Macrophage Phagocytic System Sporozoa Toxoplasma gondii Prof. Dr.Nashwa Salah Eldin

Goals of Lecture (ILOs) Toxoplasma Goals of Lecture (ILOs) Epidemiology Transmission Morphology &Life Cycle Clinical Features Immunity Diagnosis Treatment Prevention

(No organ of locomotion) Classification of the causative parasite Protozoa (Pseudopodia) Amoebae ( Entamoeba) Free living Amoebae (Naeglaria-Acanthamoeba) Intestinal…… Giardia - Blood……. Leishmania- Trypanosoma Atrial…….Trichomonas (Flagella) (Cilia) Balantidium Plasmodium - Babesia Toxoplasma Sarcocystis Cryptosporidium - Isospora (No organ of locomotion) Apicomplexa

According to locomotion Protozoa Sarcomastigophora Sarcodina (Pseudopodia) Mastigophora (Flagella) Ciliophopra (Cilia) Apicomplexa According to locomotion

Cryptosporidium - Isospora Phylum Apicomplexa Class Sporozoea Subclass Coccidia Plasmodium- Babesia Toxoplasma gondii Sarcocystis Cryptosporidium - Isospora

Characteristics of phylum Apicomplexa 1- No organs of locomotion. 2- Presence of apical complex seen by EM. 3- Presence of two-phase life cycle: ( alternation of generations) asexual cycle and sexual cycle which ends by the formation of the infective forms. Life cycle has 3 phases: Merogony (Schizogony)….schizont… ASEXUAL Gametogony… Gametes… ASEXUAL Sporogony…. Sporozoite … SEXUAL

Apicomplexan ultrustructure Apical complex: an assemblage of cytoskeletal elements and secretory organelles that helps the parasite to peneterate host cells

Phylum :Apicomplexa according to the habitat Intestinal (Cryptosporidium, Cystoisospora,Sarcocystis) Tissue (Toxoplasma, Sarcocystis) Blood (Plasmodium, Babesia)

Habitat and hosts Habitat: Toxoplasma is an obligate intracellular parasite that can invade any nucleated cell. Definitive host: Cats and other felines (harbor both sexual and asexual stages). Accidental hosts or non-specific hosts (I.H): Man, all mammals & birds (harbor only asexual stages) Man is an end-stage I.H

Morphological forms In pseudocyst in tissue cyst in oocyst They are cresentic in shape with a pointed anterior end with a conoid structure .The posterior end is rounded . Nucleus is central. ( 6×3 µ) Trophozoite Tachyzoite Bradyzoite Sporozoite In pseudocyst in tissue cyst in oocyst

Pseudocysts (parasitophorous vacuole): are macrophages or any other nucleated cell type containing several (up to 100) rapidly multiplying tachyzoites without a true cyst wall. They are seen during acute infection.

Tissuue cysts are rounded Tissuue cysts are rounded . They have well defined cyst wall and contain thousands of slowly multiplying bradyzoites (size is about 100 µ ). They are seen during chronic infection, mainly present in muscles, nervous tissues and visceral organs...

Oocysts (about 10 µ) are broadly oval ,Contain 2 round sporocysts , each containing 4 sporozoites. Are present in stool of cats. They pass immature and need few days to become infective….. Remain infective for months.

Life cycle of Toxoplasma gondii

In definitive hosts Asexual and sexual cycles take place intra-cytoplasmic in epithelial cells of the small intestine.

In accidental hosts: Only asexual development occurs: when oocysts or tissue cysts are ingested their walls dissolve tachyzoite stages peneterate intestinal wall disseminate all over the body via lymph and blood (multiply by endodyogony) with formation of pseudocysts and tissue cysts in their organs multiplication occurs according to virulence of the parasite and immunity of the host.

Infective stages and modes of infection Cats are infected by: Oocysts in soil. Tissue cysts or pseudocysts in rodents. Man and accidental hosts are infected by: Contaminated water or food by cat’s stool (oocysts). Undercooked meat (tissue cysts). Transplacental from mother to fetus. Organ transplant (rare). pseudocysts Blood transfusion (rare). N.B. All forms of the parasite are infective.

Toxoplasmosis

Epidemiology Toxoplasmosis the most prevalent zoonotic disease is found throughout the world (except extremely cold or dry climates) and tends to be more prevalent in tropical climates. Serologic prevalence rates in Egypt is around 30%. Consuming raw/undercooked meat (beef, pork& lamb) increases the risk of acquiring infection. Fresh meat is more risky as since deep freezing for several days kills most tissue cysts.

