Torch complex PART-1

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TORCH complex, forms important set of PERINATAL (Vertically transmitted infections)

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  • Greek Toxo- means a bow curved shape of trophozoites
  • Freshly passed Oocyst are non-infectious
  • The risk of maternal fetal transmission increases with gestational, whereas the incidence of severe disease decreases.
  • Toxoplasma skin test of frenkel
  • with PBS (pH.7.2) 3 times
  • After incubation and washing, chromogenic
  • The reaction was stopped was calculated as the result of Abs of wells washed with PBS-urea (U+), divided by the Abs of wells washed with PBST (U-), and multiplied with 100, based on the formula;
  • IgG - previous inf., never become -ve . no contraception. No risk of congenital Tox to fetus in next pregnancy, she can conceive at any time.
  • CVS for the prenatal diagnosis of intrauterine rubella infection is superior to assessment of amniotic fluid samples
  • Torch complex PART-1

    1. 1. Dr. Md. Ashraf Ali S. N Post graduate Dept. of Microbiology KIMS Hubli
    2. 2. The acronym for a set of vertically transmitted infections  To- TOXOPLASMOSIS R – RUBELLA C - CYTOMEGALOVIRUS H - HERPES SIMPLEX VIRUS - 2
    3. 3.  Mode of transmission in the child • Placental (chorionic villi) • Hematogenous (gestation or time of delivery)  ToRCH infections can lead to • Fetal anomalies • Fetal loss
    4. 4.  Toxoplasmosis  Rubella  Cytomegalovirus  Herpes  Torch simplex 2 test
    5. 5. 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
    6. 6.  An obligate intracellular parasite.  Human infection is dead end. Phylum Apicomlexa Class Sporozoa Sub-class Eucoccidia Order Coccidia Sub-order Eimerina Genus Toxoplasma
    7. 7. 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
    8. 8. 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
    9. 9. 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
    10. 10.  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
    11. 11. Clinical course cont……….  50% of fetuses escape  30-35% develop sub clinical infection.  Only 10% develop severe infection. (following clinical symptoms)
    12. 12. Clinical manifestation in fetus manifest with classical triad of  Hydrocephalus 20%  Chorioretinitis 86%  Intracranial calcification 37%
    13. 13. SEQUELAE If fetus develops sub clinical infection  Asymptomatic at birth .  Later on develops • Mental retardation AND Learning difficulties • Cerebral calcifications • Chorioretinitis blindness • Hydrocephalus • Epilepsy.
    14. 14.  Indirect evidences • Antibody demonstration  Direct evidences • Microscopy • Staining  Prenatal diagnosis • done when IgM & IgG positive with low avidity
    15. 15. 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.
    16. 16. 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
    17. 17. 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 .
    18. 18. 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.
    19. 19. 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.
    20. 20.  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 =
    21. 21. 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 …..
    22. 22. 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
    23. 23.  Highest dilution for which <50% of free toxoplasma have stained cytoplasm is taken as titre.  Titres of 1:128- diagnosis of acute inf. ….
    24. 24. 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
    25. 25. …………….
    26. 26. 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
    27. 27.  Amniocentesis – amniotic fluid PCR for parasite particles.  Placental tissue / Blood – inoculation into mice & isolation of parasites  USG
    28. 28. 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. ……….
    29. 29.  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.
    30. 30. Properties  Family- Togaviridae  Genus- Rubivirus  Pleomorphic,  50-70nm  ssRNA roughly spherical. in diameter genome  Enveloped with haemagglutinin polymers.
    31. 31. 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
    32. 32. C.A.
    33. 33.  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
    34. 34. 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
    35. 35. 4. Central nervous system (10–25%) • Mental retardation • Microcephaly • Meningoencephalitis 5. Characteristic purpura Blueberry muffin appearance LATE MANIFESTATIONS • Diabetes mellitus • Thyroiditis • Growth hormone deficit
    36. 36.  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
    37. 37. 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
    38. 38. Capture ELISA schematic
    39. 39. 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
    40. 40. ……………..
    41. 41. 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.
    42. 42. 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.
    43. 43. 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
    44. 44.  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)
    45. 45. 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
    46. 46. 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.
    47. 47. 1. Rubella vaccine should not be administered during pregnancy. 2. Pregnancy should be avoided for 3 months following rubella vaccination.
    48. 48.  Parasitology, 13/e K. D. Chatterjee.  Ananthanarayana & Paniker’s Textbook of Microbiology 19th edition.

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