Blood Protozoa
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BLOOD PROTOZOA II BPT
Dr Ekta Chourasia Microbiology
Protozoa - Blood protozoa Plasmodiu m
Malaria
Babesi a
Babesiosi s
Leishmani a
Leishmaniasis (Visceral, Cutaneous)
Trypanaso ma
African sleeping sickness, Chagas disease
Toxoplasma gondii
Toxoplasmosis (congenital infections)
12/03/08
Dr Ekta, Microbiology, GMCA
Genus Plasmodium Consists of 4 species:
12/03/08
P. vivax P. falciparum P. malariae P. ovale
Dr Ekta, Microbiology, GMCA
Transmission & Life Cycle Definitive host
Female Anopheles mosquito
Intermediate host
Man
Infective form
Sporozoites
Portal of entry
Skin
Mode of transmission
Bite of an infected mosquito
Site of localization
First in liver cells & then in
12/03/08
RBCs
Dr Ekta, Microbiology, GMCA
12/03/08
Dr Ekta, Microbiology, GMCA
Morphological forms seen in Humans In liver: • • •
Sporozoites Pre erythrocytic schizonts Merozoites – infect RBCs
In RBCs : 1. 2. 3.
4.
12/03/08
Trophozoites – ring form Schizonts Merozoites – released by the rupture of schizonts – infect other RBCs Gametocytes – micro and macro gametocytes Dr Ekta, Microbiology, GMCA
Morphological forms seen in Mosquito Further differentiation & development of gametocytes take place in mosquito •
•
•
12/03/08
Macro gametes (female gametes) – each macro gametocyte develops in to one macro gamete in the mid gut of mosquito Micro gametes (male gametes) – one micro gametocyte produces 6 to 8 micro gametes by exflagellation. Zygote – Ookinete – Oocyst – rupture – release of Sporozoites – predilection to salivary glands. Dr Ekta, Microbiology, GMCA
Incubation period P. vivax P. ovale P. falciparum P. malariae
12/03/08
10 to 14 days
18 days to 6 weeks
Dr Ekta, Microbiology, GMCA
Pathogenicity Infection causes intermittent fever – Malaria Each of the 4 species causes a characteristic fever: P. vivax Benign tertian/ vivax malaria P. falciparum Malignant tertian/ falciparum malaria, black water fever P. malariae Quartan malaria P. ovale Ovale malaria
12/03/08
Dr Ekta, Microbiology, GMCA
Clinical Features Series of febrile paroxysms – fever is caused by the release of merozoites & toxins from ruptured erythrocytic schizont which in turn causes the release of cytokines. Quartan malaria – every 72 hrs Tertian malaria - every 48 hrs * each paroxysm has 3 stages - cold stage (rigors), hot stage (high temp., body & joint pains, vomiting & diarrhoea) and perspiration stage (fall in temp.) 12/03/08
Dr Ekta, Microbiology, GMCA
Clinical Features Anaemia – due to breakdown of RBCs, particularly occurs in falciparum malaria Splenomegaly – all forms
12/03/08
Dr Ekta, Microbiology, GMCA
Falciparum malaria Severe falciparum malaria is associated with 1. 2. 3. 4. 5.
12/03/08
Pernicious malaria /cerebral malaria Blackwater fever Anaemia Hypoglycaemia Complications in pregnancy
Dr Ekta, Microbiology, GMCA
Malaria caused by P.vivax, P.ovale & P.malariae
Rarely life threatening Relapses/ recurrences are a feature
Recurrences in Malaria
12/03/08
May result from – reinfection or - due to persistence of infection - Occurs due to a special form of parasites called hypnozoites.
