1. بسم الله الرحمن الرحيم

2. Faculty of Allied Medical Sciences Clinical Immunology & Serology Practice (MLIS 201)

3. TORCH Prof. Dr. Ezzat M Hassan Prof. of Immunology Med Res Inst, Alex Univ E-mail: elgreatlyem@hotmail.com

4. Objectives To Know elements of TORCHTo Know elements of TORCH To know the causes of TORCH InfectionTo know the causes of TORCH Infection Describe the diagnostic methods for TORCHDescribe the diagnostic methods for TORCH

5. TORCH Infections T=toxoplasmosisT=toxoplasmosis O=other (syphilis, HBV,HIV, )O=other (syphilis, HBV,HIV, ) R=rubellaR=rubella C=cytomegalovirus (CMV)C=cytomegalovirus (CMV) H=herpes simplex (HSV)H=herpes simplex (HSV)

6. Index of Suspicion When do you think of TORCH infections?When do you think of TORCH infections? Intra-Uterine Growth Retardation (IUGR) infantsIntra-Uterine Growth Retardation (IUGR) infants Hepato-Splenomegaly (HSM)Hepato-Splenomegaly (HSM) Thrombocytopenia (Low Platelet count)Thrombocytopenia (Low Platelet count) Unusual rashUnusual rash Concerning maternal historyConcerning maternal history “Classic” findings of any specific infection“Classic” findings of any specific infection

7. TORCH - panel (IgM & IgG) ToxoplasmaToxoplasma RubellaRubella Cytomegalo virusCytomegalo virus HerpesHerpes IgM - Acute or Recent infectionIgM - Acute or Recent infection IgG - Chronic infectionIgG - Chronic infection

8. Diagnosing TORCH Infection Good maternal/prenatal historyGood maternal/prenatal history Remember most TORCH infections are mild illnesses & often unrecognizedRemember most TORCH infections are mild illnesses & often unrecognized Thorough exam of infantThorough exam of infant Directed labs/studies based on most likely diagnosis…Directed labs/studies based on most likely diagnosis…

9. Syphilis Treponema pallidum (spirochete)Treponema pallidum (spirochete) Transmitted via sexual contactTransmitted via sexual contact Placental transmission as early as 6wks gestationPlacental transmission as early as 6wks gestation

10. Clinical Manifestations Fetal:Fetal: StillbirthStillbirth Neonatal deathNeonatal death Hydrops fetalisHydrops fetalis Intrauterine death in 25%Intrauterine death in 25% Perinatal mortality in 25-30% if untreatedPerinatal mortality in 25-30% if untreated

11. Diagnosing Syphilis (Not in Newborns) Available serologic testingAvailable serologic testing RPR/VDRL: nontreponemal testRPR/VDRL: nontreponemal test Sensitive but NOT specificSensitive but NOT specific Quantitative, so can follow to determine disease activity and treatment responseQuantitative, so can follow to determine disease activity and treatment response MHA-TP/FTA-ABS: specific treponemal testMHA-TP/FTA-ABS: specific treponemal test Used for confirmatory testingUsed for confirmatory testing Qualitative, once positive always positiveQualitative, once positive always positive RPR/VDRL screen in ALL pregnant women early in pregnancy and at time of birthRPR/VDRL screen in ALL pregnant women early in pregnancy and at time of birth This is easily treated!!This is easily treated!!

12. Treatment Penicillin G is THE drug of choice for ALL syphilis infectionsPenicillin G is THE drug of choice for ALL syphilis infections Maternal treatment during pregnancy very effective (overall 98% success)Maternal treatment during pregnancy very effective (overall 98% success)

13. Rubella Single-stranded RNA virusSingle-stranded RNA virus Vaccine-preventable diseaseVaccine-preventable disease No longer considered endemic in the U.S.No longer considered endemic in the U.S. Mild, self-limiting illnessMild, self-limiting illness Infection earlier in pregnancy has a higher probability of affected infantInfection earlier in pregnancy has a higher probability of affected infant

