Microbiology: Parasitology Pg. 2

Microbiology Transcriber: Clay McEntire 10/10/2008 51:05 Parasitology

1. Parasites: protazoa (single celled) and worms:

2. Some important points for parasites: They have really complex life cycles (he probably won’t ask anything too detailed about the cycles), multiple hosts:

 Definitive host- host in which the sexual stage of the parasite occurs. This is the host that the parasite wants to “be nice to.”

 Intermediate host- parasite is “not as nice” to this host. If this host dies, then that’s good for the parasite, so it can get to the next host and reproduce.

 Reservoir host- this is the host that is anthrocentric (this means “human centered”), so this is the role that we play. Most of the time we are just an accessory host that got mixed up in the parasite’s normal life cycle.

 Vector- is the organism that transfers the parasite to us. An example would be the mosquito that transfers malaria. (In this example, the mosquito is also the definitive host and we would be an intermediate host.)

 How humans are infected is a topic of crucial importance.

 We need to think about migration patterns—where these things go in the host

 Diagnostic forms in humans. What do we look for to diagnose these? He stresses this because this is what he does for a living.

 Geographical distribution—At UAB the only suspect patients are those with a travel history.

3. So how do we look for parasites? One way is to look at stool samples. The gauze strains out the big stuff that doesn’t contain any parasites. They then add ethyl acetate (used to be ether) and formalin to the sample. It’s then centrifuged and the parasite eggs go to the bottom and the fat and other stuff is extracted from the stool. You then take a drop of the sediment and look at it under a microscope.

4. You can also do it with a saturated sodium chloride solution. The parasites will float in this solution. Then look at it under the microscope.

5. Classification of parasites:

 Protozoa are single celled parasites. An amoeba is an example (Entamoeba histalytica causes amebic dysentery). Mastigophora- Giardia lamblia is another example; it’s a flagellate. “Mast” is a whip—think flagella. This is the most common parasite seen here Microbiology: Parasitology pg. 2 Clay McEntire in Alabama. An example of sporozoa is Toxoplasma gondii, which is the cat parasite that people worry about. Cryptosporidium is also pretty common here, and malaria is another example, but we only look for malaria in people who’ve been out of the country recently.

 Helminths are multi-celled parasites. These are the worms that we really don’t see much of. Ascaris are the nice big worms (round worms- nematodes). Platyhelminths are the flat worms—schistosomes, flukes are examples. Cestodes are tapeworms.

6. Entamoeba histolytica has a pretty boring lifecycle. It’s almost like a bacterium. The cyst is what’s usually transmitted from person to person because it’s really resistant to drying and it can survive in water. When it goes into the gut of someone, it hatches and four little amoebas come out, and these multiply in the gut by binary fission. The organism will incyst and move on to the next host. It’s very difficult for the tropozoite to infect someone, but it can infect lesions. It can but it’s usually the cyst form that infects.

The picture to the right is what they look for in the lab. You can see the cyst with the chromatoidal bodies (food source in the cell) in the stool sample.

7. Entamoeba histolytica/ dispar. 90% of people infected didn’t have symptoms or disease. This is a different organism (E. dispar) that looks the same as E. histolytica, but it’s not the same—this one’s nonpathogenic. There are molecular tests that can be run to determine which organism is found in a stool sample. Humans are the only host—there’s no reservoir host—just human- human transmission. It’s all fecal-oral or water borne (because cysts can survive in water). Diagnostic forms are either the cysts or the tropozoites in the stool. You can do serology to see if you’ve ever had amebic dysentery, but this wouldn’t mean that you have it at the time. You may just have antibodies remaining from a previous case. The organisms will invade and lyse cells in the gut, and it can also spread to the liver or lungs (more rare).

8. So these are the organisms. You can see the nuclei. These slides aren’t concentrated samples. That’s just how many organisms are present.

9. Non pathogenic ameba. These are not that uncommon, and we get along with them just fine. E. dispar as we just discussed. E. harmanni- looks like histolytica but it’s smaller. Entamoeba coli- also in the gut. These don’t require treatment but we still report them because people with these organisms have been exposed to stool that hasn’t been cooked since the last person ate it, so they’re at risk for other things too.

10. This is what some of them look like. This is Entamoeba coli- instead of having 4 nuclei and a cyst, there are 8 nuclei. This is Iodamoeba butschlii with the different looking nucleus and the vacuole in it.

