Molecular Diagnostics for Ocular Infectious and Inflammatory Diseases

[MUSIC PLAYING] RUSSELL VAN GELDER: Great. Thank you, Jeff. Real pleasure to be here. I’ve been pretending to do lab
medicine for a number of years now as a complete amateur
and sort of as a hobby, and it’s fun to come here and
talk a little bit about some of the work that
we’ve been doing in terms of diagnosing ocular
infectious and inflammatory disease. I have a few disclosures. There we go. There’s a company
called NovaBay. I have no personal
stake in this, but they did support
some of the research that we’ll talk
about in this talk. So my specialty is uveitis or
ocular inflammatory disease. This is probably not
something most of you are very familiar with unless
you’ve been unfortunately affected yourself or
had a loved one who is. But it’s broadly defined as
intraocular inflammation. And this is an example of a
patient who has active uveitis. This little white blood
cell pus pocket here is called a hypopyon, and
it’s not a good thing. You don’t want that in your eye. When we talk about
uveitis, technically, this is inflammation of the uveal
tract of the eye, which is the pigmented tissues. It’s the vascular
bed of the eye. And that includes the
choroid, the ciliary body, and the visible part of the uvea
that you can see is the iris. So often, in the front of
the eye, we call this iritis, but technically these
are all uveitis. Uveitis is not that uncommon. The incidence is
about 50 per 100,000 and the prevalence
about 100 per 100,000. So at any given time, there’s
about 300,000 people in the US who have active uveitis, means
that about 1.2 million people in this country alive
today will have this at some point in their life. And it can be a royal
pain in the neck, but it can also be a
blinding eye disease. And depending on
which list you read, it’s either the fifth
or sixth leading cause of blindness in the
US, so not a trivial problem. Now, when we talk
about uveitis, people say, well, what causes it? What do we do about it? And it’s really not a
disease entity unto itself. It’s a descriptive term. It’s any intraocular
inflammation. And so it’s a very
heterogeneous collection of individual
diseases, all of which share a final common pathway
of leukocyte infiltration into the eye. So the way you know
someone has uveitis is you see an inflammatory
infiltrate in their eye. But that can be because
of an autoimmune process, an autoinflammatory process,
or occult infection. And I’m going to spend
most of the talk covering occult infection. When we think about
autoimmune diseases, the canonical one is a disease
called sympathetic ophthalmia. This was an
unfortunate gentleman who took a BB to the eye, which
basically ruptured this eye. This is what we call phthisis. So that’s an opacified
cornea, and that eye really has no visual potential. The retina is detached. But unfortunately, if
the eye is not enucleated and significant
amounts of uvea are exposed to the immune
system, because of the ocular immune
privilege, because the eye is a privileged space,
the thymus never really got a good shot at the
unique intraocular antigens. And you end up with
this terrible disease called sympathetic
ophthalmia where the good eye becomes subject
to a chronic autoimmune attack. And this is why,
when we do see people who have severe
injury to one eye, we frequently will
enucleate that eye within two weeks of the
injury, and that lessens the risk of sympathetic. Exactly what the antigens are
is not known at this point. And it’s a rare condition,
only occurs in about one in 1,000 ruptured globes,
but it’s catastrophic because it’s your only good eye. This is actually what Louis
Braille suffered from and went blind from. He had had an injury
in the early 1800s as a child in one
eye and then lost his vision in the other eye
to sympathetic ophthalmia. Most of the diseases that
we know are autoimmune are fairly rare syndromes. And they’re really
interesting diseases. Vogt Koyanagi Harada disease,
which I doubt any of you have ever heard of, is a
disease that features tinnitus, uveitis, and vitiligo. So you get patches
of skin depigmented and ringing in the
ears, and you go blind. And it’s because of
an autoimmune reaction to melanin, which is found
in your skin, your inner ear, and your eyes. Birdshot choroiditis,
another interesting, very rare disease– this is a
picture of birdshot down here– little yellow spots in
the back of the eye. It’s a retinitis. And this disease has
among the highest HLA associations of any disease. Essentially 100% of
patients with birdshot are HLA-A29 positive. And again, the
antigen is not known, but it’s a bilaterally
symmetric disease that’s highly HLA linked. And another interesting
autoimmune disease is tubulointerstitial uveitis
and tubulointerstitial nephritis and uveitis
syndrome, or TINU, which, again, is
incredibly highly linked to an HLA-DR locus, about
relative risk of 150 and causes, mostly in kids, an
acute interstitial nephritis and an acute uveitis. So there are some
pretty interesting autoimmune diseases,
but I would say this is the vast minority of
patients that I see clinically. Autoinflammatory causes
are also really interesting and perhaps even rarer depending
on how you lump things. The only really canonical
autoinflammatory disease that we know uses this
mechanism is a disease called Blau syndrome, and this is one
that you find in kids, again. It’s a bilateral chronic
granulomatous uveitis that’s usually associated
with erythema nodosum in the skin and, often,
pulmonary nodules. And it looks a lot
like sarcoidosis. The disease is due
mutation in NOD2/CARD15, which is involved in
toll receptor signaling and is in the activation pathway
between TLRs and NF-kappa B activation. So mutation there basically
causes a persistent response to normal flora, which results
in granulomatous inflammation. But what I really want to
talk to you today about are the occult infections. And this is where I think
lab medicine intersects a bit with what we do in uveitis. It’s a little bit
mind boggling to think how brief in the history
of human endeavors the knowledge of the
microbial world is. Anyone know what
this is a picture of? SUBJECT 1: Van
Leeuwenhoek’s microscope. RUSSELL VAN GELDER: Exactly. It’s van Leeuwenhoek’s
microscope. So the senior faculty were
around when this was in vogue. So and anyone know what van
Leeuwenhoek did for a living? He was not a microbiologist. SUBJECT 2: Textiles. RUSSELL VAN GELDER:
He was a textile guy. He made drapes. And he felt he was getting
ripped off on his fabric, that the thread counts on his
fabric was not up to snuff. And he was an
amateur glass blower, and so he made this little
device with a little molten spot of glass. You can barely see
it in this picture. Right up here, there’s