Dr Ekta, Microbiology, GMCA
Laboratory diagnosis of Malaria Specimen: peripheral blood smears thick and thin blood smears Leishman or Giemsa stain Trophozoites (ring forms) or gametocytes are seen within RBCs Quantitative Buffy Coat (QBC) examination P falciparum antigen detection (ELISA)
12/03/08
Dr Ekta, Microbiology, GMCA
Making of Thin & Thick films
12/03/08
Dr Ekta, Microbiology, GMCA
12/03/08
Dr Ekta, Microbiology, GMCA
Rapid Diagnostic tests HRP2 tests
detection of P.falciparum Two types of test – ParaSight F - ICT Malaria Pf
pLDH test e.g. OptiMAL test
12/03/08
Detection of P.falciparum & P.vivax Produced by all human malarial parasites Differentiation of species is based on antigenic differences between pLDH isoforms.
Dr Ekta, Microbiology, GMCA
ParaSightF test
Optimal test
ICT Malaria Pf / Pv
12/03/08
Dr Ekta, Microbiology, GMCA
12/03/08
Dr Ekta, Microbiology, GMCA
12/03/08
Dr Ekta, Microbiology, GMCA
Toxoplasma gondii Definitive host
Cat
Intermediate host
Man, sheep, birds
Infective form
Matured oocyst / tissue cyst / tachyzoites
Mode of transmission
Ingestion / intrauterine / blood transfusion/ improper handling of cat litter
Site of localization
Any organs (RE system) Eye & Brain
12/03/08
Dr Ekta, Microbiology, GMCA
12/03/08
Dr Ekta, Microbiology, GMCA
Laboratory Diagnosis –T gondii Specimens
Impression smear of LN, bone marrow, spleen, brain biopsy, blood, sputum, CSF
Microscopy
Giemsa stain, tachyzoites or cysts
Serology
ELISA / IFA – 16 fold rise in Ab titre: Acute infection Sabin-Feldman dye test: inhibition by antibody of the staining of tachyzoites by alkaline methylene blue
Prenatal diagnosis Fetal blood for IgM Ab / PCR
12/03/08
Dr Ekta, Microbiology, GMCA
Haemoflagellates
12/03/08
Dr Ekta, Microbiology, GMCA
Trypanosomes T. brucei subspecies gambiense (Chronic)
West African sleeping sickness
T. brucei subspecies rhodesiense (Acute)
East African sleeping sickness
T. cruzi (acute and chronic)
American trypanosomiasis Chagas disease
12/03/08
Dr Ekta, Microbiology, GMCA
T. brucei Definitive host
Man
Intermediate host
Tsetse fly
Infective form
Metacyclic Trypomastigote
Mode of transmission
Bite of infected tsetse fly
Site of localization
CNS
12/03/08
Dr Ekta, Microbiology, GMCA
Clinical features Sleeping Sickness Trypanosoma chancre at the site of bite Winter bottom’s sign: prominent cervical lymphadenopathy
Meningoencephalitis - Apathetic, confused, comatose
12/03/08
Dr Ekta, Microbiology, GMCA
T. cruzi Definitive host
Man
Intermediate host
Reduviid bug
Infective form
Metacyclic Trypomastigote
Mode of transmission
Feces of bug rubbed into site of bite / contamination of conjunctiva or other exposed mucous membranes with fingers
Site of localization
Autonomous nervous system of heart / GIT
Infect cardiac, smooth and skeletal muscle, reticuloendothelial cells and neuroglial cells 12/03/08
Dr Ekta, Microbiology, GMCA
Clinical features Chagoma chancre at the site of bite Acute Chagas disease
Romana’s sign: unilateral edema of face with conjunctivitis and swelling of upper & lower eyelids Fever, splenomegaly, anasarca, meningoencephalitis
Chronic Chagas disease
