Lab diagnosis of ToRCH complex

A sincere effort

A compilation of all the diagnostic methods for diagnosis of ToRCH gp of infections in Pregnant women..

Presentation is done in two parts-
part-1 includes Toxoplasmosis and Rubella virus infection

Part-2- Cytomegalovirus, HSV-1, HSV-2 are covered

microbiological, lab diagnosis of Torch complex, Laboratory diagnosis of TORCH complex, Cytomegalovirus, Herpes simplex type1, HSV-1, Herpes simplex type2, HSV-2,
Shell vial technique,

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

2. C -CYTOMEGALOVIRUS
H -HERPES SIMPLEX VIRUS –1& 2
Covered in torch complex Part-II
To-TOXOPLASMOSIS
R –RUBELLA
Covered in torch complex Part-I

3. 3.CYTOMEGALOVIRUS
4.HERPES SIMPLEX VIRUS 1
5.HERPES SIMPLEX VIRUS 2
All three belong to family herpesviridae.

4. Common characteristics:
Enveloped linear dsDNAviruses.
Haveabilitytoremainlatentintissues,leadingto
periodicreactivation.
Similarstructure.

5. Species Sub
family
Latency
Name Commonname
Human herpes virus type 1
Herpes simplex
virus type 1
alpha
Neurons
trigeminal
ganglia
Human herpes virus type 2
Herpes simplex
virus type 2
alphaNeurons
Sacral ganglia
Human herpes virus type 3 Varicella-Zoster
alphaNeurons
Human herpes virus type 4 Epstein-Barr virusgamma
Lymphoid
tissues
Human herpes virus type 5 Cytomegalovirus beta
Secretory
glands, kidneys
Human herpes virus type 6
Human B cell
lymphotropicvirus
beta
Lymphoid
tisues
Human herpes virus type 7 R K virus beta
Lymphoid
tisues
Human herpes virus type 8 gamma
Classification of herpes viruses
Human herpes virus type 2

6. Similar for all members of herpesviridaefamily.
4 structural elements
1.Core
2.Protein capsid
3.Tegument
4.Outer envelope with spikes

8. 1. Core
-contains the linear dsDNA
-DNA stabilized by a series of protein fibrils
2. Capsid-encloses the core
-A closed shell (icosahedron)
-Has capsomeres in it
-162 capsomeres-150 hexamers and 12 pentamer

10. 3.Tegument
Between capsid and envelope an amorphous
tegument is present.
Role
Important in early stages of viral replication
following entry into the host.
Shuts down host metabolic activity rapidly.

11. 4.Envelope
Trilaminar
Derived from host cell
Envelope has glycoproteins-
gB, gC, gD, gE, gG, gH, gI, gJ, gK, gL, gM.
gB, gD, gH, gL-essential for virus infectivity
gC-mediates initial attachment of virus to host in HSV-1
gB-HSV-2
gD-bonding with co-receptor

12. Salivary gland virus.
It is a double stranded
DNA virus.
Largest in herpes virus
family (Size : 150-200nm),


13. •Congenitalinfectionoccursinapproximately2%
oflivebirths.
•Itisaleadingcauseofhearingloss,mental
retardationandcerebralpalsy.
•OpportunisticpathogeninImmunocompromised .
•Accountforapprox.10%deathinsymptomatic
newborns.

14. Humansareonlyhost.
Latentinhostcells
Latent CMV -
monocytes,lungs,spleen,kidneys,secretory
glandsandcervix.
Exhibitshostspecificity.

15. Scientific classification
Family: Herpesviridae
Subfamily: Beta
Genus: Cytomegalovirus
Species: 1.Human herpes virus 5(HHV-5) -
Human cytomegalovirus
2.CeHV-5= African green monkey
cytomegalovirus
3.CeHV-8 =Rhesus monkey
cytomegalovirus
4.Pongine herpes virus 4 (PoHV-4)

17. Vertical transmission
transplacentalinfection (prenatal)
genital-tract secretions during delivery.
breast-feeding (postnatal)
Horizontal transmission(blood
transfusion, organ transplant, salivary
secretions, sexual contact)
Incubation period 28-60 days, mean-40 days.