just a tiny globule of glass in the middle
of this metal thing. And if he held his eye
close enough to it, he basically got a
simple microscope. He’d put the thread that he was
looking at here on the screw and then raise it up
into the line of sight, and that was the microscope. But what he found was not
thread count, necessarily. He drew pictures of
these things, which he called animalcules, and
these animalcules looked a lot like fungi to most of
us, and, of course, he could also see some
hints of bacteria. And it was this whole
kingdom, this whole world that had opened up because of
this the silly little device that he had made. Progress being slow, of
course, microbiology kind of stalled out for about 200
years until the mid 1800s. Anyone know what this
is as a culture medium? SUBJECT 3: Potato. RUSSELL VAN GELDER: Right. It’s anthrax growing
on a potato, which is how Koch isolated anthrax. So he did not have the
luxury of agar plates. Agar plates were actually
invented by one of his fellows. They were trying to make
something besides potatoes that they could
grow cultures on, and they were using gelatin. And the problem of
gelatin is, of course, that there’s lots of
gelatinases in bacteria, and so is dissolving the plates. And one of the spouses of one
of his fellows was a baker and said, oh, you should
try seaweed extract. It works really nicely, makes a
thing that looks like gelatin, but it isn’t. Of course, they had no idea it
was polysaccharides or anything like that. But they said, OK,
we’ll try that. We’ll use agar, and hence
the agar plate was born, and that was in about 1880. And here we are today
in 2019 and we’re still using agar plates. In fact, this could
have been a Koch plate. And viewed in the future, I
think when people look back at this era at mixtures of
seaweed extract and sheep’s blood, or brain-heart
infusion, or some of the things that we use that seem more
out of Macbeth than out of modern medicine, they’ll
kind of scratch their heads and say, well, I
guess that worked. It’s interesting. But really, was that optimal? But this is still state of the
art for a lot of microbiology. But I’m bringing coals
to Newcastle here. So all of you are
familiar with PCR. Don’t need to review
the background here except to point out Gobind
Khorana’s contributions to PCR just for history. I think Kary Mullis
won the prize for reducing PCR to practice. But if you go back
in the literature, Khorana actually described
PCR pretty accurately in a 1969 paper. But of course, he had no
thermal stable polymerase to actually do this. Khorana, of course, famous
for solving the genetic code, but also became a
vision researcher in sort of the second
phase of his career and was involved in
purification of rhodopsin. So what has PCR taught
us about uveitis? Well, we have at
least three diseases that were idiopathic uveitis. And these are all pretty
rare, but they’re interesting. This first one is called Fuchs
heterochromic iridocyclitis. And this is a really
interesting chronic uveitis that this woman has where
basically the uveitis eats away the iris over time and
changes the eye color. And it’s always unilateral. This is the affected
eye in this patient. It’s a triad of relatively
asymptomatic uveitis cataract and glaucoma
elevated pressure. And for years, people
thought, oh, there must be some missing pathogen
here because it’s always unilateral and it’s doing
funny things to the iris. Subsequent PCR and
antibody-based work actually revealed that this is
a chronic rubella infection. And it’s childhood
or congenitally acquired rubella that gets
into the lens of the eye, which can serve as a safe
harbor for this. And in about 30% of patients
with this condition, if you tap the eye
and run RT-PCR, you will find rubella virus. And all patients with
this have high titers of intraocular
rubella antibodies. A similar disease called
Posner-Schlossman, which looks very much like this
except the iris doesn’t turn as light but is associated
with much more episodic uveitis and high eye
pressures, turns out to be due to cytomegalovirus,
but in an immunocompetent host. So this is not CMV retinitis. This is actually CMV
in the normal host. Again, the eye being
immune privileged can provide a safe
harbor for the virus to replicate and persist. And so people get episodic
reactivation of CMV in an eye, and that’s Posner-Schlossman. And then PCR has also
led us to realize that HHV-6, Human
Herpes Virus 6, is associated with certain
forms of bilateral panuveitis. So we’ve had a few
small successes in identifying occult pathogens
with specific uveitic diseases. But I want to share
two cases with you that got my interest up
in this area when I was at Wash U in St. Louis. First one was this
gentleman who came to see me having gotten
something in his eye mowing his lawn about
three months prior, so just felt
something in his eye and then it went
downhill from there. He saw an optometrist,
an ophthalmologist, and then a cornea specialist. And by the time he
got referred to us, he’d had two corneal biopsies,
which were both negative for anything, no
bacteria, no fungi. It was thought maybe he had
a parasite or a protozoan, like acanthamoeba. He’d been treated
for acanthamoeba, which is basically using
pool disinfectant PHMB, and that didn’t work. So he came to us. And at that point, his
cornea looked pretty bad. Hard to see here, but
he’s kind of caved in on this side on his cornea. So he basically needed a
cornea transplant urgently. I sent him to my colleague,
Tony [INAUDIBLE],, and asked Tony if he
would share with me a piece of the cornea
and that schmutz in the front of the
eye, which he did. And I ran– this was in about
2004– ran 16S and 28S PCR. And lo and behold, the
28S band came up positive. The first band is
from his cornea. The second is from the material
in the anterior chamber. Third’s positive control. Fourth is negative control. So this was the old days. So I cut the bands out and
sequenced them and asked Tony, in the meantime– it took
about a week to do this– how’s the patient doing? And he says, oh, the
cornea transplant looks great, a
little inflammation. I’m sending him back to you. So that’s a hypopyon. The eye is 50% full of
white blood cells there. So I talked with the
patient and I said, look, it’s clearly a fungus. We got the sequence back,
and it didn’t match anything in the database. It was about an 88% match
on the 28S to fusarium, but it wasn’t fusarium. In the meantime, the
pathology came back negative, silver stain, GMS,
all negative on the cornea. So I said, I think
that this is fungal. I can’t really prove it,
but it sure looks like it. Why don’t we treat you
like this is fungus. So we put him on
Itraconazole and Natamycin, and two weeks later
he looked like this. And when I left St.
