view after download for better understanding, or else it appears quite compact and jumbled.. TORCH complex, forms important set of PERINATAL (Vertically transmitted infections)

1. Dr. Md. Ashraf Ali S. N
Post graduate
Dept. of Microbiology
KIMS Hubli

2. The acronym for a set of vertically transmitted
infections
 To-
TOXOPLASMOSIS
R
– RUBELLA
C
- CYTOMEGALOVIRUS
H
- HERPES SIMPLEX VIRUS - 2

4. 
Mode of transmission in the child
• Placental (chorionic villi)
• Hematogenous (gestation or time of delivery)

ToRCH infections can lead to
• Fetal anomalies
• Fetal loss

5.  Toxoplasmosis
 Rubella
 Cytomegalovirus
 Herpes
 Torch
simplex 2
test

6. 1.
Causative organism
2.
Life cycle
3.
Mode of infection
1. In mother
2. child
4.
Clinical course- both in the mother & child
5.
Sequel
6.
Lab diagnosis
1. Indirect evidences (imp in pregnant)
2. Direct evidences

7. 
An obligate intracellular parasite.

Human infection is dead end.
Phylum
Apicomlexa
Class
Sporozoa
Sub-class
Eucoccidia
Order
Coccidia
Sub-order
Eimerina
Genus
Toxoplasma

8. Consists of three forms
 Tachyzoites
 rapidly multiplying forms,

Bradyzoites
invade & multiply within cells
 Silver stains used to detect
Bradyzoites
 Sporozoites

 slowly multiplying forms
 Present inside tissue cysts
inside oocysts, shed in feces
 PAS positive
remian in the environment

Sporozoites
of cat and

9. LIFE CYCLE
Definitive host : Felis catus (Domestic cat)
Enteric cycle
 Gametogony and schizogony occurs in the epithelial cells of SI.
• Gametogony
Membrane--Thin
extremely resistant
Oocyst
Zygote
Cat’s feces
•Unsporulated
•Non infectious

12. MODE OF INFECTION:

Infective form : Oocyst containing sporozoites
• Ingestion
Ingestion of oocysts ( raw meat, garden products)
Contact with oocysts in cats’ feces/contaminated soil
• Contact

The incidence during pregnancy ranges from 0.3-1 %
• Of these 1 in 10 will deliver a baby with congenital Toxoplasmosis

14. 
Sporozoites penetrate epithelium of ileum
Sporozoites
Ileum
Mesenteric
LN
Lodgement is in two forms
1. Pseudocyst/intracellular form
2. Tissue cyst/ extracellularform
(proliferative stage).
•Brain, Skeletal muscle
•Cells of R.E. System, Placenta.
•Has cystozoite
•Has a crescent shaped endozoite of
6µ by 2µ
Blood & Lymph
stream
Distant
organs

18. Clinical course cont……….

50% of fetuses escape

30-35% develop
sub clinical infection.

Only 10% develop
severe infection.
(following clinical symptoms)

19. Clinical manifestation in fetus
manifest with classical triad of

Hydrocephalus
20%

Chorioretinitis
86%

Intracranial calcification
37%

20. SEQUELAE
If fetus develops sub clinical infection 
Asymptomatic at birth .

Later on develops
• Mental retardation AND Learning difficulties
• Cerebral calcifications
• Chorioretinitis blindness
• Hydrocephalus
• Epilepsy.

21. 
Indirect evidences
• Antibody demonstration

Direct evidences
• Microscopy
• Staining

Prenatal diagnosis
• done when IgM & IgG positive with low avidity

22. Antibody demonstration (Screening tests)
 ELISA
METHOD.
 Indirect
IF test (sabin-feldmen dye test).
 Goldman’s
 Fulton’s
test ( fluorescent tagged Ab.)
agglutination Test.
 Complement
Fixation Test.

23. ELISA- Routinely used
 For
IgM and IgG antibodies
 IgM IgG
Detection of specific IgM antibodies
Assays include
1. Detection of specific IgG antibodies
2. IgG Avidity testing

24. Sample
Serum.
 -20°C (if delay anticipated)

Antigen preparation

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 .

25. IgM ELISA
Aim- detect Anti-Toxoplasma IgM antibodies
Procedure

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.

26. Avidity ELISA
•
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.

27. 
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
nm.

Avidity index (AI; %) AI =

29. IgG
Negative
Negative
Positive
Positive
IgM
Negative
Positive/e •
•
quivocal
INTERPRETATION
FOLLOW UP TESTING
No serologic evidence of T.
gondii infection
acute T. gondii infection
False +ve IgM reaction
Negative
Infected with T. gondii for more
than 6 months.
Positive
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
…..

