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Lab diagnosis of ToRCH complex
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Lab diagnosis of ToRCH complex

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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,

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  • may result from the receipt of an infected organ or blood, from sexual contact,or from contact with contaminated saliva or urine
  • despite the virus's tropism for epithelial cells in vivo.
  • The degree of antibody avidity increases progressively & slowly reflecting the maturation of the immune response. Low avidity indices indicate low avidity IgG antibodies in serum caused by acute or recent primary CMV infection. Low avidity indices are encountered 18-20 weeks after the onset of symptoms in immunocompetent subjects.(in viral infections B19Virus, EB Virus, etc).
  • (sensitivity 100%).
  • specific to the major immediate-early (MIE) section of the CMV genome. The first round amplified a 416-bp region using theA DNA positive control amplification was carried out on all samples using a single-roundPCR to the GAPDH (glyceraldehyde-3-phosphate dehydrogenase) gene. The GAPDH PCR was performed concurrently with the MIE PCR under the same cycling conditions
  • Primary vs secondary infection & active lesion at time of delivery are important.

Transcript

  • 1. Dr. Md. Ashraf Ali S. N Post graduate Dept. of Microbiology KIMS Hubli
  • 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. CYTOMEGALOVIRUS 4. HERPES SIMPLEX VIRUS 1 5. HERPES SIMPLEX VIRUS 2 All three belong to family herpesviridae.
  • 4. Common characteristics:  Enveloped linear dsDNA viruses.  Have ability to remain latent in tissues, leading to periodic reactivation.  Similar structure.
  • 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.  Similar for all members of herpesviridae family.  4 structural elements 1. Core 2. Protein capsid 3. Tegument 4. Outer envelope with spikes
  • 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. 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. 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.   Salivary gland virus.  It is a double stranded DNA virus.  Largest in herpes virus family (Size : 150-200nm),
  • 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.  Humans are only host.  Latent in host cells Latent CMV - monocytes, lungs, spleen, kidneys, secretory glands and cervix.  Exhibits host specificity.
  • 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.  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. 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. • CMV infection in pregnancy can be Primary Reactivation Recurrent Leading to congenital CMV infection.
  • 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.  95% clinically inapparent.  35% transmitted to fetus.  Fetal damage more likely in first 26 weeks (32%) than later (15%). ……….
  • 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. • 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. Transplacental infection can result in  intrauterine growth restriction  intracranial calcifications  Microcephaly  hydrocephalus
  • 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. CMV Retinitis  small floaters  foggy or blurred vision  loss of central or peripheral vision Complications  loss of vision  retinal detachment
  • 24.  Jaundice (67%)  Petechiae (76%)  Microcephaly (53%)  Hepatosplenomegaly(60%)  Chorioretinitis (20%)  Seizure (7%)  Fatal outcome (10%)
  • 25.  Seizures  Chorioretinitis  Periventricular calcifications  Sensorineural hearing loss  Motor deficits
  • 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. A. In pregnant B. In fetus (prenatal diagnosis) once infection in mother proved
  • 28. 1. Microscopy 2. Virus Isolation 3. Serology 4. Demonstration of viral particles
  • 29. SPECIMENS :  From active lesions of cervix, vagina .  Blood for CMV antibody - paired serum samples Storage temperature maintained- 4 degree Celsius
  • 30. 1. Microscopy i. Direct observation---Electron microscopy ii. Staining--Haematoxylin and eosin (H &E stain)
  • 31.  Enveloped  Spherical 120 – 200 nm in diameter
  • 32. ii. H & E stain from cervical secretions of CMV infected individual. Owl‟s eye appearance
  • 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. 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. 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. 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. Method Neutralisation Sensitivity Specificity Rapidity + ++ - Radio immuno assay +++ ++ ++ Enzyme immuno assay +++ ++ ++ IFA-LA Immunofluorescent antibody against Late Antigen ++ - ++
  • 38. 4. Detection of viral components • Shell vial technique • PCR
  • 39. CMV detection methods comparison Method Sensitivity Specificity Rapidity Conventional cell culture ++ +++ + Shell Vial technique + +++ ++ PCR +++ +++ ++ Antigenemia detection +++ +++ +++
  • 40.  Viral culture 1. Amniotic fluid 2. Fetal blood  Quantitative PCR  Ultrasound  Hematological tests
  • 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. Clinical symptoms
  • 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.  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.  “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. 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. 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. 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. • 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. 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. 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.  85% via infected maternal genital tract  Ascending infection  En route  10% postpartum  5% (or less) – intrauterine infection
  • 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.  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.  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.  Long term neurologic sequelae seen in 30% of cases – even if treated  Ophthalmology involvement
  • 57. HSV 2 Arm Lesions 9 Days of Life Presenting Limb in a 34 Week Premature Infant
  • 58. Scalp Lesions 11 Days of Life HSV-2, Monitored With Scalp Lead
  • 59.  Encephalitis without visceral involvement, mainly involving the temporal lobes.  Early to 3rd week of life presentation. HSV – 2, Necrotic Brain
  • 60.  Approximately 20% of all infections  Hepatitis  Pneumonitis  DIC  Infant may be ill on first day of life  Skin lesions appear late
  • 61. 1. Microscopy 2. Virus Isolation 3. Demonstration of viral antigen 4. Demonstration of antibodies
  • 62.  Sample taken- Vesical fluid  Examination – Transmission electron Microscopy  Specimen should contain atleast 106 particles per millititre
  • 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. 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. Giemsa stain  Intranuclear type A inclusion bodies seen
  • 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. 3. Demonstration of viral antigen Fluorescent antibody technique: Immunofluorescent staining of virus by using monoclonal antibodies .
  • 68. Confirmatory Serological tests 1. Antigen Detection: ELISA. 2. Molecular Methods: PCR. DNA probes.
  • 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. THE TORCH TEST
  • 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. 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.