Louis four years later, he was 20/25 in that
eye, seeing well and the infection was cleared. So this kind of got my interest
up in occult pathogens. The next case didn’t
end as happily. This was a cardiothoracic
surgeon, also in St. Louis, who
had had a bone marrow transplant for myelodysplastic
syndrome and was out fishing, cut his arm, got some water
in it, and got a cellulitis. He was on, I think,
Mycophenolate and Tacrolimus and was admitted to the
hospital for cellulitis and started to go downhill. So he got transferred
to the ICU and was complaining about floaters
in his vision and decreased vision. So our resident service saw him. And this is a not great picture
of the back of his retina because it was taken
with a portable camera. But you can see this
little yellow dot here– there’s a couple of them– and this general
haze, and that’s what we would call a
multifocal choroiditis, multiple little choroiditis
spots plus a vitritus. So we asked the
ID service, well, what’s growing in
his blood cultures? And they said, well,
nothing’s really growing, but there’s something
kind of weird here, and we’re sending the
smear out for evaluation. So it was these
little red spherules. And I think they
sent it to AFIP, and AFIP came back and
said, we’re not quite sure, but we think it might be
prototheca wickerhamii. So how many of you have heard
of prototheca wickerhamii? Yeah, this is a great audience. I’ve asked that like 20 times
in ophthalmology audiences and no one ever, right? But this is coals to Newcastle. So prototheca wickerhamii is
a non-photosynthetic algae. This is a really bad thing
to be growing in your blood. And unfortunately, he
succumbed to the infection. His wife was very kind and let
us take the eyes at autopsy. Mort Smith, our ocular
pathologist, splayed them open. We punched out those
choroiditis lesions. I made PCR primers to prototheca
wickerhamii, and there it is– so positive for prototheca
wickerhamii in the eye. Now, the scary thing about this
case, other than what exactly you can catch from
Missouri fishing streams, is I see a lot of,
lot of patients with multifocal choroiditis
that looks like this, and in a million years,
I wouldn’t think, oh, that’s probably algae
in the eye, right? So it just takes one
or two cases like this and you start to become
a conspiracy theorist and think, well,
how much of what I see that I call idiopathic
is really an occult infection and I have no idea
what I’m looking for? So that kind of
motivated us in the lab to try to develop some
techniques for looking for occult pathogens. Now, of course, again,
coals to Newcastle, the revolution in
deep sequencing has made a lot of
approaches to this feasible that even five or
10 years ago would have been science fiction. And we’re fortunate in my lab. We have an aluminum iSeq. So we can play with this
sometimes very quickly and try things out. All of you are familiar with
Moore’s law, which basically says computing power doubles and
price halves every two years, and that’s pretty much
held for three decades. For sequencing, we’re on Moore’s
law squared at this point. The cost of
sequencing since 2008 has really dropped at the
square of Moore’s law, which, since that’s already a log
law, is really impressive. And as you all know,
that first human genome, which cost over
$100 million, now is reduced to in the
hundreds of dollars, if that. So we’ve applied
three techniques to trying to find pathogens. First is just 16S metagenomics. For reasons I’m going to
show you, we rarely use this. This is a really bad
technique if you’re dealing with a
relatively clean sample because, frankly,
16S makes stuff up. It’s just too easy
to get contaminants that are not meaningful. So we do very little
16S metagenomics. Wonderful for the
GI system where you have a rich environment,
not so great in the eye. The second technique, which
we used for a number of years, we call Biome Representational
in Silico Karyotyping, and I’ll explain
that in a minute. And more recently, we’ve just
gone to whole genome pathogen metagenomics. So 16S you’re all familiar with. The basic idea is that there’s
conserved sequences in the 16S . Ribosome you get an
amplification product that allows you to see
pretty much all bacteria if you sequence it and put it
on a high throughput platform. The Biome Representational
in Silico Karyotyping was really a technique
that we started developing at Wash U with Elaine Mardis. And when we moved in
2008, Jay Shendure was very kind to
work with us on this. And the basic idea
here was in the days when it was still too
expensive to do whole shotgun metagenomics, could we do
a representational method that approximated that? And the analogy here is let’s
say you have an Oxford English Dictionary, which is
your biopsy sample, and you think that there’s
some French contaminating your English dictionary
that is foreign DNA. You could read the
whole dictionary– that is computationally and
timewise a daunting task– or you can pull out every
50th page and read it. And if you find French,
you’re done, right? And it turns out, if you don’t
find French, statistically you can put a pretty accurate upper
limit on how much contamination there may be in your dictionary,
and it’s much less than one per every 50 page for
statistical arguments. So the way this
technique works is it takes advantage of these type
2S restriction endonucleases. In particular, we
use the one BsaX1. So BsaX1 recognizes this
AC-5N-CTCC sequence. But unlike a type
1 endonuclease that would cut inside that sequence,
the type 2S and 2B enzymes cut outside. And BsaX1 actually cuts
a big chunk outside. It cuts 33 base pairs around
that recognition sequence, leaving two random three
base pair overhangs. So what we did was we basically
digest with this enzyme. We isolate the 33
base pair fragment. And then we ligate on
the Illumina adapters directly, which
makes it very easy, then, to do high-throughput
sequencing on these. And then we do the
same trick Illumina does to get the asymmetric
adapter ligation. That is, we use one biotinylated
adapter and one non. And then we amplify that
and sequence it directly. So what does this get us? Well, the six base pair
recognition sequence, AC-5N-CTCC, occurs
once every four to the six base pairs,
or 4,096 base pairs. And we’re cutting
out 33 base pairs. We kind of throw away the
two, three base pair overhangs because we don’t know that
the ligation is perfect. And so we get 27 base pairs,
basically, out of every 4,000. And so that ends up being
about 0.7% of the genome is released, on average,
by this restriction enzyme, and then we cut it
out and sequence that. One of the advantages
of this technique is that it is, in terms
of bioinformatics, it’s extraordinarily
efficient because we can create a hash table
of every BsaX1 site in GenBank, which we’ve done. And so once we get the
sequences off of the Illumina, it takes us less than 10 minutes
to run through a billion base pairs’ worth of sequence and map
every single sequence to human, or if it’s non-human
known, or if it’s unknown, put it in its own pile. We call it karyotyping
because, as a side effect, we get a full karyotype of
the human genome out of this very rapidly, as well. So it’s a very useful technique. It’s becoming less useful as
whole genome amplification has become more affordable. But we still use it,
and I’ll show you an example where it showed
us something that we did not see with whole genome. And of course, whole genome
is just basically fractionate the DNA and sequence it all
and then put it through, crack under another pipeline
that’s designed to detect metagenomic sequences. So it’s almost like
having a new microscope. And what I want to show
you in the rest of the talk are five little
vignettes of what we’ve learned with these techniques. So here’s our new microscope. Let’s take a look. First question we want to ask
is, what’s on the normal eye? What actually– does the
eye have its own microbiome? Is it like the gut? Do we have this little colony