11. These are more Entamoeba coli cysts, and you can see the 8 nuclei within. Microbiology: Parasitology pg. 3 Clay McEntire 12. Giardia lamblia is the most common in the US. Transmission is fecal-oral or water borne. There are multiple reservoir hosts, so streams in the mountains may contain this organism as well. And yes, Dr. Waites, the beaver will have diarrhea too. This is commonly seen in day care centers. 50% of patients are asymptomatic and don’t know that they’re transmitting the organism. There is 2-15% incidence, but it’s probably more rare than that in the US. Symptoms are fatty, frothy diarrhea. Diagnostic forms are cysts and tropozoites.

13. This is the lifecycle of Giardia. Again, it multiplies by binary fission. It has a cyst form that can survive in water for weeks (survives longer in colder water). There are 4 nuclei in a cyst which will lead to 2 separate organisms with 2 nuclei each when it leaves the cyst form. The 2 nuclei look like eyes. They’re “cute little guys.”

14. This is what the cysts look like. This is not a concentrated sample. There is this many organisms —like millions per gram of stool.

15. It’s really photogenic… people like to take pictures of giardia.

16. Chilomastix mesnili is a non-pathogenic flagellate that we need to talk about. It has a single nucleus. It sort of looks like giardia, but it has a single nucleus. If seen, it should be noted that it’s present because people with one parasite often have others.

17. Toxoplasma gondii is an important parasite, and in this country about 1% of us get infected per year. There are 2 different ways to get infected. 1 is to eat meat that hasn’t been cooked well enough that was infected (this isn’t uncommon because toxoplasma infects pretty much all species in the world). The definitive host is the cat (house cats, lions, tigers, etc…) So it multiplies in the cat. The oocysts are passed in the stool of the cat, and it takes them about 48 hours to become infectious. If you clean your litter box daily, there is low risk of infection. (2) The other way to ingest the organism is to consume the oocyst.

An important thing with this organism is congenital transmission. If we’re infected with have symptoms similar to mononucleosis: fever, malaise, etc… Then you get over it, but you’re infected for life (you’ll have cysts in your brain forever). It can reactivate later in life if you’re ever immunosuppressed—like with HIV. In fact people with HIV are given sulfonamides as prophylactic treatment to prevent toxoplasmic lesions from forming in the brain.

If a pregnant woman gets infected, the organism can cross the placental barrier and cause miscarriage early in pregnancy. Later in pregnancy, it may cause hydrocephalus, mental retardation, chorioretinitis. It’s one of the more common causes of mental retardation. It’s only a risk if you’re infected during pregnancy. If you’ve been infected before, you’re ok. In the past, meat juices used to be given to pregnant women because they thought it was therapeutic. This led to high levels of congenital toxoplasmosis. Microbiology: Parasitology pg. 4 Clay McEntire 18. This is what the organism looks like. It’s about 4 microns long. It has a nucleus. The larger cells are infected macrophages that can be seen in tissues (commonly seen in spinal tap biopsies of HIV patients).

19. These are CNS lesions seen in immunosuppresed patients.

20. When you look at tissue, you can see the organisms. These are the bradyzoites, the very slowly replicating organisms that are in an immune host. They’ll just sit there for the life of the host. Occasionally they’ll come out and test to see if they can reactivate or if the immune response will beat them back. For the most part they don’t cause any trouble though unless the host becomes immunosuppressed.

21. Cryptosporidium is the other gut organism that’s fairly common in the US. We’ll run a fluorescent test for this organism and for giardia on anyone who has diarrhea if parasites are suspect. These are both fecal-oral organisms that are commonly seen in daycare centers. Zoonosis- one of the first human cases of Cryptosporidium was found in Auburn. A guy was doing research on cattle in Auburn, and began having up to 14 liters of diarrhea a day. He was the 7th case of Cryptosporidium diagnosed in humans. He diagnosed himself and also did further research on the organism. This became common in AIDS patients. This organism is seen all over. Cysts are resistant to chlorine, so it is necessary to filter water in addition to normal chlorine treatments. There was a famous case in 1993 in Milwaukee where a lot of people were infected by the city’s water supply. Cows are a normal host. There is no treatment. It lives in the epithelium of the gut (intracellular), and for the most part it causes a self limiting watery diarrhea in kids and adults.

22. This is what we look for in the lab. This is an acid fast stain (same stain you’d used for TB), and you see these little red organisms in the stool.

23. These are 5 microns across. This is Cryptosporidium parvum. You can see yeast that doesn’t stain and the cryptosporidium organisms that do stain.

