Laboratory diagnosis: - life cycle of parasits, material v laboratory dg

1. Parasitology - protosoology (protosoa), helmintology(worms), enthomology(insects) • Laboratory diagnosis: - life cycle of parasits, material v laboratory dg • Protozoa – intestinal, genital, urinary, blood, tissue • Worms - Helmints: Nematodes, Cestodes,Trematodes • Ectoparasits: louse, ticks, flies – important as vectors

2. Diagnosis • Problematic, not ususal outside endematic areas • Nonspecific clinical manigestation • eosinophilia in helmintoses – not constant sign • Importance of history – personal, travellers, social, economic, food, therapy • Conditions for successful dg: • - think on parasitosis, • - correct sampling – right sample, with good method at right time, sent in appropriate conditions to the lab that is able to identify the parasit, good interpretation: • Knowledge of life cycle is principal

3. Life cycle of parasits -terminology • Complex life cycle – key to diagnosis • Host – hosts • Definitive host – parasit finishes the growing cycle and is becoming adult in it • Not typical host - parasit cannot develop in it • Transient host – larves are developing in it but sexual multiplication is not performed • Helmints : egg - larva - (cyste) – adult worm Protozoa :trofozoit - motile, cyste – non motile - sexual multiplication - zygota - asexual- schizonts, sporogons

4. Patogenesis • Chronic infections • Protosoa – In strong immunity reaction – imunopathologic signs – symptoms hypersensitivity, cross reacting antigens (tissue devastation) and autoimunity • Worms – mechanic, big, metabolism – direct injury (elefantiosis) - imunity and inflamation – indirect immunopathological reaction

5. Imunity • production of IgE • Worms stimulates CD4Th lymfocytes – they secrete IL-4, IL-5 • ADCC – via eosinophils and IgE – elimination of worms – basic protein in granulesof eosinophils is toxic for worms • CD4 - activation of macrophages – elimination of parasits = Production of granuloms in organs, fibrosis, tissue devastation • I.c. parasits activate cytotoxic lymphocytes – interference of disseminations

6. How parasits escape human immunity • = chronicity • In-borne nonspecific mechanisms – faibly efficient • Specific mechanisms – antigenic mimicry – the surface of the parasit is covered with host antigens, komplexes of Ag-Ab, i.c.localisation of parasits, antigenic variation during infection • - size, motility, cover of albunim, extreme production of antigen imunity overload, imnosuression

7. Therapia • Less structutres • Toxic • Knowledge of the life cycle of parasit is primary • Eucaryotic cells of parasits – toxicita – selective toxicity can be achieved by changes of the molecule by metabolising of it in the body of parasit, acumulation of the molecule in the body of parasit • Chronicity – long term therapy • Tissue localisation, encystation • Socioeconomic conditions, climatic factors

8. Stool for parasitological examination • Macroscopy – blood, mucous, adult worms • Microscopy - native smear - FS, iode – motility, eggs of helmints, cysts of protosoa, ery, leu • Concentration methods – separation of cysts of protosoa and eggs of helmints from other material in the stool • Stainning - identification – smear of native stool + hematoxylin eosin, trichrome

9. Other material acc.to clinical manifestation • Perianal - Enterobius vermicularis • Sigmoidoscopy - Entamoeba histolytica • Duodenal aspiration - Giardia lamblia • Biopsia of abscesse of liver - Entamoeba histolytica • Sputum - Ascaris lumbricoides, Strongyloides, • Urine - Schistosoma • Urogenital sample - Trichomonas • Blood - (malaria, trypanosomiosis, leishmaniosis, filariosis) smear, thick drop - staining acc. Giemsa, HeO, • Serum

10. Protosoa • Amoeba - Entamoeba histolytica, Entamoeba coli, Naegleria fowleri, Acanthamoeba, Endolimax nana • Flagelata - Giardia lamblia, Trichomonas vaginalis,Leishmania, Trypanosoma • Ciliata - Balantidium coli • Coccidia a Sporosoa - Cryptosporidium, Blastocystis, Microsporidia, Plasmodia, Babesia,, Toxoplasma

11. Nematodes -worms • Enterobius vermicularis, Ascaris lumbricoides, Toxocara canis,cati, Trichuris trichiura, Ancylostoma duodenale, Strongyloides stercoralis, Trichinella spiralis, Wuchereria bancrofti, Dracunculus medinensis – aesculape´s bat • Nonsegmented body, adult worms living in the GIT - diagnosis: identification of eggs in the stool (morphology of eggs) • Filariae – thin worms parasiting in eye, skin, tissues, transmitted by insects. Larval stage - microfilariae penetrate to blood and are transmitted by suckling insect

