Congenital Toxoplasmosis Prenatal Aspect of Toxoplasma Gondii Infection

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Reproductive Toxicology 21 (2006) 458472

Review

Congenital toxoplasmosisprenatal aspects ofToxoplasma gondii infection

Efrat Rorman a,, Chen Stein Zamir b, Irena Rilkis a,c, Hilla Ben-David a

a National Public Health Laboratory, Ministry of Health, P.O. Box 8255, Tel Aviv 61082, IsraelbDistrict Health Office, Ministry of Health, Jerusalem, Israel

cNational Toxoplasmosis Reference Center, Ministry of Health, Israel

Received 26 August 2004; received in revised form 11 October 2005; accepted 24 October 2005

Available online 28 November 2005

Abstract

Toxoplasma gondii (T. gondii) is the cause of toxoplasmosis. Primary infection in an immunocompetent person is usually asymptomatic.Serological surveys demonstrate that world-wide exposure to T. gondii is high (30% in US and 5080% in Europe). Vertical transmission from a

recently infected pregnant woman to her fetus may lead to congenital toxoplasmosis. The risk of such transmission increases as primary maternal

infection occurs later in pregnancy. However, consequences for the fetus are more severe with transmission closer to conception. The timing of

maternal primary infection is, therefore, critically linkedto theclinical manifestations of the infection. Fetal infection may result in natural abortion.

Often, no apparent symptoms are observed at birth and complications develop only later in life. The laboratory methods of assessing fetal risk of

T. gondii infection are serology and direct tests.

Screening programs for women at childbearing age or of the newborn, as well as education of the public regarding infection prevention, proved

to be cost-effective and reduce the rate of infection.

The impact of antiparasytic therapy on vertical transmission from mother to fetus is still controversial. However, specific therapy is recommended

to be initiated as soon as infection is diagnosed.

2005 Elsevier Inc. All rights reserved.

Keywords: Toxoplasmosis; Toxoplasma gondii; Congenital infection; Diagnosis; Treatment; Epidemiology

Contents

1. Case report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 459

2. The parasite . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 459

2.1. Life cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 459

2.2. Mechanism of infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 460

2.3. Virulence ofT. gondii strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 460

3. Epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 460

4. Congenital toxoplasmosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 461

4.1. Incidence and prevalence in pregnant women and infants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 461

4.2. Diagnostic evaluation, manifestation and consequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 461

4.3. Prenatal laboratory diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4634.3.1. Sabin Feldman dye test (SFDT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 463

4.3.2. Enzyme immunoassays (EIA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 465

4.3.3. Immunosorbent agglutination assay test (IAAT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 465

4.3.4. Indirect fluorescent assay (IFA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 465

4.3.5. Avidity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 465

4.3.6. Animal and cell culture inoculation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 466

Corresponding author. Tel.: +972 50 6242904; fax: +972 3 6826996.

E-mail address: [email protected] (E. Rorman).

0890-6238/$ see front matter 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.reprotox.2005.10.006


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E. Rorman et al. / Reproductive Toxicology 21 (2006) 458472 459

4.3.7. Molecular diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 466

4.4. Laboratory diagnosis of infants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 467

4.4.1. Western blots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 467

5. Treatment of congenital toxoplasmosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 467

6. Prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 468

6.1. Primary prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 468

6.1.1. Vaccine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 468

6.2. Secondary prevention screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 468Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 468

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 469

1. Case report

A 26-year-old woman from a rural village in northern Israel

presented with cervical lymphadenopathy during the 13th week

of her first pregnancy. The woman was otherwise healthy and

without any symptoms. She was followed up by her primary

care physician and as the lymphadenopathy did not resolve,

was sent for surgical consultation during the 26th week of

pregnancy. The surgeon referred her to laboratory tests for Tox-oplasma gondii-specific antibodies and for various other infec-

tions. The results obtained from testing a serum sample from

the 26th week of gestation, performed at the Israel National

Toxoplasmosis Reference Center were: positive for total T.

gondii-specific immunoglobulins (Ig) (250 IU/ml by Sabin Feld-

man Dye Test) and for T. gondii-specific IgM antibodies (by

ELFA, Enzyme-Linked Fluorescent immuno-Assay) with low

IgG avidity (0.027). These results were reported and an addi-

tional serum sample, as well as an earlier sample (whether

available) were requested. Blood samples were subsequently

delivered to our laboratory; from the 12th (sample drawn as part

of the routine pregnancy follow-up) and from the 34th weeksof pregnancy. The results of the earlier sample were negative

for both total Ig and IgM antibodies. The results from the 34th

week were positive for total T. gondii-specific immunoglobu-

lins (250 IU/ml by Sabin Feldman Dye Test) and negative for

T. gondii-specific IgM antibodies (by ELFA, Enzyme-Linked

Fluorescent immuno-Assay) with low IgG avidity (0.055).

The sum interpretation of the three above tests results of

the 12th, 26th and 34th week of pregnancy was consistent

with definite recent T. gondii infection (seroconversion, constant

high T. gondii-specific immunoglobulins, emergence and disap-

pearance of IgM, low avidity and cervical lymphadenopathy).

Amniocentesis was performed during the 35th week of preg-

nancy and PCR result for T. gondii DNA in the amniotic fluidwas positive.

The woman was referred for follow-up at a high risk preg-

nancy clinic in a tertiary medical center. Anti-T. gondii therapy

including Pyrimethamine, Sulfadiazine and folinic acid was

started and continued until birth. The pregnancy course was

otherwise uneventful and fetal growth assessment through ultra-

sound follow-up did not reveal any abnormality. During the

38th week of pregnancy a female infant was born by sponta-

neous delivery. Birth weight was 2830 g and head circumfer-

ence was 33 cm. Physical examination was normal. Laboratory

tests including complete blood count, glucose, electrolytes, liver

function tests and cerebro-spinal fluid (CSF) tests were all

normal. Cranial ultrasonography, brain stem evoked response

(BERA), audiometryand eye examination were all normal. Tests

for T. gondii in the infant included: PCR of CSFnegative,

immuno-sorbent agglutination assays (IgM-ISAGA)negative

and Sabin Feldman Dye Test (SFDT)positive (250 IU/ml),

probably reflecting maternal antibodies transfer. Despite the

serological indicators of maternal infection (most probably

towards the end of the first trimester) and positive PCR of the

amniotic fluid, there was no evidence of congenital toxoplasmo-sis in the neonate. The infant was treated with the same thera-

peutic protocol as the mother planned to be continued until the

age of 1 year. Medical evaluation, auditory and ophthalmic tests

at the age of 4 and 8 months revealed normal physical growth

and development and intensive follow-up continues (at the age

of 6 months laboratory analysis was reported to be normal).

