Lecture 3.        Other Systemic Protozoa                 J. D. MacLean

Overview
Toxoplasmosis         (Toxoplasma gondii)
                        transmission:        cat feces, raw meat, in utero (vertical)
                      clinical:         lymphadenopathy
                                          congenital
                                          ocular
                                          immunocompromised
                  diagnosis:         serology, PCR, biopsy of brain

Pneumocystis (Pneumocystis carinii)
                     transmission:       airborne
                          clinical:       newborn pneumonia,
                                            immunocompromised pneumonia
                       diagnosis:       bronchoalveolor lavage

African sleeping sickness (Trypanosoma brucei)
                          transmission:     Tsetse fly vector, east African wild bovine reservoir
                            clinical:      acute febrile;
                                             chronic neurological
                        diagnosis:      blood or CSF microscopy; antigen capture, PCR

Chaga's disease (Trypanosoma cruzi) 
                  transmission:     Reduvid bug vector & spiny anteater reservoir;
                                       blood transfusion
                                       in utero (vertical)
                       clinical:     acute;
                                       chronic megavisera (heart, colon, oesophagus )
                    diagnosis:     serology, xenodiagnosis, PCR

Visceral leishmaniasis (Kala azar)  (Leishmania donovani)
                         transmission:      sand fly vector, rodent reservoir.
                         clinical:     fever, splenomegaly (visceral leishmaniasis)
                     diagnosis:     amastigotes (LD bodies) in bone marrow , PCR

Cutaneous and mucocutaneous  (Leishmania tropica, brazilianse, mexicana etc)
                 transmission:     similar to visceral leishmaniasis
                       clinical:     skin ulcer, nasal and bronchial spread (L. brazilianse)
                                       skin ulcer  (L. mexicana, L. tropica) 
                    diagnosis:    amastigotes (LD bodies) in biopsy or aspirate; culture
        species diagnosis:   PCR, culture and isoenzyme analysis

Toxoplasmosis (Toxoplasma gondii)      Phylum: Apicomplexa

Biology:  Three forms of parasite involve man:

trophozoite: the intracellular parasitic stage which proliferates by binary fission, usually seen in the early acute stage of infection - size 4-8 µm.
bradyzoite: an encysted form which is found during chronic latent (asymptomatic) infection; persists for years in human tissues (brain, retina, muscle etc.)
oocysts: the 10 µm form shed in the stool of cats, the product of the sexual reproductive cycle in cat gut epithelial cells. They  sporulate in 3 days after passage in stool and then can be infective in the environment for a year. Man swallows oocysts to become infected.

Epidemiology:

Very common throughout the world; 25-30% of adults in Montreal have become infected in their lifetime, up to 50+% in other developed or developing countries.

This is a zoonosis affecting almost all mammalian carnivores and mammals in contact with cats. The cat is the definitive host because the sexual reproductive cycle of toxoplasmosis occurs only in the intestinal epithelial cells of cats.

Humans become infected in several ways:
                - ingestion of oocysts through contamination of food, hands, etc. with cat feces.
                - ingestion of bradyzoites in uncooked meat, e.g. lamb, pork, beef, caribou.
                - transplacental when mother develops acute infection during pregnancy.
                - blood transfusion, organ transplant.
                 - rarel from contaminated drinking water (e.g. British Columbia)

Clinical:

1. majority are asymptomatic

2. acute toxoplasmosis: fever, lymphadenopathy (much like infectious mononucleosis - EBV); can rarely cause specific organ inflammation, e.g. encephalitis, myocarditis.

3. reactivation toxoplasmosis: occurs in immunosuppressed such as AIDS, transplant and cancer patients: presents with specific organ involvement e.g. encephalitis, pneumonitis.

4. choreoretinitis: occurs later in life in individuals who acquired toxoplasmosis congenitally; focal lesion  in retina presenting as decreased visual acuity; rarely occurs during acute toxoplasmosis.

5. congenital toxoplasmosis: transmission from mother to fetus when mother has developed acute toxoplasmosis during pregnancy - increased transmission rate in third trimester, but increased severity of fetal disease in first trimester. Presents as hydrocephalus, hepatomegaly, cerebral calcifications, mental retardation with death at one end of spectrum and mental retardation or just later choreoretinitis at the other end of spectrum.

toxoplasma retinal scar	toxoplasma cerebral abscess

Diagnosis:
Except for toxoplasmosis of specific organs, e.g. brain where biopsies can reveal trophozoites, serology or PCR is the usual means of diagnosis.

therefore - biopsy of organ, e.g. brain
               - biopsy of lymph nodes reveals suggestive pathology but rarely trophozoites
               - serology: in acute toxoplasmosis in routine screening of pregnancy
                            (limited value in immunosuppressed and choreoretinitis)
               - immunofluorescent staining of biopsy sections
               - PCR
               -culture

Treatment:
            - antifolates (pyrimethamine and sulfadiazine)
            - spiramycin

Pneumocystis carinii

This is a protozoan of debatable taxonomic position.