that lives in our conjunctiva? You would think that this is
a question that would have been answered a long time ago. But in fact, if you
read the literature, this question has been
debated for over 100 years, and it’s still not resolved. Does the normal
conjunctiva have any flora? The eye surface has some innate
antibacterial mechanisms. In particular, there’s
a lot of lycozyme in tears, which will
reduce bacterial load, and there are
antibacterial peptides that are secreted
by the conjunctiva. So it’s been known
for a long time that the conjunctiva is
relatively paucibacterial. But is it completely sterile
in the normal case or is there a little colonization? So to look at this,
Thuy Doan, who was a fellow resident
and fellow with us and is now assistant
professor at UCSF, and my colleagues
did a study where we took 100 normal
subjects and we swabbed all four surfaces of
their choroiditis, that is, the palpebral, which
is the lid side, and the bulbar, which
is the eye side, and then did standard culture. We did BRiSK and we
did 16S metagenomics. What we found by
culture was basically what other people have found,
which is that about 20% to 30% of the samples
yielded no growth. The remainder really only
showed four organisms in any kind of prevalence
and three of them at higher prevalence,
and that was coag negative staph, staph
epidermidis primarily, propionibacteria, diphtheroid,
corynebacteria, and strep, and mostly strep viridans. And those are the four things
that we found fairly routinely. There’s lots of other
stuff that show up in a case or two, bacillus,
Neisseria, lactobacillus, E. coli, et cetera. But for the most
part, the only things that are found in double
digit sorts of prevalence are those four bacteria. Now, when we did
16S metagenomics, we actually replicated
a result that was published in 2011, which
found 400 genera on an eye swab with metagenomics. That’s impressive
given that we only had four total bacteria
growing that we could identify and maybe another 10
that we ever identified. Getting 400 seemed like
a lot, but maybe it’s like van Leeuwenhoek
all over again, this whole new world and there’s
all these microbes that you can’t culture or whatever. But we decided to go back and
look at this a little bit more carefully, and we did
quantitative 16S PCR, qPCR. It normalized to actin NS. Well, how many bacterial
genomes are there actually on a swab when we
take it off the eye? And for controls,
we did the inside of the mouth, the
buccal mucosum. We did the skin of
the lower eyelid. And what we found
was interesting, that if we normalized
a human actin, the skin actually has
about 22 bacterial genomes per human genome on a swab. It doesn’t mean that
that’s what’s living there. It means it’s what
comes off on a swab. And the cheek was actually
a little less bacterial. I would’ve guessed it was
more, but we got about 12. But when we looked
at the conjunctiva, it flipped and we only got
0.02 bacteria per human genome. So when we calculated the human
bacterial load on the swab, it turned out that
this corresponded to about 50 bacteria per swab. So it’s very hard to imagine
how you get 400 genera on a swab when there’s 50 bacteria on it. And that’s why I say 16S
metagenomics does not work well in the
paucibacterial environment. You pick up, I think,
environmental contaminants, and I think it kind
of makes stuff up. It takes small areas
that are amplifiable, and because of
sequencing errors, you end up with what looks like
a very rich environment that I don’t think is there. So we’ve taken this
to be paucibacterial. We’ve confirmed this in
a number of other studies that there’s relatively
few bacteria, but there are bacteria. So what are the bacteria? Well, if we look at the things
that showed up in more than 1% of the samples, it’s
our four friends again. Its corynebacteria,
propionibacteria, strep, and staph. So it’s the exact same stuff. And again, we find a few
lactobacillus or Neisseria or other things, but the vast
majority are just those four. How do we know that those four
are really there of the things that we looked for? We did this
heuristic calculation based on the difference
between environmental swabs and our conjunctival
swabs over 100– actually, 400
samples, and basically did a p value on whether we
would find this by chance. And you can see the p
values on the heuristic are in the order of
10 to the minus 48th. So we’re pretty sure
they’re actually there for those bacteria. Is that niche unique? So principal component
analysis has its advantages and disadvantages. This is not a validated
set in that this is the set that we did the PCA on. But we could distinguish
a population that was conjunctival,
which is in gray here, from the skin, which is in blue,
the cheek, which is in red, and the environment,
which is in black. But you’ll notice that the
PCA, the principal component, really is on the
continuum between the skin and the environment, which
is kind of what you’d think. So it’s not like there’s a
whole different microbial world on the conjunctiva. It’s related to both
skin and environment. And if you gave me
a sample on here and asked me to characterize
it, I’d be about 90% accurate if I didn’t
know which one it was. So I think there is a
unique environment there, but it’s not bugs that
you don’t find elsewhere. So this was all
pretty disappointing. Basically our foyer into finding
the whole brave new world of the conjunctiva didn’t
really yield that much. But what we did find
was through BRiSK, and it was surprising,
which is that the ocular surface actually has a
resonant micro virome. So BRiSK is agnostic
to the DNA source. We can find
bacterial DNA, viral. Basically any kind of
DNA will show up there. And when we ran
through our samples– I realize it’s small, so
I’ll enlarge it a little bit. There’s our propionibacteria,
our corynebacteria, strep, staph again. But the arrows
show three viruses that we found in substantial
numbers of samples. And that’s a virus called
torque teno virus, which is an anellovirus that I’ll
say more about at the end, Merkel cell
polyomavirus, which we found in a fair
number of samples, and HPV, Human Papilloma Virus. So the TTV, the
torque teno virus, is shown down in the
corner here for validation. This is just looking
at 10 samples, five where we found it,
five where we didn’t. And indeed, we could confirm
that the TTV was there by direct PCR. So I’ll have more to say about
TTV toward the end of the talk. But for those of
you not familiar with this little
virus, it’s tiny. It’s 3.8 kb. No one really
knows what it does. There’s no good in vitro system
for replicating this virus. It’s nearly ubiquitous. Zero positivity rates
are north of 90%. And of course,
with any new virus, it’s been associated with
everything, chronic fatigue syndrome, other chronic
inflammatory diseases, but has not been really
definitively linked with any of these conditions. It is sort of a normal
commensal of the serum. And you can follow– you can track
immunosuppression in patients by their TTV load in
their serum, which is really interesting. So it appears that the immune
system does keep this in check for whatever reason. But when it’s not in
check, it’s not clear that it causes a
whole lot of disease. We went ahead and confirmed the
presence of this in 58 swabs, and we found it on 22. So this is not an uncommon virus
to find on the ocular surface. Merkel cell polyoma–
very similar. I think many of you are
more familiar with this. Paul Nghiem has done quite
a bit of research here on Merkel cell
polyoma for its role in Merkel cell tumors,
Merkel cell carcinoma. But it’s also a relatively
ubiquitous tiny virus, but it’s in the
polyomavirus family. So it has a large T antigen,
and it has oncogenic potential. But for the most
part, it just is found as a commensal,
not so much in the blood, but certainly on the skin. And again, almost everyone has
been exposed to this virus. We found something
interesting with this virus. I won’t go into the
study in detail. But working with
