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Dr. Md. Ashraf Ali S. N
Post graduate
Dept. of Microbiology
KIMS Hubli
To- TOXOPLASMOSIS

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

Co...
3. CYTOMEGALOVIRUS
4. HERPES SIMPLEX VIRUS 1
5. HERPES SIMPLEX VIRUS 2

All three belong to family herpesviridae.
Common characteristics:
 Enveloped linear dsDNA viruses.
 Have ability to remain latent in tissues, leading to

periodic...
Classification of herpes viruses
Species
Name

Human herpes virus type 1

Human herpes virus type 2

Common name
Herpes si...
 Similar for all members of herpesviridae family.

 4 structural elements
1. Core
2. Protein capsid
3. Tegument
4. Outer...
1. Core
-contains the linear dsDNA

-DNA stabilized by a series of protein fibrils
2. Capsid- encloses the core
- A closed...
3.Tegument
 Between capsid and envelope an amorphous

tegument is present.
 Role

 Important in early stages of viral r...
4.

Envelope


Trilaminar



Derived from host cell



Envelope has glycoproteinsgB, gC, gD, gE, gG, gH, gI, gJ, gK, gL...




Salivary gland virus.



It is a double stranded
DNA virus.



Largest in herpes virus
family (Size : 150-200nm),
• Congenital infection occurs in approximately 2%
of live births.
• It is a leading cause of hearing loss, mental
retardat...


Humans are only host.



Latent in host cells
Latent

CMV

-

monocytes, lungs, spleen, kidneys, secretory
glands and ...
Scientific classification
Family:

Herpesviridae

Subfamily: Beta
Genus:

Cytomegalovirus

Species:

1. Human herpes virus...
 Vertical transmission
 transplacental infection (prenatal)

 genital-tract secretions during delivery.
 breast-feedin...
Mother

Viraemia

Placental Trophoblasts

Infection of
the oropharynx

Virus in
amniotic fluid

Infection of fetal
endothe...
•

CMV infection in pregnancy can be
Primary
Reactivation
Recurrent

Leading to congenital CMV infection.
• Primary maternal infection leads
to fetal infection in 30-50% of
cases.

• Recurrent
less

likely

maternal
to

infectio...


95% clinically inapparent.



35% transmitted to fetus.



Fetal damage more likely in first 26 weeks (32%)

than lat...
Primary CMV
Period

Recurrent

1st
2nd
3rd
trimester trimester trimester

CMV

Risk of
infection

40%

45%

78%

0.5%

Fet...
•

Vertical transmission of CMV can occur at
any stage of pregnancy.

•

Severe sequelae are more common with
infection in...
Transplacental infection can result in


intrauterine growth restriction



intracranial calcifications



Microcephaly...
CMV in Immunocompromised Patients
• Reactivation of the dormant virus.

• Primary CMV infection in immunocompromised

- sc...
CMV Retinitis
 small floaters
 foggy or blurred vision
 loss of central or peripheral vision
Complications
 loss of vi...
 Jaundice (67%)
 Petechiae (76%)

 Microcephaly (53%)
 Hepatosplenomegaly(60%)
 Chorioretinitis (20%)
 Seizure (7%)
...
 Seizures
 Chorioretinitis
 Periventricular calcifications

 Sensorineural hearing loss
 Motor deficits
MATERNAL SCREENING

High risk of transmission

IgG POSITIVE
IgM NEGATIVE

IgG & IgM NEGATIVE

IMMUNE
REASSURANCE

UNIMMUNE...
A. In pregnant
B. In fetus (prenatal diagnosis) once

infection in mother proved
1. Microscopy

2.

Virus Isolation

3.

Serology

4.

Demonstration of viral particles
SPECIMENS :
 From active lesions of cervix, vagina .



Blood for CMV antibody - paired serum samples

Storage temperatu...
1. Microscopy
i.

Direct observation---Electron microscopy

ii. Staining--Haematoxylin and eosin (H &E stain)
 Enveloped
 Spherical

120 – 200 nm in
diameter
ii.

H & E stain from cervical secretions of CMV
infected individual.

Owl‟s eye
appearance
2. Virus isolation
Cell culture line used
 Human Fibroblast cell line cultures
 The characteristic cytopathic effect dev...
3. Serology
Antibody detection
i.

