TOXOPLASMOSIS AND PREGNANCY

PARASITOLOGY LABORATORIUM MEDICAL FACULTY JEMBER UNIVERSITY

History
Toxoplasma gondii is an obligate intracellular protozoan . It derives its name from a North African rodent the gondi, from which it was first isolated in 1908 . First case of a congenitally infected human baby was reported in 1923 Until 1969, life cycle of parasite was fully elucidate with the discovery of its definitive host, cats and other felines.

Toxoplasma gondii
Toxoplasmosis is the result of infection by Toxoplasma gondii, an obligate intracellular protozoan parasite in the phylum Apicomplexa. The major forms of the parasite are: Oocysts (containing sporozoites), which are shed in the feces. Tachyzoites, rapidly multiplying organisms found in the tissues. Bradyzoites, slowly multiplying organisms found in the tissues. Tissue cysts: walled structures, often found in the muscles and central nervous system (CNS), containing dormant T. gondii bradyzoites.

Oocyst
Oocyst are formed as a results of fertilization between male and female gametocytes and are found in the epithelial cells of the intestines of definitive host They are oval and 10-12 m in diameter Oocysts are excreted in the faeces of the cat, contamination with which results in human infection.

Oocyst of Toxoplasma gondii

Trophozoites/Tachyzoite

 Trophozoites crescent shaped with one pointed end and the other rounded end, and measure approximately 37m released from the ingested oocysts, invade epithelial cells of the intestinal tract of the host Disseminate via blood and lymph to most of the organs. Invade all mammalian cells except nonnucleated erythrocytes and are found extracellulary as well as intracellularly in various organs. multiply in a host cell by a process known as endodyogeny or internal budding. The rapidly proliferating trophozoites, as known as tachyzoites. The trophozoites are either eliminated by the immune system of the host or by a drug or they are transformed into cysts.

THE TACHYZOITE
Crescent shaped 2 by 6 m Asexual form Multiplies by endodyogeny It can infect phagocytic and nonphagocytic, nucleated cells. Organelles: pellicle, apical rings,
polar rings, conoid, rhoptries, micronemes, dense granules, apicoplast , acidocalcisome, micropore, subpellicular microtubules, mitochondrion, endoplasmic reticulum, Golgi complex, ribosomes, rough and smooth endoplasmic reticulum, nucleus.

M. Black and J. C. Boothroyd (2000). The lytic cycle of Toxoplasma gondii. Microbiology and Molecular Biology Reviews, 64 (3) p. 607

Diagram of endodyogeny
As a parasite begins to divide, two IMCs begin to develop in the middle of the cell. As the IMC extends from these structures, the nucleus (N) and mitochondrion (Mitoch.) divide into these membranous outlines. Nascent apical organelles (NO) develop within the anterior poles as the daughter cells grow. Eventually, the entire cytoplasm is divided between the daughters and the IMC of the mother dissociates. A cleavage furrow divides the cells from the anterior pole. This division continues down the length of the cells until it reaches the posterior end, where it can leave a residual body connecting the two daughters.
M. Black and J. C. Boothroyd (2000). The lytic cycle of Toxoplasma gondii. Microbiology and Molecular Biology Reviews, 64 (3) p. 607

Tissue cysts
Tissue cysts are 10-100 in diameter and contain thousands of slowly multiplying forms of the parasite, aptly known as bradyzoites. Formed within the host cells are early as 7 days after the entry of trophozoites. Predominantly found in heart and skeletal muscles and central nervous system. Known to persist within the tissue for the entire life span of the host and are responsible for the recrudescence of the infection, specially in immuno-compromised hosts.

TISSUE CYST
A highly stretched tissue cyst with more than 1,000 bradyzoites in an impression smear of brain homogenate from a rat 14 months after infection with the VEG strain of T. gondii. The cyst wall (arrow) is

barely visible.

Dubey, J.P., Lindsay, D.S. and Speer, C.A. (1998). Structures of Toxoplasma gondii tachyzoites, bradyzoites, and sporozoites and biology and development of Tissue Cysts. Clinical Microbiology Reviews, 11: 267-299.

