human body louse, which doesn't
bite, but itching smashes the louse into the skin and causes
inoculation. Don't autoinoculate with louse-borne typhus, my
momma used to say.
Note that nits (lice eggs at hair follicles) are a sign of
louse infection.
 Tends to pop up during wars and other natural disasters.
 Presents as a petechial rash on the trunk (the rash can become
necrotic or hemorrhagic); high fever and prostration, renal
failure, stupor. Note a 20-70% mortality rate if untreated
(pneumonia and circulatory collapse).
 Infection and survival confers lifelong immunity.
 There is an effective live attenuated vaccine (usually for
military only).
 Brill-Zinsser disease:
 Also Ricketssia prowazekii and similar symptoms, but milder.
Probably caused by a reactivation of epidemic typhus.
 Endemic typhus:
 Caused by Rickettsia typhi (from fleas that have bitten rats) or
Rickettsia felis (from fleas that have bitten cats).
 Milder than epidemic typhus: low-grade fever, mild headache,
joint pain.
 Scrub typhus:
 Caused by Orientia tsusugamushi; transmitted by chiggers
who've bitten rats.
 Presents without a rash, but eschar develops at site of bite like
tularemia or plague.
 Immunity is short-lived.
 Q fever:
 Caused by Coxiella burnetti: obligate intracellular bacteria that
multiply in phagolysosomes (recall that this is the only
fusogenic pathogenic intracellular bacteria). Extremely resistant
to dessication (can dry up and drift around waiting to get
inhaled).
 Reservoir: ruminants (particularly fetal membranes of
cows/sheep).
 Transmitted by aerosolized membranes/dust.
 Note that just 1 organism can be infectious (!).
 Causes a self-limited influenza-like illness. Note that this
doesn't present like other rickettsial infections at all and isn't
caused by a Ricketssia species.
5. Describe the characteristics of Ehrlichia sp. and the major features of infections
that they cause.
 Ehrlichia: obligate intracellular bacteria that infect phagocytic cells and
multiply inside vacuoles.
 Can see this on blood smear-- filled vacuoles inside leukocytes
(also called morulas).
 Note Ehrlichia more or less = Anaplasma here.
 Clinical manifestations are similar to rickettsial infections (high fever,
prostration, aches and pains) but without a rash.
 Cause leukopenia and thrombocytopenia, also high ALT/AST.
 Transmitted by the "lone-star tick."
 Note that the number of cases is increasing in the last 10 years. May
just be better diagnostics.
[Note there's a summary slide covering organisms, the diseases they cause, the
infectious vector, and the derm findings for each. Handy.]

6. Describe the characteristics of Bartonella sp. and the major features of infections
that they cause in immunocompetent and immunocompromised individuals.
 Bartonella: facultative intracellular bacteria. Three pathogenic species;
the one we tend to see more here is Bartonella henselae (cat-scratch
disease).
 Oroya fever (B. bacilliformis): can pick up through sandfly in
South America; invades RBCs and causes anemia and skin
lesions.
 Trench fever (B. quintana): transmitted by louse bite. Presents
similarly to rickettsia but with relapsing fever every five days.
 Cat-scratch disease (B. henselae): transmitted through cat
scratches, bites, or licks. Note cat is asymptomatic. Infects
capillary endothelial cells and causes fever and painful
lymphadenopathy. Usually self-limited.
 Note that in immunosuppressed patients, B. hensalae or B. quintana
can case bacillary angiomatosis: the normally self-limited trench
fever or cat-scratch disease runs amok and cause a whole lot of
painless, angioproliferative skin lesions (look like Kaposi'a sarcoma)
and hepatic blood cysts (causes abdominal pain).
Zoonotic Viral Infections
Tuesday, February 24, 2009
10:01 AM
Zoonotic Viral Infections, 2/24/09:
1. Explain why zoonotic viruses are sporadically present in the human population;
appearing, disappearing and re-emerging in either predictable or unpredictable
fashion.
 Naturally maintained outside of the human population. There's
occasional human-to-human transmission. Essentially human infection
is a dead-end for these viruses.
 Note that since they're not adapted to humans, they often cause
severe disease and death when they infect humans.
 The combination of occasional human-to-human spread and high
lethality has potential to be unpleasant.
2. Be aware of the epidemiology, prevention, diagnosis, treatment and outcomes of
zoonotic viral diseases present in Colorado (Rabies, Hantavirus, Colorado Tick Fever,
West Nile Virus, others).
 Rabies:
 Enveloped, bullet-shaped, single-strand (-)-strand RNA virus.
Has the capacity to make its own mature mRNA (replicates in
cytoplasm); packages RNA-dependent RNA polymerase in its
virions.
 Neutralizing antibodies against the one glycoprotein on the
envelope surface are protective (this is how the active vaccine
works). Only one serotype. Note you can also do passive
immunization with anti-rabies surface protein antibodies.
 Reservoir: bats, skunks, raccoons, households pets (mainly
dogs + cats). Almost always spread through bites.
Bats are the most common cause of human rabies in
the US; dogs are the most common cause worldwide
(dogs are vaccinated in the US).
 Re the boards: along with histoplasmosis, this is one of
the "cave diseases" (ie "guy goes walking in a cave and
gets sick, what is it?").
 There's a long incubation period (weeks to months), which
gives a window for effective therapy.
 The virus transmits retrograde up nerve tracts into the CNS,
which is what gives it the long incubation (has to get all the
way to the brain).
 Note this means there's no viremia, and there are no symptoms
and no immune response (nerves are shielded).
 Once it gets to the CNS, it goes into the salivary glands and
causes increased aggression and biting behavior in animals.
 In humans, present with fever, anxiety, agitation. More
specifically, see dysphagia, hypersalivation, coma, and death.
Patients often die before the diagnosis is made (it's a rare
disease and once the symptoms show up it's often too late to
treat unless the diagnosis is made rapidly).
 Histologically, see Negri bodies (dark inclusion bodies in cells
of the CNS).
 Diagnosis: can test saliva, CSF, or pull hairs from nape of neck
for immunofluorescence. Can also do PCR.
 Other than the active and passive vaccines, no real treatment.
Prevention is better-- vaccination of dogs, don't mess with
dying bats, don't bite bat heads off onstage like a big-hair 80's
dumbass (that'd be Ozzy Osbourne for those who were born
three days ago).
Hantavirus:
 Enveloped (bunyavirus), SS, (-)-strand RNA virus. Segmented
(like orthomyxoviruses and reoviruses).
 Acquired through rodent urine and feces (aerosolized and
inhaled, generally). Again, the humans are thoroughly
irrelevant to the replication cycle of the virus (it's a chronic,
largely asymptomatic infection in mice).
 Often highly fatal (15-90%). Quite rare (360 in US in 10 years).
 Presentation: largely pulmonary: fever, myalgia, N/V,
tachypnea, hypotension, crackles or rales on pulmonary exam.
CXR shows bilateral infiltrates and pleural effusions.
 Clinical progression is quite rapid:
 Prodrome (fever and malaise) shows up about 3-6 days
after infections; this goes quickly into respiratory
symptoms. Death usually occurs about 3 days after the
onset of pulmonary symptoms.
 Pathogenesis: cytokine explosion in the lungs: rapidonset pulmonary edema and shock. Can put patients on
heart-lung bypass to avoid the most severe period of
symptoms.
 Note the "Sin Nombre" virus that popped up at Four Corners a
while back was a hantavirus.
 Diagnose with serology antibodies or PCR.



Colorado Tick Fever and West Nile Virus are covered in the next
lecture.
3. Who should receive rabies vaccine and rabies post-exposure prophylaxis?
 Post-exposure: everyone bitten by rapid and potentially rabid animals.
 If the animal's available, quarantine it and observe it for 10
days to see if it dies of rabies. If so, do a full course of therapy.
While you're waiting, wash the wound very very well with soap
and water (soap kills enveloped viruses) and inject passive
antibodies around the wound site, then start vaccination shot
series (you can stop it if the animal seems okay).
 Pre-exposure: vets, spelunkers, etc (also animals!).
 Note that the vaccine isn't as painful as it used to be (more highly
purified). It is, however, pricey, at least for humans.
4. Be aware of notable zoonotic viral diseases elsewhere in the world (Ebola, SARS,
Monkeypox, Nipah, others).
 Monkeypox: looks like smallpox and the smallpox vaccine prevents
monkeypox; now that we don't vaccinate against smallpox,
monkeypox has been cropping up again.
 Reservoir is small rodents (mice and squirrels).
 Note there was an outbreak of monkeypox in 2003 from spread
of pox from imported rats to prairie dogs.
 Ebola and SARS are around (filovirus and coronavirus, respectively).
Simple health precautions and public health measures can often
contain outbreaks.
 Nipah: paramyxovirus. Evidently showed up in people with exposure to
pigs.
5. Explain how global surveillance and public health interventions for emerging and
re-emerging infectious diseases can be used to protect people in the US and
throughout the world.
 What you think.
Arboviral Diseases
Tuesday, February 24, 2009
11:03 AM
Arboviral Diseases, 2/24/09:
[General notes:]
 Arbovirus just means a virus transmitted by arthropods (ticks,
mosquitos, etc).
 These are usually alphaviruses or flaviviruses.
 Alphaviruses/flaviviruses:
 Enveloped, icosahedral, SS, (+)-strand RNA viruses.
 Neutralizing antibodies bind viral envelope glycoproteins;
protective.
 Alphaviruses are slightly more complicated: two ORFs in the
genome (replication genes are separate from capsid/envelope
proteins). Flaviviruses only have one ORF that contains
everything.
 General patterns of disease caused by arboviruses:

Encephalitis, arthralgias, sometimes hemorrhagic fevers (fever,
bleeding, hypotension, edema, shock, death).
1. Describe the transmission cycle of arboviruses. Why are blood-borne viruses like
HIV, HBV, and HCV not arboviruses; i.e. why aren’t they transmitted by mosquitoes?
 ["Arbo" = "arthropod-borne," usually referring to mosquitoes.]
 Transmission cycle of West Nile Virus:
 Generally it's mosquito to bird to mosquito to bird. Humans are
"dead-end" hosts (mosquitoes can't get infected from biting
humans).
 Mosquito ingests blood from an infected (viremic) vertebrate.
Note humans don't get viremic enough to pass on the infection
to new mosquitoes.
 This infects the mosquito. The virus matures in the mosquito
gut and spreads to the blood and saliva. There are no adverse
effects on the mosquito.
 The infected mosquito bites another vertebrate and infects it.
 [Symptoms of West Nile:]
 Generally asymptomatic; about 20% of infected patients
get mild symptoms; older patients can get meningitis,
encephalitis, or poliomyelitis.
 Yellow fever and dengue:
 Similar, but the primary hosts are monkeys and mosquitoes.
 Note the humans are not dead-end hosts for yellow fever and
dengue (get high enough titers of viremia to infect new
mosquitoes).
 Yellow fever infects the liver (thus jaundice, thus yellow).
 Only one serotype of yellow fever; there's a safe and efficacious
vaccine.
 Four serotypes of dengue but no vaccine (see below).
 Generally the progression from infection to disease is rapid (about 710 days)-- whether or not the bugs cause organ symptoms (eg.
encephalitis) depends on whether or not it can 'outrun' the host's
immune response.
 HIV, HBV, HCV, etc, aren't infectious in mosquitoes, and are thus
generally not transmissible through the arbo route.
2. What steps could be taken to thwart an arbovirus epidemic?
 Largely, mosquito control.
3. What is Colorado Tick Fever? How would you “catch it”? Where would you “catch
it”? What would you do to get rid of it? How would your doctor correctly diagnose it?
 A reovirus transmitted by ticks (not mosquitoes), generally in state
parks in Colorado. Occurs in the summer. Causes flu-like symptoms.
 Note that humans are part of the natural infection cycle (human to tick
to human).
 Generally picked up 4,000-10,000 feet in the Colorado Rockies.
 It invades RBCs to evade the host immune response.
4. How many serotypes of Dengue virus are there? Why does Dengue hemorrhagic
fever occur predominantly in regions of the world where multiple serotypes of
Dengue virus co-circulate
 He emphasized this one.
 4 serotypes.
 Transmitted by
 Dengue can cause a variety of disorders depending on serotype:
 Dengue fever
 Dengue hemorrhagic fever
 Dengue shock syndrome
 Generally with the first infection, you get dengue fever. With repeat
infections, you have a much higher incidence of hemorrhagic fever or
shock syndrome.
 Why this happens: evidently the antibody response to the first
serotype cross-binds (non-neutralizing) to the other serotypes of
dengue. This causes the bound, but not inactivated, dengue serotypes
to get into phagocytic cells (with Fc receptors) and spread more
systemically, causing high levels of cytokine release.
 There are no vaccines against dengue (don't want to cause increased
sensitivity).
5. Are vaccines available to prevent arbovirus diseases? Who should be vaccinated?
 There are vaccines against yellow fever and Japanese encephalitis.
Generally you should vaccinate people going to yellow-fever-endemic
areas (like lower Bolivia) or planning to stay in rural Asia for more
than a month, respectively.
 No vaccines against dengue.
 No vaccines against West Nile.
6. Compare the replication cycle of flaviviruses with that of picornaviruses? How is
the replication cycle of alphaviruses similar / different to that of picornaviruses?
 Picornaviruses don't have an envelope.
 We're covering picornaviruses tomorrow morning.
Picornaviruses
Wednesday, February 25, 2009
8:02 AM
Picornaviruses, 2/25/09:
[Dr. Barton does research on picornaviruses. Consequently this lecture, like most
lectures about a pet project, suffers from a lack of cohesion. For further evidence see
the Yay Polio Eradication gulag scheduled for next week.]
1. What molecules of the virus and host define picornavirus serotypes?
 Virus: surface epitopes.
 Host: neutralizing antibodies.
 This shouldn't be a surprise.
2. What significant diseases are caused by picornaviruses? Names of relevant virus?
Note well that many human enteroviruses can cause different diseases often
involving more than one organ system.
 First Aid mnemonic: PERCH: polio, echo, rhino, coxsackie B, hepatitis
A.
 Genus Enterovirus: Polio, Coxsackie, Echo, and Enterovirus
spp.
 Picornaviruses cause aseptic meningitis, paralysis, encephalitis, colds,
pneumonia, myocarditis, endocarditis, diarrhea, hepatitis, etc.
 Which causes what: there's a lot of crossover. Polio is often
associated with paralysis and coxsackie B is associated with
cardiac disease, but he made a point of saying that other
enteroviruses cause the same symptoms.
 He did it again. Paralysis doesn't = polio.
 Note that enteroviruses are known more for causing aseptic
meningitis (> 60% of cases) than they are for causing diarrhea.
 Rhinovirus is the most acid-labile of the group (which is why common
colds don't infect the GI tract).
 Echovirus: a frequently asymptomatic, cytopathic virus that generally
only cause disease in immunocompromised or infants. Echovirus is
associated with meningitis in utero.
 Poliovirus: kills motor neurons, spares sensory neurons. Generally
there's little regain of motor strength.
 Post-polio syndrome: see an exaggerated effect of aging on
loss of motor strength after a polio infection earlier in life
(there's less redundant motor innervation left).
3. How do enteroviruses infect the CNS? What "protective" immune response
prevents systemic spread of enteroviruses?
 Neutralizing IgA prevents initial mucosal infection by enteroviruses;
neutralizing IgG prevents viremic spread from the initial infection site
to the bloodstream and other organs.
 Ways of getting into the CNS: (1) get in there by being inside a cell
that crosses the BBB, or (2) go retrograde up axons. Note that
traveling retrograde up axons causes damage to the axons the virus is
traveling along.
 The nature of the paralysis in the patient reflects the route the
virus took to the CNS-- if it went across the BBB through a
carrier cell, you see bilateral paralysis; if it travels up a
peripheral axon, you see unilateral paralysis (of the axon it's
traveling up).
4. Describe the transmission cycle of enteroviruses. How does the transmission cycle
of enteroviruses relate to seasonal epidemics of enterovirus disease?
 Fecal-oral transmission. Infections peak with hot/warm temperature
(summer in temperate climates, year-round in tropical climates).
 He doesn't have a good explanation for this, actually.
 Note that you get the primary infection at the mucosal surfaces; the
virus then spreads to organs during the first round of viremia, then (if
that wasn't sufficient to get into the CNS) it undergoes a second round
of viremia to try and build high enough titer levels to get through the
BBB.
5. Describe the picornavirus replication cycle. How does the monocistronic viral
mRNA express more than one viral protein?
 Non-enveloped, icosahedral, SS, (+)-strand RNA genome.
 Picornaviruses are robust: very very stable in the environment,
extremely resistant to soap/detergents (no envelope).
 There are no viral replication proteins included in the virion-- it uses
host machinery to express its genome right off. Note it does, however,
encode viral replication genes in that genome.
 Picornaviruses express their entire genome as a long polypeptide-then they use viral proteases to chop it up and express a bunch of
different proteins. Boards likes this.
 Replicate exclusively in the cytoplasm, like all (+)-strand RNA viruses.