12/03/08
Cardiomyopathy, AV block, CCF Megaesophagus / Megacolon
Dr Ekta, Microbiology, GMCA
Laboratory Diagnosis – Trypanosomiasis
12/03/08
Specimens
Blood, CSF, Aspirates (LN)
Microscopy
Trypomastigotes in blood
Dr Ekta, Microbiology, GMCA
Leishmania Definitive host
Man
Intermediate host
Sand fly (Phlebotomus)
Infective form
Promastigote
Mode of transmission
Bite of infected sand fly
Site of localization
Reticuloendothelial system
12/03/08
Dr Ekta, Microbiology, GMCA
Leishmania Three major species:
L. donovani – kala azar/ visceral leishmaniasis
L. major – cutaneous leishmaniasis
L. braziliensis – mucocutaneous leishmaniasis
12/03/08
Dr Ekta, Microbiology, GMCA
Clinical Types Visceral leishmaniasis - fever, malaise, weight loss, anaemia and swelling of the spleen, liver, and lymph nodes Cutaneous leishmaniasis - causes 1-200 simple skin lesions which self-heal within a few months but which leave unsightly scars Mucocutaneous leishmaniasis - infection begins with skin ulcers which spread, causing dreadful and massive tissue destruction, especially of the nose and mouth
12/03/08
Dr Ekta, Microbiology, GMCA
12/03/08
Dr Ekta, Microbiology, GMCA
Laboratory Diagnosis Demonstration of parasite in clinical specimen:
Microscopy Culture Animal inoculation
Demonstartion of antibodies using
Specific leishmanial Ag – ELISA / IFA / Agglutination Non-specific Ag – CFT
Non-specific serum tests
Aldehyde test (Napier’s) Chopra’s Antimony test
Absence of hypersensitivity to leishmanial Ag Contributory lab findings – anemia, leucopenia, neutropenia 12/03/08
Dr Ekta, Microbiology, GMCA
1. Demonstration of parasite in Clinical Specimen Clinical specimens:
Peripheral blood Bone marrow aspirate Spleen aspirate Lymph node aspirate
Microscopy: Leishman, Giemsa or Wright’s stain - Amastigotes within macropahges Culture: NNN (Novy, MacNeal, Nicolle) medium for 7 days – promastigote form Animal inoculation: Hamster - Animal kept at 23-26°C 12/03/08
Dr Ekta, Microbiology, GMCA
2. Absence of Hypersensitivity to Leishmanial Ag Montenigro test – 0.1 ml of killed promastigote Ag I.D. Result read after 72 hrs.
12/03/08
Positive in Dermal leishmaniasis & recovered cases of Kala Azar. Negative in active cases of Kala Azar.
Dr Ekta, Microbiology, GMCA
Dr Ekta Chourasia Microbiology
Protozoa - Blood protozoa Plasmodiu m
Malaria
Babesi a
Babesiosi s
Leishmani a
Leishmaniasis (Visceral, Cutaneous)
Trypanaso ma
African sleeping sickness, Chagas disease
Toxoplasma gondii
Toxoplasmosis (congenital infections)
12/03/08
Dr Ekta, Microbiology, GMCA
Genus Plasmodium Consists of 4 species:
12/03/08
P. vivax P. falciparum P. malariae P. ovale
Dr Ekta, Microbiology, GMCA
Transmission & Life Cycle Definitive host
Female Anopheles mosquito
Intermediate host
Man
Infective form
Sporozoites
Portal of entry
Skin
Mode of transmission
Bite of an infected mosquito
Site of localization
First in liver cells & then in
12/03/08
RBCs
Dr Ekta, Microbiology, GMCA
12/03/08
Dr Ekta, Microbiology, GMCA
Morphological forms seen in Humans In liver: • • •
Sporozoites Pre erythrocytic schizonts Merozoites – infect RBCs
In RBCs : 1. 2. 3.
4.