PARASITOLOGIC CONSIDERATIONS
T. gondii has two of hosts: a definitive host - like cats and other felines an intermediate host - man and other . It multiplies by sexual reproduction in the definitive hosts, and by asexual multiplication in definitive as well as intermediate hosts.

 Oocysts are highly resistant to environmental conditions and can remain infectious for as long 18 months in water or warm, moist soils. They do not survive well in arid, cool climates. Tissue cysts can remain infectious for weeks in body fluids at room temperature, and in meat for as long as the meat is edible and uncooked. Tachyzoites aremore fragile and can survive in body fluids for up to a day and in whole blood for as longas 50 days at 4C.

LIFE CYCLE

Source of infection
Domestic cats
Rodents (Rats) Contaminated soil (persist in environment if moist) Farm animal (Cow, goat, sheep, rabbits)

Transmission of the infection

Occur as a result of ingestion of occysts excreted in the faeces of cats or by ingestion of udercooked meat harbouring tissue cysts. Approximately 5-35% of pork, 9-60% of lamb and 0-9% of beef contain T. gondii. Trans-placental transmission ot foetus from a mother infected during pregnancy is also a common occurrence. In days of modern medicine, the odes of transmission have attained a new perspective, like blood transfusion, leucocyte transfusion and organ transplantation.

TRANSMISSION

A Diagram showing how infection is transmitted to Non-Immune Individuals

Immunity to Toxoplasma gondii

Active infection normally occur once The risk is only from an infection caught for the first time during pregnancy, or 2-3 months before conception. Acquired immunity is life long.

Parasite remains in body as latent infection in the form of cyst in skeletal muscle, cardiac muscle and brain. Usually inactive and harmless. Reactivation occurs only in immunocomproised patients- Chemotherapy, Corticosteroid therapy, Congenital immunodeficiendy deficiency, HIV/AIDS, Patient having organ transplant

Disinfection
oocysts are resistant to most disinfectants but can be inactivated by iodine, formalin and ammonia. Can be destroyed within 10 minutes by temperatures greater than 66C (150F), and can be killed with boiling water. Tachyzoites and tissue cysts are susceptible to most disinfectants, including l% sodium hypochlorite and 70% ethanol. Tachyzoites are also inactivated at pH < 4.0. Freezing at 15C for more than three days or 20C for more than 2days destroys a high percentage of the cysts.

Geographic Distribution
Toxoplasmosis is found worldwide. Infections are particularly common in warm,humid climates and at lower altitudes. Acc. to one estimate, over 500 million humans around the world are infected with T. gondii. App 90% of the population in France, 20 to 70% in the in the USA and 30% in the U K, 1.4% - 27% in India are infected Studies shown that 30% of 30-yr-olds and 50% of 70-year-olds have had a toxoplasmosis infection It is estimated that only 15% of women booking in for antenatal care are already immune. This leaves 85% of pregnant women still at risk of contracting the infection - Tommy's, the baby charity

Incubation Period

10 to 23 days after ingesting contaminated meat, 5 to 20 days after exposure to infected cats.

Clinical Signs In immunocompetent non-pregnant individuals,

usually asymptomatic. App 10-20% develop lymphadenitis or a mild, flulike syndrome characterized by fever, malaise, myalgia, headache, sore throat, lymphadenopathy and rash. In some cases, may mimic infectious mononucleosis symptoms usually resolve without treatment within weeks to months, although some cases may take up to a year. Severe symptoms, including myositis, myocarditis, pneumonitis and neurologic signs including facial paralysis, severe reflex alterations, hemiplegia and coma, are possible but rare. Ocular toxoplasmosis with uveitis, often unilateral, can be seen in adolescents and young adults; this syndrome is often the result of an asymptomatic congenital infection or the delayedresult of a postnatal infection.

In immunosuppressed patients
Toxoplasmosis is often severe. Neurologic disease is the most common sign, particularly in reactivated infections. Symptoms are:
Encephalitis, Necrosis from multiplication of the parasite can cause multiple abscesses in nervous tissue, with the symptoms of a mass lesion. Chorioretinitis, myocarditis and pneumonitis

Toxoplasmosis during pregnancy

Tox is a part of TORCH syndrome. It is not a cause of habitual abortion. Only pregnent women with primary active infection leads to congenital tox. Development of active immunity once, protects subsequent pregnancies.