Like most RNA viruses, the virally-encoded RNA-dependent RNA
polymerase is extremely error-prone (giving rise to lots of errors in
replication).
6. Describe the difference between infection and disease? Does infection with
enteroviruses equate with disease? Are enteroviruses in a patient's feces always
associated with disease?
 Infection can be asymptomatic; disease, by definition, isn't.
 Note that you can shed and transmit virus in either state.
 This becomes important when you start talking about immunization
campaigns (see next point).
7. Describe the advantages and disadvantages of the IPV and OPV vaccines. Who
gets vaccine-associated paralytic poliomyelitis (VAPP)? What vaccine, IPV or OPV, is
associated with VAPP? How does the vaccine lead to VAPP? How does the sequential
vaccination protocol, using both IPV and OPV rather than OPV alone, prevent VAPP?
Does vaccination prevent infection or prevent disease or both? What is sterilizing
immunity? Do vaccines typically --> sterilizing immunity?
 IPV (injected polio vaccine), aka Salk vaccine:
 Killed wildtype poliotype (all 3 serotypes).
 Injected.
 No vaccine-associated disease.
 Gives IgG immunity, but only limited mucosal immunity (not
much IgA). Note this protects against disease, but not
necessarily infection (as delineated above)-- thus can still be
contagious.
 More expensive than OPV.
 OPV (oral polio vaccine), aka Sabin vaccine:
 Live, attenuated vaccine.
 Administered orally (liquid drops, usually).
 Causes both IgG and IgA immunity (protects against both
infection and disease).
 Inexpensive.
 However, note that it can cause polio in the
immunocompromised. It can also acquire a few mutations to
allow it to regain potency and cause disease in
immunocompetent people (VAPP). When that happens, the
mutated potent forms can be spread to other people.
 In the US, the CDC recommended in 2000 that IPV be the only vaccine
used on account of the 1 in 2.5 million doses that mutate into a potent
form.
 The OPV is the one most-used worldwide (better for preventing
transmission).
 The idea of having a IPV-OPV series was to give the patient systemic
immunity first (prevent disease), then give them mucosal immunity
with OPV.
 "Sterilizing immunity" is an ideal-- it effectively means that once
vaccinated, a person can never get the disease again at any exposure
level. Since vaccines generally rely on antibodies and there's only so
many antibodies to go around in the body, sterilizing immunity is kind
of a unattainable goal (if you've had the polio vaccine but you go
bathe in polio virus, you'll still get polio).
8. What is "herd immunity"?
 Essentially, the percent of potential hosts that can't get infected.
Micropathogens generally need a place to replicate in order to survive
as a species. If enough people can't get it, the pathogen dies out due
to lack of potential hosts.
 This is one reason it seems unlikely that we'll ever stamp out
influenza: the "herd" includes not just humans, but chickens, pigs, and
quite a number of other species, making the size of the herd pretty
massive.
9. What factors influence the ability to eradicate viruses from the world? Do
you think the poliovirus eradication campaign will be successful or fail?
Why?
 What you'd think.
Prions, Spongiform Encephalopathy
Wednesday, February 25, 2009
9:07 AM
Prions, Spongiform Encephalopathy, 2/25/09:
1. Identify known prion diseases and learn the epidemiology of these diseases.
 Human prion disease:
 Creutzfeld-Jacob disease, Gerstmann-Strassler-Scheinker
syndrome, fatal familial insomnia, Kuru in Papua New Guinea.
 Tend to result in spongiform encephalopathy-- neurons die out
of brain tissue leaving a bunch of vacuoles in the tissue-- thus
"spongiform."
 Animal prion disease:
 Chronic wasting disease, bovine spongiform encephalopathy,
scrapie, transmissible mink encephalopathy.
 Yes, I am writing about transmissible mink
encephalopathy. My life just reached a new low.
2. Be familiar with the pathologic features of spongiform encephalopathy.
 Note there's a long (years to decades) incubation period, during which
the prions are flipping more and more normal PrPcs to their wicked
ways.
 Note that there's a variant of CJD, almost exclusively in
England, which tends to hit younger patients and has a more
prolonged course.
 This variant CJD is associated with "M/M" genotype in the PrP
protein.
 Once symptoms begin (ataxia, vision changes, slurred speech,
myoclonus, incontinence), patients usually die in 6-12 months. No
cure.
 Note that different 'strains' of prions cause different areas of the brain
to deteriorate (thalamic initiation of sleep in fatal familial insomnia,
cortex in Creutzfeld-Jacob, cerebellum in Kuru, etc).
 Don't feed cows to cows. Don't feed sheep to sheep. Don't feed mink
to mink. Don't feed human to human. Don't feed humans to cows.
Don't feed cows who've been fed cows to humans. Don't feed minks to
cows, that's just wrong.
3. Learn evidence supporting infectious protein hypothesis.
 Not inactivated by UV light, DNase, RNase. Note it's also not
inactivated by normal autoclaving.

4.
5.
6.
7.
Prions that are protease-resistant are infectious; prions that aren't,
aren't.
 There's a gene (PrP) that encodes prions protein in all vertebrates. It's
expressed on neurons and lymphocytes.
 The normal gene product is PrPc; the spongiform form is PrPSc.
 Mice without the genes for PrPc (phenotypically normal) don't
get spongiform encephalopathy. SCID mice also are resistant
(no lymphocytes).
 Mice with human PrPc get spongiform encephalopathy when
exposed to human PrPSc.
 We can make mice die of CJD or scrapie with appropriate genetic
programming. Dr. Barton thinks it's fascinating. I think anyone whose
first reaction to ataxic dying mice is that they're fascinating has
something wrong with them.
How can an infectious protein “replicate”?
 PrPSc seems to be a conformational variant of PrPc (beta-sheet) that
has the ability to convert normal PrPc (which is alpha-helical) to PrPSc.
 PrPSc is protease-resistant and can't be readily degraded by cellular
proteasomes. Like most beta-sheets it also tends to clump together
with other beta sheets. The accumulated PrPSc causes neuronal death
by apoptosis.
 Note PrPSc seems to need some other cellular cofactors to covert PrPc.
Learn how prion diseases are acquired/transmitted. Iatrogenic exposure.
 Generally you ingest altered prion proteins, they get absorbed by M
proteins in the gut and presented to lymphocytes, and they get into
the CNS, sometimes by lymphocyte carriage, sometimes retrograde up
the vagal nerves from the gut.
 You can also have a gene that encodes a misfolding prion protein.
 You can just also have the bad luck (1/million) to have one of your
PrPc proteins spontaneously convert to the PrPSc, whence it can
convert all the other PrPc proteins.
 Iatrogenic: can transplant tissue with PrPSc; can also use surgical
equipment that is contaminated with PrPSc.
 Note you can, rarely, get CJD from donated blood from a CJD patient
(long latent period is tough for public health to control).
Can an animal prion (from a cow or elk) infect a human?
 Yes, but it's tougher than going elk-elk.
 Longer incubation period, probably due to subtle conformational
differences between species.
 Generally the infectious dose is higher.
How are prion diseases confirmed in the laboratory?
 Use protein misfolding amplification, which is what it sounds like
(amplification of misfolded proteins to levels you can detect
immunologically via Western blot).
Protozoa I + II
Monday, March 02, 2009
7:15 AM
Protozoa I + II, 3/2/09:
[Once again I've ducked Dr. Holmes. These are from the notes, slides, and
CMBMRS.]
[Also, with the greatest respect, the way these LOs are organized is asinine. I've
collated them so that you don't have to look in four different places for information
on the same bug.]
1. Define protozoa and explain why they are important for human health and
medicine.
 Protozoa: unicellular (like bacteria) eukaryotes (like us). Note they
often have truly wacky reproductive cycles. As defined here, protozoa
are parasites, although I'd be surprised if we didn't have a bunch of
commensals floating around.
 They are important for human health and medicine because they bat
.322 and can cover the infield. They cause disease, what do you think?
 Major protozoa discussed here:
 Amoebas (Entamoeba histolytica)
 Flagellates (Giarda, Trichomonas, Trypanosomes, Leishmania,
etc)
 Coccidia (Cryptosporidium, Plasmodium, Toxoplasma)
2. Describe the life cycles and diagnostic features of the major protozoa that grow in
the intestinal tract and genitourinary tract. + 3. Describe the pathogenic
mechanisms of these protozoa and compare them with those of other pathogenic
microbes + 4. Describe the major diseases caused by these protozoa and explain the
principles for preventing and treating them.
 Entamoeba histolytica:
 Amoeba; common in colon, but usually asymptomatic; third
leading cause of death from parasites. Infection is called
amoebiasis.
 The mature cyst (tetranucleated) is ingested (oral-fecal
transmission); it then excysts in the small bowel and forms
trophozoites (active, motile form), which attach to cells in the
intestine and eat a variety of things, including RBCs, WBCs, and
colonic mucosal cells.
 They replicate by binary fission in the colon; when they're
leaving, they encyst again (first to precysts with two nuclei,
then to mature tetranucleated cysts).
 Detection: look for trophozoites or cysts in the stool or colonic
tissue; there are also specific antibodies against E. histolytica
as well as an E. histolytica-specific antigen (the Gal/GalNAc
lectin antigen) that can be detected in the serum.
 (a running theme seems to be that pathogenic protozoa
look a lot like nonpathogenic protozoa and must be
distinguished therefrom.)
 Can cause colonic ulcers due to this destruction of local cells,
although again most infections are asymptomatic. With ulcers
can see some minimal bleeding along with diarrhea, but it's
rare to get severe bleeding.
 Every so often they disseminate into the bloodstream and
cause systemic disease, usually in the form of liver abscesses.
These can then travel to the lung and cause pulmonary
abscesses and death.