18. Mother Viraemia Placental Trophoblasts
Infection of fetal
endothelial cells
Fetal viraemia
Replication in
target organs (kidney)
Fetal viruria
Virus in
amniotic fluid
Infection of
the oropharynx

19. •CMV infection in pregnancy can be
Primary
Reactivation
Recurrent
Leading to congenital CMV infection.

20. •Primarymaternalinfectionleads
tofetalinfectionin30-50%of
cases.
•Recurrentmaternalinfection
lesslikelytoleadtofetal
infection(1-2%).

21. 95% clinically inapparent.
35% transmitted to fetus.
Fetal damage more likely in first 26 weeks (32%)
than later (15%).
……….

22. Primary CMV Recurrent
CMV
Period
1
st
trimester
2
nd
trimester
3
rd
trimester
Risk of
infection40% 45% 78% 0.5%
Fetal
damage 32% 16% 1% <1%

23. •Vertical transmission of CMV can occur at
any stage of pregnancy.
•Severe sequelae are more common with
infection in the 1
st
trimester, while the
overall risk of infection is greatest in the 3
rd
trimester.

24. Transplacentalinfection can result in
intrauterine growth restriction
intracranial calcifications
Microcephaly
hydrocephalus

25. CMV in Immunocompromised Patients
•Reactivation of the dormant virus.
•Primary CMV infection in immunocompromised
-scan cause serious disease.
Infection with CMV in immunocompromised patients,
Pneumonia
retinitis
gastrointestinal disease.

26. CMV Retinitis
small floaters
foggy or blurred vision
loss of central or peripheral vision
Complications
loss of vision
retinal detachment

27. Jaundice (67%)
Petechiae(76%)
Microcephaly(53%)
Hepatosplenomegaly(60%)
Chorioretinitis(20%)
Seizure (7%)
Fatal outcome (10%)

29. Seizures
Chorioretinitis
Periventricularcalcifications
Sensorineural hearing loss
Motor deficits

30. High risk of transmission
IgG POSITIVE
IgM NEGATIVE
IgG& IgMNEGATIVE IgG & IgM POSITIVE
IgGNEGATIVE
IgMPOSITIVE
REPEAT TEST
AFTER 3 WEEKS
IMMUNE
REASSURANCE
UNIMMUNE
Meticulous hand hygiene
< 20
WEEKS
FETAL WELL
BEING
USG & MRI
MATERNAL VIREMIA
BY URINARY & BLOOD
PCR
RETROSPECTIVELY
SCREEN 1
ST
TRIMESTER BLOOD
HIGH RISK OF
TRANSMISSION
INVASIVE
AMNI0TIC FLUID
PCR
HIGH CMV IgG
AVIDITY
NO FURTHER
TESTING NEEDED
> 20
WEEKS
HIGH CMV IgG
AVIDITY
LOW CMV IgG
AVIDITY
LOW CMV IgG
AVIDITY
MATERNAL SCREENING

31. A.In pregnant
B.In fetus (prenatal diagnosis) once
infection in mother proved

32. 1.Microscopy
2.Virus Isolation
3.Serology
4.Demonstration of viral particles

33. SPECIMENS :
From active lesions of cervix, vagina .
Blood for CMV antibody -paired serum samples
Storage temperature maintained-4 degree Celsius

34. 1.Microscopy
i.Direct observation---Electron microscopy
ii.Staining---
Haematoxylinand eosin (H &E stain)

35. Enveloped
Spherical
120 –200 nm in
diameter

36. ii.H & E stain from cervical secretions of CMV
infected individual.
Owl‟s eye
appearance

37. 2. Virus isolation
Cell culture line used
Human Fibroblast cell line cultures
The characteristic cytopathiceffect develops
within hours to weeks after inoculation
depending on the amount of CMV present in the
specimen.
Human fibroblasts are the only cells in which
CMV reliably grows in vitro.