24. This is where the organisms live. This is the mucosa of the gut. You can see the cells right on the surface.

25. Plasmodium (malaria) is an organism that lives in the blood. We only look for this in people with a history of travel overseas. It’s sometimes tough to detect and can survive in people’s blood for a long time. The mosquito is the definitive host (so the sexual stage of the organism occurs in the mosquito). Gametocytes (male and female) from the human combine in the gut of the mosquito to get a zygote and they multiply and you get a lot of sporozoites that spread to the salivary glands of the mosquito. Those can then be transmitted to other humans. Sporozoites go straight to the liver in the next human, where they’ll multiply initially. Then, they’ll move on and multiply in the red blood cells, causing the RBS to burst. Lifecycle is 24-72 hours. It’s a fairly intense lifecycle. It’s rare, but this can be transmitted via blood transfusion. Microbiology: Parasitology pg. 5 Clay McEntire 26. This is what the fever spikes look like. If you have a single crop of malaria, cells lyse, and you’ll get the first spike at the first day. The organism then goes into its lifecycle forming a tropozoite leading up to cell lysis, which is when the next spike will occur. Most malaria fever spikes occur every other day (48 hr cycle), but Plasmodium malaria has a 72 hr life cycle (bottom cycle)—this organism is less virulent and less common.

27. This is what you look at in the lab. The organisms’ nuclei can be seen within the cytoplasm of the RBC. These are the gametocytes from Plasmodium falciparum. These are the bad organisms that are more common and cause death pretty quickly.

28. In this case we have a big tropozoite in the cell (top left). On the bottom left you have a applicaform (?) where you have a nucleus in the cytoplasm right on the side of a cell. It may take a while to locate these on a slide, but any evidence would be very important.

We now have an antigen test where we can put blood in an ELIZA type assay and look for malaria parasites that way. This method is a little quicker than manually looking via microscope.

So malaria is pretty common all over the world. There’s about 1000-1500 cases in the US per year. Almost all of these patients had traveled to a tropical area where they were infected. If you are going to travel somewhere where there’s a malaria threat, you can take prophylactic drugs that will enable you to prevent infection.

29. Cyclospora is another gut protazoan. There were 1500 cases in 1996 of GI diarrhea that came from raspberries, which were traced to have come from Guatemalla. It was found that 1 raspberry was the infectious dose. We’re not sure what the definitive host for this organism is. We asked them to clean up the raspberry harvesting process, but there was another episode in 1997 where 90 more cases were discovered. We then stopped importation. There have been other cases too seen with basil and lettuce.

30. It was first found in Nepal and Peru and the Cook County hospital in Chicago. We still don’t know what the reservoir is. It infects the small intestinal cells. It looks like cryptosporidium on a stain, but it’s larger, so we can differentiate based on size. We do have a treatment for Cyclospora- bactrim.

31. It autofluoresces so you can see the round spores in the stool.

32. Helminths are the worms. About 1/3 of the world is infected with Ascaris lumbricoides, but it’s pretty uncommon in the US. There’s about the same amount of hook worm (Ancylostoma and Necator) infections. Trichuris is a whipworm, which also infects about 1/3 of the world. We’ll also discus Schistosomes a little bit; they’re fairly common in the world (114 million cases). Strongyloides is also something seen… 70 million cases worldwide, and this is one that we do see in the US.

33. Skipped Microbiology: Parasitology pg. 6 Clay McEntire 34. There was a study done where labs were asked to submit their next 100 stool specimens with parasite infection. They ended up with about 58,000 specimens from 61 labs. You can see that giardia, a protozoan, is the most common parasite. Cryptosporidium was the next most common. Then Dientamoeba and Ascaris.

35. Ascaris has a worldwide distribution and humans are the only definitive host. The embryonated eggs are infectious, and the parasite produces about 200,000 eggs per day. The eggs must live in the soil for weeks before they become infectious, so if someone’s a food handler and they become infected with Ascaris, they can keep handling food, but they’ll probably want to get treated at some point.

Eggs hatch in the duodenum, and larvae will penetrate the intestine. They migrate through the lungs, are coughed up, swallowed, and then mature in the small intestine where they live. These are nice big worms: 20-30 cm is possible. The lifespan is about a year, so these are constantly reinfective. Diagnostic form is eggs passed in the stool or you might even notice a long worm in the stool or it might come up the other way through vomiting. This is erratic, so if worms get old and senile, the worm may move up to the stomach and come out. Or if someone has a fever, the worm may try to escape from the host to avoid the uncomfortable temperatures.