Seroconversion

ii. IgM detection- paired sera is taken
One blood sample should be take...
IgG assays

• CMV IgG antibody – sensitive and specific for past

infection.
• CMV IgM antibody – variable sensitivity and...
The anti-CMV IgG avidity test-currently the most reliable.
 Specific------100%
 Sensitive---- 60-94%
• Low avidity- rece...
Method
Neutralisation

Sensitivity Specificity

Rapidity

+

++

-

Radio immuno assay

+++

++

++

Enzyme immuno assay

...
4. Detection of viral components

•

Shell vial technique

•

PCR
CMV detection methods comparison
Method

Sensitivity

Specificity Rapidity

Conventional cell
culture

++

+++

+

Shell V...
 Viral culture
1. Amniotic fluid
2.

Fetal blood

 Quantitative PCR

 Ultrasound
 Hematological tests
Polymerase chain reaction (PCR)
Target- MIE region of CMV genome.
Primers used
• MIE1661
• MIE1909
The cycling conditions
...
Clinical symptoms
 A vaccine to prevent CMV infections is

desperately needed but not available.

 Meticulous hand hygiene after

exposure...


Throughout pregnancy, practice good personal hygiene.



Women who develop a mononucleosis-like illness during
pregnan...


“Herpes” – from the Greek “to creep, crawl.



It is a linear double stranded DNA viruses.



Humans are primary host...
Herpes simplex virus (HSV):
 HSV type I, associated with cold sores around the

mouth;
 HSV type 2, is usually associate...
One of most prevalent infections worldwide.
 85% of adults are seropositive for HSV-1.
 20% adults seropositive for HSV-...
Scientific classification
Family:

Herpesviridae

Subfamily: Alpha
Species:

1. Human herpes virus 1(HHV-1) –
Herpes simpl...
• Prevalence 1500 – 2000 cases / year.
• 70% HSV-2 (Genital Herpes) acquired at time of
delivery.
• HSV-1 acquired through...
Risk of infection :

1. Active recurrent lesion –
risk of infection is 2-5%.

2. Active primary lesion –
risk of infection...
Primary maternal HSV infection

 1st and 2nd trimester- no significant risk

 3rd trimester- can lead to premature ruptu...
 85% via infected maternal genital tract
 Ascending infection
 En route
 10% postpartum
 5% (or less) –

intrauterine...
Clinical features

Lead to triad of symptoms:
1. Skin vesicles or scarring.
2. Eye disease (Microphthalamos, chorioretinit...
 Rare, but devastating
 Only 50 cases described
 Skin vesicles
 Chorioretinitis

 Microcephaly
 Micro-ophthalmia

 ...
 Approximately ½ of all HSV

infections limited to
skin, eye, mouth/mucous
membranes
 1st-2nd week presentation
 60-70%...
 Long term neurologic sequelae seen in 30% of cases –

even if treated
 Ophthalmology involvement
HSV 2 Arm Lesions
9 Days of Life
Presenting Limb in a 34 Week
Premature Infant
Scalp Lesions
11 Days of Life
HSV-2, Monitored
With Scalp Lead
 Encephalitis without

visceral involvement,
mainly involving the
temporal lobes.
 Early to 3rd week of life

presentati...
 Approximately 20% of all

infections
 Hepatitis
 Pneumonitis
 DIC
 Infant may be ill on first

day of life
 Skin le...
1. Microscopy

2. Virus Isolation

3.

Demonstration of viral antigen

4.

Demonstration of antibodies
 Sample taken- Vesical fluid
 Examination –

Transmission electron
Microscopy

 Specimen should contain

atleast 106 pa...
Advantage
1. Most rapid
2. Morphology described (specific)
Disadvantage
1. Not available in all diagnostic laboratories
2....
Tzanck smear


Smears prepared from base of lesion.



1% aqueous solution of toluidine blue
„O‟ for 15s

Positive smear...
Giemsa stain
 Intranuclear type A inclusion bodies seen
2. Isolation :
Cell line cultures:
Human fibroblast cells.
Vero cell line cultures.
After 1-7 days of inoculation-

Cytopa...
3. Demonstration of viral antigen
Fluorescent antibody technique:
Immunofluorescent staining of virus by using
monoclonal ...
Confirmatory Serological tests
1. Antigen Detection:
ELISA.