12. Cestodes • Head - scolex, segmented body - Hermafrodit, male and female organs are present in every segment - dif.dg. - They have not GIT, absorbtion of food. - Complex life cycle with transient host (sometimes - human – larval stage of cysticercosis, echinococcosis) • Taenia solium, Taenia saginata, Diphylobotrium latum, Echinococcus granulosus, Echinococcus multiloculare Hymenolepsis nana

13. Trematodes - • Usually hermafrodits (exception - Schistosoma) • Need transient host (hosts) • Fasciolopsis, Clonorchis, Paragonimus, Schistosoma

14. Nematodes -worms • Enterobius vermicularis, Ascaris lumbricoides, Toxocara canis,cati, Trichuris trichiura, Ancylostoma duodenale, Strongyloides stercoralis, Trichinella spiralis, Wuchereria bancrofti, Dracunculus medinensis – aesculape´s bat • Nonsegmented body, adult worms living in the GIT - diagnosis: identification of eggs in the stool (morphology of eggs) • Filariae – thin worms parasiting in eye, skin, tissues, transmitted by insects. Larval stage - microfilariae penetrate to blood and are transmitted by suckling insect

15. Enterobius vermicularis • Definitive host - human • transient - none • dg. – perianal sample – microscopy of eggs • fecal oral transmission - autoinfection

16. Enterobius vermicularis

17. Ascaris lumbricoides • Definitive host: human • Larva migrans: intestin, colon - muc.membrane - blood- lung - cough - mouth - colon • dg. Egg in stool • Infection via contaminated food • Symptoms acc.to localisation of the larva

18. Ascaris lumbricoides

19. Dracunculus

20. Cestodes • Head - scolex, segmented body - Hermafrodit, male and female organs are present in every segment - dif.dg. - They have not GIT, absorbtion of food. - Complex life cycle with transient host (sometimes - human – larval stage of cysticercosis, echinococcosis) • Taenia solium, Taenia saginata, Diphylobotrium latum, Echinococcus granulosus, Echinococcus multiloculare Hymenolepsis nana

21. Taenia solium • Definitive: pig • transient: rat • incidental: human in small intestin • dg. serology • Contaminated food

23. Echinococcus granulosus • definitive: dog • transient:sheep • incidental: human • dg serology • transmission: cyste in meat • infection: mechanic pressure from expanded cyst, prolonged growing, rupture of the cyst and dissemination

24. Echinococcus granulosus

25. Intestinal,brain and UG protosoa • entamoeba histolytica • entamoeba coli • giardia lamblia • naegleria fowleri, acanthamoeba • trichomonas vaginalis

26. Entamoeba histolytica • definitive: human • dg.trofosoits in stool, serology • dysenteria – diarhea with blood

27. Naegleria fowleri, Acantamoeba • Free Living in water • Human (via nose) • dg.microscopy in CSF- identification of invasive strains) • disease: purulent peracute meningitis

29. Trichomonas vaginalis • Definitive: human • transient: none • dg. Cultivation - microscopy trophosoit - from vagina, urine • Sexual transmission • Therapy of both (all) partners

31. Blood and tissue protosoa • Plasmodium vivax – tertiana • Plasmodium ovale – tertiana • Plasmodium malariae – quartana • Plasmodium falciparum – maligna tertiana • Toxoplasma gondii • Pneumocystis carinii – jiroveci • Leishmania donovani – kala azar • Trypanosoma brucei gambiense – sleeping disease rhodesiense

32. Toxoplazma gondii • Definitive: cat • transient: rat • incidental: human • dg. serology KFR, IgA, IgG, IgM • transmission: food borne, hand, annimal • disease: - intrauterine primoinfection - generalised lymfadenopathy, encystation in organs – abortion, eye……..

34. Life cycle • Cat swallow tissue cyst (in food) or oocyst (from excrements) – infection of epitelial cells of small intestin – asexual and sexual multiplication – formation of oocysts – excremetion during 1-2 weeks – cyst is nonsporulated = not infectious – outside the body after 1-5 days cysts sporulate – infectious during months, resistent to desinfection, freezing, drying, heating to 70 dg C for10 mins.