Prevention mainly involves sanitation. Both the notes and
CMBMRS make the point that homosexual men are often
asymptomatic carriers.
 Treatment is metronidazole.
[in passing: CMBMRS sez various other amoeba species also cause
meningitis and/or death (Naegleri, Acanthamoeba, Balamuthia).]
Giardia lamblia:
 Flagellate protozoan (singular of protozoa.. you reluctant
scholars); the only pathogenic protozoan found in the upper
parts of the small intestine.
 Cyst form is ingested (oral-fecal transmission); the stomach
acid strips off the coat, causing it to excyst; the resulting
trophozoite attaches to the small intestinal mucosa and begins
to divide and coat it.
 Since Giardia lives in the duodenum, this coating interferes with
fat absorption, which means a terrific quantity of greasy, smelly
diarrhea is heading south (from CMBMRS; class notes say the
mechanism isn't established).
 Note that there is no mucosal invasion and hence no blood
with Giardia. Note also that the infectious dose is about 10
organisms.
 Detection: look for cysts and/or trophozoites in stool or
duodenal aspirates; can also use immunofluorescence or ELISA
tests.
 Don't drink the water when you're hiking without boiling or
purifying it; other than that, general sanitation measures.
 Treatment is metronidazole.
Trichomonas vaginalis:
 Another flagellate protozoan; infects vaginal and urethral
epithelium.
 Male hosts are generally asymptomatic, although the prostate
and seminal vesicles may be infected; female hosts present
with vaginal itching, burning on urination, inflammation of the
vaginal mucosa, and a frothy, yellow-cream discharge
(CMBMRS: "thin, watery, frothy, malodorous").
 Distinguish from bacterial vaginosis, which is usually
caused by Gardnerella vaginalis, generally shows a graywhite discharge, and has a characteristic fishy odor.
 Note that cysts don't seem to have much part to play here-- all
you're looking for diagnostically are the trophozoites, either on
wet mount or by more advanced techniques (fluorescent
antibodies, culture, nucleic acid probe). The cyst form is
evidently unimportant in human pathogenesis and
transmission.
 Transmitted sexually; prevented by good personal hygiene and
safe sex.
 Treatment is metronidazole.
Cryptosporidium parvum and Cyclospora cayetanensis:
 These are coccidia; responsible for intractable diarrhea in
AIDS patients but only self-limited diarrhea in
immunocompetent individuals.


C. parvum: ingested as a cyst (fecal-oral transmission),
excysts, invades ileal epithelium and creates both further
invasion and more cysts. C. cayetanensis: details unknown.
 Detection: both of these organisms are acid-fast; they need to
be properly stained to be detected in stool. Can also use ELISA
for C. parvum.
 C. parvum is associated with spread through water; C.
cayetanensis has been associated with contaminated
raspberries.
 Prevention is sanitation and not eating Central American
raspberries.
 Treatment for C. parvum is nitazoxanide; treatment for
cayetanensis is SMX/TMP.
Plasmodium:
 Protozoa whose life cycles involve mosquito-human-mosquito
transmission:
 Mosquitoes inject sporozoites into human blood, where
the sporozoites infect liver cells, transform into round
trophozoites, and undergo lots of asexual division.
 This division creates a multinucleated mass inside the
liver cell called a schizont.
 Each nucleus of the schizont acquires a membrane and
are thus transformed into merozoites; immediately
afterwards, the liver cell bursts and the merozoites are
loosed into the bloodstream.
 Some re-infect other liver cells and repeat the cycle;
some infect red blood cells. More on this below.
 Four species cause malaria: P. falciparum, P. vivax, P. ovale, P.
malariae. Falciparum causes most of the deaths from malaria.
All of them invade and lyse red blood cells.
 These four species generally invade red blood cells of differing
ages and have differently-timed cycles of cell lysis:
 Vivax and ovale invade young RBCs and lyse them every
48 hours.
 Malariae invades old/senescent RBCs and lyses them
every 72 hours.
 Falciparum invades all RBCs and lyses them on an
irregular, 36-48 hour cycle.
 The pattern of cell invasion and lysis is similar for RBCs and
liver cells.
 Some merozoites gender-differentiate in RBCs into male and
female gametocytes; these are the ones that can infect
mosquitoes that bite the human host.
 [when they get into the mosquitoes, the male/female
gametocytes fuse (sexual reproduction) and form lots of
little sporozoites which are in turn injected into new
human hosts.]
 [To reiterate: in the human Plasmodium undergoes
asexual reproduction. In the mosquito it undergoes
sexual reproduction.]
 Note that vivax and ovale can leave dormant forms in the liver
than can reactivate. I'm guessing this will be on the exam since
it affects treatment (see next lecture and below).


Detection: get a blood smear, look at the RBCs. Morphological
features can differentiate the various species from each other.
 Recall that sickle-cell trait helps protect against P. falciparum;
Dr. Holmes also mentions that this may be the reason for
increased prevalence of G6PD deficiency and thalassemias in
people from tropical areas. CMBMRS mentions that an absence
of Duffy a/b antigens on RBCs helps protect against P. vivax.
 For you Simpsons fans: "Duffman, now with NO
maLAria! Oh yeah!"
 Clinically, see severe febrile anemia due to hemolysis (both by
Plasmodium and also by splenic macrophage activity) and bone
marrow suppression, as well as jaundice, hypotension,
tachycardia, and hepatosplenomegaly.
 P. falciparum, in particular, causes damaged, "sticky"
RBCs that stick to the endothelium and plug up venules,
causing tissue ischemia.
 Multi-organ failure is the most common cause of death
(ischemia due to anemia and/or plugged vasculature).
 Epidemiology: P. vivax is all over the place, but P. falciparum is
only in the tropics and subtropics.
 Prevention: prevent mosquito bites. Easier said than done, but
can try pesticides and mosquito nets. Prophylactic antimalarial
drugs (mefloquine, doxycycline, chloroquine, etc) are good for
travelers.
 Treatment:
 Chloroquine works, by and large, on vivax, malariae,
and ovale, but in falciparum it only works in Central
America north of the Panama canal; everywhere else
you need mefloquine or quinine to target falciparum.
 Both chloroquine and mefloquine only target
Plasmodium in the RBCs and they don't go into the liver;
to target it in the liver cells (as in vivax/ovale chronic
liver infection) you need primaquine.
Trypanosoma brucei and Trypanosoma cruzi:
 Trypanosomes are "hemoflagellates" (live in the blood and have
flagella).
 T. brucei species cause African sleeping sickness (carried by
tsetse flies); T. cruzi cause Chagas disease (South and
Central America, carried by reduviid/kissing bugs). They're
injected (as flagellated trypomastigotes, though cruzi later lose
the flagella to form amastigotes) into the bite site of the insect.
 T. brucei: a chancre appears at the bite site from the
trypomastigote multiplication; they spread to the lymph, then
the blood. You see relapsing and remitting fevers with
headache and dizziness; cyclic period is about 2 weeks.
 The cyclic nature here was the subject of an extremely
cool talk I got to see a few years back. Essentially the
bug is varying its surface (immunogenic) proteins in real
time faster than the immune system can respond-- so
you get a big IgM response (responsible for fever, etc)
that wipes out 95% of the bugs, but the other 5% now
looks like something different, and by the time you can
mount an IgM response against that it's repopulated and


another 5% now looks like something else, and so on.
This goes on indefinitely; the constant production of
immune complexes causes vasculitis and anemia.
Essentially the little guys beat our immune system at its
own game.
 In East African forms of the disease, the bugs get into
the CNS and cause drowsiness, convulsions, coma, and
death in 5-9 months. Note that when there's CNS
involvement, the treatment (fancy arsenic) kills a good
proportion of the patients.
 T. cruzi: the host autoinoculates by scratching the infected bug
bite.
 Acute disease: fever, lymphadenopathy,
hepatosplenomegaly, heart damage. Can last up to 6
months. Up to 10% mortality in kids, who are
particularly susceptible.
 Chronic disease: once you've got Chagas, you don't
generally get rid of it. Most people stay asymptomatic;
some people progress to chronic disease, which is
unpleasant. You see dilated cardiomyopathy,
arrhythmias, megacolon and achalasia. Note that this is
the most common cause of CHF in rural Bolivia, if you're
ever down there.
 Detection: IgM levels or microscopic evaluation of blood, lymph
nodes, or primary lesions works for brucei; antibody levels or
stained blood smears works for cruzi.
 Prevention: bug control, treatment of humans and/or cattle
(the more severe East African brucei disease is carried by
cattle).
Leishmania:
 Also hemoflagellates; multiply inside cellular phagolysosomes;
can cause visceral, cutaneous, or mucosal leishmaniasis.
Generally spread by sandflies.
 Cutaneous: benign, localized skin lesions.
 Mucosal: more serious, destructive mucosal lesions.
 Visceral: disseminated infection, highly lethal. Can occur
spontaneously in particular species or from a cutaneous
lesion in immunosuppressed patients. Infects
macrophages. Causes fever, diarrhea, enormous
hepatosplenomegaly, ascites, and lymphadenopathy.
 Detection: microscopic examination or culture of biopsied
tissue.
 Prevention: control sandflies.
Toxoplasma gondii:
 Coccidian; most common protozoal infection in humans.
Acquired by ingesting cysts in undercooked meat or cat feces; a
single cyst can cause infection.
 One of the ToRCHeS infections; can be transmitted in utero.
 Infects macrophages; if the macrophages become activated
they can kill the toxoplasma, but if they can't become activated
(as in AIDS patients with low CD4 counts), you're in trouble.
 When it's symptomatic in immune-competent hosts, causes a
mononucleosis-like syndrome.


