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Introduction
 These protozoa are grouped under the phylum
Apicomplexa.
 They live intra-cellularly, atleast during part of their
life-cycle.
 They do not possess any special organ of locomotion.
 They reproduce asexually by schizogony followed by
sexual union or syngamy; known as alternation of
generation.
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Classification
 Phylum Apicomplexa
 Class Sporozoa
 Order Haemosporida
 Genus Plasmodium

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 Species infecting men:
 Plasmodium malariae; Quartan malaria
 Plasmodium vivax; Benign tertian malaria
 Plasmodium falcifarum; Malignant tertian malaria
 Plasmodium ovale; Ovale tertian malaria
 Plasmodium knowlesi; Quotidian type

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 Species capable of infecting men:
 Plasmodium cynomolgi; Benign tertian
 Plasmodium cynomolgi bostianelli; Benign tertian
 Plasmodium simium; Ovale tertian
 Plasmodium inui; Quartan type
 Plasmodium brasilianum; Quartan type
 Plasmodium shortii; Quartan type
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History
 1753- Name malaria was given.
 1847- Meckel observed presence of pigment in
organs.
 1849- Virchow observed pigment presence.
 1880- Laveron discovered malaria parasite in fresh
blood unstained preparation. Given Nobel prize in
1907.
 1883- Marchiafava used methylene blue for staining
malaria parasites. 6
 1885- Golgi discovered erythrocytic schizogony.
 1891- Romanowsky give staining methods.
 1898- Ronald Ross discovered life cycle of malaria
parasite. Wins Nobel prize in 1902.
 1948- Shortt and others demonstrated pre-erythrocytic
schizogony in liver cells.
 1955- WHO launched worldwide campaign against
malaria.
 1976- Trager and Jensen; in-vitro cultivation of
parasites.
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Geographical distribution
 Malaria parasite are found in all countries.
 The tropical zone is the endemic home of all parasites.
 P. vivax is most widely distributed, being more
common in Asia, North Africa, and Central and South
America.
 P. falcifarum predominant in Africa but is rapidly
spreading to South East Asia and India.
 P. malariae is rare.
 P. ovale is confined to West Africa.
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Life cycle
 Carried out in two different hosts –
 In Man:
 Asexual stage (schizogony) inside the liver cell
and RBCs. Man is the intermediate host.
 In Female Anopheles mosquito:
 Sexual stage or sporogony. Definitive host for
malarial parasite.
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Female Anopheles mosquito

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In Humans

1. Pre-erythrocytic schizogony – inside liver.


2. Erythrocytic schizogony – inside RBCs.
3. Gametogony – inside RBCs.
4. Latent or Hepatic Stage – inside liver.

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Pre or Exo-erythrocytic Schizogony
• Sporozoites invade hepatocytes.
• Asexual replication with
generation of only one pre-
erythrocytic schizont.
• Takes 6-15 days.
• 1000-10,000 merozoites liberate
known as cryptozoites.
• No overt pathology

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Erythrocytic Stage

 Occurs inside red blood cells.


 Parasite passes through the stage of trophozoite,
schizont and merozoite.
 Asexual development.
 Responsible for clinical attack of malaria.

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Gametogony
 Some of the merozoites instead of developing into
trophozoites and schizonts, gives rise to forms which
are capable of sexual division.
 These are called gametocytes and develop in the red
blood cells of capillaries of internal organs.
 Do not cause any febrile reaction.
 Dimorphic –
 Microgametocyte
 Macrogametocyte 15
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Latent or Hepatic Stage
 Seen only in P. vivax and P. ovale.
 Parasite persist in liver cells as dormant form, with
resting stage known as Hypnozoite.
 Responsible for relapse of vivax and ovale malaria.

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In Mosquitoes
• Occurs in mosquito (9-21 d)
• Fusion of micro- and macrogametes
• Zygote  ookinete (~24 hr)

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• Ookinete transverses gut epithelium ('trans-invasion')
• Ookinete  oocyst
• Between epithelium and basal lamina
• Asexual replication  sporozoites
• Sporozoites released
• Sporozoites migrate through hemocoel
• Sporozoites 'invade' salivary glands
• Transmitted infection to man during blood sucking.