Pathogenesis and Clinical picture I.P is from 1-3 weeks Toxoplasmosis is usually minor and self- limiting but can have serious or even fatal effects on a fetus whose mother first contracts the disease during pregnancy or on an immunocompromised human. = OPPORTUNISTIC parasite

1-In immunocompetent patients: •Usually asymptomatic •Generalized symptoms: Fever Headache Anorexia Malaise (Flu- like) -Lymphadenopathy (IMN-like) - Fever+ hepatomegaly+ maculopapular rash (typhus-like) - Fever of unknown origin (FUO).

2- In HIV patients & immunosuppresed patients: Remember : Toxoplasma is an opportunistic pathogen May be newly acquired or due to reactivation of latent infection Encephalitis (most common) •Pneumonia •Chorioretinitis •Disseminated disease

3- Congenital Toxoplasmosis (3rd trimester) : Infection in first or second trimeseter results in abortion or stillbirth Is toxoplasmosis a cause of recurrent abortion? It occurs only when the mother is exposed to primary infection while she is pregnant

Manifestations of congenital toxoplasmosis: Acute Apparently healthy and may remain so for life Latent and develop manifestations later in life

Manifestations of acute congenital toxoplasmosis: Classic triad •Chorioretinitis •Hydrocephalus •Intracranial calcifications General signs •Hepatosplenomegaly & neonatal jaundice •Lymphadenopathy •Fever •Anemia and Thrombocytopenia •Apparently healthy newborn with development of chorioretinitis or mental retardation later in life

4- Ocular toxoplasmosis: May result from congenital or post-natally acquired infection. May occur in immunocompetent or immunocompromized persons. May be through acute or reactivation of latent infection. In the form of Chorioretinitis.

Differential Diagnosis Congenital toxoplasmosis: Other congenitally transmitted infectious agents (Syphilis, Rubella, CMV, & Herpes). TORCH Ocular toxoplasmosis: CMV retinitis & toxocariasis. Other forms: From causes of lymphadenopathy. From causes of fever and hepatomegaly. From causes of encephalitis.

Diagnosis of toxoplasmosis Clinical Imaging Laboratory Direct Indirect

Direct laboratory diagnostic methods 1- Isolation of T. gondii tachyzoites, pseudocysts from blood or body fluids (CSF, amniotic fluid) establishes that the infection is acute Stained smear. Detection of tissue cysts in biopsies (e.g. lymph nodes) Stained sections. 2- Isolation of the parasite can be performed by mouse inoculation intra-peritoneal injection after 2-3 weeks examination of peritoneal fluid for tachyzoites and pseudocysts or brain tissues for cysts). 3- Tissue culture It is often difficult to demonstrate tachyzoites in conventionally stained tissue sections . It is rarely used, may be in immunosuppressed patients.

Direct laboratory diagnostic methods 4- Antigen detection: in serum and body fluids is helpful especially in immunocompromized patients in which antibody titer is low or absent. 5- Molecular diagnostic methods (PCR): detection of parasite nucleic acids. PCR amplification is used to detect T. gondii DNA in body fluids (cerebrospinal fluid (CSF), vitreous and aqueous fluid, bronchoalveolar lavage (BAL) fluid, urine, amniotic fluid and peripheral blood. It has been successfully used to diagnose congenital, ocular, cerebral and disseminated toxoplasmosis.

Indirect diagnostic methods The use of serologic tests to demonstrate specific Toxoplasma antibodies is the initial method of diagnosis. 1-Sabin Feldman methylene blue dye test 2- Indirect haemagglutination test (IHAT) 3- Indirect Immuno-fluorescent test (IFAT) 4- Enzyme-linked immunosorbent assay (ELISA)

Sabin-Feldman Dye Test (DT) The DT is a sensitive and the most specific test . live organisms + patient's serum + complement + blue dye IgG T. gondii-specific antibody lyse the trophozoites so they do not take the blue stain of methylene blue = +ve reaction In contrary, in negative reaction the parasite stained blue. Its main disadvantages are its high cost and the human hazard of using live organisms.

Indirect haem-agglutination test (IHAT) Specific haemagglutination antibodies are formed later than that of the dye test. It is not a sensitive test for diagnosis of congenital infection or recent infections in general. It measures IgG antibodies.

Indirect fluorescent antibody test (IFAT) •It measures specific IgG, IgM, IgA. •In IFAT, the commercially available killed tachyzoites of Toxoplasma are used as antigen. Disadvantages of the IFAT are that a microscope with UV light is needed. +ve -ve

Enzyme linked immunosorbent assay (ELISA) It measures specific IgG, IgM, IgA Other tests: - Double sandwich IgM ELISA Immunosorbent agglutination assay IgM- ISAGA IgG avidity

Interpretation of the serological tests IgG antibodies usually: •Appear within 1 to 2 weeks of the infection •Peak within 1 to 2 months •Fall at variable rates, and usually persist for life -Can pass the placenta to the foetus. - Positive IgG indicate exposure to infection. -The titer does not correlate with the severity of illness.