This case demonstrates the complexity of establishing clin-

ical diagnosis and interpretation of laboratory results in regard

to T. gondii infection in pregnancy. The favourable outcome

despite thetiming of infectionmay be attributed to providinganti

parasitic therapy, although the specific role of therapy or other

unknown variables is unclear. Since many T. gondii infections

are sub-clinical or present with non-specific signs, physicians

should be able to integrate clinical and laboratory data in order

to make diagnostic and therapeutic decisions.

2. The parasite

T. gondii is a memberof thephylum Apicomplexa,order Coc-

cidia, which are all obligate intracellular protozoan parasites.

Other members of this phylum include known human pathogens

such as Plasmodium (malaria) and Cryptosporidium.

2.1. Life cycle

The life cycleofT. gondii consists of two stagesasexual and

sexual: the asexual stage takes place in the intermediate hosts,

which are mammals or birds. During this phase rapid intracellu-

lar growth of the parasite as tachyzoite takes place (generation

time in vitro is 68 h). The oval or crescent-shaped tachyzoites

can infect and multiply in almost any nucleated mammalian or

avian cell [1]. Following accumulation (64128), tachyzoites are

secreted into the blood stream [2] and spreadin the body, leading

to development of an acute disease (parasitemia). The normal

immune response and transformation of the tachyzoite into cyst-

forming bradyzoites limit the acute stage and establish a chronic

infection. Bradyzoites differ from tachyzoites mainly in their


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460 E. Rorman et al. / Reproductive Toxicology 21 (2006) 458472

extremely slow multiplication rate (their name reflects this slow

process) and in the distinct set of proteins they express [1,35].

The cysts are formed mainly in neural and muscular tissues,

especially brain, skeletal and cardiac muscles, and can persist,

inactivated, in the body for a very long time. In the immunocom-

promised patient the release of bradyzoites from the cyst may

cause acute encephalitis.

The sexual stage takes place in the intestine of the definitive

host. Known definitive hosts are members of the feline fam-

ily, predominantly domestic cats. When bradyzoites or oocytes

are ingested by a feline, formation of oocytes proceeds in the

epithelium of the small intestine. Several million unsporulated

oocytes may be released in the feces of a single cat over a period

318 days, depending on the stage of T. gondii ingested [1].

Under mild environmental conditions oocytes may sporulate

within a 3-week period [6], then infecting humans and other

intermediate hosts. Oocysts can spread in the environment and

contaminate water, soil, fruits, vegetables and herbivores fol-

lowing consumption of infected plant material. Investigation of

outbreaks of toxoplasmosis has led to recovery of oocytes fromsoil [7] but not from water [810]. Oocytes have been found to

be very stable, especially in warm and humid environments, and

resistant to many disinfecting agents [11], but survive poorly in

arid, cold climates [12].

2.2. Mechanism of infection

T. gondii has been shown to migrate over long distances in the

hosts body; crossing biological barriers, activelyenter the blood

stream, invade cells and cross substrates and non-permissive

biological sites such as the blood-brain-barrier, the placenta and

the intestinal wall. At the same time, the parasite minimizesexposure to the hosts immune response, by rapidly entering and

exiting cells. These two functions share common mechanisms

which depend on Ca2+ regulation [13].

Unlike many bacteria and viruses, T. gondii actively enters

the cell, in a mechanism which is mediated by the para-

sites cytoskeleton and regulated by a parasite-specific calcium-

depended secretionpathway [2,14]. Thefirst step of cell invasion

by T. gondii is recognition of an attachment point. The two

special organelles involved in this invasion process, rhoptries

and micronemes, each discharging proteins during the process

[5,15]. Following the rapid cellular invasion the parasite resides

within a vacuole, derived primarily from the host cells plasma

membrane [2,16]. The active motion of T. gondii, called glid-ing, occurs with no major changes in cell shape. It is fast (about

10 times faster than the crawling rate of amoeboid cells), and

consists of both circular gliding in a counter-clockwise direction

and clockwise helical gliding [1721]. As an obligatory parasite,

its invasive capabilities play an important role in virulence and

pathogenicity, since it can only survive intracellularly where it

gets nutrientsand escapes from thehosts immuneresponse [22].

The mostvirulent T. gondii strainhas been shown to exhibit supe-

rior migratory capacity [23] and a subpopulation of this strain

displays a special, long distance migration phenotype [14]. The

ability to cross biological barriers is associated with acute vir-

ulence and is linked to genes on chromosome VII [24,25]. The

genome ofT. gondii, consisting of 14 chromosomes, is currently

being investigated and sequenced [26] (http://ToxoDB.org).

2.3. Virulence of T. gondii strains

Clinical manifestations and severity of illness following

infection are affected by features of the interaction between the

parasite and the host and include strain virulence, inoculum size,

route of infection, competence of the hosts immune response

(both cellular and humoral), integrity of the hosts mucosal and

epithelial barriers, hosts age and genetic background [27]. Var-

ious strains of T. gondii have long been known to differ in

virulence and pathogenicity [28,29]. These strains can be classi-

fied by immunologic assays, isoenzyme analysis and molecular

analysis [3033]. There are three T. gondii clonal lineages, of

them one carries conserved genetic loci, suspected of coding

for the virulence trait [24]. Grigg et al. [34] demonstrated that a

sexual recombination, performed in vitro, between the two rela-

tively avirulent strains can give rise to the virulent strain. This is

in accordance with polymorphism analysis of the three T. gondiistrains, which indicated that they emerged within the last 10,000

years, following a single genetic cross [34,35]. Acquisition of

an efficient mechanism to spread by direct oral transmission,

bypassing a sexual phase, leads to successful clonal expansion

of this virulent lineage [35,36].

Genetic background plays a significant role in increased sus-

ceptibility to T. gondi in humans; HLA-DQ3 appears to be

a genetic marker associated with susceptibility to developing

toxoplasma-dependent encephalitis [37,38].

3. Epidemiology

T. gondii infection is most frequently caused by ingestion

of row or undercooked meat, which carries tissue cysts, by

consuming infected water or food or by accidental intake of

contaminated soil. Toxoplasmosis is also an occupational haz-

ard for laboratory workers. A total of 47 laboratory-acquired

cases have been reported, 81% of them were symptomatic cases

[39].

Tender et al. [40] collected data of nation-wide T. gondii sero-

prevalence in women at child-bearing age (19902000). The

rates of positive sero-prevalence, were 58% in Central Euro-

pean countries, 5172% in several Latin-American countries

and 5477% in West African countries. Low seroprevalence,

439%, was reported in southwest Asia, China and Korea aswell as in cold climate areas such as Scandinavian countries

(1128%). In the US 15% of females at childbearing age were

found to be seropositive [41]. It should be noted that seropositive

prevalence in the same country may differ among populations

or geographical regions and world-wide prevalence is higher in

older populations.