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Characters P. vivax P. falcifarum P. malariae P. ovale
Schizogony 48 hours 48 hours or under 72 hours 48 hours
Forms in Trophozoites, Rings and Trophozoites, Trophozoites,
peripheral blood schizonts and crescents only. schizonts and schizonts and
gametocytes gametocytes gametocytes
Trophozoites Size 2.5 µm Size 1.25-1.5 µm Size 2.5 µm Size 2.5 µm
Ring form Cytoplasm Cytoplasm fine Same as P. vivax Same as P. vivax
opposite the and regular in
nucleus is thicker outline. Often
with 2 nuclei.
Form accole.
Multiple
infection
Growing form Irregular with a Assumes a Band like. No ribbon shape.
vacuole. Actively copmact form. Slightly Slightly
amoeboid. Pigments collect amoeboid. amoeboid.
into single mass Vacuole
early. disappears early. 26
Characters P. vivax P. falcifarum P. malariae P. ovale
Schizont Size 9-10 µm. Size 4.5-5 µm. Fills Size 6.5-7 µm. Size 6.2 µm. Fills
Regular, almost two-third of RBC Regular, almost about three
completely fills fills normal RBC. quarters RBC
an enlarged RBC.
Merozoites 12 to 24. 18 to 24 or more. 6 to 12. arranfed 6 to 12.
Arranged in Arranged in grape around a central irregularly
grape like cluster like cluster mass of pigment enlarged.
like daisy
Malarial Yellowish brown, Dark brown or Dark brown Dark, yellowish
pigments fine granules. blackish; one or two coarse granules. brown; coarser
solid blocks. than P. vivax
Infected Enlarged pale. Usually unaltered. Not enlarged, not Slightly enlarged,
RBCs Schuffner’s dot Crenation, reddish pale and no oval shape,
present. violet colour and granules. fimbriated.
Maurer’s dot Ziemann’s dot. Jame’s dot.
Gametocyte Spherical or Crescentic. Larger Round or oval. Oval. Size of
globular. Larger than RBC Size of RBC RBC
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than RBC.
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Tissue phases of malaria parasite
Disease Species of Length of pre- Size of mature Number of
parasites erythrocytic schizont merozoites
schizogony
Benign tertian P. vivax 8 days 42 µm 12,000

Malignant P. falcifarum 6 days 60 x 30 µm 40,000


tertian
Ovale tertian P. ovale 9 days 80 x 50 µm 15,000

Quartan P. malariae 7 to 12 days 22 µm 2.000

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Reservoir of infection -
 Man is the only reservoir of infection.
 In some parts of Africa, chimpanzees may act as
reservoir for P. malariae.
Method of transmission –
 By the bite of female Anopheles mosquito.
 Infection is transmitted by inoculative method.

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 Transmitting agent – female Anopheles
 Some important species – Anopheles culcifacies, A.
stephensi, A. philippinensis, A. fluviatilis, etc.
 Infective forms – sporozoites
 Portal of entry – skin
 Site of localisation – first liver cells, then in
erythrocytes.
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 Other methods of transmission –
 Injection of an emulsion of salivary glands containing
sporozoites, known as sporozoite induced malaria.
 Injection of blood from malarial patient containing
asexual forms, known as trophozoite induced malaria.
 Transfusion malaria

 Congenital malaria

 Malaria in drug addicts

 Therapeutic malaria – artificially induced for treatment


of neuro-syphilis.
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Pathogenecity
 Each of four species causes a characteristic fever and
diseases are designated as follows –
 Plasmodium falciparum: malignant tertian malaria
 Plasmodium vivax: benign tertian malaria
 Plasmodium ovale : benign tertian malaria
 Plasmodium malariae: quartan malaria
 Incubation period –
 10 – 14 days: in P. vivax, P. ovale and P. falcifarum.
 18 days – 16 weeks: in P. malariae.
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Pathology of malaria -

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Plasmodium
falciparum:

Plasmodium
vivax,
Plasmodium
ovale

Plasmodium
malariae:
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Pathological changes in various organs-
 Spleen –
 Macroscopic changes –
 Organ is enlarged.
 Slate-gray or black in colour.
 Capsule is thin and stretched.
 Consistency of organ is soft.
 Cut surface appears as homogenous black area with
scattered white fibrous bands.
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 Histopathology –
 Congestion of splenic sinusoids.
 Enormous amount of pigment both haematin and
haemosiderin.
 Number of macrophages increased.
 Parasites differentiated as black dots in RBCs when
sections are stained with Hematoxylin and eosin.
 Reticulin fibres increased.

