Searching for microbes Part XII. Parasitology

1. Searching for microbesPart XII. Parasitology Ondřej Zahradníček To practical of VLLM0421c and ZLLM0421c zahradnicek@fnusa.cz

2. Picture by: Petr Ondrovčík For introduction… Are you sure, that your new clone of giant bed-bug is my favorite birthday present?

3. Survey of topics Parasites – introduction Parasites – clinical description Parasites – sampling Parasites – diagnostic methods Parasites – pictures Check-up questions

4. Parasites – introduction

5. Parasite1 × Parasite2 • „Parasite1“ (an organism performing parasitism – a type of non mutual relationship between organisms): the term may include viruses, bacteria etc. • „Parasite2“ (object of interest of medical parasitology): the term is mostly used for eucaryotic, sometimes multicelullar organismsother than fungi. • Historically they were considered microscopical „animals“ (protozoans and worms), but today unicelullar parasites are no more considered animals and they belong to taxonomically very distinct groups

6. Parasites classification The most typical groups (not necessarilly taxonomic groups) of medically important parasites are: Unicellular parasites Amoebae (taxonomically close to animals and fungi) Flagellates and other unicellular parasites (rather related with plants, especially apicomplexa/sporozoa) Multicellullar parasites Flat worms (Platyhelminthes) Flukes(liver fluke and other flukes, schistosomas etc.) Tapeworms(swine tapeworm, beef tapeworm, fish tapeworm, dwarf tapeworm, tissue tapeworms – ecchinococci and others) Roundworms (pinworm, common roundworm, Trichuris, dog and cat roundworm and other roundworms) Arthropods (insects and acarids)

7. Historical term „worms“ • The term „worms“, or its Latin equivalent „helmints“ was historically used for organisms with prolonged shape of body • For practical reasons, we use this term sometimes even now, although we know already for a long time that they are not a taxonomic unit • Majority of them are visible by a naked eye or a simple lens. Some of them are quite large (e. g. 10 m in common tapeworm). Their eggs are microscopic

8. Another parasites classification It is also common to classify parasites according to the most typical localisation: • Endoparasites • Intestinal parasites (from Giardia to tapeworms) • Blood parasites (intra- and extraerytrocytal) • Urogenital parasites (e. g. Trichomonas) • Tissue parasites (e. g. Toxoplasma) • Other parasites (e. g. eye parasites) • Ectoparasites (mostly arthropodes) This classification is important also for diagnostics. For example, we prefer indirect diagnostics in tissue parasite diagnostics, as we are usually unable to find a suitable specimen for direct diagnostics.

9. Parasites – clinical description

10. Story one • Little Nicol all the time scraped her buttocks, and it was evident to parents and teachers that something is wrong. She was also inquiet and unconcentrated. So they placed a translucent adhesive tape on her anus and sent to the laboratory. And the result did not surprise anybody. Nicol started to use drugs and in a short time everything was in order again…

11. www.medmicro.info Causative agent was Enterobius vermicularis or threadworm (UK) or pinworm (US). It is a tiny roundworm (♂: 2–5 mm, ♀: 8–13 mm) that is present in the intestine. The eggs are present in perianal area. It is present mostly in children collectives. In small children autoinfections often occur. Another related roundworm is common (or „giant“) roundworm – Ascaris lumbricoides. It is much longer (♀: 20–35 cm, ♂: 15–30 cm). It is a little similar to earthworm (Lumbricus terestris), but still slightly different (roundworms have no band – citellum). Roundworms may cause various problems, from allergic problems to mechanical obliteration of orifices of bile bladder and pancreas.

12. Threadworms/Pinworms http://www.marksimonianmd.com/pin-worms/ http://hpathy.com/cause-symptoms-treatment/worms-intestinal-worms-in-humans/

13. Enterobius: egg, worms, life cycle http://www.earthtym.net/ref-pinworms.htm http://www.tpub.com/content/armymedical/MD0842/MD08420178.htm

14. Ascarids www.sp01.com/micro/worms/imagepages/image1.htm.

15. Roundworm (Ascaris) versus Earthworm http://stanford.edu/group/esw/wiki/Image:Adult_Worm.jpg http://animals.howstuffworks.com/worms/earthworm-info.htm

16. Roundworm life cycle and world distribution http://stanford.edu/group/esw/wiki http://stanford.edu/group/esw/wiki

17. Intestinal parasites • Intestinal parasites are the most common ones. They may belong to any of endoparasital group. Some of them result in diarrheic diseases, some of them produce rather non-specific problems (dyspepsia, pruritus, tiredness…) • Most common ones:Flagellates: (Giardia intestinalis = Lamblia), Amoebae: (Entamoeba histolytica),Nematoda: Pinworm, common roundworm and others. Trematoda: e. g. Fasciolopsis buski. Cestoda: common (swine and beef) tapeworm and other tapeworms

18. Non-pathogenous amoebae in the bowel • Not allways presence of parasites, especially amoebae, is a reason for treatment. There are many species, that are considered to be rather non-pathogenic. • The most important non-pathogenous amoebas are: Entamoeba dispar (very similar to pathogenous E. histolytica!), Entamoeba coli, Entamoeba hartmanni, Endolimax nana, Iodamoeba buetschlii and others.