In a limited casecontrol study that included six large Euro-

pean centers it was shown that the consumption of undercooked

meat was the major risk factor for toxoplasmosis infection [42].

Another study aimed to determine the prevalence of T. gondii

in edible meat tested 71 meat samples from commercial sources

in the UK for the parasitepositive results were found in 27
http://toxodb.org/http://toxodb.org/


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samples. Twenty-one of these contaminated meat samples car-

ried the virulent T. gondii type I [43]. Although cats play a

definite role in the epidemiology of toxoplasmosis, no signif-

icant correlation between human toxoplasmosis infection and

cat ownership could be proven [44]. Furthermore, the oocytes

are not found on cat fur but rather are buried in the soil as they

are shed with cat faeces [4547].

Data regarding seroprevalence of specific T. gondii anti-

bodies in the Israeli population are based on several regional

surveys performed in collaboration with the Israeli National

Toxoplasmosis Reference Center. The prevalence in certain sub-

populations of pregnant women in northern Israel had been

reported to be 21% on average and the incidence rate of infection

acquired during pregnancy estimated as 1.4% [48].

Human contact with infected oocyst from contaminated soil

[7,49,50] and water[810] were associated with several reported

epidemics caused by T. gondii. Only in one case were T.

gondii oocysts recovered from the soilthe suspected source

of infection [7]. There are ongoing efforts to develop sensi-

tive detection techniques for environmental samples [11,51,52].Unfortunately, isolation of oocytes from such samples is dif-

ficult, since infectious doses are small while large volume of

sample is required for isolation of the organism. In addition,

there is a lag period between the time of infection and the time

that the contaminated source is tested, further reducing the like-

lihood of recovery of oocytes from the suspected environment

during epidemiological investigation.

T. gondii was reported to cause 0.8% of the total food-borne

illnesses attributed to a known pathogen, and 20.7% of the total

food-borne mortality caused by a known pathogen, in the United

States in 19961997.Many of these cases involved HIV-infected

patients [53].The largest reported toxoplasmosis outbreak resulting from

contaminated water occurred in British Columbia and caused

acute infection in 100 people; 19 with retinitis and 51 with lym-

phadenopathy. The likely source was a municipal water system

thatusedunfiltered,chloraminated surface water [10]. Therewas

also a seasonal correlation to rainfall and turbidity in this water

reservoir. In another small outbreak North of Rio de Janeiro,

Brazil, the sourceof theparasitewas tracedto an unfilteredwater

source. It was also linked to high prevalence of seropositivity in

this region of low socio-economic background [8].

4. Congenital toxoplasmosis

Most cases of acquired toxoplasma infection are asymp-

tomatic and self-limited; hence manycases remain undiagnosed.

The incubation period of acquired infection is estimated to be

within a range of 421days (7 days on average) [10]. When

symptomatic infection does occur the only clinical findings may

be focal lymphadenopathy, most often involving a single site

around the head and neck. Less commonly, acute infection is

accompanied by a mononucleosis-like syndrome characterized

by fever, malaise, sore throat, headache and an atypical lympho-

cytosis on peripheral blood smear [54]. In immunocompromised

patients, most commonly HIV infected and organ transplant

recipients, T. gondii may cause a severe central nervous system

disease, resulting in brain lesions or diffuse encephalitis. Other

organs, such as the heart, lung, liver, and retina may also be

involved. Most of these cases result from reactivation of latent

infection [54] although re-infection with a different T. gondii

strain in the transplanted organs may also occur.

4.1. Incidence and prevalence in pregnant women and

infants

The disease is caused by vertical transmission of T. gondii

from a seronegative pregnant woman, who is acutely infected

with T. gondii to her fetus.

The prevalence ofT. gondii and its incidence of human infec-

tion vary widely amongst various countries. Worldwide, 38

infants per 1000 live births are infected in utero [55]. Multiple

factors are associated with the occurrence of congenital toxo-

plasmosis infection, including route of transmission, climate,

cultural behaviour, eating habits and hygienic standards. This

combination leads to marked differences even among developed

nations. For example, the incidence of congenital infection inBelgium and France is 23 cases per 1000 live birthsmarkedly

higher than the US incidence, which is between 1 in 10,000 to

1 in 1000 live births [47,56].

In a research conducted in Goiania, Brazil, a region with

a relatively high seroconversion rate, pregnant women were

found to have a 2.2 times higher risk for seroconversion than

non-pregnant women of equivalent characteristics. In addition,

amongst pregnant women, adolescents were shown to have the

highest risk for seroconversion [57]. The authors hypothesized

that higher vulnerability to T. gondii infection during pregnancy

may be due to a combination of pregnancy associated immuno-

suppression as well as hormonal changes.Only a few cases of congenital toxoplasmosis transmitted

by mothers who were infected prior to conception have actu-

ally been reported [5860]. One such case published recently

involved a woman who had ocular toxoplasmosis 20 years prior

to giving birth to a newborn, who suffered from congenital tox-

oplasmosis. The mother had a toxoplasmic scar in the retina

and was tested positive for specific toxoplasma IgG antibodies.

The newborn was found to be positive for both IgG and IgM

antibodies and had a macular scar on the retina, typical to tox-

oplasmosis, as well as a calcified brain granuloma. [59]. Such

a case could be attributed to re-infection with a different, more

virulent strain or by reactivation of a chronic disease[58].

Chronically infected women, who are immunodeficienct,may also transmit the infection to their fetus; the risk of this

occurrence is difficult to quantify, but it is probably low. Latent

T. gondii infection may be reactivated in immunodeficient indi-

viduals (such as HIV-infected women) and result in congenital

transmission of the parasite [61].

4.2. Diagnostic evaluation, manifestation and

consequences

The diagnostic evaluation ofT. gondii is part of routine preg-

nancy follow-up and differential diagnosis of intrauterine infec-

tion.Intrauterine ultrasonographic findings ofT. gondii infection


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are usually non-specific and in most cases no pathological evi-

dences are revealed. In certain cases the ultrasonographic find-

ings may include: intracranial calcifications, echogenic streaks,

microcephalus, ventricular dilatation and hydrocephalus [62].

Gay-Andrieu et al. [63] described two cases of intrauterine

infection in which the diagnosis was based upon hydrocephalus

in fetal ultrasound, even though PCR of amniotic fluid was

negative in both cases. The authors emphasized that hydro-

cephalus is the most frequent lesion detected by fetal ultra-

sound, reflecting the pathological process taking place within

several months post-infection in cases of intrauterine infection

ofT. gondii. Additional ultrasonographic findings may include

hepatomegaly, splenomegaly, ascitic fluid, cardiomegaly and

placental abnormalities [55,64]. Safadi et al. [65] followed 43

children with congenital toxoplasmosis for a period of at least

5 years. Most of them (88%) had sub-clinical presentation

at birth. The most common neurological manifestation was a

delay in neuro-psychomotor development. Half of the children

developed neurological manifestations, 7 children had neuro-

radiologic alterations in skull radiography, and 33 children intomography. Notably, cerebral calcifications were not associated

with an increased incidence of neurological sequelae. Choriore-

tinitis was the main ocular sequelae, found in almost all children

and noted years after birth, despite specific therapy in the first

year of life.