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 Liver –
 Macroscopic changes –
 Organ is enlarged.
 Slate-gray or black in colour.
 Cut surface shows dilated lobular veins.
 Histopathology –
 Central veins of lobules and sinusoidal capillaries are
dilated and often filled with parasitised RBCs.
 Kupffer cells increased.
 Parenchyma shows fatty degeneration, atrophy and
necrosis.
 Parasites are found in various stages of development. 38
Clinical features -
1. Febrile paroxysm –  Throbbing headache
1. Cold stage  Lasts 2-6 hours
 Feeling of intense 3. Sweating stage
cold  Profuse sweating
 Vigorous shivering  Declining
 Lasts 15-60 minutes temperature
2. Hot stage  Exhausted and weak
 Intense heat → sleep
 Dry burning skin  Lasts 2-4 hours

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2. Anaemia – Anaemia of microcytic or normocytic
hypochromic type develops as a result of breaking
down of red blood cells during segmentation of
parasites.
3. Spleenomegaly – Enlargement of spleen is one of the
most important physical sign of malaria.

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Relapses in malaria -
 Results from –
 Persistence of blood infection as erythrocytic form.
Known as Recrudescence and feature of P.
falcifarum.
 Persistence of hypnozoites form in liver. Known as
Recurrence or true relapse. Seen in P. vivax and P.
ovale.

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Pernicious malaria -
 Refers to a series of phenomena occurring during the
course of infection of P. falcifarum which if not
effectively treated threatens the life of patients.
 Chiefly caused by capillary blockage results from
agglutination of parasitized RBCs within internal
organs, thus decreasing the effective circulating blood
volume.

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 Clinical types –
1. Cerebral malaria – hyperpyrexia, coma, paralysis.
2. Algid malaria – cold and clammy skin with
vascular collapse.
3. Septicemic malaria – continuous high temperature
with pneumonia and cardiac syncope.

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Black water fever -
 Manifestation of falcifarum malaria occurring in
previously infected patients, characterised by massive
intra vascular haemolysis followed by fever and
haemoglobinuria.
 Now rare due to newer drugs.
 It occurs in person who has taken inadequate doses of
quinine for suppressive prophylaxis and treatment.
 Patient with G-6-PD deficiency may develop this
condition after taking oxidant drugs.
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 Pathogeneis:
 It is attributed to intra-vascular hemolysis with
rapid destruction of red blood cell caused by auto-
antibodies leading to haemoglobinaemia and
haemoglobinuria.
 Following repeated attacks of falcifarum malaria, a
hypersenstivity state is produced, which when
stimulated by heavy falcifarum infection or
administration of quinine leads to production of
anti-erythrocyte antibodies.
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 Effects of intra-vascular hemolysis-
1. Methaemalbuminaemia –

Oxy-haemoglobin in blood
Broken into
globin + haematin (ferrous)
Oxidation
Serum albumin + ferric state
Methaemalbumin
Not excreted in urine. Retained in plasma resulting in
methaemalbuminaemia.
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2. Hyperbilirubinaemia –
The bilirubin formed by R.E. system is in excess of
what liver can excrete and hence retained in plasma.
3. Haemoglobinuria –
Excess of haemoglobin in circulation and when
haptoglobin is unable to bind the free Haemoglobin. It
is excreted through the kidneys.
Pigment in blood:
 Oxyhaemoglobin, methaemalbumin and bilirubin.
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Pigment in urine:
 Oxyhaemoglobin (gives red colour)
 Methaemoglobin (dark brown or black)
 Haematin
 Urobilin
Clinical features:
 Fever with rigor, aching pain, hemoglobinuria,
icterus, bilious vomiting, circulatory collapse, acute
renal failure.
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Immunity -
 Influenced by
 Genetics
 Age
 Health condition
 Pregnancy status
 Intensity of transmission in region
 Length of exposure
 Maintenance of exposure