IgM,IgA and IgE are formed soon after exposure and disappear earlier than IgG. Positive IgM and IgA indicate recent infection In patients with recently acquired infection, IgM T. gondii antibodies are detected initially and, in most cases, these titers become negative within a few months .

Serologic evidence for an acute acquired infection IgG titers or total antibody rise by a factor of 4 to 16 in serum taken 2 to 4 weeks after the initial serum collection. Or IgG-avidity test The finding of antibody in even undiluted serum is useful in the diagnosis of ocular toxoplasmosis because patients with this disorder usually have low T gondii antibody titers . or when specific IgM antibody is detected.

IgG-avidity index (test): Avidity is commonly applied to antibody interactions in which multiple antigen-binding sites simultaneously interact with the target antigenic epitope. IgG-avidity could be measured by ELISA, IFAT or other assays. It differentiates between acute and chronic infections at first during acute infection the avidity is low then it becomes high with chronicity.

Diagnosis of congenital infection: IgM and IgA are important markers of infection in the newborn. IgG can cross the placenta from mother to foetus, so it is not a marker for transmission of infection

Imaging diagnostic methods Plain x ray on the skull ……… intracraneal calcifications. - CT and MRI for toxoplasmic encephalitis.

Treatment of toxoplasmosis Indications of treatment: Symptomatic patients. Immuno-suppressed patients: for 4-6 weeks after symptoms subside followed by prophylaxis as long as immuno-suppression lasts. Asymptomatic congenitally infected children under 5 for 4-6 weeks to avoid latent eye affection. Symptomatic congenitally infected children for one year. Pregnant women with seroconversion.

Drug therapy: Pyrimithamine ( folic acid antagonist) 25-50 mg orally/day + Triple sulfa (Sulfadiazine, sulfamethazine, sulfamerzaine). 2-6 gm daily for one month. Pyrimethamine is teratogenic and should not be given in the first trimester of pregnancy. Folinic acid 15 mg twice weekly or yeast tablets to overcome thrombocytopenia and leucopenia which are side effects of the above treatment Corticosteroides is given in addition in ocular toxoplasmosis to minimize the inflammatory reaction

Treatment of pregnant females: Spiramycin (Rovamycin) 2-3 gm /day for one month and repeated every 2 weeks all through pregnancy. It is indicated in the first trimester to avoid the teratogenisty of Pyrimethamine.

Prevention and control Newly married and pregnant women Pet cat care General Measures

Prevention and control General Measures: oocysts - Wear gloves when handling soil. - Peel or carefully wash all fruits and vegetables. Tissue cysts - Fully cook all meats. Wash hands with soap and water after handling meat. deep freezing for several days. Pseudocysts or tachyzoites: Proper screening of blood donors. Proper screening of organs before transplantation.

Pet cat care: Wear gloves when changing cat litter. •Wash hands carefully after changing litter box . •litter box should be changed frequently (at least every 2 days) so that the cysts don't have time to sporulate. •Keep cat inside and avoid strays. •Use only commercial or cooked cat’s food.

Newly married and pregnant women: If serologically positive they are immune. If serologically negative and got pregnant they should: 1- avoid intimate contact with cats. 2- take care while handling and cutting meat. 3- retested every 2-3 months to detect seroconversion . 4-they should be counseled on prophylaxis ttt if seroconverted.

A 25y old female returns home from France with a diagnosis of toxoplasmosis. She reports that she developed fever, sore throat, muscle aching and headache. Her physician noted diffuse adenopathy and an enlarged spleen. Testing showed an elevated IgM and IgG antibody for Toxoplasma gondii. Each of the following but one is correct regarding toxoplasmosis. Which statement is WRONG? She may have contracted this parasite by ingesting food or water contaminated with cat feces containing oocysts. b. It would be important to do a pregnancy test since contracting toxoplasmosis during pregnancy may result in fetal infection and birth defects. c. She may have contracted this parasite by eating undercooked pork or beef. d. She should not be allowed around her pregnant sister to avoid giving her the illness during the pregnancy. e. She would be at increased risk of developing Toxoplasma encephalitis should she become immunocompromised even years later.

Interpret ( Give reason): 1. You can’t depend on measuring IgG to diagnose congenital toxoplasmosis. 2. It is advised to repeat the high tittered T. gondii test after 2-3 weeks before confirming your diagnosis. 3. Pyrimithamine is forbidden in the 1rst trimester. 4. Most probably, repeated abortion isn’t a symptom of toxoplasmosis.

Thank you