1-2:Chapters 66-71, You Should Read Those

MicrobiologyBrian Bolerjack

Mycology57 minutos

10-10-08

1-2:Chapters 66-71, you should read those.

3:How are fungi different from other groups you are studying? Bacteria is the comparison I want to stick with. You need to know the epidemiology, where they live, how they are encountered and colonized or cause disease. You can classify them as endemic- areas of the country where the infections are at a low level at all times- or worldwide. The worldwide are the common ones like aspergillous, penicillium. They may be in the room right now and aren’t going to bother us. There is a differentiation on geography, and most fungal infections that are serious are respiratory, they start out there. For example systemic mycoses. You can get cutaneous enoculation working with wood products or flowers, can cause sporotrichosis. There are things we carry around with us, candida species in our GI tract. If you become immunosuppressed, take broad spectrum antibiotics that suppress the normal flora, you can get problems. Very few are contact between persons. Some of the dermatophytoses are (on) the skin. One is tenia capitis to hair. That’s one reason persons shouldn’t share hats.

4:Then you have to look at the host. Most of the serious things we see are opportunist. There are risk factors depending on where you travel or your occupation. If you have never been outside the SouthEast, you should never have coccidioid mycosis, called Valley Fever, which is in the SouthWest. Occupation, if you are doing something that exposes you to the environment, if you tear down an old chicken coup or clear bats out of an attic you might get a histoplasmosis. Most of the stuff down here (on the slide) causes serious infections. So host defense is mostly cell mediated host defense (T cells, macrophages, and neutrophils, which are very important in candidiasis and aspergillosis). If you are neutropenic for a period of time you are at high risk for infection for those organisms. There are a variety of mechanisms for immunsuppression (HIV, AIDS, transplants and malignancies). Diabetes as well, diabetics are susceptible to certain diseases based on their condition.

5:Be aware where certain organisms live, and that will guide you to the probability of cause. Are they endogenous, ubiquitous, only in certain areas? This is a map for endemic distribution of Blastomycosis, with higher incidence along the great river valleys and St. Lawerence pathways. If you have never been here, you shouldn’t have the disease.

6:Fundamentally, they are different from bacteria. They are eukaryotes. They have a membrane and defined nucleus. They lack chlorophil, they are not plants. For the most part they are aerobic. They are saprophytic, maybe one exception that lives on humans. For the most part they live in the environment and we are incidental hosts. They have a great variation in morphology, and we use that to identify them.

7:They have a cell wall made of complex polysaccharides, things we don’t have like cellulose and chitin, great targets for antifungal therapy. Cell membrane also varies. The predominant sterol in our membranes is cholesterol. The predominant sterol in fungi is ergosterol, and is related to chemotherapy. Because there are similarities between the sterols, the toxicity to us is high.

8:So morphologically, anytime you see an organism with a capsule, that’s antiphagocytic. It makes it hard for neutrophils or macrophages to latch on and engulf them. Cryptococcus neoformans is the main example for fungi that infect humans, an amorphous, water soluble polysaccharide coating. It can elicit antibody but its not a good opsonizer for these.

9:Growth forms are varied. Everyone knows what yeast is, makes beer, wine and bread rise. They are round cells that bud off. Cryptococcus falls here. Then there are molds, and they have hyphae, which are tubular structures. The mass of hyphae is a mycelium. Sometimes they have septations, partitions, crosswalls. Other times they don’t have that or have very few. Another slightly different is pseudohyphae, false hyphae, and that’s made only by Candida species. It’s not really important to know the difference, but you will run across it. They have a thermal dimorphism, they grow as different forms at different temperatures/environments (yeast in the tissue and mold in the environment).

10:This table summarizes what we just said. Think of fungi as ten times bigger than bacteria. 5-50 microns in diameter versus 1-5 microns. Generalities between fungi and bacteria.

11:Some examples of the terms. This is a yeast, Candida species. The round cell buds off and is called Blastoconidia. It extends out like this and is referred to as pseudohyphae, like linked sausage. It’s pinched at the junction point. It’s formed because something budded off and then grew straight. If you pseudohyphae that is almost without exception a Candida, so it is very helpful to know that.

12:True hyphae, there is a septate, or a wall. There is no pinching. True hyphae have parallel walls and then crosswalls. That is the only distinction between true and pseudohyphae. True hyphae grow at the tip, called apical extension.

13:Non-septate hyphae, at higher magnification, is a big ribbony thing with one septation. If you see something like this you know it belongs to a certain group of fungi.

14:Dimorphism. Blastomyces dermatitidis causes blastomycoses in the US and exists in two forms. On the right is the environment. It has hyphae with all these lollipop things, called conidia. They are small, 3-5 microns, and they are dispersed in air, we inhale those into the alveolar spaces and they turn into the yeast phase (on the left) in the tissue, large broad based budding yeast.

