Introduction to Clinical Mycology: Part 1 [Hot Topic]

Welcome to Mayo medical laboratories
hot topics.

These presentations provide short
discussion of current topics

and may be helpful to you
in your practice.

Our speaker for this program is
Dr. Glenn Roberts

a professor of laboratory medicine
and pathology and microbiology

as well as a consultant in the division
of clinical microbiology at Mayo Clinic

in Rochester Minnesota.

Dr. Roberts provides a
four part introduction to

clinical mycology including culture
and identification

of organisms encountered
in the clinical practice.

This is part one in the series,
thank you Dr. Roberts.

Thank You Sharon for that introduction.

I have nothing to disclose.

Introduction to clinical mycology.

This is the first in a series
of four presentations on

clinical mycology.

Today is part one.

Discusses what the clinical laboratory
does to make the diagnosis

of a fungal infection.

Discusses the classification of fungal
infections in the fungi related to them.

It also discusses the general features
of the fungi their importance in the basic

morphological features of the molds.

I think we periodically need to think
of why we're working in the field

that we are in
and that is to support patient care.

And a lot of activities go on
behind the scenes that

we're not aware of for the most part.

And one is that a patient with
some symptoms of something

whether it's a fungal
infection or something else,

and for our purposes
we'll say it's a fungal infection

"come in with signs and
symptoms of infection".

The clinician has to decide what
he or she suspects might be the problem.

Ask pertinent questions,
like a history of travel.

And then investigate the immune status
of the person,

because fungal infections take advantage
of people who are immunosuppressed.

They then have to decide
if they're going to culture something,

and they select appropriate specimen
for that.

They have to order a direct microscopic
examination if they think it's appropriate,

So that we can make
a rapid diagnosis in some instances.

And then sometimes it even
goes a lot further than that,

where they involve in basic procedures
like bronchoscopy or

fine needle biopsy or something like that.

And other things that can be done
or serologic tests for antigen antibody

and some metabolites
of some of these fungi

that assist in making a diagnosis prior
to maybe doing an invasive procedure.

If a culture is ordered
and something grows,

they will perhaps order
as and a fungal susceptibility test.

And then the laboratory sometimes is
involved interpretation of those results,

whether it's a serologic test or whether
it's anti fungal susceptibility test.

And then the most important thing
and it will reemphasize

this again in just a moment
is communication with the physician.

The laboratory must communicate with
a physician and not be afraid to call

the condition up and give them results
of patient care

for about the patient result,

so that we don't delay therapy
for these patients.

And oftentimes we have to also go back
and communicate with others

in a laboratory to make sure
we have all this information straight.

Well what is the laboratory involved
with?

Well oftentimes we're to ask
questions about a collection

of specimens by the clinician and we need
to be aware of what those rules are.

We need to be proficient at the direct
microscopic examination of specimens.

We know about culturing,
we identify the yeast and molds

in the laboratory.

We either refer out or do
antifungal susceptibility tests

and fungal share logic testing.

We're involved in reporting results
and again here we are,

we're communicating with physicians again.

And this is the key part of what
we do is communication with our

colleagues and with our physicians.

Well what are fungal infections;
what are some general features?

Well first of all these
fungi may affect normal

and immunocompromised patients.

Many of them are chronic and duration
they last for years like a ringworm

some things like that,

but in the immunocompromised
patient these can be acute infections

and progressed very quickly to death.

They're not transmitted from
patient to patient

they are not susceptible
to the usual antibacterial agents

that we know about.

However they are susceptible
to several groups

of antifungal drugs called
polyenes, azoles, and some others.

And the therapy for treating
fungal infections

is not easy for the patient -
very difficult.

Side effects are substantial
and we need to make sure that we make

a accurate diagnosis before these patients
are placed on therapy.

And again these infections
may range from being

just chronic type things to some that
are acute and progressed to rapid death.

