TORCH COMPLEX

Dr. Md. Ashraf Ali S. NPost graduateDept. of MicrobiologyKIMS HubliToRCH COMPLEXPart-I

The acronym for a set of vertically transmitted infectionsTo- TOXOPLASMOSISR RUBELLAC - CYTOMEGALOVIRUSH - HERPES SIMPLEX VIRUS - 2

Mode of transmission in the childPlacental (chorionic villi)Hematogenous (gestation or time of delivery)

ToRCH infections can lead toFetal anomaliesFetal loss

ToxoplasmosisRubellaCytomegalovirusHerpes simplex 2Torch test

TOXOPLASMOSISCausative organismLife cycleMode of infectionIn motherchildClinical course- both in the mother & childSequelLab diagnosisIndirect evidences (imp in pregnant)Direct evidences

CAUSATIVE ORGANISM-Toxoplasma gondiiAn obligate intracellular parasite.Human infection is dead end.PhylumApicomlexa ClassSporozoaSub-classEucoccidiaOrderCoccidiaSub-orderEimerinaGenusToxoplasma

Greek Toxo- means a bow curved shape of trophozoites7

MORPHOLOGY AND LIFE CYCLEConsists of three forms

Tachyzoites rapidly multiplying forms,invade & multiply within cellsSilver stains used to detect

Bradyzoitesslowly multiplying formsPresent inside tissue cystsPAS positiveSporozoites

Bradyzoites

Sporozoites inside oocysts, shed in feces of cat and remian in the environment

Definitive host : Felis catus (Domestic cat)Enteric cycleGametogony and schizogony occurs in the epithelial cells of SI.

Gametogony

ZygoteOocystMembrane--Thinextremely resistant

Cats fecesUnsporulatedNon infectiousLIFE CYCLE

MODE OF INFECTION:Infective form : Oocyst containing sporozoitesIngestion of oocysts ( raw meat, garden products)Contact with oocysts in cats feces/contaminated soilThe incidence during pregnancy ranges from 0.3-1 %Of these 1 in 10 will deliver a baby with congenital ToxoplasmosisIngestionContact

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Freshly passed Oocyst are non-infectious13

Exoenteric cycle (man & other intermediate hosts)Sporozoites penetrate epithelium of ileum

Tissue cyst/ extracellular formBrain, Skeletal muscleHas cystozoiteLodgement is in two formsPseudocyst/intracellular form (proliferative stage).Cells of R.E. System, Placenta.Has a crescent shaped endozoite of 6 by 2

CLINICAL COURSE

The risk of maternal fetal transmission increases with gestational, whereas the incidence of severe disease decreases.17

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

Clinical manifestation in fetus manifest with classical triad of Hydrocephalus 20%

Chorioretinitis 86%

Intracranial calcification 37%

SEQUELAE If fetus develops sub clinical infection - Asymptomatic at birth . Later on developsMental retardation AND Learning difficultiesCerebral calcificationsChorioretinitis blindnessHydrocephalusEpilepsy.

LABORATORY DIAGNOSISIndirect evidencesAntibody demonstration

Direct evidencesMicroscopyStaining

Prenatal diagnosisdone when IgM & IgG positive with low avidity

Toxoplasma skin test of frenkel

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Indirect evidencesAntibody demonstration (Screening tests)ELISA METHOD.Indirect IF test (sabin-feldmen dye test).Goldmans test ( fluorescent tagged Ab.)Fultons agglutination Test.Complement Fixation Test.

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ELISA- Routinely usedFor IgM and IgG antibodiesIgM- Detection of specific IgM antibodiesIgG Assays includeDetection of specific IgG antibodiesIgG Avidity testing

SampleSerum.-20C (if delay anticipated)

Antigen preparationTachyzoites 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= 4C overnight later -20C .

with PBS (pH.7.2) 3 times24

IgM ELISAAim- detect Anti-Toxoplasma IgM antibodiesProcedureSera is diluted seriallyadd to T. gondii antigen-coated microtiter plateAdd anti-human IgG conjugated with horseradish peroxidase. Chr. substrate -- o-phenylenediamine (OPD)Read by means an automated ELISA-reader.

After incubation and washing, chromogenic25

Avidity ELISAMicrotitre plates pre-coated with Toxoplasma antigensSerum diluted to 1/200 100 l/well on 2 rows(row A and row B), incubation for 45 min at 37C and washrow A wash 3 times with modified PBST buffer containing 6 M urea, fourth time with PBSTrow B - wash 3 times with PBST.

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; 26

The anti-human IgG conjugated with HRP addedChromogenic substrate, o-phenylenediamine (OPD) Sulfuric acid 20%. The absorbance (Absb) read by an automated ELISA reader at 492 nm. Avidity index (AI; %) AI =

IgGIgMINTERPRETATIONFOLLOW UP TESTINGNegativeNegativeNegativePositive/equivocalPositiveNegativePositivePositive

No serologic evidence of T. gondii infectionacute T. gondii infection False +ve IgM reaction Obtain a new specimen for IgG and IgM testing orretest this specimen using a different assayAll Indeterminate resultsfurther testing in a reference laboratory for the diagnosis of toxoplasmosis.T. gondii inf. within past 1 yr or false +ve IgM reaction.Infected with T. gondii for more than 6 months.second specimen aft 2-3 weeks for IgG and IgM testing; if same results, then its false +ve IgM reaction..