19. Story two • Bianca, who took care of several cats, had enlarged lymphonodes for some time, and long time it was a question, what is the reason. The throat swabs showed nothing, and also results or other investigations had no results. • Bianca planned to have a child, and so she was afraid. And it was shown, that it is really so: the causative agent responsible for her lymphonode syndrome is really sometimes dangerous for pregnant women…

20. The causative agent was Toxoplasma gondii, a coccidium, that is transmitted by cats, although people say that people with dogs are in risk more than people with cats (they can wear the particles of cat faeces in their fur) In immunocompetent persons, majority of infections is asymptomatical, or only with mild symptomas (enlarged lymphonodes). Rarely, ocular form may occur In pregnant women toxoplasmosis may lead to abortion or embryonic organ defects Even asymptomatic cases may lead to carriership of cysts, that is also asymptomatic. Toxoplasma may only reactivate in case of immunity problems (HIV+), especially in decrease of CD4+ lymphocyte count

21. Rare, but important: toxoplasma retinitis may occur… http://web.indstate.edu http://fullmal.hgc.jp

22. Toxoplasma life cycle Down: toxoplasma cyst in brain www.antoranz.net webdb.dmsc.moph.go.th

23. Toxoplasma – how to get infected http://www.dpd.cdc.gov/dpdx/html/Toxoplasmosis.htm

24. Toxoplasma – life cycle http://en.wikipedia.org/wiki/File:Toxoplasmosis_life_cycle_en.svg

25. Tissue parasites • Some parasites may live inside the organism. • Some unicellular parasites, like toxoplasma, produce parasital cysts (formations containing living parasital cells) • Some nematodes may be present in tissue, e. g. Toxocara canis or T. cati • Some cestodes (e. g. swine tapeworm) may produce cysticercs in the tissue • Symptomatology is variable. In toxoplasmosis, cysts are clinically quite mute. Cysticercs of tapeworms may be dangerous e. g. by pressing to important organs • Diagnostics is difficult, because it is usually nothing to take for direct diagnostics.

26. Leishmaniasis • One of worldwide important tissue parasitosis is leishmaniosis. It occurs in the whole tropical and subtropical band. It is caused by a unicellular organism Leishmania • Vector is a tiny blood sucking insect (mothfly) of genera Phlebotomus or Lutzomyia • There exist some twenty important species, that may be classified into “Old world“ leishmaniae and „New world“ leishmaniae, and also to skin, skin-mucosal and visceral • They may cause diseases from skin mutilation to inner organ damage, often lethal • There are no cases of leishmaniosis in Central Europe (except imported ones)

27. http://web.indstate.edu/thcme/micro/parasitology Leishmaniasis

28. http://web.indstate.edu/thcme/micro/parasitology

29. Story three Joddie had again gynaecological problems. It was quite common in her, but this time bacteriological examination was of no help. So, C. A. T. swabwas sent for examination, and so the result was found. Foto: Ondřej Zahradníček

30. Causative agent was • Trichomonas vaginalis,a flagellate, that is transmitted sexually, although exceptionally other ways of transmission are possible, too • Discharge of typical smell and colour is typical • In men, the disease is usually asymptomatic • As treatment for anaerobic infections (metronidazol) is effective for trichomonosis, too, the number of trichomonad disease decreasesin recent times.

31. Trichomonad discharge holebi.info/gids.php depts.washington.edu

32. So called „strawberry cervix“ webmedia.unmc.edu

33. Urogenital parasites Among sexually transmittedparasital diseases, trichomonosis is the only really important (if not discussing the pubic louse). Usually its diagnostic is done together with bacteriology examination, as the ethiology is rarely clear at the first moment In the urinary tract, most important parasites are some flukes (schistosomas), i. e. Trematodes. Diagnostic is partially microbiological, partially histological.