An important step in the diagnosis of congenital toxoplasmo-

sis and evaluation of time of infection is achieved by laboratory

techniques, monitoring the immune response: titer and affin-

ity of specific antibodies (Fig. 1). Other laboratory tools focus

on direct detection of the parasite by animal or tissue inocula-

tion or more commonly, by molecular techniques. Carvalheiro et

al. studied the incidence of congenital toxoplasmosis in Brazil,based on persistence of anti-Toxoplasma IgG antibodies beyond

the age of 1year. Disease incidence was estimated to be 3.3 per

10,000. A definitive diagnosis wasconfirmed in five infants with

both serum IgM and/or IgA antibodies, and clinical abnormali-

Fig. 1. Laboratory diagnosis of congenital toxoplasmosis.

ties. They concludedthat positive screening results must be care-

fully confirmed [66]. Laboratory methods and their implications

in supporting evidence-based diagnoses are discussed below.

The risk of fetal infection is multifactorial, depending on the

time of maternal infection, immunological competence of the

mother during parasitemia, parasite load and strains virulence

[40]. The probability of fetal infection is only 1% when pri-

mary maternal infection occurs during the preconception period

but increases as pregnancy progresses; infection acquired dur-

ing the first trimester by women not treated with anti-T. gondii

drugs results in congenital infection in 10 to 25% of cases.

For infections occurring during the second and third trimesters,

the incidence of fetal infection ranges between 3054% and

6065%, respectively [54].

The consequences are more severe when fetal infection

occurs in early stages of pregnancy, when it can cause miscar-

riage (natural abortion or death occurs in 10% of pregnancies

infected with T. gondii [67]), severe disease, intra-uterine growth

retardation or premature birth. A multi-centre prospective cohort

study evaluated the association between congenital toxoplas-mosis and preterm birth, low birth weight, and small size for

gestational age [68]. Freeman et al. reported that infected babies

were born earlier than uninfected babies and that congenital

infection was associated with an increased risk of preterm deliv-

ery when seroconversion occurred before 20 weeks of gestation.

Congenital infection was not associated with low birth weight

or small size for gestational age. The cause for shorter gestation

is not yet known. The highest frequency of severe abnormalities

at birth is seen in children whose mothers acquired a primary

infection between the 10th and 24th week of gestation [67]. The

likelihood of clinical symptoms in the newborn is reduced when

infection occurs later.Clinical manifestations in newborns with congenital toxo-

plasmosis vary and can develop at different times before and

after birth. Most newborns infected with T. gondii are asymp-

tomatic at birth (7090%) [61]. Whenclinical manifestations are

present they are mainly non-specific and may include: a mac-

ulopapular rash, generalized lymphadenopathy, hepatomegaly,

splenomegaly, hyperbilirubinemia, anemia and thrombocytope-

nia [69]. The classic triad of chorioretinitis, intracranial calcifi-

cations andhydrocephalus is found in fewerthan 10%of infected

infants [47]. Hydrocephalus and/or microcephaly may develop

when intra-uterine infection results in meningo-encepahlitis

[69]. All these signs and symptoms are included in the general

work-up of suspected congenital TORCH infections: toxoplas-mosis, other (syphilis, varicella-zoster, parvovirus B19), rubella,

cytomegalovirus (CMV) and herpes infections. Cerebral cal-

cifications can be demonstrated by cranial radiography, ultra-

sonography or computerized tomography. Neurologic impair-

ment may initially present as seizures, necessitating specific

evaluation and treatment.

The most prevalent consequence of congenital toxoplasmosis

is chorioretinitis.

Chorioretinitis is diagnosed based on characteristic retinal

infiltrates. Vutova et al. [70] investigated eye manifestations of

congenital toxoplasmosis in 38 infants and children. The most

frequent finding was chorioretinitis (92%), together with other


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ocular lesions in 71% of cases, and the second most common

finding was microphthalmia with strabismus. Lesions of the

anterior segment of the eye included iridocyclitis, cataracts and

glaucoma. Other uncommon findings were diminished visual

acuity and neurological sequelae such as hydrocephalus, calci-

fication in the brain, paresis, and epilepsy.

Wallon et al. [71] reported the clinical evolution of ocular

lesions and final visual function, in a prospective cohort of 327

congenitally infected children in France. The children were iden-

tified by maternal prenatal screening and monitored for up to

14 years. After 6 years, 79 (24%) children had at least one

retinochoroidal lesion. In 23 of them a new lesion was diag-

nosed within10 years, mainly in a previously healthy location.

Normal vision was found in about two thirds of children with

lesions in one eye, half the children with lesions in both eyes

and none had bilateral visual impairment. Most of the mothers

(84%) had been treated. A combination of pyrimethamine and

sulfadiazine had been prescribed in all the children (38% before

and 72% after birth). Late-onset retinal lesions and relapse can

occur many years after birth, but the overall ocular prognosis ofcongenital toxoplasmosis seems satisfactory, when infection is

identified early and appropriately treated. Early diagnosis and

treatment are believed to reduce the risk of visual impairment.

Relevant laboratory tests include complete blood count

(CBC), liver function tests and specific T. gondii diagnostic tests

as described in details below. IfT. gondii infection is suspected

at the time of birth, diagnostic work-up includes ophthalmic,

auditory and neurological examinations, lumbar puncture and

cranial imaging [69].

In a large percentage of children the disease sequelae may

become apparent and present withvisual impairment, mental and

cognitive abnormalities of variable severity, seizures or learningdisabilities only after several months or years [55].

Infants born to women infected simultaneously with HIV

and T. gondii should be evaluated for congenital toxoplasmosis,

considering the increased risk of reactivation of parasitemia and

disease in these mothers.

In a casecontrol study in Israel, Potasman et al. tested 95

children with variable neurological disorders: cerebral palsy,

epilepsy and nerve deafness compared with a control group of

109 healthy children, for the presence ofT. gondii-specific anti-

bodies in the serum. They found that children with any of the

neurological disorders were significantly more likely to have

T. gondii specific IgG antibodies, especially those with nerve

deafness (relative risk 2.5 and 7.1, respectively) [72].A definite diagnosis cannot be made in the following situa-

tions: (1) the infant is older than one year of age and was not

tested for toxoplasmosis previously, (2) either the child or the

mother is seronegative, or (3) the mother was known to be sero-

positive prior to conception.