Chris Chambers, one of our oculoplastic
surgeons, we wanted to look at whether
the microbiome and virome of the eye surface
changed when there’s no eye. So he does enucleations
on patients who have tumors and other
reasons for their eye to be removed. And when we remove the eye,
we restore the conjunctiva, but there’s no longer
a cornea there. It’s an acrylic implant
underneath the conjunctiva. So we took swabs of these
patients, of their healthy eye and their enucleated
conjunctiva, and ran PCR for
the various things that we’d been finding
on the surface. And interestingly, if
you’ve had your eye out, you shed Merkel
cell from that eye. So we found Merkel polyomavirus
in pretty much every sample of the fellow of
the enucleated eye, and in the same patients,
in the majority of them, we did not find it
in the healthy eye. So interesting finding–
don’t know what it means. But this is a pretty
ubiquitous virus in humans. And then HPV– a
little plug for those of you doing metagenomic work. My colleagues Aaron
and Cecilia Lee kind of tackled the Kraken problem. Those of you who use
Kraken as your pipeline for looking for
metagenomic sequences know it has its limitations. It’s not super
efficient, and it often pulls up stuff that’s
not necessarily correct. So they devised a new pipeline
that’s really interesting. It uses the Google search
algorithm, basically, to create hash tables of chopped
up 30-mers of your sequence and can then assign
pathogen sequences very quickly that way. And using that, we were
able to fully reconstruct this HPV that we found on an eye
surface, which is shown here. So there are 7,000 bases
of HPV and obviously very deep coverage on this,
more than 30x coverage. So we’re pretty sure
we got the whole virus. But when we ran it
through GenBank, it did not fully
match any known HPV. It had bits and pieces. Now, whether that’s a
recombinant, I don’t know. But the tiling really
looked like it’s one virus. And so our suspicion
is that this is a new variant
on the eye surface, but we haven’t
confirmed that to date. All right. So that’s the normal surface. Let’s talk a little bit about
what we find in disease states, and I’ll give you
three examples of this. First one is
microbial keratitis. This is a picture of a
patient with a corneal ulcer. This is still a pretty
common clinical problem. You can see the ulcer here. Basically that’s an infiltrate. The epithelium has
been eroded, and you have infectious keratitis with
an immune reaction around it. Very serious problem. Obviously, if that’s
in your visual axis, you’re not going to see
well out of that eye. Still a leading reason for
us to do cornea transplants. In the US, the
main reason people get these is they abuse their
contact lenses, sleep in them, wash them in tap water,
things like that. Worldwide this is
a huge problem, and there’s over 1
and 1/2 million cases of blindness a year worldwide
due to corneal ulcers, many of them in India and
many of them due to fungi. Thankfully, in this
part of the world, we don’t see a lot
of fungal ulcers. So we wanted to know if PCR– so one of the surprises
in this condition is the patient shows up like
this in the emergency room. We take a sterile spatula. We go right here where
we know it’s infected. We scrape it. We put it on our sheep’s
blood and agar mix, and nothing grows. 40% of the time, these
are culture negative, cultured Gram stain negative. Really weird because
it’s infected. There’s no question this
is an infectious process. So we wanted to see if PCR
would help us figure out what these pathogens were. This is an older study that I
did when we were in St. Louis. Elma Kim, who was a medical
student at the time with us, was first author. And we wanted to test
100 corneal ulcers as to whether they had infectious PCR
evidence of pathogen or not. At Barnes Hospital, which is
a 1,400-bed hospital in St. Louis, it would have taken
us two years to collect 100 corneal ulcers. We got about one a week. And that was going
to take too long. So I talked to my friends
Jack Whitcher and Tom Lietman at UCSF who had a collaboration
with the Aravind Eye Hospital in Madurai, India. Aravind is an amazing facility. It’s an 800-bed hospital, so
the size of Harborview and UW hospitals put together
for eyes and only eyes. And they see about 50 corneal
ulcers a week, not a year, coming into their ED. So I sent Elma over
with the UCSF team who’s doing a study there,
and I told her, come back when you have
100 corneal ulcers. I really thought it
would take her a month. Two weeks, she
emails me and says, I have 100 corneal ulcers
and really bad diarrhea. Can I come home now? I said, yes, you can come home. So she brought 108 ulcers home. This is their operating
room, by the way. When they do cataract
surgery, there’s five patients in there
at a time, each– or six, actually. You can’t see the
sixth one here. Each surgeon is doing
surgery on two tables. So they’re operating on
one while they’re swapping the other patient in and out. And the surgeon just
does this loop all day where does a cataract surgery,
turns around, rescrubs, rotates, does the next
surgery while they move the other patient
out, turns around, scrubs. They’re very efficient,
and their outcomes are actually a little
better than ours in terms of complications. It’s an amazing place. So what’d we find with
PCR in these cases? The answer was that all of
them showed either– nearly all of them showed either bacterial
or fungal bands by PCR, but not both, and that’s important. So these do not look to
be commensal because we found either one or the other. Most of them in India or 3/4
of them are fungal, 2/3 to 3/4. Only one sample, 107, was
positive for both a bacteria and a fungus. When they were culture positive,
which was, in our study, about 55% of the time, the
PCR agreed 95% of the time with the cultured organism. So we’re pretty sure that
we’re capturing the pathogen. So what did we find in
the culture negatives? Well, there were 46 of them
altogether in the study. 17 of them amplified bacteria. 29 of them amplified fungus. The bacterial ones were
not that interesting. So we found stuff
that we usually culture, corynebacteria, strep,
pneumo, pseudomonas, staph epi. These are all common
causes of corneal ulcers. But we did find two, quote,
“uncultured bacteria” in the database. So maybe two out of 17 of
those were new and interesting things. The fungal side was a
lot more interesting. So out of the 29 fungal, a
little over half of them, 17, were either fusarium
or aspergillus or both. But the rest of them
were stuff that we just don’t see very often– Sordaria, Phythium,
Botryodiplodia. If you go to the
literature, all of these have been associated
in one or two case studies with a corneal ulcer,
but no big series on any of these. So altogether, if
you take the ones in orange and red here, which
are either weird bugs that didn’t grow or uncultured
fungi or bacteria, that ended up accounting for
about a quarter of the culture negatives were bugs that
we wouldn’t have thought of or necessarily detected. So we do think that there is
this little world of stuff that doesn’t grow well that shows up. In India, this would be
a very useful technique because obviously treatment
of fungal and bacterial ulcers is quite different,
and you can’t tell them apart just looking at them. So a quick point of
service test that said fungal or
bacterial worldwide would be hugely helpful in
endemic parts of the world where corneal ulcer is endemic. Fourth lesson– this is actually
something that I’m guessing about a third of you
in the audience have– you may not know it– blepharitis. So blepharitis is a
condition where you basically get a scaly excrescence
of your eyelashes. Bacteria grow in the
roots of the lashes or in the meibomian glands,
tend to secrete some toxins. Those get into your
eye and give you a chronic feeling of grit,
foreign body sensation, a little redness,
itchiness of your eyes. And it’s due to this condition. This is exacerbated