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 Innate immunity –
 Red cell polymorphisms associated with some
protection –
 Hemoglobin S sickle cell trait or disease
 Hemoglobin C and hemoglobin E
 Thalessemia – α and β
 Glucose – 6 – phosphate dehydrogenase
deficiency (G6PD)
 Red cell membrane changes –
 Absence of certain Duffy coat antigens improves
resistance to P. vivax. 50
 Acquired immunity –
 Transferred from mother to child
 3-6 months protection
 Then children have increased susceptibility
 Increased susceptibility during early childhood
 No complete immunity
 Can be parasitemic without clinical disease
 Need long period of exposure for induction
 May need continued exposure for maintenance
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1. Microscopy -
 Blood smear examination
 Gold standard technique
 Thick and thin smears made
 Stains commonly used are:
 JSB stain
 Giemsa stain
 Leishman stain
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Morphology of malarial parasite on leishman stain

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2. Quantitated Buffy coat/ QBC II system –
 Centrifuge patient’s blood in capillary tubes blood
in capillary tubes pre-coated with Acridine Orange
(AO)
 Molded plastic float presses the parasitized RBCs.
 Viewed by strong ultraviolet light microscopy.

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3. Culture of malaria parasites –
 1912- Bass and Johns first attempted in-vitro culture
of malaria parasite.
 1976- Trager and Jensen discovered simple method
for continuous cuture of P. falcifarum.
 Original method- Petridish culture in candle jar with
3% Oxygen and 10% CO2 and relatively simple
culture medium or RPMI1640 supplemented with
human, rabbit or calf serum.
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 Fresh RBCs are added periodically for continuation of
growth and multiplication of Plasmodium.
 Computer- controlled culture system introduced.
 Several culture lines established using blood of
infected Aotus monkey or directly from human
patients.

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4. Serological test for malaria –
 Antigen detection
 Immuno-chromatograpic
 Antibody detection
 Indirect fluorescent antibody

 Enzyme immune assays

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5.Fluorescent microscopy –
 Modification of light
microscopy
 Fluorescent dyes detect
DNA and RNA present in
parasites
 Stain thin film with
acridine orange (AO)

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6. Rapid diagnostic tests (RDTs) –
 For malaria antigen detection
 Target antigens-
1. pLDH ( plasmodium lactate dehydrogenase)

2. HRP2 ( Histidine rich protein 2)

3. Aldolase

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Plasmodium Lactate Dehydrogenase Card Test

Pan LDH
Pf LDH

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7. Molecular diagnosis –
 DNA probe.
 Polymerase chain reaction

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8. Other tests –
 Measurement of hemoglobin and packed cell volume
(PCV)
 Total WBC and platelet count
 Blood glucose for hypoglycemia
 Coagulation tests like anti thrombin III level, plasma
fibrinogen, etc
 Free hemoglobin in urine- if Blackwater fever suspected
 Blood urea and serum creatinine- renal failure
 G6PD- before giving drugs like Primaquine
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Treatment -
 Anti-malarial drugs are used –
 Therapeutic – to eradicate erythrocytic cycle.
 Radical cure – to eradicate exo-erythrocytic cycle.
 Gametocidal – to destroy gametocytes.
 Chemoprophylaxis – to prevent infections in non-
immune person.

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 Therapeutic drugs –
 Chloroquine – 600 mg stat followed by 300 mg
after 8 hours and 300 mg for next two days.
 Pyrimethamine 25mg + sulfonamide 500mg – 3
tablet in single dose.
 Mefloquine – 15-25mg/kg
 Quinine – 600mg for 7 days along with
pyrimethamine+sulfonamide or doxycycline.

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 Artesunate (oral) – 100mg BD 1st day followed by
100mg OD for 5 days.
 Artesunate (i.v.) – 120mg on 1st day followed by 60
mg daily for 4 days.
 Artemether – 80 mg BD for 1st day then OD for 4
days.
 Arteether – 150 mg OD for 3 days.
 Radical cure –
 Primaquine – 15 mg daily for 2 weeks.
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 Gametocidal –
 Primaquine – 45mg single dose immediately after
clinical cure.
 Prophylaxis –
 Proguanil
 Primaquine
 Chloroquine
 Mefloquine
 Doxycycline
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Prophylaxis -
 Malaria vaccine – SPf66
 Vector control strategies –
 Residual spraying with DDT, malathion, etc.
 Space sprays application with Pyrethrum extracts.
 Individual protection by use of repellents, protective
clothing, bed nets, etc.
 Anti-larval measures –
 Dusting the water with paris green.
 Source reduction. 74
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History and Distribution
 Toxoplasma gondii is an obligate intra-cellular
coccidian parasite.
 First discovered by Nicolle and Manceaux in 1908 in
a small rodent of North America called gondii.
 It is widely distributed in man and animal all over the
world.