Biology - in two forms, cysts (5 mu), a dormant form which contains 8 trophozoites (5 x 1 m). -appears to be asexual binary division of trophozoites.

Silver stain of P. carinii in bronchoalveolar lavage (BAL)

Epidemiology:
Acquired in childhood by almost everyone, probably airborne and possibly (unknown) without overt illness. Remains in latent nonpathogenic form in human pulmonary interstitium and produces overt illness in the immune compromized (premature infants, AIDS)

Clinical:
1. interstitial plasma cell pneumonia of infants (especially premature). Occurs in epidemics, usually in hospitals; 50% mortality if untreated.
2. pneumonia in immunocompromized - e.g. leukemia, lymphoma, AIDS; 100% mortality if untreated. Most common cause of mortality in AIDS patients.

Diagnosis:
Open lung biopsy, brush biopsy, and at times sputum stains:
                        ..Grocot, silver stain will stain thick cyst wall.
                        ..Giemsa stain will stain trophozoites.
Treatment:
Antifolates (e.g. Septra (trimethoprim and sulfamethaxazole))
Pentamidine
Clindamycin and primaquine
Atovaquone

Hemoflagellates                  Phylum:   Sarcomastigophora

Genera:    2 important medically - Trypanosoma and Leishmania

Overview: 
- Important pathogens of humans and livestock
- All pathogenic species transmitted by insects
- All have more or less complex cycles alternating from one morphologic form to another
- Distinguishing structural characteristics: flagellum, kinetoplast
- Basic morphologic stages: -flagylated trypomastigote, promastigote or epimastigote
                                          - amastigote                                                        

Genus Trypanosoma
     - 2 different species of medical importance
            - Trypanosoma cruzi: causes Chagas' disease in South America
            - Trypanosoma brucei: causes African trypanosomiasis (African sleeping sickness)

Genus Leishmania
      many species grouped by clinical presentation: cutaneous, mucocutaneous and visceral
           - visceral  Leishmania donovani
          - cutaneous Leishmania tropica, major, mexicana etc
           - mucocutaneous Leishmania braziliensis etc

Trypanosoma cruzi (Chagas' Disease, American Trypanosomiasis)

Phylum: Sarcomastigophora

Biology
A zoonosis with reservoirs of armadillo, opossums, raccoons; transmitted to man by the reduvid bug (e.g. in Mexico Rhodnius prolixus). The bug can live and be infective for two years and hides in adobe walls.

Reduvid bug bites human while asleep and passes feces containing parasite (trypomastigote form, 20 m) onto skin near the bite. Human rubs faeces and parasite into wound or eye.

Parasite causes local infection and inflammation (chagoma) and then circulates in blood of host (doesn't multiply intravascularly) and then invades tissue cells (muscle and mononuclear phagocytic system of spleen, liver, 1ymph nodes and CNS) where it changes into amastigotes (2-4 mu) and divides by binary fission.

T.cruzi trypomastigote

Epidemiology:
Transmission: ..by reduvid bug bite
                       ..vertical (in utero)
                       ..blood transfusion

     12 million persons affected; 35 million at risk; Central and South America.                                                                                                                                                        

Clinical:
Acute: local inflammation at site of innoculation (chagoma) generalized lymphadenopathy, hepatosplenomegaly fever.
Chronic: up to several decades after acute stage myocardopathy - heart failure, arrhythmias megaintestine - megaoesophagus, achalasia, megacolon

Diagnosis:
                           - blood smear for trypanosomes (Giemsa stain)
                           - serology, PCR
                           - biopsy
                           - xenodiagnosis

Treatment:   - Nifurtimox (Lampit) a nitrofurone, poorly effective in chronic Chagas

Control:  - Concrete walls and floors prevent reduvid bug infestations
                         - antiparasitic agents added to transfused blood products

African Trypanosomiasis (African Sleeping Sickness)
A hemoflagylate found only in Africa, a zoonosis with a resevoir, in the East African form of disease, of bovines (antelope, cattle) where transmitted from resevoir animal to man by the vector tsetse fly (e.g. Glossina palpalis). In west African form it is transmitted by tsetse human to human..