14. “Blueberry muffin” spots representing extramedullary hematopoesis

15. Diagnosis Maternal IgG may represent immunization or past infection - Useless!Maternal IgG may represent immunization or past infection - Useless! Can isolate virus from nasal secretionsCan isolate virus from nasal secretions Less frequently from throat, blood, urine, CSFLess frequently from throat, blood, urine, CSF Serologic testingSerologic testing IgM = recent postnatal or congenital infectionIgM = recent postnatal or congenital infection Rising monthly IgG titers suggest congenital infectionRising monthly IgG titers suggest congenital infection Diagnosis after 1 year of age difficult to establishDiagnosis after 1 year of age difficult to establish

17. Treatment Prevention…immunize, immunize, immunize!Prevention…immunize, immunize, immunize! Supportive care only with parent educationSupportive care only with parent education

18. Cytomegalovirus (CMV) Most common congenital viral infectionMost common congenital viral infection ~40,000 infants per year in the U.S.~40,000 infants per year in the U.S. Mild, self limiting illnessMild, self limiting illness Transmission can occur with primary infection or reactivation of virusTransmission can occur with primary infection or reactivation of virus

19. Clinical Manifestations 90% are asymptomatic at birth!90% are asymptomatic at birth! Up to 15% develop symptoms later,Up to 15% develop symptoms later, Symptomatic infectionSymptomatic infection HSM, petechiae, jaundice, neurological deficitsHSM, petechiae, jaundice, neurological deficits >80% develop long term complications>80% develop long term complications Hearing loss, vision impairment, developmental delayHearing loss, vision impairment, developmental delay

20. Diagnosis Maternal IgG shows only past infectionMaternal IgG shows only past infection Infection common – this is uselessInfection common – this is useless Viral isolation from urine or saliva in 1 st 3weeks of lifeViral isolation from urine or saliva in 1 st 3weeks of life Viral load and DNA copies can be assessed by PCRViral load and DNA copies can be assessed by PCR Less useful for diagnosis, but helps in following viral activity in patientLess useful for diagnosis, but helps in following viral activity in patient Serologies not helpful given high antibody in populationSerologies not helpful given high antibody in population

21. Herpes Simplex (HSV) HSV1 or HSV2HSV1 or HSV2 Primarily transmitted through infected maternal genital tractPrimarily transmitted through infected maternal genital tract

22. Clinical Manifestations Most are asymptomatic at birthMost are asymptomatic at birth 3 patterns of symptoms between birth and 4wks:3 patterns of symptoms between birth and 4wks: Skin, eyes, mouth (SEM)Skin, eyes, mouth (SEM) CNS diseaseCNS disease Disseminated disease (present earliest)Disseminated disease (present earliest)

23. Presentations of congenital HSV

24. Diagnosis Culture of maternal lesions if present at deliveryCulture of maternal lesions if present at delivery Cultures in infant:Cultures in infant: Skin lesions, oro/nasopharynx, eyes, urine, blood, rectum/stool, CSFSkin lesions, oro/nasopharynx, eyes, urine, blood, rectum/stool, CSF CSF PCRCSF PCR Serologies again not helpful given high prevalence of HSV antibodies in populationSerologies again not helpful given high prevalence of HSV antibodies in population

25. Treatment High dose acyclovir 60mg/kg/day divided q8hrsHigh dose acyclovir 60mg/kg/day divided q8hrs X21days for disseminated, CNS diseaseX21days for disseminated, CNS disease X14days for SEMX14days for SEM Ocular involvement requires topical therapy as wellOcular involvement requires topical therapy as well

26. Taxoplasmosis (Toxoplasma gondii Infection)

27. Toxoplasma gondii Worldwide Intracellular paradite.Worldwide Intracellular paradite. All parasite stages are infectious. All parasite stages are infectious. Domestic Cat is the Definitive Host Domestic Cat is the Definitive Host Infects animals (cattle, birds, rodents, pigs& sheep)and humans as Intermediate Hosts.Infects animals (cattle, birds, rodents, pigs& sheep)and humans as Intermediate Hosts.

28. Causes the disease Toxoplasmosis.Causes the disease Toxoplasmosis. Toxoplasmosis is leading cause of abortion in sheep and goats.Toxoplasmosis is leading cause of abortion in sheep and goats. Risking group: Pregnant women, meat handlers (food preparation) or anyone who eats the raw meat Toxoplasma gondii (Cont.)

