Mode of infection
1. In mother
Clinical course- both in the mother & child
1. Indirect evidences (imp in pregnant)
2. Direct evidences
An obligate intracellular parasite.
Human infection is dead end.
Consists of three forms
rapidly multiplying forms,
invade & multiply within cells
Silver stains used to detect
slowly multiplying forms
Present inside tissue cysts
inside oocysts, shed in feces
remian in the environment
of cat and
Definitive host : Felis catus (Domestic cat)
Gametogony and schizogony occurs in the epithelial cells of SI.
MODE OF INFECTION:
Infective form : Oocyst containing sporozoites
Ingestion of oocysts ( raw meat, garden products)
Contact with oocysts in cats’ feces/contaminated soil
The incidence during pregnancy ranges from 0.3-1 %
• Of these 1 in 10 will deliver a baby with congenital Toxoplasmosis
Sporozoites penetrate epithelium of ileum
Lodgement is in two forms
1. Pseudocyst/intracellular form
2. Tissue cyst/ extracellularform
•Brain, Skeletal muscle
•Cells of R.E. System, Placenta.
•Has a crescent shaped endozoite of
6µ by 2µ
Blood & Lymph
Clinical course cont……….
50% of fetuses escape
sub clinical infection.
Only 10% develop
(following clinical symptoms)
Clinical manifestation in fetus
manifest with classical triad of
If fetus develops sub clinical infection
Asymptomatic at birth .
Later on develops
• Mental retardation AND Learning difficulties
• Cerebral calcifications
• Chorioretinitis blindness
• Antibody demonstration
• done when IgM & IgG positive with low avidity
Antibody demonstration (Screening tests)
IF test (sabin-feldmen dye test).
test ( fluorescent tagged Ab.)
ELISA- Routinely used
IgM and IgG antibodies
Detection of specific IgM antibodies
1. Detection of specific IgG antibodies
2. IgG Avidity testing
-20°C (if delay anticipated)
Tachyzoites of T. gondii (RH strain)
Grown in peritoneal cavity of mice 3 days.
Tachyzoites - washed, centrifuged at 12,000g -1 hr
supernatant has soluble antigen.
Coated on microtiter plates= 4°C overnight later -20°C .
Aim- detect Anti-Toxoplasma IgM antibodies
Sera is diluted serially
add to T. gondii antigen-coated microtiter plate
Add anti-human IgG conjugated with horseradish peroxidase.
Chr. substrate -- o-phenylenediamine (OPD)
Read by means an automated ELISA-reader.
Microtitre plates pre-coated with Toxoplasma antigens
Serum diluted to 1/200
100 µl/well on 2 rows(row A and row B),
incubation for 45 min at 37°C and wash
row A – wash 3 times with modified PBST buffer
containing 6 M urea, fourth time with PBST
row B - wash 3 times with PBST.
The anti-human IgG conjugated with HRP added
Chromogenic substrate, o-phenylenediamine (OPD)
Sulfuric acid 20%.
The absorbance (Absb) read by an automated ELISA reader at 492
Avidity index (AI; %) AI =
FOLLOW UP TESTING
No serologic evidence of T.
acute T. gondii infection
False +ve IgM reaction
Infected with T. gondii for more
than 6 months.
T. gondii inf. within past 1 yr or
false +ve IgM reaction.
second specimen aft 2-3 weeks for IgG
and IgM testing; if same results, then its
false +ve IgM reaction
All Indeterminate results
further testing in a reference laboratory
for the diagnosis of toxoplasmosis.
Obtain a new specimen for IgG and IgM testing or
retest this specimen using a different assay
Live Tachyzoites + Accessory factor + Test serum (Serial dilutions)
Incubation at 37 C for 1 hour
Alc. soln of Meth. Blue (pH-11)
Examine under 40x
Highest dilution for which <50% of free toxoplasma
have stained cytoplasm is taken as titre.
Titres of 1:128- diagnosis of acute inf.
DIRECT EVIDENCES - CONFIRMATORY TESTS
Presence of tachyzoites in clusters : Acute infection
Tachyzoites in smears of lymph
nodes, brain, bone marrow .
