1. Treasa James

2. Purpose To discuss about Toxoplasmosis’ Laboratory Diagnosis
Epidemiology
Treatment
Prevention

3. Laboratory Diagnosis May be made by the following: Microscopy
Isolation of parasite
Serology
Polymerase chain reaction
Serolgy is the most common method used.

4. specimens Blood Cerebrospinal fluid Amniotic fluid
Bone marrow aspirate
Cerebrospinal fluid
Amniotic fluid
Bronchoalveolar lavage( esp. in HIV and immunocompressed patients)
Biopsy material from lymph node , spleen and brain

5. Microscopy
Giemsa or PAS( periodic acid Schiff) used for staining of smears
Tachyzoites of T.gondii can be demonstrated in stained smear
Tissue cysts seen as spherical and cyst wall stains well with silver stain
Bradyzoites strongly PAS positive
Monoclonal antibody based fluorescent stains used to detect T.gondii in tissues

6. Isolation of the parasite
Animal inoculation :
Intraperitoneal inoculation of the body fluid or ground tissue in laboratory bred mice free from infection
Suspected material show trophozoite after 7 to 10 days
Confirmed by demonstration of tissue cyst in brain of inoculated animal
2. Tissue culture
tissue culture may be inoculated by specimen for isolation of parasite

7. Serology Most common method which includes Sabin –Feldman dye test
Other serological test
Indirect fluorescent antibody test ( IFA)
Enzyme linked immunosorbent assay (ELISA)
Indirect haemagglutination test
Complement fixation test (CFT)
Latex agglutination test

8. a) Sabin – Feldman dye test
The test is based on presence of certain antibodies that prevent methylene blue dye from enetering cytoplasm of toxoplasma organisms
Patient serum is treated with trophozoites and complements as activator then incubated
Then methylene blue is added
If antitoxin antibodies are present in serum because these antibodies are activated by complements and lyse parasite membrane
Toxoplasma trophozoites not stained ( positive result)
If there are no antibodies trophozoites are stained and appear blue(negative result)
It is complement mediated antigen antibody reaction
Becomes positive 2 weeks after infection
Not widely used nw

9. b) Other serological test
IFA ( Indirect fluorescent antibody) , ELISA ,
CFT (complement fixation test) , indirect haemagglutination and latex agglutination tests
used for antibody detection in serum
IFA , ELISA and latex agglutination-widely used for diagnosis
IgM ELISA is more sensitive and specific than IgM-IFA

10. TSP( T. gondii serological profile)
TSP is used in lab to diagnose if the infection is acquired recently or in the past. It includes
The Sabin – Feldman dye test to detect IgG antibodies
ELISAs for – IgM , IgA, IgE antibodies
Immunosorbent agglutination assay(ISAGA) measure level of IgE
Differential agglutination test to measure levels of IgG antibodies
Detection of antigen: ELISA detects Toxoplasma antigen in serum, aqueous fluid or amniotic fluid. Useful in detection of Toxoplasmosis in AIDS.
Detection of antigen in in amniotic fluid is useful for diagnosis of congenital toxoplasmosis

11. Interpretation of serological tests
In immunocompetent patients :
A rising antibody titre helps to diagnose
2 samples are collected at an interval of 2 to 4 weeks
A 16 fold rise in antibody titre indicates acute infection
Detection of IgM is indicative of recent infection
2)In pregnant women:
Positive antitoxoplasma IgM Titre- acute infection n consequent risk of foetus
Rising titre of IgG antibodies – recent infection and risk of foetus

12. 3) In congenital infection: positive antitoxoplasma IgM titre or rising titre of IgG antibodies in neonate determines congenital toxoplasmosis 4) In immunocompromised patients Diagnosis done when there is a rising titre, an IgM-IFA titre of 1:64 and IgM –ELISA titre of 1:256

13. Polymerase Chain Reaction
PCR of amniotic fluid to detect B1 gene of T.gondii – diagnosis of congenital toxoplasmosis
recommended test to establish intrauterine diagnosis of congenital toxoplasmosis

14. Radiological Methods
Ultrasonography is useful for prenatal diagnosis of congenital toxoplasmosis
toxoplasma is one of the component T of TORCH term in congenital infections
It can cause miscarriage, stillbirth or damage to baby’s brain esp eyes
Other components : 0 – others incl. syphyllis
R – rubella
C – cytomegalovirus
H- herpes

15. Epidemiology Infection is present worldwide wherever there are cats
Full natural cycle is maintained by mice and cats – mice eats material contaminated with oocyst shed by cat
Mice get infected and develop cyst in their tissues
When such mice eaten by cat they get infected
Infected cats shed oocyst in faeces
Human toxoplasmosis is zoonosis. Acquired through contaminated food or water or undercooked meat.
Flies and cockroaches may act as mechanical vectors
Rarely acquired through blood or leucocyte transfusion or organ transplantation.
Toxoplasmosis may be acquired by laborartory infection
Incubation period is 1 to 3 week

16. Outcome of infection depends on the immune status of infected person
Outcome of infection depends on the immune status of infected person. Active progression of infection in immunocompromised patients
Toxoplasmosis is one of major fatal complication in AIDS
Incidence of congenital toxoplasmosis is approx. 1:1000 live births.
Because of public health importance of congenital toxoplasmosis , serological surveys for toxoplasma antibodies conducted in many advanced countries in women of childbearing age , antenatal women and newborn.

18. Treatment
Pyrimethidine ( daraprim) combined with sulphadiazepine have been found to be effective remedy
a) Pyrimethidine 50mg orally 6-8hrs later 25 mg on first day followed by 25 mg daily for 2 weeks.
in severe case treatment prolonged to 6 months
Should be avoided in first trimester pregnant women
b) Sulphadiazepine 2g orally ,stat; followed by 1g 6 hourly for same period. Folic acid and vit B complex administered concurrently.
Prednisolone is given in ocular toxoplasmosis
Spiramycin ( 3/4gm/day for 3 to 4 weeks) either alone or in combination with other drugs.
Treatment is effective against trophozoites and not against cysts.

19. Prevention
Avoidance of human contact with cat’s faeces and uncooked meat.
Washing of fruits and vegetables before consumption
Proper cooking of meat
Wasing of hands before eating to avoid soil contamination of fingers
At present there is no effective vaccine for immunization against T.gondii

20. Bibliography Medical parasitology by C P Baveja
Parasitology by K Chatterjee
Medical parasitology by Panicker
Book of parasitology by Chakraborty
Wikipedia
Google Images