because a lot of people, probably 30% to 40%, also
have demodex, the mite, living in their hair follicles. I didn’t put my demodex picture
in here, but it’s really cool. These alien-looking
skin mites actually live in your hair follicles
and they’re thought to contribute to blepharitis. So blepharitis is the bane of
ophthalmologists’ existence because we don’t have
good treatment for it, it drives patients nuts, and
it doesn’t blind anybody. So people take up a lot of chair
time with their blepharitis, and you want to help them, but
you can’t really help them, and they’re not
going to go blind, and the next patient
sitting in the waiting room is going to go blind. So if we had a cure
for blepharitis, everyone would be happier. Why don’t we have a cure
for something this simple? Well, I had the opportunity to
work with a company, NovaBay that I mentioned earlier,
who designed a drug that was intended to
treat, initially, adenoviral conjunctivitis, but
ultimately they kind of pivoted it to blepharitis. And it’s an interesting drug
because it’s not a drug. The drug that they sell,
which is called Avenova, is actually preserved saline. That’s the product. But it’s preserved with 0.01%
hypochlorous acid, which basically, when it’s
applied to the skin, converts to hypochloric
acid for about 20 seconds and then is oxidized
away into harmless stuff. So it’s actually a really
good antiseptic for the eye. In fact, in their studies, it’s
comparable to povidone iodine in terms of its killing
power on bacteria. And it has a little bit of
antiviral activity, as well. So they wanted to test it on
blepharitis as a lid scrub. And they did a very small pilot
study where they took three patients and treated them with
these scrubs where they took them for a week and then
two patients with, I guess, non-preserved saline– that’s the control–
and they wanted to know what changed on
the flora of the skin. So they sent us the
swabs that they did. In the study, they
swabbed people before they used it, 20 minutes
after their first scrub, a week after using
it, and then a week after they stopped using it. And what they wanted
to see, I think, was that all the
bacteria went away and the patients were happy. Well, patients were
happy, actually, the results on patient comfort. Whether it’s placebo
or not is hard to say, but they do feel
better with this. But when we looked by PCR– and this was done with BRiSK– we didn’t really
find a huge change. So this is just pie graphs here. Blue is staphylococcus in the
subject, which is number 16. Left eye is treated. Right eye is untreated. And basically there’s not a
whole lot of change there. It’s staph, staph, staph, staph. There’s a little bit
of corynebacteria and a little bit of P. acnes,
but it didn’t really change. Now, of course, BRiSK or any
PCR can’t tell live from dead. We do know that this stuff
kills four logs of bacteria when it goes on the eye. But we call this the mowing
the lawn effect, which is you kill all your
bacteria in the morning and then by night
they’re all back. It’s the same people. It’s the same lawn. You’ve just mowed the lawn. So it didn’t really
seem to work. Here’s another patient. Same type of result,
but the weird thing is that the software just colors
the dominant organism blue. And now it’s corynebacteria
in this patient. And in fact, there’s no staph
to be seen, or very little. I think one sample here
had a little bit of staph. And that was a surprise to us. I mean, why does
this patient have all corynebacteria and the
other one have all staph? And all of the results were
kind of weird that way. So we decided to just
validate this by direct PCR and see what’s going on. So these are the five subjects,
and this is their actin PCR. And you can see, we
got good recovery on almost all the swabs. The key to these experiments
is it’s the left, right eye, and visit one before
they scrubbed, visit two is 20 minutes
after their first scrub, visit three is a
week after scrubbing, visit four is a week
after they stop scrubbing. So that’s the key here. And this is the
staphylococcal result. And that patient I
showed you was number 16. And you can see,
indeed, that confirms its staph, staph, staph, staph. But if you look at 12, who is
the second one I showed you, indeed, there’s very
little staph there. And same with 13,
very little staph. But 14, 15, 16, lots of staph. What about the corynebacteria? Well, here’s 12, who
I showed you before, lots of corynebacteria. Same with 14 and same with 15. Again, not much in
13, not much in 16. What about strep? Well, strep we find
in 12, 15, and 16. And for amusement,
we thought we’d run our torque teno
because we know torque teno’s on
the eye surface, and we find it here
in 12, 14, and 16. So you need a scorecard to
keep track of all of this. Here’s the scorecard. And the answer is to
each his own or her own. There’s no consistent
patterns here. Some people are
corynebacteria dominant. Some are staph. We find some who have both
corynebacteria and staph. We have some who have no
corynebacteria, but show strep. No patterns here. Everyone’s unique,
and I think this is why we have such a hard
time treating this condition is it’s not one condition,
it’s a whole bunch of different conditions
with different bacteria that probably
respond differently to different treatments. All right. Last lesson and this is probably
the most interesting one and the most serious one. So this is an eye that
looks like that first eye I showed you with uveitis,
but this is a particularly bad form of uveitis. This is post-operative
endophthalmitis. This is someone who
had cataract surgery and then 48 hours later
called in complaining their vision had
suddenly dropped and the eye was red and painful. Thankfully, this
is extremely rare. In the current cataract surgery,
the rate of this condition is 0.03%, three per 10,000. So it’s rare we see this. But when we do, it’s
a serious problem. The incidence of
endophthalmitis, despite it being very rare
nowadays, is actually going up, and that’s because, in
the treatment of macular degeneration, we have
to inject drugs directly into the vitreous of the
eye, the anti-VEGF drugs, and so there’s actually more
intravitreal injections being done now annually than
cataract surgeries. And there’s already 3.3 million
cataract surgeries a year done in the US. There’s about five
million injections a year now done for macular
degeneration. So even in a 1 per
10,000 risk, when you’re doing 10
million surgeries, it ends up you have
thousands of cases of this. So it’s a serious condition. So we published a
study a couple of years ago where we did
our BRiSK analysis on these endophthalmitis cases. Now, just like with
the corneal ulcer, we have a problem in that
we know the eye is infected, and yet, if we tap the
eye, acutely infect it, and immediately
culture it or Gram stain it, about half the time
we get nothing, 40% to 50%. Are these infected? Is it the same
story as the cornea? Are we just not
detecting the pathogen? So collaborating with
my friend and colleague at Wills Eye Hospital in
Philadelphia, Sunir Garg and his group, we took 21
consecutive endophthalmitis cases that presented to retina
specialists who treat this, and we did culture 16S
metagenomics in BRiSK. And 11 of these were
culture positive, 10 were culture negative. For the ones that were culture
positive, for the most part, both 16S and BRiSK
recapitulated the organism. So there were seven of
these that grew staph epi. Five of them we
detected molecularly by either 16S or by
BRiSK, and they all agreed that it was staph epi. Two of them we didn’t,
actually, which was interesting. And I don’t– it’s challenging
to know, with staph epi, whether, when you
just go in and sample, you’ve gotten a little bit of
the stuff that’s on the conj or was it really the
stuff in the syringe. The streps were interesting. So the streps came back with
the lab medicine microbiology strep intermedius and viridans
that are descriptive but not really genera or
species-level descriptions. And again, molecularly,