15:Know a little bit of the definition of these terms to help with your reading. If you see blastospores, that means budding yeast. Remember BLAST! Like you are blasting or budding out. Chlamydospores are thick walled survival cells. Arthospores are chains that look like a freight train, square boxes. They are liberated easily and associated with this organism (Coccidioides immitis). And Sporangiospores. These are all based on how they make spores, and that’s how we identify fungi.

16:You don’t need to be a taxonomist. Here are the major group. These three are based on how they sporulate. Organisms that cause us harm are distributed throughout. There is not one subgroup with all the pathogens. These three have a sexual phase and these are asexual. They have plus and minus strains that will “mate”, you go from 1n to 2n, and then recombination and meiosis, reduction division. These probably have those characteristics but no one has found them. (sorry about all the these, he never says any genus).

17:Where are they located? Superficial, in the skin, subcutaneous, systemic. Cutaneous are generally inconsequential, i.e. Athlete’s foot, jock itch (dermatophytoses) caused by organisms in this genus (Trychophiton) and sometimes the Candida species. Subcutaneous, Sporothrix schenckii, causes Rose handler’s disease, florist’s disease. Lives in the wood or plant, or under your skin if you get stuck. Generally does not cause systemic disease. Immune compromised patients break all the rules though. Systemics are put into pathogenic and opportunist fungi. Oppurtunists shouldn’t hurt anyone unless immunocompromised. Everyone is susceptible to infection of them, but not disease (in the pathogenic group). May have mild flu, think it’s a virus, buts its actually histoplasmosis or blastomycosis.

18:Candida causes a lot of problems. Patients can have disseminated candidiasis. Patients become fungemic, they can get endopthamitis. It settles other places. It gets in there. Everyone in the hospital is immunocompromised (IV insertion, broad antibiotics, etc.). Antibiotics wipe out the gut flora, Candida says more room for me, grows, gets on skin, gets on catheter, gets in blood stream. Fungemia! 20 years ago that was called catheter associated fungemia. Treatment was to pull the line, and reinsert if necessary. Well, people would come back with endopthamitis. Now the standard of care for fungemia or bacteremia, they get treated. There is no harmless –emia. There is some hypersensitivity, for example thermophilic molds usually with people who work with agricultural areas where there is compost. They develop a lot of spores. There is some contact transfer (dermatophytes), and we won’t talk about mycotoxins. But it might be in your peanut butter. One mycotoxin causes hepatomas.

19:What can the lab do? Microscopic exam is most useful on direct tissue. You wouldn’t do a specific Gram stain, but they will stain with the Gram stain. Geimsa. India ink is a specific negative stain, stains for seeing the capsule. The ink is particles of black material and are excluded from the area around the fungi because of the capsule. It lets you visualize the capsule. Sometime you have to digest away parts of the host, do that with a strong base like potassium hydroxide. These are done on paraffin embedded tissue. So H&E, PAS, GMS, and Mucicarmine. H&E is the mainstain of histology. Some fungi you can see on H&E others you can’t. The silver stain is the preferred stain. PAS and GMS are based on the complex polysaccharides in the wall. They attach a silver compound (GMS) which makes them black or a red dye (PAS).

20:This is what it looks like. Similar to Staph, but giant in size compared to them. They are also budding, which staph don’t do. This is Candida, which will Gram stain.

21:Here’s the India Ink. This is mainly done on CSF, Cryptococcus neoformans is an opportunist that causes pulmonary infection, and has a predilection to CNS infection. So crytococcal meningitis. You can do an immediate diagnosis by putting the CSF in India Ink, and you get a halo around the budding yeast. That’s diagnostic.

22:This is a little less distinct, a test by Potassium Hydroxide digestion. It will often cause artifacts. It’s hard to see the fungi. This is a Broad-base Budding Yeast in a direct respiratory sample, which is diagnostic for a particular organism, Blastomyces dermatididis.

23:H&E, you can see them, but it’s not the method of choice. You might miss them if there aren’t many organisms.

24:This is the PAS.

25:This is the silver stain, the preferred stain for fungi. Here is the crosswall, septations. They aren’t all in the same plane because of their size. This is with Aspergillosis. So acutely branching hyphae with crosswalls.

26:Mucicarmine Stain, this is Cryptococcus neoformans in the tissue. The red stuff is the capsule and the cells around it are host cells trying to do something about it. The macrophages can’t do anything. There often isn’t an inflammatory response at all.

27:Histopathology. This is the whole spectrum. You can have acute pyogenic abscess (Candida), chronic granuloma formation, which is indistinguishable from TB. Before there were good techniques to distinguish the two they would misdiagnose people with TB that had histoplasmosis. Chronic, localized dermal inflammation, that’s the superficial dermatophytes, athlete’s foot. It gets red, irritated, and erothymatous. Blastomyces is mixed pyogenic and granulomatous inflammation. These two groups (Mucor and Aspergillus) like to invade blood vessels. You get obstruction, clot formation, necrosis around it and cell death. Hypersensitivity responses like allergic bronchopulmonary aspergillosis.