In immunocompromised hosts, see encephalitis and brain mass
lesions (as per small groups last week). In utero, results in
retinitis, malformations, and neurological damage.
Prevention: lives mainly in cats. Don't scoop cat litter, and
wash your hands well, if you're pregnant around cats. For the
love of God don't give your cats up to a shelter or throw them
out onto the street, that's overreacting. Yes, people do this.
Helminths
Tuesday, March 03, 2009
7:56 AM
Helminths, 3/3/09:
[He didn't get close to finishing all this-- so once again, who knows what we're
responsible for. Best guess follows.]
1. What are some of the distinctive properties of helminths especially in comparison
to other types of infectious agents (e.g. bacteria, viruses, fungi)?
 From CMBMRS: "Within the normal human host there is usually no
immune reaction to living worms. However, there is often a marked
response to dead worms or eggs."
 Worms (helminths) can be pretty huge (25m) or very small (though
usually only the eggs are microscopic).
 They have a reproductive system (reproduce sexually). They have a
sexual stage in the human host but generally can't multiply inside one
host-- if you get one egg, you get one worm; if you get three eggs,
you get three worms. Those worms put out eggs but the eggs they put
out generally don't mature inside ya.
 Exception is Strongyloides, which can reproduce by
parthenogenesis.
 So the worm life cycle generally requires several different hosts or at
least stages (for many species the egg maturation process involves
being in the soil for a while).
 Generally you get them by eating eggs or cysts, or by the larva
burrowing into the skin. Can also get it from an insect bite. Human-tohuman transmission is fairly rare.
 To clear up the human-to-human transmission thing: as far as I
can tell (which isn't far), eggs are excreted by helminths that
live in humans. The eggs, as mentioned, generally have to
mature somewhere outside the human in order to become
infectious to humans again. This means that most of them you
can't pass to other humans directly. The exceptions are the
ones where you can get sick from exposure to the immature
eggs alone (eg. Taenium solium or pinworms) or the ones
where the pathogenesis comes from eggs that mature to
cysts/larvae inside the human body (Strongyloides).
 Pathogenesis: blood loss and anemia (as per hookworms), immune
reaction damage (granulomas surrounding schistosome eggs), or
mechanical blockage (as per Ascaris blocking the bile duct).
Malnutrition can be a result as well in situations where the worm load
is really high or the patient isn't getting much in the way of nutrients
in the first place.
 They evade the immune response in particular ways: they encyst
themselves, they live in intraluminal spaces where there isn't a good
immune response, they can cause immunosuppression, and they can
acquire host antigens to disguise themselves.
 Diagnosis relies heavily on travel and eating history. Eosinophilia is
also often indicative but only occurs in species that get into the blood.
Can look for worms or ova in stool or tissue biopsies. Can also use
DNA detection and immunological tests. Note Charcot-Leyden crystals
(conglomerations of eosinophils, we've seen them before in the
bronchi of asthmatics) can show up in stool.
2. Consider learning the major factors associated with the following intestinal
helminths. A. Where is the organism acquired (i.e. its reservoir in nature)? B. What
is its mode of transmission? Is it only transmitted human to human or is it acquired
by contact with animals? C. Where can the worm be located in the human body?
Does it invade beyond the intestine? D. What are the major symptoms associated
with human disease? How is a laboratory or clinical diagnosis made?
 ["Consider learning?"]
 Tapeworms:
 Flatworms; have no intestinal tract, have to absorb predigested nutrients.
 Acquired when the larval cysts are ingested in undercooked
meat (beef for Taenia saginata, pork for Taenia solium, etc).
For T. solium you can also get the worm from direct ingestion
of ova from an infected person.
 Taenia saginata: mainly asymptomatic, some weight loss, can't
be passed directly from person to person.
 Taenia solium: Humans are the definitive hosts; eating the
cysts in pork causes the tapeworm (asymptomatic as above),
but eating the ova (which pass directly from person to person)
causes cysticercosis (cyst formation in muscle or brain,
dangerous). Note that this means you can get cysticercosis
without ever actually eating the infected meat.
 Tapeworms hook their mouths onto the intestinal wall, stay
there, and grow. Since they don't get into the blood there's no
eosinophilia with tapeworms, but there's a big inflammatory
response to the cysts in cysticercosis.
 Diagnosis: look at worm segments in feces (evidently one type
has a different number of uterine lateral branches.. yeah,
really.. T. solium has less uterine branches if anyone is anal
enough to ask).
 Note that you can get tapeworms from a wide variety of
animals; tapeworms acquired from freshwater fish can cause
B12 deficiency.
 Ascaris:
 Roundworms (have intestinal tract). Get pretty big.
 Live in contaminated food or dirt; acquired when the larval
eggs are ingested in food.
 Eggs get to the small intestine, hatch, burrow into the wall of
the GI tract, get to the bloodstream, travel to the lung, grow in




alveoli, get coughed up and then swallowed, and go back to the
small intestine to mature there.
 Can be mild or asymptomatic; with lots of worms, kids can get
intestinal obstruction or malnutrition (the worms eat the
nutrients before the kid can get them). More severe infections
can see the worms invading up the biliary tree and blocking it.
Can also see eosinophilia (worms going through the blood).
 Can't be directly passed human to human (eggs need 2 weeks
in soil to mature).
 Diagnosis: passage of a worm, ova in stool, and eosinophilia
during pulmonary or extra-intestinal stages.
Pinworms:
 Roundworms.
 Acquired when the larval eggs are ingested. As far as I can tell
this means you can get human-to-human transmission.
 Hatches, matures, and lives in the colon.
 Mature females migrate to the anus at night and lay their eggs
there-- patient can autoinfect with these eggs (cause itching) or
transmit them to others. No other symptoms.
 Diagnosis is made by putting tape on the perianal region-- this
picks up the eggs and you can see them under the microscope.
This is called the Scotch tape test. No, really.
Whipworms:
 Roundworms.
 Live in contaminated food or dirt; acquired when the larval
eggs are ingested (have to mature in the soil, thus are not
transmitted human-to-human).
 Worms hatch in the upper GI and burrow into the intestinal
mucosa; they migrate to the large intestine and live there.
 Mainly asymptomatic but can cause mild GI symptoms. See gut
eosinophilia (since they're burrowing into the GI wall).
 Note in severe cases (as per small group) they can cause rectal
prolapse.
Strongyloides:
 Roundworms.
 Live in the soil. Acquired when the larva penetrate through the
skin, usually of the foot. No human-to-human transmission (I
think).
 Note that eggs aren't shed into the stool (either reproduce in
the host or shed larvae).
 Severe infections generally occur only in immunocompromised
individuals (classically the guy on chronic corticosteroids who
ups his dosage).
 They burrow into the mucosa of the small intestine, go to the
lung, and are coughed up and swallowed again as before; as
mentioned, this is the only worm mentioned that can multiply
by its lonesome inside the body-- once it gets outside of the GI
tract it can be lethal (goes to lung, obstruct the intestine, etc).
 Cause diarrhea, vomiting, and anemia.
Hookworms:
 Roundworms.

Live in the soil. Acquired when the larva penetrate through the
skin, usually of the foot. Again, no human-to-human
transmission.
 They get into the bloodstream, travel to the lungs, break into
the airways, and again are coughed up and swallowed to get
back to the intestine, where they hook onto the intestinal wall
and suck blood. Can cause anemia. Cause eosinophilia.
4. Consider learning the major factors associates with the following blood and deep
tissue helminths. A. Where is the organism acquired (i.e. its reservoir in nature)? B.
What is its mode of transmission? Is it only transmitted human to human or is it
acquired by contact with animals? Is there an insect or animal vector that is required
for its transmission? C. Where can the worm or other form of the organism (e.g.
cyst) be located in the human body? Does it invade beyond the intestine? How are
bacteria associated with the symptoms associated with certain filarial diseases (e.g
Wuchereria bancrofti, Elephantiasis) D. What are the major symptoms associated
with human disease? How is a laboratory or clinical diagnosis made?
 Cysticercus and Echinococcus:
 Cysticercus: Cysticercus isn't actually an organism, but the
result of ingesting ova from porcine tapeworms (larvae go to
the CNS). See earlier discussion.
 Echinococcus is obtained from ingesting the ova of a canine
tapeworm; can cause large cysts in the liver. Note if you break
the cyst open you can cause anaphylaxis (lots of sudden IgE in
blood).
 Trichinella:
 Get the disease (trichinosis) from ingesting cysts in raw pork.
The larvae invade the GI tract and spread systemically.
Specifically they go into the muscle and cause myalgias. Can
also see periorbital edema and conjunctivitis.
 Disease state is caused by larval presence in heart, skeletal
muscle, and brain as well as GI tract.
 (don't confuse the Latin Trichinella with the Latin name
of whipworm,
Trichuris.)
 Schistosoma:
 Flatworms.
 Major worldwide problem (not in US): lives in certain species of
freshwater snail.
 Complicated life cycle- larva penetrates the skin, go to a variety
of sites in the body, causes extensive inflammation and Th2
immune response.
 Causes dermatitis, Katayama fever, fibro-obstructive disease.
 Clinically: lymph node enlargement,
hepatosplenomegaly (often with decreased liver function
due to inflammation), and eosinophilia. Also look for
hematuria with or without dysuria.
 What happens: the larvae get into the intestinal or bladder
veins; there they shed their eggs (which, unlike the larva, are
very immunogenic) into the bloodstream and they go into the
lung, liver, or brain. In the liver, in particular, the eggs cause
an immune reaction and granulomas and fibrosis in the
hepatic/portal veins, resulting in portal hypertension.
 Filaria:



Roundworms.
Caused by Wucheria bancrofti-- invades blood and lymphatic
channels and cause elephantiasis.
(note that Wucheria bancrofti has a bacterium, confusingly
named Wolbachia, that's carried by the worm-- the bacteria
secrete a toxin that seems to help the worm survive and cause
elephantiasis.)
Antiparasitics
Tuesday, March 03, 2009
8:54 AM
Antiparasitics, 3/3/09:
[Again: LOs = know drugs.]
[As ultra-condensed through CMBMRS: For most clinically significant protozoa use
metronidazole. For roundworms generally use the bend-drugs (see below). For
flatworms generally use praziquantel. Malaria use chloroquine or quinine for RBC
treatment and primaquine for liver treatment.]