And so we need to be
thinking about a rapid

reporting of results as we do our work.

Well we try to classify
fungal infections into

groupings and it's hard to do,
because it's an artificial thing.

But we divide them up into
superficial infections, subcutaneous,

systemic,
and opportunistic infections.

The superficial infections
you can remember by

just thinking about the fact that
they involve the keratinized tissue.

The hair, the skin, and the nails,
and that your matter fights

are good examples
of those and some others.

Subcutaneous infections
involve the skin and the contiguous

subcutaneous tissues
that includes the lymphatic vessels.

And most of the infections that
are involved in subcutaneous tissue

are acquired by trauma to a site
using the extremity.

Systemic infections
can involve any organ system

and generally they're caused by
certain groups of fungi that

have specific geographic niche
at an environment.

And they live in certain
locales within the world,

and we know where those are
and so we ask appropriate questions

when the clinician does anyway
ask some appropriate questions.

But these are organisms that
are involved in this

actually can infect any organ system,

and actually do that
and the patients may be

totally asymptomatic and unaware of this.

Or they may be very very ill
if they happen to be a know compromised.

And then the last
group is very important one,

opportunistic fungal infections.

These are infections caused by fungi that
normally don't cause disease in humans.

Their environmental flora.

And if we have a breakdown
of our immune system,

then we - and we happen to be
in the wrong place at the wrong time.

Then we acquire these infections and
they progress very quickly particularly
in transplant patients, and so we need to
be aware of what organisms cause these
infections.

The hardest part of mycology
is the terminology, It's a language.

And I'm just going to go through very
quickly here some of the names, so you
can see them.

Superficial infections of
dermatophytes involved primarily causing
these infections.

Another northern
organism in however that does cause
infection in the College tinea
versicolor is called malassezia furfur.

Dermatophytes belong the three groups
Trichophyton Microsporum and Epiduralphyton.
And these are just here to show
you what the names look like.

Subcutaneous infections: Sporotrichosis,
Mycetoma, Chromoblastomycosis, and Phaeohyphomycosis.
It caused by Sporothrix schenckii, Pseudalleschria boydii, Phialaphora verrucosa, Cladophialophora carrionii
respectively.

And you can see the names
get a little complicated sometimes
they're not that hard once you learn.

Terms of systemic infections:
Histoplasmosis, Blastomycosis
Coccidioidomycosis, and Paracoccidioidomycosis.

or the infections
that are grouped into this particular
heading, and they're extra to another one
which is not listed here and I mentioned
it to you in a minute.

The fungi that are involved in causing
those infections Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis, Paracoccidioides brasiliensis.

An one called Penicillium marneffei that causes Penicilliosis in
places like Cambodia, Vietnam,
Thailand.

We don't see it in this country
very often, but we do see it.

Opportunistic infections
examples of Candidiasis, Cryptococcosis,
Aspergillosis, Zygomycosis,
and a whole lot more.

The list could go on and on because we see
organisms that we have never thought
would cause disease.

Cause disease in
patients here and cause substantial
infection in the elephants as leads to
death.

Some of the organisms related to
that Candida albicans, Cryptococcus
neoformans, Aspergillus,
rise up as Mucor,
and a number of other of the Mucorales
Fusarium, Acremonium, Trichosporon and many
others.

Let's talk about the fungi and - and in
general here just a moment that they are
eukaryotic cells. They have a cell wall.

They have a cell membrane, and the cell
walls made up of poly-n-acetylgucosamine and the cell membrane contains
ergosterol in contrast to maeín cells
which contains cholesterol and the
reason for that I mention in this is
that there are certain of these
anti-fungal agents that actually bind to
the cell membrane that has ergosteroll in it.

The heterotrophic; They break down an
organic matter to get energy sources for
themselves decaying matter. They are not
susceptible again to the usual
antimicrobials. They reproduce sexually
or asexually most of the time in the
laboratory, we see the result of asexual
reproduction with spores.