38. 3.Serology
Antibody detection
i.Seroconversion
ii.IgMdetection-paired sera is taken
One blood sample should be taken on suspicion
of CMV, another within 2 weeks.
IgM-marker of active or recent CMV infection
Merits
Negative test will rule out infection
Demerits
Persistent IgM
False positive results

39. IgGassays
•CMV IgG antibody –sensitive and specific for past
infection.
•CMV IgM antibody –variable sensitivity and
specificity.
•Antibody avidity testing can increase accuracy of
detection of primary infection.

40. The anti-CMV IgG avidity test-currently the most reliable.
Specific------100%
Sensitive----60-94%
•Low avidity-recent/past infection
16
th
-18
th
wk of gestation, sensitivity 100%.
After 20 wks of gestation, sensitivity is reduced
(62.5%).
•A high avidity index during the first 12-16 weeks of
gestation indicator of past infection.

42. Method SensitivitySpecificityRapidity
Neutralisation + ++ -
Radio immunoassay +++ ++ ++
Enzyme immunoassay +++ ++ ++
IFA-LA
Immunofluorescent
antibodyagainst Late
Antigen
++ - ++

43. 4.Detection of viral components
•Shell vial technique
•PCR

47. CMV detection methods comparison
Method SensitivitySpecificityRapidity
Conventional cell
culture
++ +++ +
ShellVial technique + +++ ++
PCR +++ +++ ++
Antigenemiadetection +++ +++ +++

48. Viral culture
1.Amniotic fluid
2.Fetal blood
Quantitative PCR
Ultrasound
Hematological tests

49. Polymerase chain reaction (PCR)
Target-MIE region of CMV genome.
Primers used
•MIE1661
•MIE1909
The cycling conditions
•denaturationat 94°C for 2 min,
•30 cycles
94°C for 30 s,
58°C for 40 s,
72°C for 50 s.
A final incubation at 72°C for 3 min
Other primers
•MIE1517
•MIE1932

50. Clinical symptoms

51. A vaccine to prevent CMV infections is
desperately needed but not available.
Meticulous hand hygiene after
exposure to urine or saliva from
infants, toddlers and
immunocompromised patients.
Anti-CMV antibody may be effective.

52. Throughout pregnancy, practice good personal hygiene.
Women who develop a mononucleosis-like illness during
pregnancy should be evaluated for CMV infection.
Laboratory testing for antibody to CMV can be performed
to determine if a women has already had CMV infection.
The demonstrated benefits of breast-feeding outweigh the
minimal risk of acquiring CMV from mother.

53. “Herpes” –from the Greek “to creep, crawl.
It is a linear double stranded DNA viruses.
Humans are primary host.
Latent in nerve root ganglia.

54. Herpes simplex virus (HSV):
HSV type I, associated with cold sores around the
mouth;
HSV type 2, is usually associated with genital sores.
However, either type can infect either the mouth or
genitals and both can be passed on to the newborn.

55. One of most prevalent infections worldwide.
85% of adults are seropositivefor HSV-1.
20% adults seropositivefor HSV-2.
More are infected than symptomatic disease
would indicate.
50% HSV-1 infected individuals asymptomatic.
20% HSV-2 individuals asymptomatic.

56. Scientific classification
Family: Herpesviridae
Subfamily: Alpha
Species: 1.Human herpes virus 1(HHV-1) –
Herpes simplex virus type 1
2.Human herpes virus 2(HHV-2)-
Herpessimplex type 2

58. •Prevalence 1500 –2000 cases / year.
•70% HSV-2 (Genital Herpes) acquired at time of
delivery.
•HSV-1 acquired through postnatallyby contact
with oro-labial disease.
•30-40% of all adult women are seropositive.

59. 25
Risk of infection :
1.Active recurrentlesion –
risk of infection is 2-5%.
2.Active primarylesion –
risk of infection is 33-50%.
Intrauterine infection occurs with Primary
lesions of mother.

