الله بسمالرحيم الرحمن

Faculty of Allied Medical Sciences

Clinical Immunology & Serology Practice

(MLIS 201)

TORCH

Prof. Dr. Ezzat M HassanProf. of ImmunologyMed Res Inst, Alex UnivE-mail: elgreatlyem@hotmail.com

Objectives

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

TORCH Infections

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

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

TORCH - panel (IgM & IgG)

• Toxoplasma• Rubella• Cytomegalo virus• Herpes

• IgM - Acute or Recent infection• IgG - Chronic infection

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…

Syphilis• Treponema pallidum (spirochete)• Transmitted via sexual contact• Placental transmission as early as 6wks

gestation

Clinical Manifestations

• Fetal:• Stillbirth• Neonatal death• Hydrops fetalis

• Intrauterine death in 25%• Perinatal mortality in 25-30% if

untreated

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!!

Treatment• Penicillin G is THE drug of choice for ALL

syphilis infections• Maternal treatment during pregnancy very

effective (overall 98% success)

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

“Blueberry muffin” spots representingextramedullary hematopoesis

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

Treatment

• Prevention…immunize, immunize, immunize!

• Supportive care only with parent education

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

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

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

Herpes Simplex (HSV)

• HSV1 or HSV2• Primarily transmitted through infected

maternal genital tract

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)

Presentations of congenital HSV

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

Treatment

• High dose acyclovir 60mg/kg/day divided q8hrs• X21days for disseminated, CNS disease• X14days for SEM

• Ocular involvement requires topical therapy as well

Taxoplasmosis

(Toxoplasma gondii Infection)

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.

• 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

Toxoplasma gondii (Cont.)

Transmission Contaminated water or food by oocystsUndercooked infected meat.Mother to fetus.Organ transplant (rare).Blood transfusion (rare).

Toxoplasma gondii

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.

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

Lab Diagnosis1) 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 TestAll measure circulating antibodies to Toxoplasma.

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.

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.

SABIN –FELDMAN DYE TEST

Live tachyzoites +Complement+Test serum Methylene Blue 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

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.

indirect fluorescent antibody test (IFAT)

• 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.

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.

Toxoplasmosis IHA 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 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.

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.

• INTERPRETATION OF RESULTS:A. Negative : < 0.500 (arbitrary units)B. Equivocal : 0.500 - 0.599C. Positive : ≥ 0.600.

This applies to the diagnosis of Acute T. gondii infection acquired during pregnancy

• 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 IgG testing 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

COMMENTS

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

Treatment• Treatment of cases with acute toxo

• Spiramycin aantibiotic daily

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

Assignment

• Diagnostic methods for Toxoplasmosis

عثمان – – – – نجاتو يحيى منى الزهراء فاطمة ابراهيم ريوان رزق روان

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