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Morphology
 3 forms –
1. Trophozoite
Asexual stage (schizogony)
2. Tissue cyst
3. Oocyst Sexual stage (gametogony or sporogony)

 All 3 forms occur in domestic cats and are infectious


to man.
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 Trophozoite –
 Crescent shaped.
 3-7µm in length with
ovoid nucleus.
 Stains well with giemsa
stain. Cytoplasm – blue
and nucleus – red.
 Seen intracellularly in
various tissues during
acute phase of
infection.
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 Tissue cyst –
 Resting form
 Found during chronic
stage of infection in
brain (most common
site).
 Round or oval, 10-20
µm in size and
contain numerous
bradyzoites.
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 Oocyst –
 Develops only in
definitive host.
 Oval in shape with 10-
12 µm in diameter.
 Formed by sexual
reproduction.
 Undergo sporulation in
soil with formation of 2
sporocysts and 4
sporozoites. 80
Life cycle -
 Definitive host – cats and other felines.
 Intermediate host – man and other mammals.

 2 types of life cycles –


 Enteric cycle – in definitive host
 Exoentric cycle – in intermediate host.

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Life cycle of Toxoplasma gondii
Pathogenecity -
 In majority of cases, infection may pass unnoticed.
 Via blood stream disseminate to various organs.
 Pathological lesion characterised by area of focal
necrosis, surrounded by inflammatory cells.
 A large number of parasites may be found inside the
cell which is enlarged, forming pseudocyst.

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Clinical features -
 Mainly two forms –
 Congenital
 Acquired
 Congenital toxoplasmosis –
 Results when T. gondii is transmitted trans-
placentally from mother to foetus.
 Generally fatal with widespread lesions.
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 Present as chorioretinitis, cerebral calcifications,
convulsions, deafness, blindness, mental retardation,
microcephaly and hydrocephalus.
 Acquired toxoplasmosis –
 Mostly asymptomatic.
 Most common manifestation is lymphadenopathy
(Cervical lymph nodes).
 Fever, headache, myalgia and spleenomegaly are
often present.
 Illness may resemble mild flu and is self limited.
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 Toxoplasmosis in immuno-compromised patients –
 It is most serious and often fatal in immuno-
compromised patients, particularly in AIDS.

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Laboratory diagnosis -
 Direct evidences –
1. Microscopic examination –
 Tachyzoites and tissue cysts detected in blood, bone
marrow aspirates, centrifuged deposits of CSF, and
smears made from biopsy or autopsy material.
 Stains used are – Giemsa, PAS, GMS

2. Animal inoculation –
 In laboratory animals likemice, guinea pig or
hamsters. 87
 Indirect evidences –
1. Serological tests –
a) Antibody detection –
i. For IgG antibody –
 ELISA
 Indirect fluorescent antibody test
 Latex agglutination test
 Sabin-Feldman dye test

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ii. For IgM antibody –
 Double sandwich IgM ELISA
 IgM immunosorbent assay
iii. For IgA antibody –

 Double sandwich IgA ELISA

b) Antigen detection –

 ELISA

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2. Molecular diagnosis –
 PCR
3. Imaging –
 MRI and CT scan of
central nervous system
 Ultra-sonography for
congenital toxoplasmosis
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Treatment and Prophylaxis -
 For congenital infection –
 Pyrimethamine and sulfadiazine.
 Systemic corticosteroid to reduce chorio-retinitis.
 Alternate drug – spiramycin.
 For primary prophylaxis –
 Trimethoprim – sulfamethoxazole is the drug of
choice.
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 For prevention –
 Individuals at risk especially pregnant women
avoid contact with cats and its faeces.
 Proper cooking of meal
 Proper washing of hands and of fruits and
vegetables before eating.
 Screening of blood and blood products.

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