4.3. Prenatal laboratory diagnosis

The principle method used to diagnose and evaluate tim-

ing of congenital infection relies on indirect evidence, and is

based on detection of specific antibodies, by monitoring the

immune response. Direct evidence is obtained by animal or tis-

sue inoculation or more commonly, by molecular techniques. It

is important to combine all available clinical and laboratory data

during the evaluation of toxoplasmosis diagnosis and providing

treatment recommendations.

Infection during gestation may cause serious damage to

the fetus and hence, a major objective of the diagnosis is to

estimate the time of maternal infection. IgG antibodies usu-

ally appear within two weeks of infection, peak within 68

weeks and persist in the body indefinitely [67]. IgM antibod-

ies are considered the indicators of recent infection and can

be detected by enzyme immunoassay (EIA) or immunosor-

bent agglutination assay test (IAAT) relatively earlywithin

2 weeks of infection. Uncertainty may arise as IgM may per-

sist for years following primary infection [73]. IgA antibodies

may also persist for more than a year [67] and their detection

is informative mainly for the diagnosis of congenital toxoplas-

mosis. The level of specific IgE antibodies increases rapidly

and remains detectable for less than 4 months after infection,

which leaves a very short time to be used for diagnostic pur-

poses [74]. However, IgE serology is not useful in samples fromnewborns.

When serology alone is insufficient direct evidence for

toxoplasma infection should be sought. Both the laboratory

performing the tests and the referring physician should be

aware of the limitations and select the best combination of

tests available to timely evaluate the stage of toxoplasma

infection [75]. Laboratory tests available are summarized in

Table 1.

4.3.1. Sabin Feldman dye test (SFDT)

This is the first test developed for the laboratory diagnosis of

T. gondii infection [76], it is still considered the gold standard.SFDT detects the presence of anti-T. gondii specific antibod-

ies (total Ig) and is performed only in reference centers. The

change in antibody titer as determined in SFDT in consecu-

tive serum samples taken at least 3 weeks apart is important

for the evaluation of infection during pregnancy. A significant

change is considered to be at least a four-fold difference. The

absolute antibody titer is also importantvalues over 250IU/ml

are considered high suggestive of recent infection. The tested

sera are serially diluted and incubated with live tachyzoites

(carrying toxoplasma-specific antigens) in the presence of sep-

arated human plasma from sero-negative donors (providing

complement components). The antigenantibodycomplement

complexes formed are subsequently lysed in the presence ofthe dye methylene blue. End-point titer is established by count-

ing the numbers of dead (unstained) and live (stained) para-

sites. The reported titer is that producing lysis of 50% of the

organisms. End-point titer can be converted to international

units (IU): additional standardization is achieved by prepara-

tion of a standardised control serum (consisting of a pool of

sera), tested by numerous reference centers, and adjusted so

that the SFDT value of this control serum is set at 1000 IU/ml

[77]. Recently, the WHO recognized the first international stan-

dard for human anti-toxoplasma IgG, with an assigned potency

of 20 IU per ampoule of total anti-toxoplasma antibodies

[78].


7/15


Table 1

Laboratory diagnostic tests for congenital toxoplasmosis

Test Matrix Results Interpretation Time Degree of

expertise

equired

Other remarks Suggested use

Sabin Feldman Dye

Test (SFDT)

Serum Titer in international

units (IU) of total

specific Ig

Quantitative data:

detection of high

(250 IU) antibodies

titers and significant

changes (4) in

titer in consecutive

samples important

for evaluation of

recent infection

Routine =24

week

High, reference

center only

Gold standard Confirmation of

infection

hands

on= several

hours

Live parasites

and animal

injection risk

to lab employee

Standardized

assay

(international

effort)

Follow-up

change in titer

EIAa/total Igb Serum Positive/negative for

total specific Ig

Exposure to T. gondii Several hours Low =simple

automated test

Possible false

negative very

early infection

Screening test

IFAc-total Ig Serum Titer (in IU) of total

specific Ig

Exposure to T. gondii Several hours High, reference

center only

Very

subjective and

difficult to

standardize

When SFDT is

unavailable

IgG by EIA Serum Positive/negative for

specific IgG Abs

Exposure to T. gondii Several hours Low =simple

automated test

Partial results

(combine with

IgM detection)

Screening test

IgM/IgA or IgE by

EIA

Serum Positive/negative for

specific IgM, IgA or

IgE Abs

Possible recent

infection with T.

gondii

Several hours Low =simple

automated test

Requires

further testing,

IgE not in

newborn

IgM

Screening

IgM/IgA

Newborn

IgE Earlier

IgM/IgA or IgE by

IAAT



IgE Abs

Possible recent

Infection with T.

gondii

Several hours Relatively high Most sensitive

and specific

test

IgM/IgA

Newborn

Western blot

should be

considered if

contamination

with maternal

blood is

suspected

IgM/IgA or IgE by

IFA



IgE Abs

Possible recent

Infection with T.

gondii

Several hours High, reference

center only

Very

subjective and

difficult to

standardize

When ISAGA is

unavailable

IgG avidity Serum Avidity = f unctional

affinity

High avidity supports

past infection (4

months)

Several hours Relatively

simple

Supportive

evidence

When only a

single serum

sample is

available, in the

beginning of

pregnancy

Mice Body flu-

ids/tissue

Positive/negative Presence of parasite 36 weeks High, reference

center only

Low

sensitivity

Strain isolation

Live parasites

and animal

injection risk

to lab employee


8/15


Table 1 (Continued)

Test Matrix Results Interpretation Time Degree of

expertise

equired

Other remarks Suggested use

Cells Body flu-

ids/tissue

Positive/negative Presence of parasite 36 days Very high

reference center

only

Low

sensitivity

When available

fora directproof

of infection

Live parasitesPCR Body flu-

ids/tissue

Positive/negative Presence of parasites

DNA

Several hours High High

sensitivity

Amniotic fluid

Western blot IgG,

IgM

Serum Identical/unidentical

to maternal Ig

Fetal/newborn

infection

1 day High, reference

center

Infrequent

availability

Confirmatory

test or

fetal/newborn

infection

a EIA: enzyme immunoassay.b Ig: immunoglobulin.c IFA: indirect fluorescent assay.

4.3.2. Enzyme immunoassays (EIA)

The most common laboratory tests for toxoplasmosis

infection, also available as commercial kits and/or auto-mated platforms, are EIA. These tests include: enzyme-linked

immunosorbent assay (ELISA) and enzyme linked fluorescent

immuno-assay (ELFA) which test for the presence of IgG and/or

IgM antibodies specific for the parasite in human sera. EIA are

useful as fast, low-cost screening tests and have been improved

over the years to avoid false positive results due to non-specific

detection of interfering factors such as rheumatoid factor and

antinuclear antibodies.