SABIN FELDMAN DYE TEST- for cytoplasm mod. Ab.Serial dilutions of patients sera 1:16, 1:32.., 1:256Live Tachyzoites + Accessory factor + Test serum (Serial dilutions)

Incubation at 37 C for 1 hour

Alc. soln of Meth. Blue (pH-11)Examine under 40x

Highest dilution for which 1 year

IgG AVIDITYLow IgG Avidity HIgh IgG Avidity

OLD INFECTION16wks 1 yrRECENT INFECTION < 16WKSRepeat test after 2 wks to confirm before intervention

IgG & IgM POSITIVE

Prenatal Diagnosisdone when IgM & IgG positive with low avidityAmniocentesis amniotic fluid PCR for parasite particles.

Placental tissue / Blood inoculation into mice & isolation of parasites

USG

IgG - previous inf., never become -ve . no contraception. No risk of congenital Tox to fetus in next pregnancy, she can conceive at any time.35

Treatment of toxoplasmosis Indication Treatmentonly IgG positive No treatment Suspected acute infection after pregnancySpiramycin till confirmationEvidence of fetal infectionAmniotic fluid PCR positiveUltrasound signsPyrimethamine + sulphadiazine X 3 wks. Alternating with Spiramycinconsider MTP before 20 weeks of gestation THERE IS NO VACCINE FOR TOX PREVENTION. .

Rubella (German measles)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.

PropertiesFamily- TogaviridaeGenus- RubivirusPleomorphic, roughly spherical.50-70nm in diameterssRNA genomeEnveloped with haemagglutinin polymers.

MODE OF TRANSMISSIONperson to person contactDroplet secretions of the infected.Rash appears 2-3 weeks following exposure & persist for three days.Infection can be communicated 7days before and 4 days after appearance of rashTransmission to fetus- transplacental

C.A.

Congenital Rubella SyndromeCataract- 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

Table 2. Congenital defects & late manifestations of rubella infectionPRESENT AT BIRTH1.Audiologic anomalies (6075%)Sensorineural deafness2.Cardiac defects (1020%)Pulmonary stenosisPatent ductus arteriosusVentricular septal defect

3.Ophthalmic defects (1025%)RetinopathyCataractsMicrophthalmiaPigmentary and congenital glaucoma

4. Central nervous system (1025%)Mental retardationMicrocephalyMeningoencephalitisCharacteristic purpuraBlueberry muffin appearanceLATE MANIFESTATIONSDiabetes mellitusThyroiditisGrowth hormone deficit

Diagnosis of maternal rubella infectionSerologic studies best performed within 7 to10 days after the onset of the rash and should be repeated two to three weeks later.IgM AssaysIgG AssaysViral isolation

IgM assaysIgM captureIgM 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

Capture ELISA schematic

2. Indirect EIA for virus-specific IgMPre-treatment step: rheumatoid factor absorbent is used for the complexing of IgG antibodies from test sera.First stepabsorption of virus antigen onto the solid phaseSecond stepThe patient's serum is then added virus-specific Ab. (IgM & IgG) binds to the Ag.Third stepAdd enzyme-labeled anti-human IgM monoclonal antibody A chromogen substrate is added to reveal the presence of virus-specific IgM in the test sample

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Rubella IgG Assays1. Indirect EIAThe 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.

2. IgG avidity assaysAVIDITY: Antibody avidity is the strength of interaction of an antibody with a multivalent antigen.

..IgG avidity testing differentiates between primary & secondary rubella inf. excludes possibility of residual IgM which r present months or years after primary infection. Presence oflow-affinity antibodies ---------early stage of infectionhigh-affinity antibodies -------reflects past immunity.

IgG avidity assays are difficult to establish, standardize, quality control and interpret, hence recommended only for experienced laboratories.

CULTURE Specimens-nasopharyngeal, blood, throatswab , urine, and cerebrospinal fluid from pregnant women.Cell culture lines: Vero cells- currently recommended incubated at 35C for 3 or 5 days. RK -13Detection of rubella E1 glycoprotein in infected Vero cells using monoclonal antibodies by Immunofluorescentimmunocolorimetric assay

Summary: Diagnosis of maternal rubella infectionA 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)

Diagnosis of Fetal InfectionSamples takenCVS (10-12wks of gest)Amniotic fluid (14-16 weeks of gest)Fetal blood (18-20 wks of gest)PCRRubella-specific PCRAdvantageAllows early detectionCVS as early as 10-12 weeks of gestation

CVS for the prenatal diagnosis of intrauterine rubella infection is superior to assessment of amniotic fluid samples54

PREVENTION OF CONGENITAL RUBELLA

Universal infant immunization.strain- RA 27/3, live attenuateds/c injection2.Screening for immunity and vaccination before conception.3.Screening of all pregnant women to determine susceptibility.

RecommendationsRubella vaccine should not be administered during pregnancy.

Pregnancy should be avoided for 3 months following rubella vaccination.

ReferencesParasitology, 13/e K. D. Chatterjee.Ananthanarayana & Panikers Textbook of Microbiology 19th edition.