we confirmed all three of them as strep, but
as strep gordonii, agalactiae, or mitis, which
were the true bugs in each of those three cases. We had one where the lab
called it light growth strep. We didn’t find anything
molecularly in that one. The Moraxella case
we all agreed on. Interestingly, the lab came
back with one Prevotella. But molecularly, both
molecular mechanisms showed it was not Prevotella. It was a strep species
that came up positive. 16S did not find any
detectable pathogenic organisms in the remaining
cases, and BRiSK found one that might have had
a tag or two for pseudomonas and one that had a strep. But they were very, very rare. So we went back and did our
quantitative 16S analysis again. And what I’m showing you is
the raw results here of the 16S normalize to actin and
then the quantitative PCR that was done separately. And we take two
bacteria, two genomes, as sort of our background
level because you can’t go into an eye
completely sterilely. Even when you’ve
done povidone iodine, there’s dead bacteria
on the surface. What do we find? Well, if we take
two as our limit, every culture positive
was above the line. Every culture negative
was below the line. So it means that the
culture negatives really are bacteria negative
or pretty close to it. It’s not that there’s this whole
world of unculturable bacteria in these cases. We just didn’t find
bacteria there. So what do we find? Well when we did
BRiSK, we found a ton of tags for our friend
torque teno, and not a few in some of these cases, but
a lot in some of these cases. So this is number
of tags recovered, which is– because there’s only
one or two tags per genome, it’s a rough measure of the
number of genomes recovered. And we were getting 1,000 and,
in one case, almost 10,000 tags back. So this is not a
small amount of virus. This is a lot of tags. We found torque teno in
every culture negative case. And we found it in half
the culture positive cases. But we had seven
controls of eyes that underwent vitrectomy
surgery for macular holes and diabetes,
and in none of those did we find torque teno. So it was only found
in the infected eyes. We’ve subsequently done a
larger study with outcomes. We didn’t have
outcomes in this study. And in the larger study,
we find torque teno present in 21 of the 33 vitreous
samples, so about 63%. And interestingly,
we find our friend Merkel cell polyomavirus virus
in about 25% of these samples, although rarely both,
usually one or the other. Here’s the one sample where
we clearly found both viruses in the same sample. Now, I do not want
anyone to leave here thinking torque teno is a
cause of endophthalmitis. We have no data to support that. We don’t know if it gets
in the eye and replicates, if it’s coming out of the
serum because the eye is very inflamed, if it’s being
carried by white blood cells. No idea, but it’s a biomarker. So we decided to look at
outcomes in the study. And the first
outcome we looked at is how people did for vision. Now, vision here is
listed as logMAR, which is logged minimal
angle of resolution, which is a standardized
way of showing vision. 0.1 is a normal logMAR. So 20/20 vision is
0.1 on the scale. A one is 2200 vision,
which is legal blindness. And a two is really bad. That’s can you count fingers
or see a hand moving? So most of these patients come
in with a logMAR pretty close to two. And many of them
get a lot better. So you can see that the average
outcome in these two groups ends up being
about 0.7, which is a visual acuity of
about 20/70, which is almost legal driving vision. And I certainly have
patients whose vision is worse than that
who are on the road. So be careful when
you’re out there. But it’s a fair outcome, not a
great outcome, but it’s fair. And those were the ones
who either had staph epi or were culture negative. They had about
the same outcomes. But the ones that were culture
positive for everything else, the strep, the Moraxella,
they did not do so well. So they ended up with
vision worse than 2200, which is legal blindness. So we do know that what bug
you have in the eye matters. And I’ll come back to
that in the slider, too, when I have my
list for you guys. But the interesting thing
is when we look just by TTV, torque teno virus. What we found is
that the patients who presented with really
bad vision were the ones who tended to have
the torque teno virus, which, again, doesn’t say
whether it’s causative or whether it’s just
reactive to patients that have more inflammation. But they came in with
much worse vision, and they ended up with
much worse vision. So the group that was torque
teno negative actually ended up with
visual acuities that were in the 20/40
range, which is pretty– most people’s cataracts
come out when they’re 20/40. So they don’t do too badly. But the ones who were
torque teno positive end up with a much poorer
vision on their outcome. So at least we do consider
this a biomarker, potentially, for risk stratification. We thought we had a
clever result here. And I was at a meeting with
one of my colleagues, Todd Margolis, who’s now the chair
at Wash U of Ophthalmology, and I said, we found
this interesting virus in endophthalmitis
called torque teno virus. I didn’t expect that. And he says, that anello virus? And I said, yeah, I think
it’s an anello virus. He said, didn’t I
just publish that? And he did, and I’d missed it. So there’s this
really unusual uveitis in Nepal called seasonal
hyperacute panuveitis, and it looks just
like endophthalmitis. It happens in kids every fall. And it’s coincident with the
eclosion of this moth that has this giant
caterpillar that tends to crawl on people’s
faces and really likes hanging out and
drinking their tears. So the thought is
that these little moth hairs, these little caterpillar
hairs penetrate the eye. This happens with
tarantulas, also, by the way. Don’t get a tarantula as a
pet because their little hairs get into your eye and
can cause a bad uveitis. But it never cultures anything. It’s always sterile. And Todd and George
[INAUDIBLE] in Holland analyzed the fluid
from these kids. And what did they find? 90% of them had
torque teno virus. And they looked in a cohort
of endophthalmitis cases and found 50% very close
to what we found, as well. So really the
credit for this goes to George and Todd’s groups. But it suggests that this
is a worldwide phenomenon. So some lessons learned from
our foyer into deep sequencing– first, the conjunctiva turns
out to be an interesting surface that’s paucibacterial. But it’s got a resident
virome, and that’s not something we think about. But in chronic
inflammatory disease, which is a lot of what we
deal with on the eye surface, it kind of makes sense. If you’re shedding virus all
the time and fighting it, doesn’t that give rise
to chronic inflammation? And maybe that’s
some of the dry eye, blepharitis stuff like that
that we see in these patients. When we see keratitis and
it’s culture negative, PCR says usually interesting
bugs, or often interesting bugs, whether it’s
fungus or bacteria, but there’s something there. The blepharitis story is
that everyone’s different, and what looks like one
disease by just phenotype ends up having lots of different
microbes associated with it. Maybe we need to do some
personalized medicine here to say yours looks viral,
yours looks bacterial. I didn’t tell you
the EKC story today. That’s a similar story
for conjunctivitis where it turns out that what
we thought was all adenoviral is not, and there’s other
things responsible there. But the culture negative
endophthalmitis, unlike the keratitis,
is actually bacterial negative in
most of these cases. But surprisingly, we find
this virus in the eye that we did not
expect, and it seems to be a biomarker for
presentation and outcome. So I put together
a little wish list for working together
with lab medicine. This is, I think, what
Jeff was alluding to. He gave a wonderful talk
for the chairs at MSEC on your department,
and it’s amazing the work that’s going on