Anti-protozoals:
 Metronidazole:
 Administered as a prodrug, converted to active form only by a
unique protozoal enzyme and thus relatively nontoxic for host
(causes an unpleasant metallic taste, but that's about it for
most people).
 Drug interactions: alcohol plus metronidazole produces a
disulfiram-type effect. Disulfiram plus metronidazole
produces a delusional state.
 Mechanism: it breaks up DNA (free radical activity).
 Used for trichomoniasis, amebiasis, and giardiasis. Of bacterial
note it's also used for C. difficile and H. pylori.
 Paromamycin:
 Aminoglycoside that's only effective against parasites in the
lumen (no effect on systemic infections): oral administration
only, no systemic absorption, concentrated in gut (and
parasite). Like all aminoglycosides, binds to A site of ribosome
and blocks protein synthesis.
 Well tolerated; some GI side effects.
 Used for luminal amebiasis and visceral/cutaneous
leishmaniasis.

Anti-opportunistics:
o Toxoplasmosis: use pyrimethamine/sulfadiazine. This inhibits
folate synthesis in the protozoa (like TMP and SMX respectively).
o Pneumocystis: use SMX/TMP. These also inhibit folate synthesis.
 Note both of these have sulfa components, so watch out for
allergies.
o Pentaminide can also be used if there's a sulfa allergy-- it can be
used against the milder form of trypanosomiasis or visceral
leishmaniasis. It screws up target DNA.
Pentaminide is used in inhaled form for PCP (no side effects).
When given IV or IM, however, get a very rapid accumulation
of dead or dying microorganisms and a corresponding increase
in histamines, which leads to anaphylaxis. Also get severe, lifethreatening diabetes. Also seems to inhibit human dihydrofolate
reductase, so watch out if co-administering methotrexate.
Atovaquone: used for PCP, toxoplasmosis, and most significantly
malaria.
 Inhibits a parasite-unique enzyme, prevents electron transport.
 Very well tolerated but doesn't play well with rifampin.

o


Anti-helminthics:
o Vs. roudworms (Ascaris, pinworms, hookworms, whipworms):
 Benzimidazoles (thiabendozole, mebendazole, albendazole-they all have bend in them):
 Bind to parasite tubulin, inhibit microtubule formation.
 Easy to acquire resistance (single point mutation)-widespread resistance.
 Results in parasite paralysis and usual subsequent
expulsion.
 Note that albendazole is the drug of choice for invasive
cystic infection (cystic hydatid disease caused by dog
tapeworm, cysticercosis caused by pork tapeworm) and
is absorbed and distributes well.
 Mebendazole targets GI tract infections only (no
absorption).
 Teratogens.
 Pyrantel:
 ACh receptor agonist; only given orally and not
absorbed (otherwise it would cause host paralysis and
death).
 Causes complete spastic paralysis of worms.
o Vs. tapeworms and liver flukes:
 As mentioned, albendazole is used for invasive tapeworm cyst
infection from dogs/pork.
 Praziquantel:
 Used for tapeworm and flukes except the dog/pork
cystic diseases.
 Induces paralysis of the worm/fluke's nervous system;
also damage the outer shell of the worm and induce
local host immune activity. This is why you don't use it
in dog/pork cystic diseases-- the cysts are in sensitive
tissue (eg. brain) that will be damaged by an immune
response (can cause permanent optic damage,
meningeal damage).
 Used orally only.
Anti-malarials:
o Malarial drug resistance is extremely widespread and develops rapidly.
Killing the mosquitoes is probably the best way to go.
o Need to think about two things with treatment: treat the febrile stage
of the infection and also eradicate the latent stage in hepatocytes (if
present) to prevent reinfection.
o
o
o
o
o
o
o
Recall that P. falciparum is the most dangerous (infects all RBCs, is
often fatal) but has no latent stage. Recall also that P. vivax and P.
ovale are much milder but have a latent stage. Blood smears can tell
the various species apart.
Chloroquine is the drug of choice for the strains that are sensitive to
it. It inhibits the parasite's heme polymerization enzymes (interferes
with feeding process).
 Chloroquine distributes slowly but has a long half-life. Good
absorption and well-tolerated; most adverse reactions are
associated with IV injections (cardiac and CNS disturbances).
 It will cause some hemolysis in G6PD-deficient patients.
 For species with latent infections, you have to use primaquine
in addition (primaquine targets latent Plasmodium in liver cells,
chloroquine targets them in RBCs only!).
Quinine/Quinidine is the drug for chloroquine-resistant strains.
 Used in conjunction with an antibiotic (which one depends on
which region the malaria is from). Derived from cinchona bark
(Peruvian tree). Quinidine is the injected form, quinine is the
oral form.
 Similar MoA to chloroquine (inhibits heme polymerization).
 Good absorption and distributed. Note that its bioavailability is
much lower during the febrile stage than the afebrile stage
(dose higher during febrile attacks, dose lower during afebrile
stages to avoid drug toxicity).
 Adverse: cinchonism: visual impairments, tinnitus and
deafness, nausea and vomiting. You also get hypoglycemia and
hypertension. Causes some hemolysis in G6PD deficiency.
Mefloquine: alternative drug for prophylaxis of malaria. Fallen out of
favor due to psychological effects (having been on it for 12 weeks last
summer, I can vouch for that).
 MoA unknown, probably similar to chloroquine/quinine.
 Very long half-life (about 20 days)-- you can't give quinine and
mefloquine at the same time, which means you can't give
quinine for quite some time after discontinuing mefloquine.
 Teratogenic.
Proguanil: another alternative drug for malaria. Inhibits dihydrofolate
reductase; used along with atovaquone (together = "malarone") for
synergistic effects.
 "Wonder-drug": 98-100% effective against multidrug-resistant
malaria.
 $35/shot, very expensive.
 Safe to use in children. Minimal side effects.
Primaquine: the only malaria drug that targets the hepatic stage of
malarial infection.
 Unclear MoA.
 Resistance develops rapidly; with resistance, there is no other
drug to target hepatic stages of malaria.
 Causes massive, potentially fatal hemolysis in patients with
G6PD deficiency! Must test patient before drug administration.
Artemisinin: component of Chinese medicine; clinical cure rates of
about 90% of P. falciparum. Recommended to be used in combination
therapy with other antimalarials or antibiotics. Not specifically FDA
approved for malaria treatment yet but occasionally used.
Protozoa and Worm Cases
Tuesday, March 03, 2009
10:04 AM
Protozoa and Worm Cases, 3/3/09:
[This was a clinical rehash of the preceding lectures. The LOs have no new questions.
General notes on the presentation follow.]