36. Clinical manifestation • 1941 - Sabin – meningoencefalitída • - Pinkerton a Henderson – tyfoidné príznaky • 1951 - Siim – generalised lymphadenopathy - lymphocyto-monocytes proliferation in periferal blood • gained toxoplasmosis • gained or reactivated toxoplasmosis in IDS • In borne – congenital toxoplasmosis • Eye toxoplasmosis

37. Gained toxoplasmosis • Usually asymptomatic • 10% - 20% cervical lymphadenopathy + flue like symptoms • Clinicallly selfliminting – reactivation in IDS, in gravidity

38. Gained toxoplasmosis in IDS • Primary infection or reactivation of anamnestic infection • CNS , myocarditis, pneumonia • AIDS pacients – encephfalitis, intracerebral lesions

39. Congenital toxoplasmosis • In acute primoinfection of mother during pregnancy • Symptoms depends on the lenghth of gravidity during primoinfection • Therapy can decrease the symptomatology • Acute diagnosis is important • New borne can have - subclinical symptoms – without therapy usually getting worse - sy i.u. toxoplasmosis – hydrocefalus, calcifications in brain and liver, cataracta, microcephalus

40. Eye toxoplasmosis • Ofthen asymptomatic untill the 2nd-3rd decenium • Symotoms – rupture of the cyst in eye, tachysoits and bradysoits are released • Chorioretinitis – unilateral – after gained infection – bilateral – after i.u. infection

41. Toxoplasma gondii protozoa i.c. parasit • Infiects different warm-blood annimals • Cat is host for sexual stages of Toxoplasmy gondii (schisonts) – main source • 3 stages – tachysoits (trofosoits) – quick multiplication and destruction of the invaded tissue – bradysoits – slowly multipliing in tissue cysts – sporosoits (male and female gamonts) in oocysts – in cat excrements

42. developmental stages of Toxoplasma gondii Neonsporulating adn sporulating cyst from cat stool Tachysoits free and i.c. Bradysoits in tissue cysts

43. Infection in men • 1) food borne with inappropriately boiled food containing tissue cysts • 2) transplantacentarly • 3) accidentally by innoculation of tachysoits • 4) swallowing of oocysts (sandy playgrounds., contaminated hands) • 5) kontaminated trasfusion or transplantation

44. Pathogenesis • Swallowing of the cyst (bradysoit) or oocyst (sporosoit) • Releasing of microorganism • Invasion to the small intestin epitelium • Dissemination and multiplication intracelularly • Death of infected cells, releasing od tachysoits that invade other cells • Reaction of immunity system, change of tachy to bradysoits, formation of tissue cysts (in muscles, heart, brain) • Reactivation during IDS – rupture of cysts, releasing of parasits

45. Direct proof • Seldom positive • Staining acc. Giemsa • Immunofluorescence • ELISA for antigen detection • Tissue cultures • Innoculation of mouses • PCR

46. Laboratory results IgG IgM specific antibodies: – – without serological proof – + probable acute infection (+IgA from the same sample) or false positive IgM reaction (repetition of IgG and IgM from neuw sample – no changes) + + probable infection 6 – 12 mnths ago. ++ – probable infection 1 year ago KFR – total antibody detection

47. CFR titres above 8 are suspect for infection dynamicity – increase of the titre after 14 days - acute – maps decrease of the infection after therapy and possible reactiovation of the infection CFR antibodies are life long persistent

48. Serology of toxoplasma gondi in pregnancy • I. trimestre – test for IgM antibodies – detection of i.u. infection – positivity – indicate recetn infection current IgA positivity – acute infection – therapy of mother and screening of the baby • IgG antibodies positive (seldome with IgM positivity) – not indicating acute infection, can result in reactivation of possible tissue cyst, thah has no relation to pregnancy and cannot be eliminated by therapy • Increase of IgG antibodies – reactivation of past infection without threat of fetus health

49. Antenatal diagnosis of inborne toxoplasmosis • Acute infection in pregnant mother • Therapia • Ultra sonography • Amnionic fluid + fetal blood: PCR innoculation do mouses and tissue cultures • Fetal blood: Toxo IgM and IgA, activity of liver ensyms

50. Newborne infection dg • Isolation from placenta, umbilicus leucocytes • Clinical and laboratory test for tissue injury detection • Špecific antibodies – newborne serum CSF – mother serum • IgG,M,A IgM,IgA • PCR • tachysoit