DR. RAAFAT MOHAMED

UMM AL-QURA UNIVERSITY

FACULTY OF MEDICINEParasitology Department

PracticalCLASS NO. 2

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* Indicate the parasites of this lab

Protozoa

Flagellate : blood & tissue :

Leishmania spp

Trypanosoma spp

Sporozoa :

Plasmodium spp

Toxoplasma gondii ( coccidia )

arthropoda

Myasis

Scabieiasis

Lab Slides 12

1. Plasmodium falciparum ( Ring , gametocyte )

2. Plasmodium vivax ( Ring , Gametocyte , schizont)

3. Leishmania donovi smear from infected spleen ( amastigoites )

4. Leishmania donovi smear from culture ( promastigoites )

5. Trypanosoma gambiense smear(large number , trypomastigoite stage )

6. Trypanosoma cruzi in blood smear( scanty, trypomastigoite stage )

7. Trypanosoma cruzi in Cardiac muscles ( amastigoite stage )

8. Toxoplasma gondii tissue smear with parasite

9. Toxoplasma gondii tissue smear ( with peritoneal cells )

• Don’t move the slides please • Cover the microscopes after you finish please

Plasmodium spp The available types in the lab :• Plasmodium falciparum ( the most

dangerous )• Plasmodium Vivax ( the most common in

KSA NOT avaliable in the lab :• Plasmodium Ovale• Plasmodium malariae

Infection Sporozoites

Liver

Asexualcycle

Gametocytes

Merozoites

Transmissionto mosquito

MALARIA LIFE CYCLE

Diagnostic stages in human are:

- Ring stage ( early trophozoite )

-mature trophozoites

- schizonts

- gametocytes

Diagnosis of plasmodium spp

• Thin blood film :• Show small amount of the parasite • Helps in Determining the spp

• Thick blood film :• Large drop of blood + RBCs haemolysis

Plasmodium falciparum slides1. Ring stage ( early trophozoite ): • ( ring of chromatin+cytoplasm+ parasite

nuculus )• appears like a pair of stereo-head phones • more than one ring in the Same RBCs (multiple infection).• Accolè form (marginal ring).

2. Gametocyte • ( banana shape )

P. falciparum

Accolè form

Plasmodium falciparum keys The size of the infected RBC = non infect. Infects all stages of RBCs Maurer’s dots present

the mature trophozoite & schizont stage:

1. Rarely seen in peripheral blood because they are sticking to the vascular endothelium inside the internal organs capillaries.

2. Seen only in the end stage patients

Recrudescence : reappearance of the symptoms in falciparum + malariae due to insufficient R + ↓ pt immunity .

Plasmodium falciparum – ring stage = early Trophozoite

P. falciparum - gametocyte

P. falciparum Black water Fever • Autoimmune disease due

to R of the pt with strong medications.

• Leads to massive heamolysis of RBCs :

1. Severe anemia

2. Severe schock

3. Jundice

4. Heamoglobinuria which leads to :

a. Acute renal faliure

b. Dark urine

Normal urineHaemoglobinuria

Cerebral malariadue to blockage of brain capillaries

“ P . falci. Adhere to the blood vessels endothelial lining by sticky surface knob”

Plasmodium Vivax slides

1. Ring stage ( the chromatin ring is thicker than the p .falciparum )

2. Trophozoite (amoeboid) NOT IN THE LAB

3. Schizont ( fragmented chromatin ) , forming 12-24 merozoites

4. Gametocyte ( powdered pigment + chromatin mass ) , fills the enlarged RBC

Plasmodium vivax – Key PointsThe size of the infected RBC > non infect.

Infects reticulocytes (large RBCs) only

Schüffner's dots present

Relapse occurs in vivax + ovale due to

dormant Hypnozoites = merozoites within liver cell which become activated after a period of time = recurrence of clinical pictures .

P. vivax Ring stage

P. Vivax (amoeboid) trophozoitenot in the LAB slides

Mature Trophozoite (late trophozoite): In all palsmodium spp The parasite develops by increasing the amount of cytoplasm. Digested haemoglobin gives rise to malaria pigments (haemozoin). Parasitised cells become osmotically fragile.