Rate of Transmission
Develop infection at least 6-9 months before pregnancy Pt immune rare transmission within 23 months before conception - 1% or below risk of transmission but a high risk of miscarriage The first trimester - 15% chance but Severity of disease in neonate is more Second trimester - 25% risk Third trimester - 65% chance but Severity of disease in neonate is less usually asymptomatic

Congenital toxoplasmosis
The consequences of the infection of foetus can be very different between subclinical to very serious. Abortion or still birth. Overt disease - Symptoms with classical triad. Hydrocephalus Intracranial calcification Chorioretinitis Sub clinical infection - Usually asymptomatic at birth Later on develops hearing defects, visual defects, mental retardation and learning disabilities, even severe, life-threatening infections later in life, if left untreated

 Up to 90 percent of infected babies appear normal at birth, 80 to 90 percent will develop sightthreatening eye infections months to years after birth. About 10 percent will develop hearing loss and/or learning disabilities, 60% of infected may suffer from Long Term Sequella

Contd
mild cases with only slightly diminished
vision. Ocular disease is usually bilateral; The most common symptom is chorioretinitis but strabismus, nystagmus and microphthalmia may also be seen. Infants infected late in gestation may have a fever, rash, hepatomegaly, splenomegaly, pneumonia or a generalized infection.

Diagnosis of toxoplasmosis
May be established by
Serological tests, Polymerase chain reaction (PCR), Histological demonstration of the parasite and/or its antigens (i.e. immunoperoxidase stain), Or isolation of the organism

Serodiagnosis
Presence of IgM antibodies or a four fold rise in IgG titres at 2-3 wks interval indicates a relatively recent infection. Significant levels of IgM antibodies indicate infection acquired within the past 3 months. IgG antibodies usually appear within 1 to 2 wks of infection, peak within 1 to 2 months, fall at variable rates, and usually persist for life The titer does not correlate with the severity of illness

Specific Toxoplasma Antibody Titre : Some interpretations Antibody Titre

IgM
IgM-IFA > 1:80
IgM-ELISA > 1:256

IgG

IFA/Dye IgG and IgM

IFA/Dye Titre < 1:1000
IgM-IFA/ELISA Negative

Single high titre Rising titers at

IFA > 1:1000 3 week interval

Recent Acute

Probably Recent Definite Recent

Exclude Recent

Infection

Acute Infection

Acute Infection

Acquired Infection

Serological Tests
IgM test
Determine recent infection or in the distant past. Significant potential of misinterpretation of +ve result, should be confirmed by other tests. Kits often have low specificity IgM antibodies can persist for months to more than one year. Persistence of these IgM antibodies does not appear to have any clinical relevance

IgA Antibodies
IgA antibodies may be detected in sera of acutely infected adults and congenitally infected infants using ELISA or ISAGA methods. May persist for many months to more than one year. Of little additional assistance for diagnosis of the acute infection in the adult. Has increased sensitivity of IgA assays over IgM assays hence useful for diagnosis of congenital toxoplasmosis

IgE Antibodies
Detectable by ELISA in sera of acutely infected adults, congenitally infected infants, and children with congenital toxoplasmic chorioretinitis. The duration of IgE seropositivity is less than with IgM or IgA antibodies and hence appears useful as an adjunctive method for identifying recently acquired infections

confirmatory test, the Toxoplasma Serological Profile (TSP)

TSP, differentiate between recently acquired and chronic infection, is superior to any single serological test. TSP consist of Sabin-Feldman Dye Test (DT) double sandwich IgM ELISA or IgM-immunosorbent (IgM-ISAGA) , IgA ELISA, IgE ELISA, and AC/HS test.