29. Transmission Contaminated water or food by oocysts Undercooked infected meat. Mother to fetus. Organ transplant (rare). Blood transfusion (rare). Toxoplasma gondii

30. Tissue phase (intermediate hosts). Intermediate host gets infected by ingesting sporulated oocysts. Oocytes do not become infectious until they sporulate, sporulation occurs 1- 5 days after that the oocyte is excreted in the feces. Intermediate host Human, cattle, birds, rodents, pigs, and sheep.

31. CLINICAL MANIFESTATIONS Acute toxoplasmosis is usually asymptomatic and self-limited.Acute toxoplasmosis is usually asymptomatic and self-limited. In some case of acute toxoplasmosis cervical lymphadenopathy, headache, malaise, fatigue, and fever may appearIn some case of acute toxoplasmosis cervical lymphadenopathy, headache, malaise, fatigue, and fever may appear It causes sever complications in eyes and brains of infected new born babiesIt causes sever complications in eyes and brains of infected new born babies Toxoplasmosis causes repeated abortion in pregnant females Toxoplasmosis causes repeated abortion in pregnant females

32. Lab Diagnosis 1)Microscopic demonstration of the T. gondii organism in blood, body fluids, or tissue. 2)Detection of T. gondii antigen in blood or body fluids by ELISA technique. 3)Serological diagnosis for antibodies by  Sabin-Feldman dye test  IHA  ELISA  IFAT  Latex agglutination Test All measure circulating antibodies to Toxoplasma.

33. Lab Diagnosis (Cont.) 6)Polymerase Chain Reaction (PCR) on body fluids, including CSF, amniotic fluid, and blood. 7)Skin test results showing delayed skin hypersensitivity to Toxoplasma gondii antigens. 8)Antibody levels in aqueous humor or CSF may reflect local antibody production and infection. 9)Animal inoculation: inoculation of suspected infected tissues into experimental animals. 10)Culture: inoculation of suspected infected tissues into tissue culture.

34. Sabin-Feldman dye test Live virulent tachyzoites of T gondii are used as antigenLive virulent tachyzoites of T gondii are used as antigen The parasites are mixed with dilutions of the test serum + complement obtained from Toxoplasma-antibody free- human serum + Methylene blue dye.The parasites are mixed with dilutions of the test serum + complement obtained from Toxoplasma-antibody free- human serum + Methylene blue dye. After one hour incubation at 37 o C the parasites are examined microscopically for dye stainingAfter one hour incubation at 37 o C the parasites are examined microscopically for dye staining organisms are lysed if the patient has T gondii-specific IgG antibody and they do not stained with the dyeorganisms are lysed if the patient has T gondii-specific IgG antibody and they do not stained with the dye Parasites stained with dye NegativeParasites stained with dye Negative This test is sensitive and specific for toxoplasmosis.This test is sensitive and specific for toxoplasmosis. It is available mainly in reference laboratoriesIt is available mainly in reference laboratories A negative test result practically rules out prior T gondii exposureA negative test result practically rules out prior T gondii exposure Its main disadvantages areIts main disadvantages are  high cost  human hazard of using live organisms.

35. SABIN –FELDMAN DYE TEST Live tachyzoites +Complement+Test serum Methylene Blue Dye Methylene Blue Dye Incubation at 37 0 C for one hr. +ve -ve +ve -ve If Abs are present If Abs are absent If Abs are present If Abs are absent <50% of tachyzoites 90-100 % <50% of tachyzoites 90-100 % do not stain. tachyzoites Stain do not stain. tachyzoites Stain

36. indirect fluorescent antibody test (IFAT) Overcomes some of the disadvantages of the dye test.Overcomes some of the disadvantages of the dye test. In IFAT, killed tachyzoites of Toxoplasma, which are available commercially, are used as antigen.In IFAT, killed tachyzoites of Toxoplasma, which are available commercially, are used as antigen. Titers obtained by IFAT are similar to those from the dye test.Titers obtained by IFAT are similar to those from the dye test. Disadvantages of the IFAT areDisadvantages of the IFAT are  Fluorescent microscope is needed, fluorescent  false-positive titers may occur in hosts with anti- nuclear antibodies.