P. vivax schizont

• Erythroytic Schizont: • The chromatin break into fragments = merozoites range (14-24,

mean 16) . • The pigments remains as a single mass in the center. •  The Schizont occupies the entire R.B.C

P. vivax gametocytes

powedered pigement + chromatin mass

P. falciparum P. vivax .

Rings

Trophozoites

Schizonts

Gametocytes

Summary

Protozoa cont

Flagellate : blood & tissue

Leishmania spp

Trypanosoma spp

Diagnosis of Leishmania• Direct parasitology :

1. Gimsa stain : to detect the intracelluar Amastigoite

2. Culture on N.N.N media : to detect promastigoite ( animal inoculation )

• Biopsy :

1. Cut. Muct. : biopsy from the ulcer

2. Visceral L. : Biopsy from ( B.M “ sternum , iliac crest ” , L.N , blood buffy

coat , Spleen “ risk of bleeding & rupture “ ) If the pt has ( diarrehea + dysentry ) Amastigoite in stool• Montenegro skin test : intradermal skin test , reflect type 4

hypersensitivity ( cellular immunity ) , +ve for life ( induration after 48 hrs ) , - ve in acute stage.

• PCR , ELISA ,direct agglutination .., ect

Diagnosis of Leishmania

Montenegro skin test

Skin biopsy

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Slides of Leishmaniaboth are in the lab

• Amastigote in splenic tissue• No flagella• Kinetoplast : is the flagella

origin• Intracellular to mononuclar

phagocytes• Differentiate from yeast by

presence of nucleus and kinetoplast

• Promastigote from culture• Spindle shape• only available in the vector

salivary glands & culture media ( N.N.N ) “ in the lab”

Promastigote stage in vector or culture

Amastigote stage in host (Giemsa stain)

LEISHMANIASIS

amstigote

Leishmania donovani amatigotes in splenic biopsy

( visceral )

Leishmania donovani amatigotes in bone marrow biopsy

Leishmania cutaneous ulcer

• Types of cutaneouse leishmaniasis:

1. Simple ( dry “single”& wet “multiple”)2. Disseminated

3. Chronic relapsing

Leishmania amastigote in skin biopsy

Mucocutaneous leishmaniasis• L.braziliensis• Destroy : skin, MM, cartilage • Site : eye ,nose,

mouth ,larynx ,pharynx

• Type of lesion :

1. Non ulcerative : edema + hypertrophy

2. Ulcerative (Espundia ) : painful & destructive

3. 2ry bact. Infect → enlarged L.N

• Montenegro skin test : +ve• Treatment : chemotherapy

Depigmented areas

Butterfly pigmentation

Skin changes of visceral Leishmania = kala-azar = black fever = Dum-dum fever

• Early: dark pigmented erythematous areas distributed over the body. It takes a butterfly distribution over the nose.

• Late (PKDL): In late stage of disease or after incomplete treatment, the skin is invaded by de-pigmented non-ulcerative nodules [contain many parasites, act as a source of infection for vector, mainly over the face & extensor surface of limbs and common in India & Africa]

• Usually occur in patients from the old world

Post Kala-Azar dermal leishmanoid

(PKDL)

visceral Leishmania = kala-azar = black fever = Dum-dum fever

exhibits splenomegaly, distended abdomen , severe muscle wasting , and jaundice .

Protozoa cont

Flagellate : blood & tissue

Leishmania spp

Trypanosoma spp

TRYPANOSOMIASIS African Trypanosomiasis (African sleeping sickness)• Trypanosoma brucei complex:

1. Trypanosoma gambiense (West Africa)

2. Trypanosoma rhodesiense (Eest Africa)• Vector: Glossina spp ( tsetse fly )

American Trypanosomiasis (Chagas’ disease)

1. Trypanosoma cruzi (south and central America)

• Vector: Reduviid bugs spp ( Triatoma bug ) ( wing / kissing / barber bug )

Slides of Africans Trypanosoma

Trypomastigotes•Will be seen in the lab•Spindle shaped•Central nuculuse•Posterior kinetoplast•Free flagellum•Undulating membrane ( whole length )•Extracllular ( Bl , Lymph , CSF, tissue )•3 forms ( long slender,short stumpy,intermed )

Epimastigote ( crithidia )•Will not be seen in the lab•Seen only in the vector salivary gland or in the media

T. Gambiense trypomastigotes in blood film

•African Try. Characters in blood film :

1. polyomorphic ( 3 forms of trypomastigote )

2.Large number in film

3.Exttracellurar ( between RBCs )

Clinical picture of AfricanTrypanosoma

Winterbottom's sign • seen in the early phase of African trypanosomiasis• the swelling of lymph nodes  along the back of the neck, in the posterior cervical

 chain of lymph nodes• may be suggestive of cerebral infection

T. Gambiense sleeping sickness

Diagnosis of AmericanTrypanosoma

• Mainly is by pateint’s blood sample Examined by :• Direct thick Smear to detect trypomastigotes ( polyomorphic )• Culture on suitable media to detect Epimastigote

• Chagoma speiciemen (acute skin granulomatouse lesion at the bite site )

• Muscle biopsy to detect the amastigoites

• Cruzin test : intra-dermal skin test looking for Ab

• Xenodiagnosis : using starving healthy vector to diagnose suspected cases.