2. Molecular Methods:
PCR.
DNA probes.
 Active primary herpes should be

treated with oral acyclovir.
 Suppressive therapy in last four weeks

of pregnancy may...
THE TORCH TEST
• The test is ordered when a pregnant woman is
suspected of having any of the TORCH infections.
• Used for screening.
• Re...
REFERENCES:
 K. D. Chatterjee parasitology, 13th edition.
 Ananthanarayana & Paniker‟s textbook of

Microbiology, 9th ed...
Lab diagnosis of ToRCH complex
Lab diagnosis of ToRCH complex
Lab diagnosis of ToRCH complex
Lab diagnosis of ToRCH complex
Lab diagnosis of ToRCH complex
Lab diagnosis of ToRCH complex
Lab diagnosis of ToRCH complex
Lab diagnosis of ToRCH complex
Lab diagnosis of ToRCH complex
Lab diagnosis of ToRCH complex
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Lab diagnosis of ToRCH complex

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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,

Published in: Health & Medicine, Technology
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Lab diagnosis of ToRCH complex

  1. 1. Dr. Md. Ashraf Ali S. N Post graduate Dept. of Microbiology KIMS Hubli
  2. 2. To- TOXOPLASMOSIS R – RUBELLA Covered in torch complex Part-I  C - CYTOMEGALOVIRUS  H - HERPES SIMPLEX VIRUS – 1& 2 Covered in torch complex Part-II
  3. 3. 3. CYTOMEGALOVIRUS 4. HERPES SIMPLEX VIRUS 1 5. HERPES SIMPLEX VIRUS 2 All three belong to family herpesviridae.
  4. 4. Common characteristics:  Enveloped linear dsDNA viruses.  Have ability to remain latent in tissues, leading to periodic reactivation.  Similar structure.
  5. 5. Classification of herpes viruses Species Name Human herpes virus type 1 Human herpes virus type 2 Common name Herpes simplex virus type 1 Herpes simplex Sub family alpha Latency Neurons trigeminal ganglia alpha Neurons Sacral ganglia alpha Neurons virus type 2 Human herpes virus type 3 Varicella-Zoster Human herpes virus type 4 Epstein-Barr virus gamma Human herpes virus type 5 Cytomegalovirus beta Secretory glands, kidneys Human herpes virus type 6 Human B cell lymphotropic virus beta Lymphoid tisues Human herpes virus type 7 R K virus beta Lymphoid tisues Human herpes virus type 8 gamma Lymphoid tissues
  6. 6.  Similar for all members of herpesviridae family.  4 structural elements 1. Core 2. Protein capsid 3. Tegument 4. Outer envelope with spikes
  7. 7. 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
  8. 8. 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.
  9. 9. 4. Envelope  Trilaminar  Derived from host cell  Envelope has glycoproteinsgB, 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
  10. 10.   Salivary gland virus.  It is a double stranded DNA virus.  Largest in herpes virus family (Size : 150-200nm),
  11. 11. • Congenital infection occurs in approximately 2% of live births. • It is a leading cause of hearing loss, mental retardation and cerebral palsy. • Opportunistic pathogen in Immunocompromised. • Account for approx. 10% death in symptomatic newborns.
  12. 12.  Humans are only host.  Latent in host cells Latent CMV - monocytes, lungs, spleen, kidneys, secretory glands and cervix.  Exhibits host specificity.
  13. 13. 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)
  14. 14.  Vertical transmission  transplacental infection (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.
  15. 15. Mother Viraemia Placental Trophoblasts Infection of the oropharynx Virus in amniotic fluid Infection of fetal endothelial cells Fetal viraemia Fetal viruria Replication in target organs (kidney)
  16. 16. • CMV infection in pregnancy can be Primary Reactivation Recurrent Leading to congenital CMV infection.
  17. 17. • Primary maternal infection leads to fetal infection in 30-50% of cases. • Recurrent less likely maternal to infection (1-2%). lead infection to fetal
  18. 18.  95% clinically inapparent.  35% transmitted to fetus.  Fetal damage more likely in first 26 weeks (32%) than later (15%). ……….
  19. 19. Primary CMV Period Recurrent 1st 2nd 3rd trimester trimester trimester CMV Risk of infection 40% 45% 78% 0.5% Fetal damage 32% 16% 1% <1%