Stain : PAS- Bradyzoites
Toxoplasma specific IgG
Test for Toxo. SPECIFIC IgM ANTIBODY
IgG +ve, IgM -ve
IMMUNE INFECTED FOR > 1 year
IgG & IgM POSITIVE
Low IgG Avidity
RECENT INFECTION < 16WKS
Repeat test after 2 wks to confirm before
HIgh IgG Avidity
16wks – 1 yr
amniotic fluid PCR for
tissue / Blood –
inoculation into mice &
isolation of parasites
only IgG positive
Suspected acute infection after pregnancy
Spiramycin till confirmation
Evidence of fetal infection
1. Pyrimethamine + sulphadiazine X 3 wks.
1. Amniotic fluid PCR positive
2. Ultrasound signs
Alternating with Spiramycin
2. consider MTP before 20 weeks of gestation
THERE IS NO VACCINE FOR TOX PREVENTION.
Rubella, caused by the rubella virus.
Minor infection in absence of pregnancy.
But during early pregnancy it is directly responsible for
abortion and severe congenital malformations to fetus.
Acquired immunity is life long.
It is Vaccine-preventable disease.
MODE OF TRANSMISSION
• person to person contact
• Droplet secretions of the infected.
Transmission to fetus- transplacental
Rash appears 2-3 weeks following exposure & persist for three
Infection can be communicated 7days before and 4 days after
appearance of rash
4. Central nervous system (10–25%)
5. Characteristic purpura
Blueberry muffin appearance
• Diabetes mellitus
• Growth hormone deficit
Serologic studies –
best performed within 7 to10 days after the onset of the rash and
should be repeated two to three weeks later.
• IgM antibody in serum is bound to anti-human IgM antibody adsorbed onto a
solid phase. This step is non virus specific.
• Removal of other Igs & serum proteins.
• Viral antigen, added to detect virus-specific IgM present.
• Wash and add anti-virus monoclonal antibody conjugated with an
enzyme., to detect the bound Ag..
• A chromogen substrate is added
2. Indirect EIA for virus-specific IgM
rheumatoid factor absorbent is used for the complexing of IgG antibodies from
absorption of virus antigen onto the solid phase
• The patient's serum is then added
• virus-specific Ab. (IgM & IgG) binds to the Ag.
Add enzyme-labeled anti-human IgM monoclonal antibody
A chromogen substrate is added to reveal the presence of virus-specific IgM in
the test sample
1. Indirect EIA
The most widely used are indirect EIAs.
Purified virus antigen is adsorbed onto a solid phase
patient's serum added.
Virus-specific Ab. in serum binds to Ag, and this virus-specific IgG
detected using enzyme conjugated anti-human IgG.
The binding of virus-specific IgG is measured by a detector system
using a chromogen substrate.
2. IgG IgG avidity testing
1. differentiates between primary & secondary rubella inf.
AVIDITY: Antibody avidity is the strength of interaction of an
excludes possibility of residual IgM which r present months or years
antibody with a multivalent antigen.
after primary infection.
low-affinity antibodies ---------early stage of infection
high-affinity antibodies -------reflects past immunity.
IgG avidity assays are difficult to establish, standardize, quality control
and interpret, hence recommended only for experienced laboratories.
Specimens-nasopharyngeal, blood, throatswab , urine, and
cerebrospinal fluid from pregnant women.
Cell culture lines:
• Vero cells- currently recommended
• incubated at 35°C for 3 or 5 days.
• RK -13
Detection of rubella E1 glycoprotein in infected Vero cells using
monoclonal antibodies by
• immunocolorimetric assay
A fourfold rise in rubella IgG antibody titer between acute
and convalescent serum samples.
A positive rubella-specific IgM antibody.
A positive rubella culture (isolation of rubella virus in a
clinical specimen from the patient)
CVS (10-12wks of gest)
Amniotic fluid (14-16 weeks of gest)
Fetal blood (18-20 wks of gest)
Allows early detection
CVS as early as 10-12 weeks of gestation
1. Universal infant immunization.
strain- RA 27/3, live attenuated
2. Screening for immunity and vaccination before conception.
3. Screening of all pregnant women to determine susceptibility.
1. Rubella vaccine should not be administered
2. Pregnancy should be avoided for 3 months
following rubella vaccination.
Parasitology, 13/e K. D. Chatterjee.
Ananthanarayana & Paniker’s Textbook of
Microbiology 19th edition.