Slides of Americans Trypanosoma

Trypomastigotes•Will be seen in the lab ( blood smear slide)

•C or U shape•Large Central nuculuse•Large kinetoplast•Free flagellum•Undulating membrane •Extracllular ( Bl , Lymph , tissue )

Epimastigote •Will not be seen in the lab•Seen only in the vector salivary gland or in the media

Amastigoites•Will be seen in the lab in Cardiac muscles•Same shape for lieshm.•Intracellular tissue ( cardiac ms , sk ms , nerve ganglion , GIT cells )

T. cruzi trypomastigote in blood film( in the lab )

•AmericanTry. Characters in blood film :

1.C or U shape

2.scanty number in film

3. polyomorphic ( 2 forms of trypomastigote )

4.Exttracellurar ( between RBCs )

T. cruzi Amastigotes in cardiac muscles

( in the lab )

• Leads to : • Arrythmia• Rt bundle branch block • Cardiomegaly • Cardiomyopathy• Heart faliure

Cardiomegaly ofChagas disease by the amastigoite stage

T. Cruzi clinical signs

Romana’s sign• the unilateral painless periorbital swelling associated with the

• acute stage of Chagas' disease.

•Chagoma•a skin tumor occurring in Chagas'

disease.

Protozoa cont

Sporozoa

coccidia

Toxoplasma gondii

Slides of Toxoplasma gondiiTrophozoite

• The only slide in the lab ( from rats peritoneal fluids ) ( 2 slides )

• Crescent in shape • Centeral nuculus• 2 forms ( tchyzoites & bradyzoites )• Obligatory intracellular in pahgocystic

cells

True cyst• Contains bradyzoites• Site ( cardiac & sk ms , brain , eye )

Pseudocyst• Contains Tachyzoites• Site ( heart , braine , eye , lung , RES )

Oocyst • In D.H = cats only

Slides of Toxoplasma gondii trophozoite

Toxoplasma gondii sporulated oocyst

• Oocyst contains:

• 2 sporocyst

• 4 sporozoites

• Called : disporocystic tetrazoic

Toxoplasma gondii brain cyst

Congenital toxoplasmosis

Microcephaly

Hydrocephalus

Arthropoda

Myasis

Scabiasis

Myasis

• Definition : is the invasion of human or animal tissue by Larvae of Dipterous flies ( maggots ) where they feed on living or necrotic tissues.

• Classification according to :

1. Habit of flies ( specific , semi-specific , accidental )

2. Habitat of invaded tissue ( cutaneous , occular , aural , nasopharyngeal , urogenital , intistinal & gastric )

Nodular Cutaneous Myiasis

The larva burrow in the skin forming nodule

Occular myiasis

Sarcoptes Scabies

• Caused by : a mite • Life cycle : takes 2 weeks ( rapid )• Mode of infection :

1. Direct contact with infected animals & humans

2. Using infected towels , clothing , beddings

Diagosed by :

3. Clinical manifestation

4. Seeing the skin burrows by a hand lens

5. Demonstration of mite microscopically

Sarcoptes scabieimite

Scabies characteristic skin papules &vesicles

Scabies characteristic skin burrows

“the pereferential sites are interdigital space“

Lab Slides 121. Plasmodium falciparum Ring stage

2. Plasmodium falciparum gametocyte stage

3. Plasmodium vivax Ring stage

4. Plasmodium vivax Gametocyte stage

5. Plasmodium vivax schizont stage

6. Leishmania spp amastigoites stage from infected spleen

7. Leishmania spp promastigoites stage from culture

8. Trypanosoma gambiense in blood smear ( trypomastigoite stage )

9. Trypanosoma cruzi in blood smear ( trypomastigoite stage )

10. Trypanosoma cruzi in Cardia muscle ( amastigoite stage )

11. Toxoplasma gondii tissue smear with parasite ( trophozoite )

12. Toxoplasma gondii tissue smear with peritoneal cells ( trophozoite , tachyzoite )

• Don’t move the slides please • Cover the microscopes after you finish please