2 species:   Trypanosoma brucei gambiense (Africa west of Rift valley)
                  Trypanosoma brucei rhodesiense (Africa east of Rift valley)

As much importance to man are the trypansomes (e.g. Trypanosoma brucei brucei) which produce disease in cows making cattle raising in a quarter of Africa impossible.

Pathogenesis

Tsetse bites man and injects saliva containing trypanosomes into the wound. These multiply locally producing a local lesion and then invade intravascular space where the trypanosome multiplies by binary fision extracellularly producing fever and lymphadenopathy and then eventually reaches the central nervous system producing a meningoencephalitis.

Clinical picture - trypanosomal chancre - parasitemia with lymphadenopathy fever - central nervous system signs of variable focal nature and eventually coma and death

encephalitis of African Sleeping Sickness.

Diagnosis 
    - motile organisms on wet preparations of fresh blood or CSF
    - stained smears (Giemsa) may reveal organism (organism 14-33 x 1.5-3.5 µm)
    - high levels of protein (IgG and IgM) in CSF                    
    - lymphocytosis in CSF
    -antigen capture or PCR

Treatment    - Pentamidine
                          - Suramin
                          - Eflornithine
                          - Melarseprol

Major problems 
  - how to control Tsetse fly (killing off all wild animal reservoirs?)
   - vaccine production very difficult because of antigenic variation of trypanosome

Leishmaniasis
A protozoan infection involving a number of species of the genus Leishmania. There are two distinct groups of clinical presentations.

Visceral leishmaniasis:presenting with a systemic illness of fever, splenomegaly and lymphadenopathy (caused by Leishmania donovani) -- other names Kala azar, Dum Dum fever.
Cutaneous leishmaniasis:presenting with a skin ulcer or ulcers and caused by Leishmania tropica, mexicana, and braziliensis mainly: A varient can also involve mucous membranes of nose and throat.
These are almost always zoonoses with a dog or rodent reservoir, and are transmitted to man by sandfly bite (Phlebotomus sp). They are distributed throughout the tropics in circumscribed foci.

Organism:
Exists in 2 morphological forms, the promastigote which is found in the sandfly vector and the amastigote in man, intracellularly in macrophages in skin lesions (in cutaneous leishmaniasis) or in the reticuloendothelial system of blood, liver, lymphnodes, spleen (in visceral leishmaniasis).

Promastigotes	Amastigotes, also called Leishmania Donovani bodies

Pathophysiology/life cycle:
The sandfly bites human and injects promastigotes which invade macrophages, becoming amastigotes. These multiply, invade new macrophages, and with the attraction of lymphocytes to the local infection, a local subcutaneous 1-3 cm. nodule is produced which ulcerates and is contained locally by the cell mediated immune system in cutaneous leishmaniasis or spreads systemically in visceral leishmaniasis.

sandfly vector

Clinical picture:
visceral leishmaniasis: (L. donovani)
early nodule at site of sandfly bite seen at times; usually presents with chronic fever, splenomegaly (very large), lymphadenopathy and dark skin = Kala, azar. High mortality from anemia and bacterial superinfections.

cutaneous leishmaniasis:
several variants basically with skin nodules which ulcerate.
    L. tropica complex - in Asia, Africa, and Mediterranean (called Oriental sore, Delhi or Bagdad boil).
    L. mexicana complex and others - in Central and South America

cutaneous leishmaniasis	throat mucocutaneous leishmaniasis (2 years duration)

mucocutaneous leishmaniasis:
begins as a cutaneous ulcer and metastasizes to mucosa of nose and pharynx leading to destruction, obstruction and death.

L. braziliensis - in South  and Central America (called espundia).

Diagnosis:
Cutaneous and mucocutaneous
1. aspirate material from edge of ulcer and stain (Giemsa).
2. biopsy and stain with Giemsa - pathology sections show granulomatous lesion and organisms (amastigotes = Leishmania donovani bodies =LD bodies) are seen in macrophages.
3. culture aspirate or biopsy material in special media producing promastigotes.
4. serology
5. PCR

Visceral
1. aspirate bone marrow or spleen and stain (Giemsa) or culture material.
2. serology.
3. PCR

Treatment:
- relatively toxic drugs, e.g. antimonials (Pentostam), Pentamidine, Amphoteracin B.
- resistance to all drugs in some geographic areas.

Scientific Challenges:
      - defining the many variants in species.
            - finding less toxic drugs.