They may be
monomorphic which means they have one
form a yeast or a mold form, they may be
dimorphic and that is if they have a
yeast and a mole form or another type
form. And then some of them actually
produced more than two or three forms,
and they're called polymorphic.

So reproduction by these is used by asexual
spores and that's what we see all the
time when we looked at these organisms
underneath the microscope. Examples of
the fungi that you'll be familiar with
our yeast and molds which we'll talk
about. Mushrooms, puffballs, shelf fungi,
the morels, which I'm sure some of
you have collected.

Plant pathogens and animal pathogens.

And some of these are
very substantial things that are
involved in causing disease of plants
and animals.

One of the things that the
fungi are very have a very important
role in doing is keeping the carbon
cycle going, they break down organic
matter decompose it into carbon. They
cause disease and trees for example a
Dutch elm disease, which is a devastating
thing for a population of trees and
that we lose them all whenever
they get Dutch elm.

Crop diseases;
Wheat, corn, barley.
Corn smut is one of
those examples of that and we actually
have seen patients who have had disease
caused by corn smut.

Wood and timber degradation;
If you look at wood, you'll see that there's some purple
to blue material sometimes on there
where the wood gets broken down by
these fungi and it damages it.

Another very important thing is these fungi
can cause spoilage of food particularly
grain. And developing countries
where grain as a primary staple. The
grain will get infected with a fungus,
and produce a toxin called aflatoxin if
it happens to be infected with aspergillus,
and actually caused substantial disease
in these patients
and oftentimes liver cancer.

And where would we be
without fungi and the brewing and baking
industry? We would be nowhere, because the
yeast of the things that cause
fermentation. So their play a major role
in that.

Well what are these fungi in
general? We mentioned their heterotrophic.
They break down organic matter They
have chitin in the walls, ergosterol in the cell membrane,
they have an organized nucleus, they
reproduce by asexual spores again most
often. And if it happened to be a mold
they produce hyphae, which you can see on
this left-hand photograph. They're
nothing more than filaments that look
like garden hoses underneath the
microscope.

And if it happens to be a yeast
they produce single cells that
reproduce by budding,
and oftentimes a
few more structures in there.

So just to give you an idea
of some of the terms.
Some more language if you will to learn.
But we have terms that we use to help us
describe what we see under the
microscope to make it a little easier
for others to understand.

Well if you look at a mold on a culture plate you'll
see the colony there, a fluffy colony
and our laboratory we call them
fuzzies.

If you look underneath the
microscope you'll find out they're made
up of these hyphae, these are filaments
that have parallel walls you look like
small garden hoses. The collective name
for the colony is mycelium, and I don't
think many people use the term mycelium anymore but it's there
for you to see.

The hyphae; these garden hose
type structures may be divided up in
compartments by some structures called
septa, and so we talked about septate
hyphae.

And we talked about non-septate
hyphae which don't have any of these
division these compartments in them.

And sometimes we just refer to non septate
hyphae as pauciseptate, because
certainly these fungi that are that are
thought to be non septate

have a few of these separations and I'll
show you shortly here.

Spores; most of the
fungi that we deal with produced
spores that are called conidial. And
they're produced on specialized
structures whether it's a short to long
stalk or really elaborate structure
called a conidiophore.

Some of the spores
are small, some of them are large. And so
we define them by being macroconidia
or microconidia. And sometimes these
spores or hyphae may be pigmented or they
may not be and that leads us to
different groups.

Well just some more
terms here the hyphae we talked about these
are structures that make up with a mold
colony. The septa are the cross walls
that break down the hyphae into compartments.
Non-septate means that they're lacking
septa like we said while ago.

And hyaline is another term you haven't
talked about yet that is and that the hyphae may be
non-pigmented. In other words if you look
at them underneath the microscope
without any stains at all they'll be
clear.