36. This is the lifecycle. You eat the embryonated eggs, which hatches in the duodenum. It goes into the blood stream to the lungs—coughed up—swallowed. The picture to the right shows the diagnostic form: an egg in the stool.

37. Sometimes the worms themselves are actually brought in.

38. This is a vomit worm from a 19 year old kid. This was brought in, and this was probably the only worm that he was infected by because there was no eggs in the feces after the worm was removed from his system.

The worm actually helped the kid, because he was diagnosed with tuberculosis. In fact it was probably the fevers that sent the worm into the stomach anyway.

39. Strogyloides is one that’s here, and it’s one that can be pretty dangerous and even fatal. The infectious form are the filariform larvae. These are in the soil. These are the reason that grandma said don’t go around barefooted. If someone defecates and releases some of the larvae, the larvae can then borough through another host’s intact skin. This is a similar means of infection to hook worms.

40. The worm will then get into the blood, move up the lungs—coughed up—swallowed and move down into the lower GI tract to mature. The worm grows to be about 2mm long. They are tiny little worms that lay eggs in the gut. The eggs, however, are not seen in the stool. The larvae are what’s seen—diagnostic form is larvae!

These are free living in the soil. Microbiology: Parasitology pg. 7 Clay McEntire These can reactivate in immunosuppressed people. Within the last 2 years there have been a couple deaths. They are located mainly in tropical areas; they need a wet, warm environment. Autoinfection can lead to a hyperinfection syndrome, so these things can remain infective decades. One sign that you can look for is a high eosinophil count.

41. You can see them in the sputum as they’re traveling through the lung, or you’ll see them in the stool (more common).

42. Sputum stain

43. Schistosomes aren’t really in the US. They can live in people for 10-15 years. There are 3 main species. They’re located in tropical areas. Adults are about 1 inch long. They live in venules around the gut or bladder, so these are blood flukes. All flukes have snails as an intermediate host. We defecate in the water, and the snails will become infected through the water, the worm will multiply in the snail, kill the snail, and be released. They’re then ready to borough through intact skin.

There are schistosomes in the US, but they’re not infective to humans. They’re common with water fowl (like geese). You can get swimmer’s itch though where they borough into the skin before being eradicated by the immune system.

44. It's about 1/3 of a mm long (very small), boroughs through the skin, goes into the liver and matures, it goes into the venules of the large intestine. The 3 different species have 3 different egg types which end up in the snail where multiplication occurs. The picture on the right shows the organism in the venules.

45. The eggs are very distinctive for Schistosoma mansoni. It has a big hook on the side.

46. These are the worms. Male is flat and female is round. They live together in the venules for up to 15 years.

47. There are a lot of tape worms. Taenia solium is the pork tape worm. It’s found wherever people eat undercooked pork. Humans can be the definitive host and the intermediate host. Cysts can form in the brain cause epilepsy (or worse). The infectious form is cysticerci in pork. If you get this, you can get adult worms in your system for about 10 years. They can be up to 10 feet long. Diagnostic forms are ova in the brain or you can even pass worms segments out the anus. Cysticerci can be seen in brain with CAT or MRI scans.

48. This is what the worm looks like—head is small.

49. These are the cysts in the pork. You can see the heads of the worms poking out the holes. If you eat the worm, you’ll be the definitive host and it’s not that serious. Problems arise when the cysticerci get in and cause damage especially in the brain. Microbiology: Parasitology pg. 8 Clay McEntire 50. Other cestodes: Taenia saginata- beef tapeworm has a lot of the same life cycle as solium except for the fact that we can’t be the intermediate host. We’re only the definitive host, having the tapeworm, so it’s not as dangerous. You get this from eating undercooked beef. Diphyllobothrium latum is a broad fish tapeworm. Sushi chef got this here in Bham. Hymenolepis nana is a dwarf tapeworm that you get if you eat insects or roaches (he was trying to be funny I think—poor guy).

51. This is one that’s about eradicated from the world, so we won’t discuss this. It should be extinct. It’s a meter long and wonders around subcutaneously for a while.

He said the key to this info is to remember how to be infected and how to be diagnosed.

Dr. Waites asked him to go over river blindness. He did so without the microphone, but I took some notes and did some other research too: Basically river blindness is caused by a nematode —Onchocerca volvulus. It is spread to humans by a black simulium fly that acquires worm larvae from the water. Fly then bites human and the worms’ larvae spread into the skin and larvae breed and spread throughout the body including the eyes where it can cause blindness.