بسم الله الرحمن الرحيم

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 TORCH • To know the causes of TORCH Infection • Describe the diagnostic methods for TORCH

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

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

7. TORCH - panel (IgM & IgG) • Toxoplasma • Rubella • Cytomegalo virus • Herpes • IgM - Acute or Recent infection • IgG - Chronic infection

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

9. Syphilis • Treponemapallidum (spirochete) • Transmitted via sexual contact • Placental transmission as early as 6wks gestation

10. Clinical Manifestations • Fetal: • Stillbirth • Neonatal death • Hydrops fetalis • Intrauterine death in 25% • Perinatal mortality in 25-30% if untreated

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

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

13. Rubella • Single-stranded RNA virus • Vaccine-preventable disease • No longer considered endemic in the U.S. • Mild, self-limiting illness • Infection 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! • Can isolate virus from nasal secretions • Less frequently from throat, blood, urine, CSF • Serologic testing • IgM = recent postnatal or congenital infection • Rising monthly IgG titers suggest congenital infection • Diagnosis after 1 year of age difficult to establish

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

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

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

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

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

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

23. Presentations of congenital HSV

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

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

26. Taxoplasmosis(Toxoplasma gondii Infection)

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

28. Toxoplasma gondii (Cont.) • Causes the disease Toxoplasmosis. • 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

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

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

31. CLINICAL MANIFESTATIONS • Acute toxoplasmosis is usually asymptomatic and self-limited. • In 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 babies • Toxoplasmosis causes repeated abortion in pregnant females

32. Lab Diagnosis • Microscopic demonstration of the T. gondii organism in blood, body fluids, or tissue. • Detection of T. gondii antigen in blood or body fluids by ELISA technique. • 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.) • Polymerase Chain Reaction (PCR) on body fluids, including CSF, amniotic fluid, and blood. • Skin test results showing delayed skin hypersensitivity to Toxoplasma gondii antigens. • Antibody levels in aqueous humor or CSF may reflect local antibody production and infection. • Animal inoculation: inoculation of suspected infected tissues into experimental animals. • Culture: inoculation of suspected infected tissues into tissue culture.

34. Sabin-Feldman dye test • Live 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. • After one hour incubation at 37o 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 dye • Parasites stained with dye Negative • This test is sensitive and specific for toxoplasmosis. • It is available mainly in reference laboratories • A negative test result practically rules out prior T gondii exposure • Its main disadvantages are • high cost • human hazard of using live organisms.

35. SABIN –FELDMAN DYE TEST Live tachyzoites+Complement+Testserum MethyleneBlue Dye Incubation at 370 C for one hr. +ve -ve If Abs are present If Abs are absent <50% of tachyzoites 90-100 % do not stain . tachyzoites Stain

36. indirect fluorescent antibody test (IFAT) • Overcomes some of the disadvantages of the dye test. • 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. • Disadvantages 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. • 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. • It 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 method is simple, relatively inexpensive, and excellent for screening pregnant patients, • It should not be used to measure IgM antibodies specific for T gondii.

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

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

42. Comments • 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. • Maternal IgGtesting indicates past infection (but when…?) • The parasite can be isolated in culture from placenta, umbilical cord, infant serum • PCR testing on WBC, CSF, placenta • Not standardized

43. Comments • Persisting 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. • This applies to the diagnosis of Acute T. gondii infection acquired during pregnancy

44. Treatment • Treatment of cases with acute toxo • Spiramycinaantibiotic daily

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

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

47. Thanks