If you stain them with a dye like we
do in the laboratory lactophenol and on
blue, you can see they'll turn blue with
it with a dye. But they still don't have
any definable pigment to them, so they're
called hyaline.

And then we have other
fungi that are dematiaceous, and these fungi
are the ones that contain a dark pigment.
Either kind of a chestnut brown pigment
or very black.

They belong to a whole
different group of fungi sometimes some
of which are very difficult to identify.

And then there are a lot of structures
that we have to deal with, and some of
them are just nonspecific kind of things
that don't tell you anything about the
organism.

And one of those is called a
Chlamydoconidium. It's a big round
spore found right up within the hyphae strand
or on the end of the hyphae strand.

And it's the primary function is to protect the
organism so that if it gets - becomes
under adverse conditions it'll round up
and form these spores so they can
survive. We say are non specifically in
the lots of organisms.

And then the Conidia.

The conidia are those asexual
spores you mentioned produced by molds
that have septae. And the reason I
mentioned with septae is that there are a
whole group of fungi that do not produce
asexual spores and have
type septate hyphae. They happen to have non
septate hyphae, and the spores are produced in
a different way and we'll talk about that.

This is a typical mold colony.
Colonies are things that we see that we
have to work from to make them out in
the laboratory to be able to identify
the organism. However looking at a
culture plate sometimes gives you a
little information, but many times
doesn't help you at all. The colonies can
be multicolored. They can be white,
off-white, brown, tan, green, yellow, pink,
brown, black, there could be all sorts of
colors. You may get a ballpark idea of
what the organism is, but it's not going
to tell you specifically what it is.

This is another one or you can see the
colonies are smooth adherent to the
augur. And they have different
morphological forms when you start
looking at these colonies. They look
either very fluffy or very adherent to
the augur, and other appearances as well.

So you look at molds you'll be surprised
at the variety of things that you see,
and you have to kinda you have to with
experience you can tell sometimes which
are the ones you think are going to be
important.

now this is the example of the
high P this is an electron micrograph
where you can see these garden hose
structures I told you about and there is
a septum the cross wall that divides
that organism up and there'd be another
septum probably down below it there and
it allows that organisms to be
compartmentalized so that if it happens
to be a break apart each compartment can
then grow and start a new colony so it's
a matter it's kind of a motor survival
for these organisms this is what it
looks like under the microscope when you
really look at it you can see the septum
there to the right of the arrow that
divides it up and they're not usually so
hard to see as this one but sometimes
they are this is an organism that is
DeMatteis in other words it's pigmented
and one of the things that you do in the
laboratory is to recognize these moles
sometimes by the when you look at the
way the spores are produced you look at
the scores to recognize the size and
shape of the spores and so on and you
can look at this one and say ah I know
what this is this is by Polaris because
of the features that you'll be familiar
with as we go through the few other
sessions but the arrow here shows those
septic and you can see the
this Highfill strand at the bottom left
hand corner is divided up into
compartments so that each one of those
is an exception and can survive and grow
and produce in your colony and then the
image here is one of non septate havi
these are very large they're not
compartmentalized at all and when the
laboratory process is something that's
suspected to have one of these organisms
that has non cefotetan - if you grind it
up like people do in the laboratory that
Highfill strand will pop open all the
cytoplasm will leak out and it'll die so
what you have to do is be very careful
you end up having to not grind the
culture up you end up having to cut
pieces of whatever it is tissue or
whatever it is so that you can allow
survival for this organism but these
high PR non septate and occasionally
they will have cept Asians and we call
them policy sceptic this completes part
1 the introduction of clinical mycology
future presentations will be part 2
reviews the basic structures of moles
and yeast presents a brief introduction
to the direct microscopic examination of
clinical specimens and also media that
are useful for culture part 3% specific
information on the culturing and
incubation of cultures for the off more
recovery of fungi and part 4 presents
the methods for identification of fungi
primarily moles and some helpful hints
for working within the clinical
laboratory