34. Story four • Mr. Hikerused to travel throughout the world. After coming back from the last trip he started to have some problems, he caugth fever, then it finished, but during two days it started again. The GP sent him to infection department. They took his blood and made a smear to two slides, using two various methods. And really the causative agent was…

35. Causative agent was • Plasmodium vivax,one of four species of malaric plasmodia • Malaria is worldwide one of the most serious diseases. It causes illness of many persons every day, including many coming from Europe. • The worst course is in „tropical malaria“ or „malignant tertiana“, caused by P. falciparum. Fever mostly does not decrease. • Milder are both „benign tertianas“, caused by P. vivaxand P. ovale. Fever comes in 48 h interval here (day 1, 3, 5, 7…  „malaria tertiana“). P. vivax is more common, P. ovale occurs in western Africa. • The malaria quartana, caused by P. malariae, is rare. Fever comes in 72 interval here (day 1, 4, 7…)

36. web.indstate.edu

37. Bloostream parasites • Among bloodstream parasites, of course, malarial plasmodia are the most important, but not the only ones. • A flagellate, Trypanosoma brucei lives in bloodsteam, causing sleeping disease. Trypanosoma cruzi lives there too, producing Chagas disease • Nematodes– filariae may live in bloodstream, too (in tropical countries). Nevertheless, filariae may also occur in lymphatic vessels, eye, skin etc. • Symptomatology:in all of them fever appears, other symptomas are related to the agent

38. Elefantiasis www.sp01.com/micro/worms/imagepages/image1.htm.

39. Dracunculiasis www.sp01.com/micro/worms/imagepages/image1.htm.

40. Parasites – sampling

41. Sampling • For intestinal parasitesrectal swab is not sufficient, a bit of stool is needed (see more ) • For Trichomonas either a slide for Giemsa staining is sent (alone or in pair with another one for Gram staining), or a C. A. T. swab • For Acantamoeba used contact lenses are sent in their own fluid, eventually corneal scraping might be performed • For tissue parasitesserum is sent usually • In otherparasites we sample according to the situation (urine, content of a cyst)

42. Sampling for intestinal parasites • To send stool for parasitological sampling (usually using Kato and Faust methods), we need sample of stool sized like a hazelnut. A vessel for sampling need not be sterile. Unlike virology examination the sample does not need low transport temperature Coconut-sized specimens (as sometimes students use to say) are not recommended 

43. Sampling for blood parasites • For blood parasites, it is recommended to send a thick smear and a thin smear. Thick smear is just a drop mixed by a corner of another slide, thin smear is spread to the slide by special movement. Thick smear is not fixated. Pictures taken from CD-ROM „Parasite-Tutor“ – Department of Laboratory Medicine, University of Washington, Seatle, WA

44. Some more sampling methods

45. C. A. T. swab for urethral and vaginal sampling for Candida (yeast) and Trichomonas Here the swab is broken to fit into the test tube Foto: Ondřej Zahradníček

46. Parasites – diagnostic methods

47. Parasites: diagnostic methods in general • Microscopy is important, eitherwet mount,or staining (trichrom, Giemsa stain, Ziehl Neelsen for intestinal coccidia) • Cultureis rarely used, in practice only in Trichomonas and Acantamoeba. • Among other direct methodsPCR is used recently • Indirect detectionis used in tissue parasitoses, mostly toxoplasmosis, larval toxocarosis etc.

48. Intestinal parasites diagnostics • As a basis, we use methods based on modified wet mount: • In Kato methodcounterstain with malachite green is used, to make parasites better visible • Faust methodis a concentration one (see later) • Graham methodis used in pinworms only (see later) • Wet mount „sensu stricto“ and stained preparations (e. g. trichrom) are used in increased suspicion for intestinal protozoa (either primarilly, or after seeing Faust and Kato)

49. Faust method In the second half, Kato is already prepared • Principle: stool is repeatedly mixed with ZnSO4 solution, centrifuged and supernatant taken for the next step.Finally, the solution is filled up to the top of the test-tube and covered by a coverslip. The parasites adhere to the coverslip from below. Then coverslip is removed onto the slide with allready prepared Kato method.

50. Methods for diagnostics of intestinal protozoa • Helmint eggs are found directly in Faust and Kato methods. When something resembling cysts (of trophozoites) of protozoa is found, more methods are used. We use here • Wet mount, just stool mixed with a drop of saline, eventually a drop of Lugol solution is added after first observation to see better some structures • Trichrom staining. Fixation using alcohol-sublimate and further 70% alcohol, proper trichrom, 96% alcohol and carbolxylene. Or haematoxylinstain. • for intestinal coccidia eventually Ziehl Neelsen, or , in Czechia, Miláček staining (Mr. Miláček was a laboratory, assistant in parasitology in České Budějovice)