He has a couple diagrams at the back of his handout for classification of
malarial blood smear findings and eggs in stool examination.
He emphasized travel history for worms and direct visualization for
protozoans.
Amastigotes = hemoflagellates that have lost their flagella (eg. trypanosome
cruzi).
Flukes (Schistosoma) and tapeworms are flatworms (most of the rest are
roundworms).
Roundworms have a digestive tract; flatworms (being flat) don't.
Case 1: Splenomegaly + travel to desert regions + bitten by flying insects =
Leishmania.
Case 2: Lymphadenopathy + facial edema + hepatosplenomegaly + South America
= T. cruzi.
(acquired from kissing bug)
Case 3: Mexico + mucosy-diarrhea w/ blood specks + slow onset = Entamoeba
histolytica.
(very central nucleus with well-distributed chromatin.)
(in non-fresh stool samples, see encysted, tetranucleated form of amoebas.)
(can cause liver abscesses.)
Case 4: Multiflagellated single-cell organism on wet mount of vaginal discharge:
Trichomonas.
(treat with metronidazole.)
(no cysts in humans from Trichomonas, just flagellated forms.)
Case 5: Travel to West Africa (Togo) + chronic diarrhea (usually fatty with lots of
diarrhea) and weight loss = Giardia.
(can also visualize with immunofluorescence or ELISA tests.)
Case 6: Immunosuppression + chronic diarrhea + confusion (multiple brain lesions)
+ acid-fast organisms in stool sample = Cryptosporidium parvum (diarrhea) and
Toxoplasma (CNS symptoms).
(Toxoplasma is carried by cats; cysts in food can be infectious.)
Case 7: Achlorhydria + raspberries + flu-like symptoms + chronic GI upset + acidfast in stool = Cyclospora cayetensis.
(looks very similar to Cryptosporidium: acid-fast, coccidium. Cayetensis is
bigger.)
Case 8: Vietnam + paroxysmal febrile episodes + splenomegaly + 2 episodes in four
days + single "engagement-ring" ring form in RBCs + > 12 merozoites in schizonts
in peripheral smear = Plasmodium vivax.
(two fever episodes in four days implies a 48-hour febrile cycle, which
implicates P. vivax, ovale, or falciparum.)
(note sickle-shaped gametocytes = falciparum; recall falx = L. sickle.
Falciparum also shows more than one ring form per RBC.)
Case 9: Japan + ascites/liver failure + splenomegaly + umbilical hernia = Japanese
schistosomiasis (Schistosoma japonicum).
(lots and lots of egg production = lots and lots of immune reaction = lots and
lots of tissue damage, as in the liver.)
Case 10: Egypt + blood in the urine + eggs in the urine = another schistosome,
Schistosoma haematobium.
(presumably would also find eggs in liver.)
Case 11: Lymphatic blunting/stasis + Jamaica + extreme lower leg edema = Filaria
(invasive helminth) plus Wucheria bacteria infection in the helminth = elephantiasis.
(Filarial species invade into and live in the lymphatics; the chronic
inflammatory response and bacteria toxin cause blockage of the lymphatics
and edema.)
Case 12: Fever, aches and pains, periorbital edema and conjunctivitis after eating
uncooked pork = Trichinella.
(larvae and cysts in muscle produce aches and pains. Has a predilection for
the muscles of the eyes.)
(other types of meat associated with Trichinella: bear, whales, seals.)
Case 13: Kid with constant perianal itching with positive scotch tape test: pinworm.
(very common in US, largely asymptomatic.)
Case 14: Passage of a worm through stool after cramps and abdominal pain =
Ascaris.
(acquired through ingestion of cysts which hatch in GI tract.)
Case 15: Asthma/COPD patient on chronic corticosteroids + dyspnea,
nausea/vomiting: Strongyloides.
(increased steroid dose allow organism to divide and get into lungs, etc)
(other nemotodes with a lung phase: hookworms, Ascaris.)
Case 16: Seizures + Central America + multiple calcifications and cystic lesions in
brain: Taenia solium (pork tapeworm) that's progressed to cysticercosis.
(ingestion of ova, not cysts in meat, cause cysticercosis-- thus the guy here
got this from fecal-oral contact, not ingestion of pork.)
Hospital Infection Control
Monday, March 09, 2009
9:08 AM
Hospital Infection Control, 3/9/09:
[Conveniently enough, the LOs for this lecture are in her notes. I've copied and
pasted them here. Yes, I'm being lazy, but most of this is pretty simple and I'm
about a year and a half past the point where I would have been excited about typing
it all up myself.]
[Note that she made a point of saying that gown and glove contamination occurs
regardless of patient contact-- if you're in the room of someone with contact
precautions and you're touching anything at all, you're probably accumulating bugs.
She also likes gowns and gloves (vs. just gloves) for preventing VRE spread. Note
the distinction between precautions with pneumonia (droplets, can use surgical
mask) and precautions with TB (airborne, have to use N-95).]
A.
Understand the proper indications and use of hand hygiene.
1.
Define hand hygiene.
a) Hygiene usually refers to cleanliness and especially to any practice
that leads
to the absence or reduction of harmful infectious agents.
Applies to
handwashing, antiseptic hand rub, or surgical hand
antisepsis.
2. Describe the different methods of hand hygiene and when each method
should be employed.
a) Hand hygiene methods include:
1) Handwashing (washing with soap and water) and includes hand
drying.
2) Antiseptic hand rub (rubbing hands with an antiseptic hand
rub which
include alcohol based gels and foams.)
3) Surgical hand antisepsis (preoperative antisepsis handwash
or hand rub
performed by surgical personnel.)
b) Indications for handwashing and hand antisepsis:
1) Decontaminate hands before and after having direct contact with
patients.
2) Decontaminate hands after contact with inanimate objects
(including
medical equipment, furniture and environmental
surfaces) in the occupied
patient room.
3) Decontaminate hands before donning sterile gloves while
inserting a
central intravascular catheter.
4) Decontaminate hands if moving from a contaminated-body site to
a clean-body site during patient care.
5) Decontaminate hands after removing gloves.
6) Decontaminate hands prior to the placement of indwelling urinary
catheters, peripheral vascular catheters, or other invasive devices that
do
not require a surgical procedure.
7) Decontaminate hands after smoking, or applying makeup/lipstick/lip balm.
8) Decontaminate hands after coughing, sneezing and/or blowing your
nose.
c)
Which method should be employed?
1) When hands are visibly dirty or contaminated with
proteinaceous material
or are visibly soiled with blood or
other body fluids, wash hands with
either a non-antimicrobial
soap and water or an antimicrobial soap and
water. In
addition, this applies to contact with patients with Clostridium
difficile or Bacillus anthracis. (Alcohol has poor activity against
spores.)
2) If hands are not visibly soiled, use an antiseptic hand rub for
routinely
decontaminating hands.
3) Before eating and after using a restroom, wash hands with
a nonantimicrobial soap and water or with an
antimicrobial soap and water.
4) Surgical hand antisepsis is performed prior to operative
procedures.
5)
Antimicrobial-impregnated wipes are not a substitute for
using an alcohol- based hand rub or antimicrobial soap.
3. Explain why health care workers with patient contact are not allowed to
have artificial nails or extenders.
a) Artificial nails or extenders can become colonized with bacteria
and are not
easily eradicated using hand hygiene. Outbreaks of
Pseudomonas aeruginosa
and other bacteria have been associated
with the use of artificial nails.
b) University of Colorado Hospital (UCH) policy forbids the use of
artificial
nails or extenders by providers with patient contact.
B. Understand the various types of barrier precautions and why they
are utilized.
1. Explain the rationale for standard precautions and what it entails.
a) Standard precautions are designed to reduce the risk of
transmission of
microorganisms from both recognized and
unrecognized sources of infection.
b) Standard precautions refers to the avoidance of contact with blood
and other
potentially infectious materials through the use of
mechanical safety devices,
modifications in performing
techniques/procedures and/or the wearing of
physical barriers to
exposure (this can include gloving, gowns, masks, and eye
protection.)
2. Explain what transmission based precautions are.
a) Transmission based precautions are systems of specific activities
based on
identified modes of transmission designed to contain
and/or prevent transfer
of organisms from a source (patient and/or
environment) to a susceptible host
(patient, health care provider,
visitor, etc.)
b) Three recognized types of isolation/transmission based
precautions are
practiced in addition to Standard Precaution:
Airborne, Droplet and Contact
Precautions.
3. Explain what airborne precautions entail.
a) Airborne isolation refers to the isolation of patients infected with
organisms
spread via airborne droplet nuclei <5mm in diameter.
b) Patient should be placed in a private room and the isolation area
must have >
6-12 air changes per hour and is under negative
pressure (the direction of the
airflow is from the outside adjacent
space into the room).
c) The use of personal respiratory protection (N-95 masks) is also
indicated for
persons entering the rooms in addition to standard
precautions.
3.
Explain what contact precautions entail.
a)
In addition to standard precautions, contact precautions are used
for patients
known or suspected to be infected or colonized with
epidemiologically
important microorganisms transmitted by direct
contact with the patient, with
environmental surfaces, or with
patient care items in the environment.
b) Patients must be placed in a private room and when entering the
room, gowns
and gloves must be worn. Gowns and gloves must be
removed prior to
exiting the room and hand hygiene should be
performed both before and after.
4.
Explain what droplet precautions entail.
a)
Droplet precautions refer to the isolation of patients infected with
organisms
that can be transmitted by droplets (large-particle
droplets [larger than 5 Fm in
size] that can be generated by the
patient during coughing, sneezing, talking,
or the performance of
procedures.
b) Patients must be placed in a private room, and in addition to
standard
precautions, anyone entering the room must wear a
surgical mask.
5.
Give an example of an organism(s) that would require standard
precautions versus airborne precautions versus contact precautions versus
droplet precautions.
a) Standard precautions - Human Immunodeficiency Virus (HIV),
Aspergillus
spp., methicillin sensitive Staphylococcus aureus
(MSSA), Streptococcus spp.,
etc.
b) Airborne precautions - Mycobacterium tuberculosis (mTB),
Measles, and
Varicella zoster virus (chicken pox) (including
disseminated Varicella zoster
(aka, shingles)).
c) Contact precautions - methicillin resistant Staphylococcus aureus
(MRSA),
vancomycin resistant enterococcus (VRE), and multidrugresistant gram- negative bacilli such as Acinetobacter spp.,
Pseudomonas spp., (after
assessment of antibiotic resistance
report), Clostridium difficile, and others.
d) Droplet precations - Respiratory syncitial virus (RSV), Influenza,
Neisseria
meningitides, etc.
C.
Blood Borne Pathogens and Bodily Fluid Exposures
1.
List 3 organisms that can be transmitted via needle sticks.
Human Immunodeficiency Virus (HIV), Hepatitis B virus, Hepatitis C
virus
2. Explain the proper procedure that should occur after exposure to a bodily
fluid.
a) Wash needlesticks and cuts with soap and water.