  20. 20. • Vertical transmission of CMV can occur at any stage of pregnancy. • Severe sequelae are more common with infection in the 1st trimester, while the overall risk of infection is greatest in the 3rd trimester.
  21. 21. Transplacental infection can result in  intrauterine growth restriction  intracranial calcifications  Microcephaly  hydrocephalus
  22. 22. 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.
  23. 23. CMV Retinitis  small floaters  foggy or blurred vision  loss of central or peripheral vision Complications  loss of vision  retinal detachment
  24. 24.  Jaundice (67%)  Petechiae (76%)  Microcephaly (53%)  Hepatosplenomegaly(60%)  Chorioretinitis (20%)  Seizure (7%)  Fatal outcome (10%)
  25. 25.  Seizures  Chorioretinitis  Periventricular calcifications  Sensorineural hearing loss  Motor deficits
  26. 26. MATERNAL SCREENING High risk of transmission IgG POSITIVE IgM NEGATIVE IgG & IgM NEGATIVE IMMUNE REASSURANCE UNIMMUNE Meticulous hand hygiene < 20 WEEKS HIGH CMV IgG AVIDITY NO FURTHER TESTING NEEDED IgG NEGATIVE IgM POSITIVE IgG & IgM POSITIVE REPEAT TEST AFTER 3 WEEKS > 20 WEEKS LOW CMV IgG AVIDITY LOW CMV IgG AVIDITY HIGH RISK OF TRANSMISSION MATERNAL VIREMIA BY URINARY & BLOOD PCR FETAL WELL BEING USG & MRI HIGH CMV IgG AVIDITY RETROSPECTIVELY SCREEN 1ST TRIMESTER BLOOD INVASIVE AMNI0TIC FLUID PCR
  27. 27. A. In pregnant B. In fetus (prenatal diagnosis) once infection in mother proved
  28. 28. 1. Microscopy 2. Virus Isolation 3. Serology 4. Demonstration of viral particles
  29. 29. SPECIMENS :  From active lesions of cervix, vagina .  Blood for CMV antibody - paired serum samples Storage temperature maintained- 4 degree Celsius
  30. 30. 1. Microscopy i. Direct observation---Electron microscopy ii. Staining--Haematoxylin and eosin (H &E stain)
  31. 31.  Enveloped  Spherical 120 – 200 nm in diameter
  32. 32. ii. H & E stain from cervical secretions of CMV infected individual. Owl‟s eye appearance
  33. 33. 2. Virus isolation Cell culture line used  Human Fibroblast cell line cultures  The characteristic cytopathic effect develops Human fibroblasts are the only cells in which CMV reliably grows in vitro. within hours to weeks after inoculation depending on the amount of CMV present in the specimen.
  34. 34. 3. Serology Antibody detection i. Seroconversion ii. IgM detection- 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
  35. 35. IgG assays • 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.
  36. 36. The anti-CMV IgG avidity test-currently the most reliable.  Specific------100%  Sensitive---- 60-94% • Low avidity- recent/past infection  16th-18th 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.
  37. 37. Method Neutralisation Sensitivity Specificity Rapidity + ++ - Radio immuno assay +++ ++ ++ Enzyme immuno assay +++ ++ ++ IFA-LA Immunofluorescent antibody against Late Antigen ++ - ++
  38. 38. 4. Detection of viral components • Shell vial technique • PCR
  39. 39. CMV detection methods comparison Method Sensitivity Specificity Rapidity Conventional cell culture ++ +++ + Shell Vial technique + +++ ++ PCR +++ +++ ++ Antigenemia detection +++ +++ +++
  40. 40.  Viral culture 1. Amniotic fluid 2. Fetal blood  Quantitative PCR  Ultrasound  Hematological tests
  41. 41. Polymerase chain reaction (PCR) Target- MIE region of CMV genome. Primers used • MIE1661 • MIE1909 The cycling conditions Other primers • MIE1517 • MIE1932 • denaturation at 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
  42. 42. Clinical symptoms
  43. 43.  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.
  44. 44.  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.
  45. 45.  “Herpes” – from the Greek “to creep, crawl.  It is a linear double stranded DNA viruses.  Humans are primary host.  Latent in nerve root ganglia.
  46. 46. 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.
  47. 47. One of most prevalent infections worldwide.  85% of adults are seropositive for HSV-1.  20% adults seropositive for HSV-2. More are infected than symptomatic disease would indicate.  50% HSV-1 infected individuals asymptomatic.  20% HSV-2 individuals asymptomatic.