30. 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

31. 
Highest dilution for which <50% of free toxoplasma
have stained cytoplasm is taken as titre.

Titres of 1:128- diagnosis of acute inf.
….

32. DIRECT EVIDENCES - CONFIRMATORY TESTS
Presence of tachyzoites in clusters : Acute infection
Microscopy :

Tachyzoites in smears of lymph
nodes, brain, bone marrow .
Stain : PAS- Bradyzoites
Wright’s
Giemsa- Tachyzoites

33. …………….

34. Toxoplasma specific IgG
IgG POSITIVE
IgG NEGATIVE
NONIMMUNE
susceptible
Test for Toxo. SPECIFIC IgM ANTIBODY
IgG +ve, IgM -ve
IMMUNE INFECTED FOR > 1 year
IgG & IgM POSITIVE
IgG AVIDITY
Low IgG Avidity
RECENT INFECTION < 16WKS
Repeat test after 2 wks to confirm before
intervention
HIgh IgG Avidity
OLD INFECTION
16wks – 1 yr

35.  Amniocentesis
–
amniotic fluid PCR for
parasite particles.
 Placental
tissue / Blood –
inoculation into mice &
isolation of parasites
 USG

36. Indication
only IgG positive
Treatment
No treatment
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.
……….

37. 
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.

38. Properties
 Family-
Togaviridae
 Genus-
Rubivirus
 Pleomorphic,
 50-70nm
 ssRNA
roughly spherical.
in diameter
genome
 Enveloped
with haemagglutinin polymers.

39. 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
days.

Infection can be communicated 7days before and 4 days after
appearance of rash

40. C.A.

41. 
Cataract- if infection betn 3rd and 8th week of gestation.

Deafness betn 3rd and 18th week.

Heart abnormalities betn 3rd and 10th week.
VSD, PDA, PS, and coarctation of aorta

42. Table 2. Congenital defects & late manifestations of rubella infection
PRESENT AT BIRTH
1. Audiologic anomalies (60–75%)
•
Sensorineural deafness
2. Cardiac defects (10–20%)
•
Pulmonary stenosis
•
Patent ductus arteriosus
•
Ventricular septal defect
3. Ophthalmic defects (10–25%)
• Retinopathy
• Cataracts
• Microphthalmia
• Pigmentary and congenital
glaucoma

43. 4. Central nervous system (10–25%)
•
Mental retardation
•
Microcephaly
•
Meningoencephalitis
5. Characteristic purpura
Blueberry muffin appearance
LATE MANIFESTATIONS
• Diabetes mellitus
• Thyroiditis
• Growth hormone deficit

44.  Serologic studies –
best performed within 7 to10 days after the onset of the rash and
should be repeated two to three weeks later.
 IgM Assays
 IgG Assays
 Viral isolation

45. IgM assays
1.
IgM capture
• 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

46. Capture ELISA schematic

47. 2. Indirect EIA for virus-specific IgM

Pre-treatment step:
rheumatoid factor absorbent is used for the complexing of IgG antibodies from
test sera.

First step
absorption of virus antigen onto the solid phase

Second step
• The patient's serum is then added
• virus-specific Ab. (IgM & IgG) binds to the Ag.
•
Third step
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

48. ……………..

49. 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.

50. 2. IgG IgG avidity testing
avidity assays
1. differentiates between primary & secondary rubella inf.
AVIDITY: Antibody avidity is the strength of interaction of an
2.
excludes possibility of residual IgM which r present months or years
antibody with a multivalent antigen.
after primary infection.
Presence of
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.

51. CULTURE

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
• Immunofluorescent
• immunocolorimetric assay

52. 
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)

54. Samples taken
1.
CVS (10-12wks of gest)
2.
Amniotic fluid (14-16 weeks of gest)
3.
Fetal blood (18-20 wks of gest)
PCR
Rubella-specific PCR
Advantage
Allows early detection
 CVS as early as 10-12 weeks of gestation

55. 1. Universal infant immunization.
strain- RA 27/3, live attenuated
s/c injection
2. Screening for immunity and vaccination before conception.
3. Screening of all pregnant women to determine susceptibility.

56. 1. Rubella vaccine should not be administered
during pregnancy.
2. Pregnancy should be avoided for 3 months
following rubella vaccination.

57.  Parasitology, 13/e K. D. Chatterjee.
 Ananthanarayana & Paniker’s Textbook of
Microbiology 19th edition.