37. indirect fluorescent antibody test (IFAT)

38. Other serologic tests including the hemagglutination test, the latex agglutination test and ELISA offer some advantages.Other serologic tests including the hemagglutination test, the latex agglutination test and ELISA offer some advantages. For example, agglutination tests are easy to perform and cheap.For example, agglutination tests are easy to perform and cheap.

39. Agglutination IgG test This test uses formalin-preserved whole tachyzoites to detect IgG antibody.This test uses formalin-preserved whole tachyzoites to detect IgG antibody. It is sensitive to IgM antibodies, which can cause a nonspecific agglutination in seraIt is sensitive to IgM antibodies, which can cause a nonspecific agglutination in sera This problem is avoided by pretreatment of samples with 2-mercaptoethanol.This problem is avoided by pretreatment of samples with 2-mercaptoethanol. This method is simple, relatively inexpensive, and excellent for screening pregnant patients,This method is simple, relatively inexpensive, and excellent for screening pregnant patients, It should not be used to measure IgM antibodies specific for T gondii. It should not be used to measure IgM antibodies specific for T gondii.

40. Toxoplasmosis IHA Test APPLICATION: To detect Toxoplasma IgM antibodies by indirect haemagglutination test.APPLICATION: To detect Toxoplasma IgM antibodies by indirect haemagglutination test. The reagent for this test consisted of stabilized human red cells coated with a Toxoplasma gondii heat-stable alkaline-solubilized extractThe reagent for this test consisted of stabilized human red cells coated with a Toxoplasma gondii heat-stable alkaline-solubilized extract react predominantly with IgM antibodies found in serum samples from patients with a recent infectionreact predominantly with IgM antibodies found in serum samples from patients with a recent infection INTERPRETATION OF RESULTS:INTERPRETATION OF RESULTS: Results will be reported as:Results will be reported as:  Positive  Doubtful  Negative Doubtful results should be retested within 2 weeks.Doubtful results should be retested within 2 weeks. In ocular Toxoplasmosis, titres of antibodies may be very low.In ocular Toxoplasmosis, titres of antibodies may be very low.

41. Toxoplasma IgM Elisa APPLICATION: For measurement of the IgM antibodies to toxoplasma gondii in human serum and plasma to aid in the diagnosis of primary infection.APPLICATION: For measurement of the IgM antibodies to toxoplasma gondii in human serum and plasma to aid in the diagnosis of primary infection. INTERPRETATION OF RESULTS:INTERPRETATION OF RESULTS: A.Negative:< 0.500 (arbitrary units) B.Equivocal:0.500 - 0.599 C.Positive:≥ 0.600. This applies to the diagnosis of Acute T. gondii infection acquired during pregnancy

42. Diagnosis of acute infection with T. gondii can be established by detection of the presence of IgG and IgM antibody to Toxoplasma in serum.Diagnosis of acute infection with T. gondii can be established by detection of the presence of IgG and IgM antibody to Toxoplasma in serum. The presence of circulating IgA favors the diagnosis of an acute infection.The presence of circulating IgA favors the diagnosis of an acute infection. Maternal IgG testing indicates past infection (but when…?)Maternal IgG testing indicates past infection (but when…?) The parasite can be isolated in culture from placenta, umbilical cord, infant serumThe parasite can be isolated in culture from placenta, umbilical cord, infant serum PCR testing on WBC, CSF, placentaPCR testing on WBC, CSF, placenta Not standardizedNot standardized COMMENTS

43. Comments Persisting IgM levels may be detected long after the onset of acquired infectionPersisting IgM levels may be detected long after the onset of acquired infection Thu,s the use of a single serological test result must be used with caution in those cases when it is critical to establish the time of infection.Thu,s the use of a single serological test result must be used with caution in those cases when it is critical to establish the time of infection. This applies to the diagnosis of Acute T. gondii infection acquired during pregnancyThis applies to the diagnosis of Acute T. gondii infection acquired during pregnancy

44. Treatment Treatment of cases with acute toxoTreatment of cases with acute toxo Spiramycin aantibiotic dailySpiramycin aantibiotic daily

45. Study Questions: Write a short note on:Write a short note on: Diagnostic methods for CMV.

46. Assignment Diagnostic methods for ToxoplasmosisDiagnostic methods for Toxoplasmosis روان رزق – ريوان ابراهيم – فاطمة الزهراء – منى يحيى – نجاتو عثمان

47. Thanks