There is no standardization of these tests, which causes high

variability in results obtained with different kits and/or in differ-

ent laboratories. Consequently, and also as a result of the high

incidence of false-positive results even in reference centers, the

US Food and Drug Administration (FDA) issued a health advi-

soryto physicianson July, 1997.The FDArecommends avoiding

reliance of results obtained with any single commercial kit for

the detection of toxoplasma-specific IgM, as the sole deter-

minant of recent toxoplasma infection in pregnant women. In

our experience at the Israeli National Toxoplasmosis Reference

Center, during the years 19972002, in an average of 747 sam-

ples (range: 652816) received annually for confirmation, only

17% 2.6% were indeed positive for T. gondii-specific IgM. It

is therefore recommended that patient follow-up would be per-

formed by a reference center, and that commercial kits would be

locally evaluated to achieve the highest degree of accuracy and

repeatability possible for screening tests.In general, when toxoplasma infection is suspected based on

detection of specific IgM antibodies specimens are referred for

confirmation by a reference center where SFDT, PCR and other

advanced assays can be performed.

4.3.3. Immunosorbent agglutination assay test (IAAT)

IAAT is highly specific in detection of anti-T. gondii IgM,

IgA or IgE antibodies [79]. This assay utilizes the entire tachy-

zoite and is the most sensitive commercially available method

[8082]. Unfortunately, it is expensive, requires a high degree of

expertise and is not automated. It is consequently seldom used in

reference centers, usually in neonates suspected of having con-

genital infection (where the expected levels of antibodies are

very low) [74,83].

Toxoplasma-specific IgE antibodies can be detected by EIAor IAAT in sera of recently infected adults, congenitally infected

infants, and children with congenital toxoplasmic chorioretinitis

[84]. IgE detection is, however, ineffective in evaluating fetal or

newborn samples where IgA tests are most informative.

4.3.4. Indirect fluorescent assay (IFA)

The IFA was widely used to demonstrate T. gondii-specific

antibodies: serially diluted serum samples are incubated with

live, inactivated toxoplasma fixed to a glass slide. T. gondii-

specific antibodies present in the serum would bind to the inacti-

vated parasite, and the complex is thendetected usingfluorescein

isothiocyanate-labeled anti-human Ig (or anti-IgG or anti-IgM).

IFA is safer to perform and more economical than the SFDT. It

appears to measure the same antibodies as the dye test, and its

titers tend to parallel dye test titers [47,85]. However, the IFA

interpretation is subjective and time consuming. False positive

results may occur with sera containing antinuclear antibodies

and rheumatoid factor [86], and false negative results of IFA for

IgM may occur due to blockage by T. gondii-specific IgG [87].

4.3.5. Avidity

IgG avidity testing was developed by Hedman et al. and is

based on the increase in functional affinity (avidity) between

T. gondii-specific IgG and the antigen over time, as the host

immune response (and specific B cell selection) evolves [88].Dissociation of the antigenantibody complexes reflects the

lower avidity closer to primary infection. Pregnant women with

high avidity antibodies are those who have been infected at least

35 months earlier, which makes the avidity test most useful and

reliable in the first trimester when high-avidity is detected [89].

In one study, 35 out of 63 patients (55%) who were classified

by toxoplasma-specific serology as having recent or border-

line infection showed high avidity-antibodies and were therefore

treated as chronic patients [90]. Lappaplainen et al. [91] were

able to follow 13 women who showed high-avidity antibodies in

thefirst trimester andconfirmedthat none of the born infants was

found to be infected with T. gondii (as determined serologically


9/15


after birth). The avidity test is most important when only a sin-

gle serum sample is available at the time when critical decisions

must be made. To the best of our knowledge, commercial IgG

avidity kits have been licensed in Europe but not in the US [92].

When avidity is low or borderline it may be misleading and

a more careful interpretation of all laboratory tests results in

conjunction with other clinical findings, should then be under-

taken. Several studies have shown that this test is reliable and

valuable in diagnosis of recent infection during early pregnancy

[88,9398].

Accurate and definitive serologic diagnosis of recently

acquired toxoplasma infection is still difficult and depends on

testing of more than one sample. Efforts to develop better

diagnostic approaches continue based on antigens specifically

expressed either during the primary phase (i.e. GRA7, GRA4)

or the latent phase (i.e. GRA1) of infection. These antigens can

be produced by recombinant DNA technologies and may lead to

a more informative serologic diagnosis, based on a single serum

sample [47,99,100].

4.3.6. Animal and cell culture inoculation

A definite laboratory confirmation of active toxoplasmosis

infection (especially in immunocompromised patients and preg-

nant women) can be established by inoculation of body fluids or

tissue into mice or cell culture [47].

Mice are injected intraperitoneally or subcutaneously with

1030 ml of sediment from amniotic fluid or whole fetal blood.

The mice are bled prior and 36 weeks following inoculation.

Antibody detection by SFDT establishes infection and final

proof is obtained by staining to demonstrate brain cysts [101].

Cell culture inoculation with amniotic fluid or blood uses

indirect IFA to detect the parasite in monolayers within 36days following inoculation [102]. When compared, inoculation

of both blood and amniotic fluid from an infected fetus resulted

in toxoplasma isolation from both cultures in 70% of cases.

However, in 40% of the cases T. gondii isolation is successful in

only one of the samples [100]. Derouin et al. [100] demonstrated

similar sensitivities comparingcell culture and mice inoculation.

Thulliez et al. [102] reported that the sensitivity of amniotic

fluid cell culture inoculation is only 53% compared with 73%

sensitivity in mice inoculation.

Currently, the principle role for these methods may be con-

firmation of PCR as they are complex, expensive and relatively

insensitive. [103].

4.3.7. Molecular diagnosis

Replacing fetal blood analysis, which is a high risk proce-

dure for the fetus, with molecular evaluation of amniotic fluid

has provided a low risk diagnosis of congenital toxoplasmosis.

Polymerase chain reaction (PCR) is currently the most common

molecular technique routinely usedfor diagnosis of toxoplasmo-

sis, although, it has not yet been standardized. No attempts have

been made to standardize either the sample preparation process

or the PCR amplification itself, and numerous laboratories use

multiple in-house methods of varying sensitivities and relia-

bility [104,105]. Recently, a commercial PCR proficiency test

became available.

As in all diagnostic tests based on amplification of DNA,

a few technical aspects are of crucial importance in achieving

reliable results. Therefore, PCR based test should be carefully

designed to include negative, positive and internal control, target

DNA for amplification should be specific, sample preparation

techniques should be perfected to extract minute parasite DNA

[105] and to prevent cross contamination.