12/03/08
Trophozoites – ring form Schizonts Merozoites – released by the rupture of schizonts – infect other RBCs Gametocytes – micro and macro gametocytes Dr Ekta, Microbiology, GMCA
Morphological forms seen in Mosquito Further differentiation & development of gametocytes take place in mosquito •
•
•
12/03/08
Macro gametes (female gametes) – each macro gametocyte develops in to one macro gamete in the mid gut of mosquito Micro gametes (male gametes) – one micro gametocyte produces 6 to 8 micro gametes by exflagellation. Zygote – Ookinete – Oocyst – rupture – release of Sporozoites – predilection to salivary glands. Dr Ekta, Microbiology, GMCA
Incubation period P. vivax P. ovale P. falciparum P. malariae
12/03/08
10 to 14 days
18 days to 6 weeks
Dr Ekta, Microbiology, GMCA
Pathogenicity Infection causes intermittent fever – Malaria Each of the 4 species causes a characteristic fever: P. vivax Benign tertian/ vivax malaria P. falciparum Malignant tertian/ falciparum malaria, black water fever P. malariae Quartan malaria P. ovale Ovale malaria
12/03/08
Dr Ekta, Microbiology, GMCA
Clinical Features Series of febrile paroxysms – fever is caused by the release of merozoites & toxins from ruptured erythrocytic schizont which in turn causes the release of cytokines. Quartan malaria – every 72 hrs Tertian malaria - every 48 hrs * each paroxysm has 3 stages - cold stage (rigors), hot stage (high temp., body & joint pains, vomiting & diarrhoea) and perspiration stage (fall in temp.) 12/03/08
Dr Ekta, Microbiology, GMCA
Clinical Features Anaemia – due to breakdown of RBCs, particularly occurs in falciparum malaria Splenomegaly – all forms
12/03/08
Dr Ekta, Microbiology, GMCA
Falciparum malaria Severe falciparum malaria is associated with 1. 2. 3. 4. 5.
12/03/08
Pernicious malaria /cerebral malaria Blackwater fever Anaemia Hypoglycaemia Complications in pregnancy
Dr Ekta, Microbiology, GMCA
Malaria caused by P.vivax, P.ovale & P.malariae
Rarely life threatening Relapses/ recurrences are a feature
Recurrences in Malaria
12/03/08
May result from – reinfection or - due to persistence of infection - Occurs due to a special form of parasites called hypnozoites.
Dr Ekta, Microbiology, GMCA
Laboratory diagnosis of Malaria Specimen: peripheral blood smears thick and thin blood smears Leishman or Giemsa stain Trophozoites (ring forms) or gametocytes are seen within RBCs Quantitative Buffy Coat (QBC) examination P falciparum antigen detection (ELISA)
12/03/08
Dr Ekta, Microbiology, GMCA
Making of Thin & Thick films
12/03/08
Dr Ekta, Microbiology, GMCA
12/03/08
Dr Ekta, Microbiology, GMCA
Rapid Diagnostic tests HRP2 tests
detection of P.falciparum Two types of test – ParaSight F - ICT Malaria Pf
pLDH test e.g. OptiMAL test
12/03/08
Detection of P.falciparum & P.vivax Produced by all human malarial parasites Differentiation of species is based on antigenic differences between pLDH isoforms.
Dr Ekta, Microbiology, GMCA
ParaSightF test
Optimal test
ICT Malaria Pf / Pv
12/03/08
Dr Ekta, Microbiology, GMCA
12/03/08
Dr Ekta, Microbiology, GMCA
12/03/08
Dr Ekta, Microbiology, GMCA
Toxoplasma gondii Definitive host
Cat
Intermediate host
Man, sheep, birds
Infective form
Matured oocyst / tissue cyst / tachyzoites
Mode of transmission
Ingestion / intrauterine / blood transfusion/ improper handling of cat litter
Site of localization
Any organs (RE system) Eye & Brain
12/03/08
Dr Ekta, Microbiology, GMCA
12/03/08
Dr Ekta, Microbiology, GMCA
Laboratory Diagnosis –T gondii Specimens
Impression smear of LN, bone marrow, spleen, brain biopsy, blood, sputum, CSF
Microscopy
Giemsa stain, tachyzoites or cysts
Serology
ELISA / IFA – 16 fold rise in Ab titre: Acute infection Sabin-Feldman dye test: inhibition by antibody of the staining of tachyzoites by alkaline methylene blue