28:Cultures, how do they differ from bacteria? You can isolated fungi from bacterial cultures, but they might get overgrown by bacteria, so that’s not the method of choice. So we put them on different media and temperature of growth. We look at tolerance of elevated temp, how fast they grow, what they look like grossly and microscopically, sporulation, and occasionally biochemicals. Sabouraud agar with antibiotics (antibacterials to let the slower growing fungi grow) is the mainstay.

29:Candida species are the most rapidly growing, grows in 24 hrs. Cryptococcus grows in 3-5 days. So if you just keep a bacterial plate 2 days, you are going to miss Cryptococcosis if you just rely on that. Aspergillosis, rapid growing 24-48 hours. The systemic fungi though, histoplasmosis and blastomycosis, 7-14 days. You must order the right tests. Unfortunately, serology is not very helpful. Cryptococcal antigen is the most sensitive way of detecting CNS disease with Cryptococcus via latex agglutination (attached polysaccharide). Some role for antibodies in certain niches. Coccidioides mycosis, can be in central nervous disease. You can look for its involvement. And skin tests, there are a few developed but are no value for diagnosis. Most people (80%) in the indigenous/endemic area will be positive.

30:Candidiasis, you name it, it does it. Normal flora, GI tract, oral cavity, you can get vaginitis, esophagitis, oral thrush. It is hanging around and given the opportunity will expand and cause problems. Persistent oral thrush indicates an underlying problem. There are cutaneous, chronic mucocutaneous (a group endocrine abnormalities that compromise macrophage functionality, causes a nasty disease), systemic, and UTI. The longer on IV or antibiotics, higher chance you will get cystitis and UTIs. Systemically you can get fungemia, hepato-spleenic syndrome, endophthalmitis, and renal problems. This is dependent on the host.

31:Oral candidiasis, an extreme example, maybe an AIDS patient. The white is the fungus.

32:This is more reasonable, white plaques on the palate. This might be on older patients with dentures. You need to look at it under the microscope to confirm the diagnosis.

33:This is a semi-Darkfield, nice tubular structures, pseudohyphae, budding yeast- those two things means Candidiasis. If you treat them and they don’t come back all is good, but if it does come back the Candida may have become drug resistant.

34:Candidiasis, in diabetics yet to be diagnosed. Looks like the dermatophytes. Its between the fingers. It grows in the folds of skin.

35:Mucocutaneous candidiasis, rare infection. It doesn’t cause invasive disease. Though their macrophages can’t do anything their neutrophils protect against invasive disease. It’s quite odd.

36:This is a much better silver stain. It’s about the right darkness. These are the linked sausages, pseudohyphae, budding yeast throughout, that is a Candida species.

37:Aspergillosis. Most problems we see are pneumonia and disseminated multiorgan involvement. Aspergilloma occurs with people with previous cavitary disease, bollous emphysema, cavitary TB etc. They have cavity in their lung. It says up house there and grows. People develop allergies. And this comes before this (I think he pointed to pneumonia comes before multiorgan).

38:Aspergillomas, localized in a preformed cavity, they are not invasive, but will continue to get larger. They may erode into a blood vessel and cause massive bleeding. They are not harmless.

39:This is a mass of fungus and host material.

40:Allergic bronchopullmonary Aspergillosis causes mucous plugging. This is because there is a continual stimulus. Here, the organism colonizes and grows on the surface of the bronchial tube. You get high levels on IgE. Here’s the bronchus and here’s the plug typical of this. Nor harmless.

41:This is the nemesis of the immunocomprimised host, disseminated disease, especially in the CNS. You generally do not cure these. There is a chance if it’s singular in an approachable part of the brain. But if it’s peppered throughout that’s the end of the story.

42:Aspergillus. KOH prep. Parallel walls, crosswalls. If you see this on a direct exam it means it is actually growing.

43:Aspergillus, high powered silver stain. You can’t tell from this level if it is Aspergillus or Candida.

44:If you get down close, there is no budding yeast, so not Candida.

45:Zygomycosis, another mold, and cause things, all in immunocomprimised host. Usually start in the sinuses and can spread to rhinocerebral or pulmonary or renal.

46:This is the classic disease- Rhinocerebral Mucormycosis in Diabetic who is Ketoacidodic. They are out of control. The organism also likes to live with lots of iron, so sometimes you see it in people with high iron. You can see there is a droop on the side, which may mean it’s involved in the brain. It is rapidly growing.

47:It progresses rapidly. It invades blood vessels, infarcts them, kills all the tissue around them. That’s why it’s hard to get antifungal agents in to treat it. The roads are blocked. Treatment is generally surgical.

48:If you do a biopsy, this is different from Aspergillus. It’s not very regular, it’s running all around. That’s how to tell them apart.

49:Histoplasmosis, dimorphic organism endemic in certain areas, similar to TB.

50:Residual calcified lesions in people who live in the region, probably have no clinical problems. Can become reactivated if immunosuppressed.

51:Disseminated histoplasmosis, involves the mucous membranes, determine with biopsy.