  48. 48. 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)Herpes simplex type 2
  49. 49. • Prevalence 1500 – 2000 cases / year. • 70% HSV-2 (Genital Herpes) acquired at time of delivery. • HSV-1 acquired through postnatally by contact with oro-labial disease. • 30-40% of all adult women are seropositive.
  50. 50. Risk of infection : 1. Active recurrent lesion – risk of infection is 2-5%. 2. Active primary lesion – risk of infection is 33-50%. Intrauterine infection occurs with Primary lesions of mother. 25
  51. 51. Primary maternal HSV infection  1st and 2nd trimester- no significant risk  3rd trimester- can lead to premature rupture of membranes  Significant risk Vaginal delivery in a woman with active lesions
  52. 52.  85% via infected maternal genital tract  Ascending infection  En route  10% postpartum  5% (or less) – intrauterine infection
  53. 53. Clinical features Lead to triad of symptoms: 1. Skin vesicles or scarring. 2. Eye disease (Microphthalamos, chorioretinitis, keratoconjunctivitis). 3. Microcephaly, encephalitis and cerebral calcifications. 21
  54. 54.  Rare, but devastating  Only 50 cases described  Skin vesicles  Chorioretinitis  Microcephaly  Micro-ophthalmia  IUGR Archival Photo: HSV “In Utero” Healed by Time Of Birth – With Microcephally
  55. 55.  Approximately ½ of all HSV infections limited to skin, eye, mouth/mucous membranes  1st-2nd week presentation  60-70% of untreated patients progress to CNS/disseminated disease Groin Vesicles 16 Days of Life HSV-1, This Infant Had a Cardiac Cath (Groin Line) At 3 Days of Life
  56. 56.  Long term neurologic sequelae seen in 30% of cases – even if treated  Ophthalmology involvement
  57. 57. HSV 2 Arm Lesions 9 Days of Life Presenting Limb in a 34 Week Premature Infant
  58. 58. Scalp Lesions 11 Days of Life HSV-2, Monitored With Scalp Lead
  59. 59.  Encephalitis without visceral involvement, mainly involving the temporal lobes.  Early to 3rd week of life presentation. HSV – 2, Necrotic Brain
  60. 60.  Approximately 20% of all infections  Hepatitis  Pneumonitis  DIC  Infant may be ill on first day of life  Skin lesions appear late
  61. 61. 1. Microscopy 2. Virus Isolation 3. Demonstration of viral antigen 4. Demonstration of antibodies
  62. 62.  Sample taken- Vesical fluid  Examination – Transmission electron Microscopy  Specimen should contain atleast 106 particles per millititre
  63. 63. Advantage 1. Most rapid 2. Morphology described (specific) Disadvantage 1. Not available in all diagnostic laboratories 2. Relatively insensitive (specimen should contain atleast >= 106 particles per millititre) 3. Cannot differentiate between HSV-1 and HSV-2
  64. 64. Tzanck smear  Smears prepared from base of lesion.  1% aqueous solution of toluidine blue „O‟ for 15s Positive smear multinucleated giant cells with faceted nuclei and homogenously Stained ground glass chromatin
  65. 65. Giemsa stain  Intranuclear type A inclusion bodies seen
  66. 66. 2. Isolation : Cell line cultures: Human fibroblast cells. Vero cell line cultures. After 1-7 days of inoculation- Cytopathic effects:  ballooning  polykaryotic cells
  67. 67. 3. Demonstration of viral antigen Fluorescent antibody technique: Immunofluorescent staining of virus by using monoclonal antibodies .
  68. 68. Confirmatory Serological tests 1. Antigen Detection: ELISA. 2. Molecular Methods: PCR. DNA probes.
  69. 69.  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 25
  70. 70. THE TORCH TEST
  71. 71. • The test is ordered when a pregnant woman is suspected of having any of the TORCH infections. • Used for screening. • Results given as positive or negative, indicates the presence or absence of IgG and 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.
  72. 72. REFERENCES:  K. D. Chatterjee parasitology, 13th edition.  Ananthanarayana & Paniker‟s textbook of Microbiology, 9th edition.  Principles and practice of clinical virology 5th edition Wiley publications.  Williams Obstetrics : 23rd edition.

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