HYDROCEPHALUS

INTRACRANIAL CALCIFICATION

CHORIORETINITIS

Treatment

Non-pregnant women desirous of pregnancy:

If IgM is positive, they should conceive after it becomes negative and until IgG titres should be stable and less than or equal to 1:1000. IgM positive titres and rising IgG titres mean recent infection. If these women are asymptomatic, no treatment required.

Treatment of pregnant women

Spiramycin is drug of choice can be given before 26 weeks

no teratogenic effects If fetus is infected there can be replaced by or addition of

Sulfadiazine + pyrimethamine
Clindamycin or Dapson : If patient has sulfa drug allergy. Higher dose therapy, continue for 4-6 weeks. Lower dose maintenance therapy given there after :

Drug
Spiramycin

Toxo during pregnancy

Remarks
safest amongst the antitoxoplasma agents Pyrimethamine is teratogenic. used in pregnancy only if potential benefit justifies the risk to foetus.

6-9 MIU/day in 2 or 3 divided doses for 3 wks followed by 2 wks intervals until parturition Pyrimethamine After 16 weeks in combination pyrimethamine 25 with mg/day with sulphonamides sulphadiazine 1-2 gm gid alternating with the cycles of spiramycin 6-9 MIU/day for 3 weeks until parturition

Drug Spiramycin

Congenital toxoplasmosis

0.15-0.30 MIU/Kg/day for 12 months Pyrimethamine 15 mg/square metre or 1 mg/kg/day in combination (maximum 25 mg/day), with with sulphadiazine or sulphonamides trisulphapyrimidine, 50-100 mg/kg/day alternation with the cycles of spiramycin 0.15-0.3 MIU/kg/day for 3 wks for 12 months

Pyrimethamine and sulfadoxine

Sulfadoxine: The sulfonamides are structural analogues of para-aminobenzoic acid (PABA), and competitive inhibitors of dihydropteroate synthetase. Pyrimethamine: An antifolate, a highly selective and powerful competitive inhibitor of dihydrofolate reductase.

TOXOPLASMA
Pteridine + PABA
Dihydropteroate synthetase

Folate metabolism
HUMAN
sulfadoxine

Dihydropteroic acid
GLUTAMATE

Dihyrofolate
NADPH NADP Dihydrofolate reductase

pyrimethamine

Preformed dietary folates

NADPH NADP Dihydrofolate reductase

Tetrahydrofolate

Tetrahydrofolate

FAH4 cofactors
Thymidine Purines Methionine Glycine f-met-tRNA

FAH4 cofactors
Thymidine Purines Methionine Glycine f-met-tRNA

DNA

DNA RNA

PROTEINS

DNA

DNA RNA

PROTEINS

Parasites synthesize their own folic acid; unlike man, they cannot import preformed folic acid. Tetrahydrofolic acid is an essential cofactor for the synthesis of nucleic acid precursors, and some amino acids.

Avoiding toxoplasmosis
only eat meat which has been thoroughly cooked (i.e. with no trace of blood or pinkness) avoid raw cured meat, like Parma ham wash hands, chopping boards and utensils thoroughly after preparing raw meat wash all fruit and vegetables thoroughly to remove all traces of soil dont drink unpasteurised goats milk or eat dairy products made from it

 wear gloves when gardening and wash hands and gloves afterward
if you eat while gardening wash your hands first, and try to avoid gardening in areas which may have been soiled with cat faeces cover childrens sandpits to prevent cats using them as litter boxes remove faeces from cat litter tray every day wearing rubber gloves and wash gloves and hands afterwards or have someone else do this do not handle lambing ewes and do not bring lambs into the house

Conclusion
Toxoplasmosis remains a serious disease although recent advances in diagnosis and treatment have greatly ameliorated the prognosis for the affected infants. Routine screening is currently controversial If IgM +ve or 3-4 fold increase in IgG, start Spiramycin When infection in utero is documented, using PCR on an amniotic fluid, Add or replace a combination of pyrimethamine and sulfadiazine with folinic acid supplementation to antibiotic. Infected infants should be treated postnatally up to one year of age with the same drugs,

Education programmes may be preventing acquisition of toxo cases, only routine screening of all pregnant women or all newborn infants at birth would prevent or detect a higher proportion of congenital infections.