In a small (5 laboratories) inter-laboratories comparative

work[106] followed by a largerstudy(15 laboratories) [104] sig-

nificant differences in test performances were obtained, includ-

ing false negatives and false positives. These results should

definitely urgeoptimization and standardization of the test.More

recently, three PCR protocols were optimized prior to a compar-

ative study, using three different targets: 18S ribosomal DNA,

B1 gene and AF146527. No significant difference was observed

between the results of the three protocols [107].

Chabbert et al. [108] used two different primer sets of the B1

gene to compare PCR performance followed by Southern blot,

on various sample types (including amniotic fluid, blood and

tissues). For amniotic fluid both PCR conditions produced sim-ilar results. The fragments produced by one of the primer sets

had to be confirmed by specific hybridization, otherwise non-

specific results were obtained. The PCR product of the same

amplification procedure was sequenced by Kompalic-Cristo and

suspected of originating from human DNA, as predicted by

bioinformatics analysis [109].

Different protocols influence the sensitivity and specificity

of PCR assays. The specificity and positive predictive value of

PCR tests on amniotic fluid samples is close to 100% [110,111].

However, the sensitivity of these PCR tests varies and estimated,

based on a large number of studies, to be 7080% [105]. One

report showed that the sensitivity of PCR from amniotic fluid isaffected by the stage of pregnancy in which maternal infection

occurs: best sensitivity was detected when maternal infection

occurred between 17 and 21 weeks of pregnancy [89,111,112].

In addition, treatment with anti-toxoplasma drugs may also

affect the sensitivity [89,112]. However, the reliability of a PCR

test performed on amniotic fluid prior to the 18th week of preg-

nancy requires further evaluation [110,111]. It should also be

noted, that testing amniotic fluid for T. gondii was found to be

effective about 4 weeks following infection, which is already

during the parasitemic stage in the infected mother. Therefore,

PCR test should not be performed in the absence of serologic or

other clinical/sonographic data indicative of infection.

In the last 4 years there have been reports on the use of RealTime PCR, a sensitive and specific technique, which enables

rapiddetection of amplification products as well as hybridization

of amplicon-specific probes, similar to PCR followed by South-

ern blot analysis. The method, which will ultimately replace

traditional PCR, enables an overall time for amplification and

detection of less than two hours. In addition, cross contamina-

tion is prevented by elimination of the need to handle amplified

amplicons. In Real Time PCR it is possible to perform a quanti-

tative study and follow the parasite load, allowing determination

of parasite count and its correlation with clinical symptoms and

impact of treatment. The technique permits linear range over 6

logs of DNA concentrations [113,114].


10/15


The most popular target gene for PCR diagnosis ofT. gondii

is the 35-fold repetitive gene B1. A variety of primers have been

used for amplification, some of which include nested primers.

The second common locus is the single copy gene P30 also

known as SAG1, which encodes for a surface antigen. Another

PCR target is the 18S ribosomal DNA. As reviewed by Bastien

[105], two other target loci have been examined but are currently

not used by most laboratories. Recently, some laboratories have

shown success in amplification of a DNA fragment, AF146527,

which is repeated 200300 times [89,113,114].

4.4. Laboratory diagnosis of infants

Laboratory diagnosis of Toxoplasma infection in infants is

based on a combination of serologic tests, parasite isolation,

and nonspecific findings [112].

When suspected, serologic follow-up of the newborn is rec-

ommended for the first year of life [90]. Evaluation for direct

evidence as described above should be repeated as well during

this period.Serologic tests should follow total (or IgG) T. gondii specific

antibodies titer (taking into account that closely after birth these

are maternal in origin, transferred through the placenta), IgM

and IgA titers. Though passively transferred maternal IgG has a

half life of approximately 1 month, it can still be detected in the

newborn for several months, generally disappearing completely

within one year [112]. Appearance of autonomous IgG antibod-

ies in a congenitally infected newborn begins, in an untreated

patient, about 3 months after birth. Anti-parasitic therapy may

delay antibody production for about 6 months and, occasionally,

may completely prevent antibodies production [86].

4.4.1. Western blots

Remington et al. introduced Western blots (using T. gondii-

specific labeled antigens to detect antibodies, separated by elec-

trophoresis and transferred to a membrane) to compare newborn

versus maternal antibodies [115117]. Western blotting could

potentially separate maternal from fetal/newborn antibodies.

The test is not widely used mainly because of its technical com-

plexity and high price.

5. Treatment of congenital toxoplasmosis

Anti T. gondii treatment initiation generally requires con-

firmatory laboratory tests in a reference center, followed byconsultation with experts. Treatment is indicated in the fol-

lowing conditions: infection during pregnancy and congenital

infection as well as infection of an immunocompromised host

(e.g. HIV/AIDS) and in case of an invasive disease. In pregnant

women and infected neonates, both symptomatic and asymp-

tomatic, specific treatment of T. gondii infection is indicated

immediately following established diagnosis. The combination

of pyrimethamine, (adult dosage 25100 mg/d 34 weeks),

sulfadiazine adult dosage 11.5 g qid 34 weeks) and folinic

acid (leucovorin, 1025 mg with each dose of pyrimethamine,

to avoid bone marrow suppression) is the basic treatment pro-

tocol recommended by the WHO [118] and CDC [119]. Other

drugs such as spiramycin (adult dosage 34 g/d 34 weeks)

and sometimesclindamycin are recommended in certain circum-

stances. Spiramycin is used to prevent placental infection; it is

used in many European countries especially France, Asia and

South America. In the US, spiramycin is currently not approved

by the FDA but, available as an investigational drug, requiring

special approval. Treatment with pyrimethamine and sulfadi-

azine to prevent fetal infection is contraindicated during the first

trimester of pregnancy due to concerns regarding teratogenicity,

except when the mothershealth is seriously endangered. During

the first trimester sulfadiazine can be used alone.

As recently reviewed by Montoya and Liesenfeld [112], treat-

ment protocols vary among different centers. The effectivity of

anti-T. gondii treatment is evaluated based on two criteria: rate

of mother to child transmission and prevalence and severity of

sequelae. The majority of the studies are retrospective or cohort

studies of various populations and case definitions. The differ-

ence in study patterns and methodologies affects the reliability

and validity of the results and thus prevents issuing further rec-

ommendations.Wallon et al. [120] reviewed studies comparing treated and

untreated concurrent groups of pregnant women with proved

or likely acute toxoplasma infection. Outcomes data of the

offspring were reported. The results showed treatment to be

effective in five studies but ineffective in four. Gras et al. [121]

reported that the effect of prenatal pyrimethaminesulfadiazine

combination treatment on the cerebral and ocular sequelae of

intrauterine infection with T. gondii was not beneficial in 181

children of infected mothers. Neto reported the outcome of

patients with congenitaltoxoplasmosis who were all treated with

pyrimethamine,sulfadiazine and folinic acid; of 195 patients 138

(71%) were asymptomatic until the age of 2 years. The authorssuggest that for six patients with sequelae because of the delay

in anti-toxoplasma treatment (614 months post diagnosis) the

disease was not prevented [122]. Gratzlet al. [123] reported vari-

able concentrations of spiramycin and its metabolites in serum

and amniotic fluid of 18 pregnant women following treatment.