here and the innovation. And so, from our
perspective, there’s some stuff we would
love to innovate around. The main one is the eye is
small and the fluids we get out of the eye are tiny small. If I tap an eye, I’m lucky
if I get 50 microliters to 100 microliters. You really don’t want to suck
5 milliliters out of an eyeball because there’s not much
left when you do that. So we need micro-scale
analyses in order to do things that, for everyone
else, is easy, like serology. I would love to know if someone
has toxoplasmosis antibodies, anti-toxoplasma
antibodies or anti HSV or VZV in certain cases of
inflammatory eye disease. But we can’t analyze small
samples well enough to do that. That would be a
godsend if we could do micro analysis for serology. Cytokine analysis–
again, a lot of the stuff that we’re looking
at, we don’t know if it’s infectious,
autoinflammatory, autoimmune, or even a masquerade tumor. IL6, IL10 levels, for
example, completely different from a tumor in the
eye and uveitis. So IL6 will be sky high
in uveitis, IL10 sky high in a lymphoma. We would love to be able to do
a 25 or 50 microliter cytokine analysis on those samples. The deep sequencing stuff
that I’ve shown here, I think, is great. It’s all under
research protocol. Obviously can’t
use it clinically. It would be wonderful if we
could do deep sequencing using the techniques and get
it under a CLIA license so we could actually use
this stuff clinically. The second wish list
is the endophthalmitis. Again, many of these
cases are negative, but if they’re either PCR
negative or staph epi, we don’t worry as
much about them. They’re going to do
OK, for the most part, with just injecting
antibiotics into their eye. The ones I worry about are the
streps and the pseudomonases and those that can
really completely destroy an eye within a day or two. And we would want to take those
patients to the operating room and clear all their jelly
out, do a vitrectomy, just bathe the
eye in antibiotics and treat it like an abscess. But I don’t know who’s
who when they present, and they all look the
same when they present. So a rapid point of service
diagnostic for staph epi or a suite of bacteria would
be, again, a godsend for us. So that’s an area we’d love
to work with you guys to help develop that kind of a test. And then what we’d really
love is one or more people who are fascinated
by tiny things, like tiny fluid
volumes, and would want to be a point of
contact with our department. So if you’re interested,
please talk to me. We have lots of
interesting projects, and we’d love to work with you. So we’ve come a long way from
van Leeuwenhoek to Illumina. We still have a long way to go. But I think it’s an
interesting era right now where we can re-examine many of these
diseases with tools that just weren’t available 10
years ago and find some interesting things in
terms of the microbiology. Lots of folks
contributed to this work. I want to particularly
highlight Thuy Doan who was our resident and fellow
who did the ocular surface microbiome– she now has a wonderful group
at UCSF working with Joe DeRisi, continuing the work in deep
sequencing and eye diseases– my colleague Cecilia and Aaron
Lee who are both assistant professors in the department
who have done much of the bioinformatics
work that I’ve shown– and Cecilia really has done
all of the endophthalmitis project– and then a number
of medical students, UW medical students, who
contributed to this, including Dallin Anderson and Michael
Gutowski, both of whom are now ophthalmology
residents who did other aspects of this work. Lots of great
collaborators, as well. Jay Shendure has been
wonderful to work with on the representational
deep sequencing, colleagues at Wills, Bascom, which is
University of Miami and UCSF. And of course, none of this
happens without funding. I’m very fortunate to have
good NIH funding and also some foundation in philanthropy
funding for this work, as well. So I appreciate the
opportunity to present, and I’d be happy to
take some questions. Thank you. MARK: Thank you
for a great talk. What’s known about local
production of antibodies versus systemic and ratios? How can we help
you in that regard, and what can you
tell us about it? RUSSELL VAN GELDER:
Great question. Mark is the only person here
that I really work directly with because we share
some patients together with autoimmune diseases
that affect the eye. That’s been pretty well studied. And if you sample
intraocular fluid in a case where you know the
eye is infected, for example, toxoplasmosis
where you can see the scar and reactivate it,
if you normalize to either IGG, total IGG, or
you pick a ubiquitous antibody, like an anti-mumps or
something like that, the ratio of
intraocular antibody titer to the normalization
relative to serum to the normalization
is always above three. So if you draw a line at
three-fold higher antibody production, you
basically get a 100% area under the curve for
local production. So it’s a very useful
diagnostic test for us, and it’s been used, in
addition to toxoplasmosis, for HSV and VZV, and CMV
infections in the eye. Obviously, AIDS patients
it kind of falls apart because their immune
responses are weaker. But in general, it’s a
very useful technique. There’s a lab in
Rotterdam that provides the service for the EU,
Aniki Rothova’s lab. And so in Europe, they
do this test routinely. And in fact, they do it
in preference to PCR, in many cases,
because often there’s a transient bump in viral load,
but as the immune system comes in, it becomes harder to
detect for the viruses. MARK: And if I could,
the related question is are there B cells
and plasma cells, then, within the eye that are
making those antibodies? RUSSELL VAN GELDER:
Yeah, there are. They’re rare, but
they can be found. If you actually
do flow cytometry on a patient with
an active uveitis, you find a small CD19 positive
or B220 20 positive population. To date, no one’s done
the single-cell cloning, which I would love to do in some
of these idiopathic diseases. And if anyone’s
doing single cell B cell cloning and reconstitution,
like the Don Gilden type thing in MS or SSPE, we would
love to work with you on that because that’s
something we’ve been trying to do for a long
time, or hoping to do. MARK: Sean. SEAN: That was really
interesting talk. I’m interested in this
cutoff between background and true positives. So you said in your
microbiome studies, you’re getting 50
bacteria, let’s say, off the surface of the eye. And so there are plenty of PCR
tests that would pick that up, and yet it wouldn’t be
pathogenic presumably. So when you go to a keratitis,
is there a big jump? RUSSELL VAN GELDER: Yeah. That’s a great question. The answer is, yes, there is. There’s a huge increase
in the number of bacteria that are recoverable, and it
becomes dominant bacteria, like 1,000 to one over human
cells in an active case where it can be recovered. So that’s why we didn’t have– well, we did that study,
the PCR study in India, before we were doing a
lot of deep sequencing. So that was manual. In the ancient
days, we actually, I think, took 12 clones
of each 16S product and sequenced each of them
from each of those 100. And all of them agreed
on one organism. So you don’t find this
proliferation of organisms and it’s dominant to one. So we’re pretty sure that,
in those cases, it’s there. It’s just not growing
on the culture plate. And whether it’s the immune
system has already killed them off by the time you see
them and they’re not viable or what is hard to know. But the flip side is it is true
that the PCR techniques are not great for speciation when
you’re in this paucibacterial environment. I would guess that
would apply to CSF, too, that if you did sort of
wholesale 16S metagenomics on lumbar punctures, you would
get the same type of results that we’re seeing here,
which is a lot of bugs that you just scratch
your head and go, there shouldn’t be
rhodococcus in a CSF. [MUSIC PLAYING]

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