b) Flush splashes to the nose, mouth, or skin with water.
c) Irrigate eyes with clear water, saline, or sterile irrigants.
a. There is no scientific evidence that using antiseptics or
squeezing the wound will reduce the risk of transmission of a
bloodborne pathogen.
b. Report the exposure to your supervisor and to the department
responsible for managing exposures.
1. Describe 3 methods used to prevent exposures to blood borne
pathogens or body fluids.
a) Do not recap needles by hand and dispose of them in the
appropriate sharps
disposal containers.
b) Use medical devices with safety features designed to prevent
injuries.
c) Use appropriate barriers such as gloves, eye and face protection,
or gowns
when contact with blood or bodily fluids is expected.
D.
Understand the utility of and methods used in infection control
surveillance.
1.
Describe why surveillance for nosocomial (hospital acquired) infections
is important.
a) Nosocomial infections affect more than 2 million patients, cost
$4.5 billion, and contribute to 88,000 deaths in the US annually.
b) Surveillance is aimed at identifying patients with infections with
high level of
morbidity, pose recurrent epidemic problems, and are
potentially preventable.
On-going analysis of infection rates helps to
determine whether control efforts
are succeeding and whether
increased education or control measures are
needed.
2.
List 3 methods used for surveillance.
Review of microbiology laboratory results, “shoe leather” epidemiology
[ie. go around and look for similar cases], standar (that's all that's in
the notes.)
3.
Describe the process of an outbreak investigation.
a) Verify the diagnosis.
b) Determine the case definition.
c) Preliminary determination if outbreak exists.
d) Case finding and line listing.
e) Identify denominator of “at risk” population and develop rates.
f) Formulate hypothesis.
g) Institute control measures.
h) Epidemiologic analysis of hypothesis.
4.
Describe methods used to prevent common nosocomial infections.
a) Use of hand hygiene in conjunction with appropriate standard
precautions or
transmission based precautions.
b) Use of aseptic techniques when inserting and handling invasive
devices.
c) Removal of invasive devices as soon as possible.
d) Appropriate and judicious use of antimicrobial agents.
e)
Reduction of risk factors associated with patient care (e.g.,
antibiotic
prophylaxis prior to surgery, minimizing aspiration pronesupine positions for
patients on ventilators, cleaning of surfaces
with appropriate disinfectants,
etc.)
E.
Understand the role of infection control in emerging infections and
bioterrorism.
1. Identify bacteria or viruses that are considered potential bioterrorism
threats.
Anthrax, plague, small pox, tularemia, viral hemorrhagic fevers,
botulism toxin, etc.
2. Define the role of infection control in emerging infections and bioterrorism.
The key to the defense against bioterrorist attacks is a highly
functioning system of public health surveillance and education so that
attacks can be quickly recognized and effectively contained. Syndromic
surveillance and recognition of unusual clustering of infections is the
first step in identifying potential issues.
3.
Describe the impact of SARS on hospitals and medical staff and the role
of infection control.
Refer to required reading assignment.
[They like SARS as a good example of these kinds of control measures
preventing outbreaks.]
Bioterrorism/Poxviruses
Tuesday, March 10, 2009
7:31 AM
Bioterrorism/Poxviruses, 3/10/09:
[She says that new viruses are indistinguishable from bioterrorism and that you have
to respond in the same way to both.]
1. Identify the key features that make a microbiological agent a potential bioweapon.
Evaluate each of these factors for smallpox, anthrax, ebola, and plague.
 High mortality/morbidity, human-to-human spread, low infectious
dose, aerosolizable, lack of rapid diagnostic tests/effective
drugs/vaccines/immunity, able to be cultured in large quantities,
stable in the environment, able to be weaponized (put into stable form
for storage), and able to cause mass casualties (which would seem to
follow from the rest).
 Evaluation of individual agents in any practical depth gone over neither
in notes nor lecture. Most of this should be familiar from other parts of
the course (eg. tularemia isn't able to be spread human-to-human,
plague has a transmissible pneumonic flavor and a non-transmissible
bubonic flavor, etc). Vaccines and treatment are discussed below.
2. Describe the characteristics of organisms classified by CDC as Category A agents,
and explain what makes each of them so dangerous.
 Easily transmissible, high mortality rates, "might cause public panic,"
require special action for public health preparedness.
 This seems to argue for naming new deadly micropathogens after
cutesy things. "It's a Fluffy Bunny Virus epidemic" just doesn't stir up
a lot of public panic.
3. For each Category A agent, evaluate what resources are available for prevention,
diagnosis and treatment.
 Anthrax: new vaccine is in trials; antibiotics are effective.
 Plague: no effective vaccine; antibiotics effective if given both before
and after exposure.
 Tularemia: no vaccine; antibiotics effective if given both before and
after exposure.
 Smallpox: vaccine exists but scant herd immunity; no antiviral drug is
effective.
 Viral hemorrhagic fevers: vaccines are in development (yellow fever
vaccine exists but not in bulk); no antiviral drugs are effective.
 Botulism: vaccine exists but not in bulk, new vaccine under
development; antitoxin exists but is only effective against unbound
toxin.
4. Identify mechanisms for manipulation of pathogenic mechanisms, virulence,
antigenicity and antibiotic resistance of existing microbiological pathogens for use as
biothreat agents.
 She seems to say that you too can make novel and deadly bioweapons
in your garage, but you can't weaponize them on a large scale without
additional support.
 Virulence enhancement (changing antigenicity, creating antibiotic
resistance, etc) could be either genetically tailored or naturally
selected for (with heat, small amounts of antibiotics, etc).
5. Describe how new or chimeric pathogens could be created as bioweapons, and
how recombinant DNA technology could be used to recognize such novel organisms.
 Essentially you take anthrax genes and stick them into, say, staph
aureus, or your organism of choice.
 She's optimistic about our ability to quickly recognize a novel organism
with PCR and antitoxins. You can also sequence any organism's DNA
and then run it through genomic databases to see what parts of a new
microorganism come from what.
6. Describe how clinicians can rapidly recognize a bioweapons incident, and how the
health care network can respond.
 Look for unusual clinical presentations, clusters of cases of previously
rare diseases, or agricultural diseases in an urban area.
 There are also surveillance networks, response teams, and diagnostic
labs in place along with isolation and quarantine protocols.
 I remember being a few blocks away from Times Square on
New Year's Eve of 1999. The police had set up barricades to try
and limit the number of people going in, but there were so
many people that, inevitably, one of the barricades got tipped
over, and after that there was no stopping the flood of people
that poured through-- the cops just melted away. Containment
protocols are nice but I wonder if we'll see the same thing.
Normal Flora
Tuesday, March 10, 2009
9:01 AM
Normal Flora, 3/10/09:
1. • identify body sites that have normal flora and describe typical bacterial species
that are likely to be isolated from clinical specimens collected at such sites (review
and integrate the relevant content from other lectures, labs and small group
exercises).
 More or less all exposed body surfaces contain normal flora. Have
about 10 times as many NF bacteria than human cells in the body.
 Note that genetic predisposition to obesity may be related to different
colonic flora (lean = Bacterioides, obese = Firmicutes in mice) and
different flora may also predispose to obesity (Firmicutes in genetically
neutral mice cause increased weight gain, presumably by more
efficient energy extraction).
 You know what I'm going to review and integrate? A cold beer.
2. • describe how molecular methods can be used to complement traditional culture
methods for analyzing the complexity of microbial flora in health and disease.
 You use PCR sequencing of various 16S rRNA segments (a portion of
which is extremely well-conserved) to distinguish between different
bacteria.
 Normal flora is pretty complex (on a first pass they found about 240
new species of bacteria in the colon alone). However, notice that a few
large bacterial groups make up most of that population (in particular
Bacteroidetes and Firmicutes).
 Note that E. coli isn't one of them (~0.1% of colonic bacteria).
 Note also that "Firmicutes" sounds like Latin for "nice butt."
Bacteriologists need to get out of the lab and stop naming
organisms as icebreakers to their co-workers.
 No 'normal flora' population (in us or nature) has been sampled to
completion.
3. • explain how bacteria can use quorum sensing for adaptive responses to signals
that reflect the density of homologous and heterologous bacterial populations.
 Quorum sensing: bacteria secrete signaling molecules; the
concentration of those molecules lets the bacteria 'know' how many of
their colleagues are nearby.
 Note that these signals can be species-specific (Lux I/R proteins in
Gram-negatives, oligopeptide sequences in Gram-positives) or signal
the presence of a variety of species, presumably commensal with each
other (AI-2, Lux S). Note that human signaling molecules (EPI/NE) can
also be signaling molecules, although I'm not sure how that counts as
a quorum.
 This affects bacterial behavior in various ways, including biofilm
production (see next point) and toxin production.
4. • explain how bacterial biofilms are formed, how they disperse, and how the
properties of sessile bacteria in biofilms differ from those of planktonic bacteria.
 Biofilms (secreted polymeric matrix containing bacteria) are often
quorum-stimulated things-- without sufficient bacterial numbers, they
aren't secreted. This is presumably why small amounts of (for
example) Pseudomonas don't get stuck in biofilms all the time.
 They disperse either because something's happened to cause the bugs
to dissolve out of them or because an external stimulus has detached
them (which would really be the only two possibilities).
 Bacteria in biofilms divide more slowly and are more resistant to host
defenses and antibiotics than non-biofilmed (ie. planktonic) bacteria.
Note that degranulation of phagocytic cells doesn't much bother the
biofilm but can cause significant host tissue damage.
5. • describe the importance and role of bacterial biofilms in infectious diseases.
 I think we've been over this enough.
6. • define probiotics, indicate conditions for which controlled data support their use,
and indicate what additional studies will be needed to support their use for other
conditions.
 Probiotics: supplemental, live microbial organisms administered with
the intent of benefiting the host. I imagine the point is to establish
'ideal normal' flora in the gut in order to minimize the likelihood of
overgrowth of unpleasant minorities (eg. c-diff).
 Currently a controversial subject; some studies say it's good for
diarrhea.
 Probiotics are supplements, thus not well-regulated at the moment.