All the drug concentrations were below the level reported to

inhibit parasite growth in vitro. The authors suggested that the

possible reasons being individual pharmatokinetic variability

and patients treatment compliance. Gilbert et al. [124] reported

the effect of prenatal treatment in 554 infected women and

their offspring. In this study comparison of early versus late

treatment and of combination treatment (pyrimethamine, sulfa-

diazine) with spiramycin or no-treatment, were all statisticallyinsignificant. The possible interpretation is that delayed treat-

ment initiation led to failure to prevent parasite transmission.

Another European multicenter study comparing transmission

rates and clinical outcomes in 856 motherinfant pairs, found

no significant association between the outcome and the intensity

of treatment protocol in pregnancy [125]. Bessieres et al. [126]

studied the effect of treatment during pregnancy in a cohort of

165 women and found that cases could be identified during preg-

nancy as well as during the neonatal period. They also noted

that T. gondii was less frequently isolated in women treated

with pyrimethamine and sulfadoxine than in women treated

with spiramycin only. Foulon et al. [127] reviewed the measures


11/15


of prevention of congenital toxoplasmosis and concluded that

treatment during pregnancy significantly reduces sequelae and

treatment of infected children has a beneficial effect when ther-

apy is begun soon after birth.

In conclusion, the efficacy of anti-T. gondii treatment in preg-

nancy is still an unsettled matter. It is difficult to find the effect

of treatment when comparing the different studies because of:

different treatment regimes and timing (for small groups of

patients), the pharmacokinetics patterns of drugs (concentra-

tion in amniotic fluid and fetal CSF), patient (none) compliance

with treatment and different methodologies of follow-up in each

study.

As concluded by Peyron et al. [128] and others, further large

scale, carefully controlled studies are necessary in order to clar-

ify this controversial issue. At present the anti-parasite treatment

recommended for toxoplasmosis as outlined above, should be

considered as the guideline for good medical practice.

6. Prevention

6.1. Primary prevention

In the United States efforts at prevention of congenital tox-

oplasmosis have been primarily directed towards health educa-

tion, focused to avoid personal exposure to theparasite(hygienic

and culinary practice during pregnancy). In Poland, an extensive

health education campaign, increased toxoplasmosis awareness

and knowledge of preventive behaviour significantly within the

4 years of the reported study [129]. Many other countries have

introduced educational programs aimed at reducing the inci-

dence of congenital toxoplasmosis. Such programs depend on

careful identification of unique target-populations and tailoringappropriate approaches of education. To evaluate the success of

such programs it is important to measure incidence rate before

onset and at pre-determined intervals after introducing the cam-

paign.

6.1.1. Vaccine

Development of a vaccine for toxoplasmosis can prevent

human disease by immunization of human as well as animals

(the source of infection). Both attenuated parasite and immuno-

genicantigens are considered as potential agents for vaccination.

Live attenuated S48 strain is in use for vaccination of sheep in

Europe and New Zealand but is unsuitable for human use due to

its expense, short shelf life and most importantly, to the ability oftheattenuated parasite to revert to a pathogenic strain[130133].

Much of the work has been focused on SAG1, a surface anti-

gen expressed on tachyzoites, in attempts to induce protective

immune response (mainly T-helper response) when introduced

to the host with various adjuvants [134136]. Development of

vaccine using antigens expressed by bradyzoites and oocytes is

also under investigation[134,137].

6.2. Secondary prevention screening

Routine toxoplasmosis screening programs for pregnant

women have been established in France, in Austria and in the

State of Goias,Brazil as recommended by experts [127]. Screen-

ing of women should begin prior to conception with follow-up

monthly tests during pregnancy to detect seroconverion. This is

the basis for the French [138] screening program and the Aus-

trianToxoplasmosis Prevention Programs,bothrecommendrou-

tine serologic testing, in Austria three times during pregnancy:

in the first, second and third trimesters and in France six times

following the initial finding [139]. Treatment is recommended

if one of the tests suggests definite or probable primary mater-

nal infection [140]. In Massachusetts, USA, where there is low

seroprevalence in the population, only newborns are screened

for the presence of T. gondii-specific IgM [141]. IgM detec-

tion is followed by an extensive clinical evaluation and a one

year treatment regimen combination of pyrimethamine and sul-

fadiazine [140]. A recent study screened 364,130 neonates in

the United States for T. gondii specific IgM and confirmed 195

cases of congenital toxoplasmosis (1 in 1867). Moreover, a 7-

year follow-up of the treated patients revealed no symptoms or

at least no progress of the disease. Based on these findings, the

authors suggest including toxoplasmosis in neonatal screeningprograms [122].

In the United Kingdom a national committee concluded that

no prenatal or neonatal screening for T. gondii should be per-

formed, which brought out controversy among specialists [142].

A survey conducted in Italy reported 35/1000 pregnant

women with primary T. gondii infection and recommended

maternal screening during pregnancy rather than neonatal

screening [143]. In Norway, screening of pregnant women was

recommended until 1977 when the National Institute of Public

Health discouraged it, following a large study that showed low

(0.17 %) incidence of primary infection during pregnancy [144].

Two years following this change in policy, a study by Eskild etal. [145] showed that despite the recommendations, 81% of the

pregnant women were still routinely tested for T. gondii-specific

antibodies.

In Finland, a cost-benefit analyses of screening programs for

pregnant women as well as educationprograms revealed theben-

eficial effect of such programs in both low and high incidences

of toxoplasmosis [146].

Cost-effectiveness of optional screening programs (no

screening, pre-conception or neonates screening, frequency of

tests during pregnancy) depends on local factors: incidence of

congenital toxoplasmosis, available diagnostic and therapeutic

services, and the population compliance with screening. It is

important to promote public, as well as professional, knowledgeregarding the disease, in order to effectively prevent, diagnose

and treat congenital toxoplasmosis.

In conclusion, it is highly recommended to educate the pub-

lic and professionals to minimize risk of infection. Screening

programs of women at childbearing age and upon gestation or

at least newborn screening is highly effective for early treatment

and prevention of sequelae.

Acknowledgments

Dr. Irena Volovik Sub-district Health Officer, Hadera, Israel,

for providing data of the presented case.


12/15


Mrs. R. Kaufman and Mrs. R. Avni for excellent laboratory

work.

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