Prenatal diagnosis Fetal blood for IgM Ab / PCR
12/03/08
Dr Ekta, Microbiology, GMCA
Haemoflagellates
12/03/08
Dr Ekta, Microbiology, GMCA
Trypanosomes T. brucei subspecies gambiense (Chronic)
West African sleeping sickness
T. brucei subspecies rhodesiense (Acute)
East African sleeping sickness
T. cruzi (acute and chronic)
American trypanosomiasis Chagas disease
12/03/08
Dr Ekta, Microbiology, GMCA
T. brucei Definitive host
Man
Intermediate host
Tsetse fly
Infective form
Metacyclic Trypomastigote
Mode of transmission
Bite of infected tsetse fly
Site of localization
CNS
12/03/08
Dr Ekta, Microbiology, GMCA
Clinical features Sleeping Sickness Trypanosoma chancre at the site of bite Winter bottom’s sign: prominent cervical lymphadenopathy
Meningoencephalitis - Apathetic, confused, comatose
12/03/08
Dr Ekta, Microbiology, GMCA
T. cruzi Definitive host
Man
Intermediate host
Reduviid bug
Infective form
Metacyclic Trypomastigote
Mode of transmission
Feces of bug rubbed into site of bite / contamination of conjunctiva or other exposed mucous membranes with fingers
Site of localization
Autonomous nervous system of heart / GIT
Infect cardiac, smooth and skeletal muscle, reticuloendothelial cells and neuroglial cells 12/03/08
Dr Ekta, Microbiology, GMCA
Clinical features Chagoma chancre at the site of bite Acute Chagas disease
Romana’s sign: unilateral edema of face with conjunctivitis and swelling of upper & lower eyelids Fever, splenomegaly, anasarca, meningoencephalitis
Chronic Chagas disease
12/03/08
Cardiomyopathy, AV block, CCF Megaesophagus / Megacolon
Dr Ekta, Microbiology, GMCA
Laboratory Diagnosis – Trypanosomiasis
12/03/08
Specimens
Blood, CSF, Aspirates (LN)
Microscopy
Trypomastigotes in blood
Dr Ekta, Microbiology, GMCA
Leishmania Definitive host
Man
Intermediate host
Sand fly (Phlebotomus)
Infective form
Promastigote
Mode of transmission
Bite of infected sand fly
Site of localization
Reticuloendothelial system
12/03/08
Dr Ekta, Microbiology, GMCA
Leishmania Three major species:
L. donovani – kala azar/ visceral leishmaniasis
L. major – cutaneous leishmaniasis
L. braziliensis – mucocutaneous leishmaniasis
12/03/08
Dr Ekta, Microbiology, GMCA
Clinical Types Visceral leishmaniasis - fever, malaise, weight loss, anaemia and swelling of the spleen, liver, and lymph nodes Cutaneous leishmaniasis - causes 1-200 simple skin lesions which self-heal within a few months but which leave unsightly scars Mucocutaneous leishmaniasis - infection begins with skin ulcers which spread, causing dreadful and massive tissue destruction, especially of the nose and mouth
12/03/08
Dr Ekta, Microbiology, GMCA
12/03/08
Dr Ekta, Microbiology, GMCA
Laboratory Diagnosis Demonstration of parasite in clinical specimen:
Microscopy Culture Animal inoculation
Demonstartion of antibodies using
Specific leishmanial Ag – ELISA / IFA / Agglutination Non-specific Ag – CFT
Non-specific serum tests
Aldehyde test (Napier’s) Chopra’s Antimony test
Absence of hypersensitivity to leishmanial Ag Contributory lab findings – anemia, leucopenia, neutropenia 12/03/08
Dr Ekta, Microbiology, GMCA
1. Demonstration of parasite in Clinical Specimen Clinical specimens:
Peripheral blood Bone marrow aspirate Spleen aspirate Lymph node aspirate
Microscopy: Leishman, Giemsa or Wright’s stain - Amastigotes within macropahges Culture: NNN (Novy, MacNeal, Nicolle) medium for 7 days – promastigote form Animal inoculation: Hamster - Animal kept at 23-26°C 12/03/08
Dr Ekta, Microbiology, GMCA
2. Absence of Hypersensitivity to Leishmanial Ag Montenigro test – 0.1 ml of killed promastigote Ag I.D. Result read after 72 hrs.
12/03/08
Positive in Dermal leishmaniasis & recovered cases of Kala Azar. Negative in active cases of Kala Azar.
Dr Ekta, Microbiology, GMCA
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