60. Primary maternal HSV infection
1
st
and 2
nd
trimester-no significant risk
3
rd
trimester-can lead to premature rupture of
membranes
Significant risk
Vaginal delivery in a woman with active lesions

61. 85% via infected maternal genital tract
Ascending infection
En route
10% postpartum
5% (or less) –
intrauterine infection

62. 21
Clinical features
Lead to triad of symptoms:
1.Skin vesicles or scarring.
2.Eye disease (Microphthalamos, chorioretinitis,
keratoconjunctivitis).
3.Microcephaly, encephalitis and cerebral
calcifications.

63. Rare, but devastating
Only 50 cases described
Skin vesicles
Chorioretinitis
Microcephaly
Micro-ophthalmia
IUGRArchival Photo:
HSV “In Utero”
Healed by Time
Of Birth –With
Microcephally

64. Approximately ½ of all HSV
infections limited to
skin, eye, mouth/mucous
membranes
1
st
-2
nd
week presentation
60-70% of untreated
patients progress to
CNS/disseminated diseaseGroin Vesicles
16 Days of Life
HSV-1, This Infant
Had a Cardiac Cath
(Groin Line)
At 3 Days of Life

65. Long term neurologic sequelaeseen in 30% of cases –
even if treated
Ophthalmology involvement

66. HSV 2 Arm Lesions
9 Days of Life
Presenting Limb in a 34 Week
Premature Infant

67. Scalp Lesions
11 Days of Life
HSV-2, Monitored
With Scalp Lead

68. Encephalitis without
visceral involvement,
mainly involving the
temporal lobes.
Early to 3
rd
week of life
presentation.HSV –2, Necrotic Brain

69. Approximately 20% of all
infections
Hepatitis
Pneumonitis
DIC
Infant may be ill on first
day of life
Skin lesions appear late

70. 1.Microscopy
2.Virus Isolation
3.Demonstration of viral antigen
4.Demonstration of antibodies

71. Sample taken-Vesicalfluid
Examination –
Transmission electron
Microscopy
Specimen should contain
atleast10
6
particles per millititre

72. Advantage
1.Most rapid
2.Morphology described (specific)
Disadvantage
1.Not available in all diagnostic laboratories
2.Relatively insensitive (specimen should contain
atleast>= 10
6
particles per millititre)
3.Cannot differentiate between HSV-1 and HSV-2

73. Tzanck smear
Smears prepared from base of lesion.
1% aqueous solution of toluidineblue
„O‟ for 15s
Positive smear
multinucleated giant cells with
faceted nuclei and homogenously
Stained ground glass chromatin

74. Giemsastain
Intranucleartype A inclusion bodies seen

75. 2. Isolation :
Cell line cultures:
Human fibroblast cells.
Vero cell line cultures.
After 1-7 days of inoculation-
Cytopathiceffects:
ballooning
polykaryoticcells

76. 3. Demonstration of viral antigen
Fluorescent antibody technique:
Immunofluorescent staining of virus by using
monoclonal antibodies .

77. Confirmatory Serological tests
1.Antigen Detection:
ELISA.
2.Molecular Methods:
PCR.
DNA probes.

78. 25
Active primary herpes should be
treated with oral acyclovir.
Suppressive therapy in last four weeks
of pregnancy may prevent recurrence
at term and prevent LSCS.
NO LESION NO LSCS

79. THE TORCH TEST

80. •Thetestisorderedwhenapregnantwomanis
suspectedofhavinganyoftheTORCHinfections.
•Usedforscreening.
•Resultsgivenaspositiveornegative,
indicates the presence or absence of IgGand IgM
antibodies for each of the infectious agents tested
for with the panel.
A "normal“ result is negative(undetectable) IgM
antibody in the blood of the mother or newborn.

81. REFERENCES:
K. D. Chatterjeeparasitology, 13
th
edition.
Ananthanarayana& Paniker‟stextbook of
Microbiology, 9
th
edition.
Principles and practice of clinical virology 5
th
edition
Wiley publications.
Williams Obstetrics : 23rd edition.