congenital toxoplasmosis—prenatal aspects of toxoplasma gondii infection

15
Reproductive Toxicology 21 (2006) 458–472 Review Congenital toxoplasmosis—prenatal aspects of Toxoplasma 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, Israel b District Health Office, Ministry of Health, Jerusalem, Israel c National 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 50–80% 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 linked to the clinical 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 of T. 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 ....................................................................................... 463 4.3.1. 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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Page 1: Congenital toxoplasmosis—prenatal aspects of Toxoplasma gondii infection

Reproductive Toxicology 21 (2006) 458–472

Review

Congenital toxoplasmosis—prenatal aspects of Toxoplasma 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, Israelb District Health Office, Ministry of Health, Jerusalem, Israel

c National Toxoplasmosis Reference Center, Ministry of Health, Israel

Received 26 August 2004; received in revised form 11 October 2005; accepted 24 October 2005Available 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 50–80% in Europe). Vertical transmission from arecently infected pregnant woman to her fetus may lead to congenital toxoplasmosis. The risk of such transmission increases as primary maternalinfection occurs later in pregnancy. However, consequences for the fetus are more severe with transmission closer to conception. The timing ofmaternal primary infection is, therefore, critically linked to the clinical 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 ofT. 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, provedto 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 recommendedto 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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4592. The parasite . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 459

2.1. Life cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4592.2. Mechanism of infection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4602.3. Virulence of T. gondii strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 460

3. Epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4604. Congenital toxoplasmosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 461

4.1. Incidence and prevalence in pregnant women and infants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4614.2. Diagnostic evaluation, manifestation and consequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4614.3. Prenatal laboratory diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 463

4.3.1. Sabin Feldman dye test (SFDT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4634.3.2. Enzyme immunoassays (EIA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4654.3.3. Immunosorbent agglutination assay test (IAAT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4654.3.4. Indirect fluorescent assay (IFA). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4654.3.5. Avidity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4654.3.6. 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) 458–472 459

4.3.7. Molecular diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4664.4. Laboratory diagnosis of infants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 467

4.4.1. Western blots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4675. Treatment of congenital toxoplasmosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4676. Prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 468

6.1. Primary prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4686.1.1. Vaccine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 468

6.2. Secondary prevention – screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 468Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 468References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 469

1. Case report

A 26-year-old woman from a rural village in northern Israelpresented with cervical lymphadenopathy during the 13th weekof her first pregnancy. The woman was otherwise healthy andwithout any symptoms. She was followed up by her primarycare physician and as the lymphadenopathy did not resolve,was sent for surgical consultation during the 26th week ofpregnancy. 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 fromthe 26th week of gestation, performed at the Israel NationalToxoplasmosis 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 (byEItadoofwl

normal. Cranial ultrasonography, brain stem evoked response(BERA), audiometry and eye examination were all normal. Testsfor T. gondii in the infant included: PCR of CSF—negative,immuno-sorbent agglutination assays (IgM-ISAGA)—negativeand Sabin Feldman Dye Test (SFDT)—positive (250 IU/ml),probably reflecting maternal antibodies transfer. Despite theserological indicators of maternal infection (most probablytowards the end of the first trimester) and positive PCR of theamniotic 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 theage of 1 year. Medical evaluation, auditory and ophthalmic testsat the age of 4 and 8 months revealed normal physical growthand development and intensive follow-up continues (at the ageof 6 months laboratory analysis was reported to be normal).

LFA, Enzyme-Linked Fluorescent immuno-Assay) with lowgG avidity (0.027). These results were reported and an addi-ional serum sample, as well as an earlier sample (whethervailable) were requested. Blood samples were subsequentlyelivered to our laboratory; from the 12th (sample drawn as partf the routine pregnancy follow-up) and from the 34th weeksf pregnancy. The results of the earlier sample were negativeor both total Ig and IgM antibodies. The results from the 34theek were positive for total T. gondii-specific immunoglobu-

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

This case demonstrates the complexity of establishing clin-ical diagnosis and interpretation of laboratory results in regardto T. gondii infection in pregnancy. The favourable outcomedespite the timing of infection may be attributed to providing antiparasitic therapy, although the specific role of therapy or otherunknown variables is unclear. Since many T. gondii infectionsare sub-clinical or present with non-specific signs, physiciansshould be able to integrate clinical and laboratory data in orderto make diagnostic and therapeutic decisions.

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. The parasite

T. gondii is a member of the phylum Apicomplexa, order Coc-idia, which are all obligate intracellular protozoan parasites.ther members of this phylum include known human pathogens

uch as Plasmodium (malaria) and Cryptosporidium.

.1. Life cycle

The life cycle of T. gondii consists of two stages—asexual andexual: the asexual stage takes place in the intermediate hosts,hich are mammals or birds. During this phase rapid intracellu-

ar growth of the parasite as tachyzoite takes place (generationime in vitro is 6–8 h). The oval or crescent-shaped tachyzoitesan infect and multiply in almost any nucleated mammalian orvian cell [1]. Following accumulation (64–128), tachyzoites areecreted into the blood stream [2] and spread in the body, leadingo development of an acute disease (parasitemia). The normalmmune response and transformation of the tachyzoite into cyst-orming bradyzoites limit the acute stage and establish a chronicnfection. Bradyzoites differ from tachyzoites mainly in their

T. gondii-specific IgM antibodies (by ELFA, Enzyme-LinkedFluorescent immuno-Assay) with low IgG avidity (0.055).

The sum interpretation of the three above tests results ofthe 12th, 26th and 34th week of pregnancy was consistentwith definite recent T. gondii infection (seroconversion, constanthigh 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 therapyincluding Pyrimethamine, Sulfadiazine and folinic acid wasstarted and continued until birth. The pregnancy course wasotherwise uneventful and fetal growth assessment through ultra-sound follow-up did not reveal any abnormality. During the38th 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. Laboratorytests including complete blood count, glucose, electrolytes, liverfunction tests and cerebro-spinal fluid (CSF) tests were all

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460 E. Rorman et al. / Reproductive Toxicology 21 (2006) 458–472

extremely slow multiplication rate (their name reflects this slowprocess) and in the distinct set of proteins they express [1,3–5].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 maycause acute encephalitis.

The sexual stage takes place in the intestine of the definitivehost. Known definitive hosts are members of the feline fam-ily, predominantly domestic cats. When bradyzoites or oocytesare ingested by a feline, formation of oocytes proceeds in theepithelium of the small intestine. Several million unsporulatedoocytes may be released in the feces of a single cat over a period3–18 days, depending on the stage of T. gondii ingested [1].Under mild environmental conditions oocytes may sporulatewithin a 3-week period [6], then infecting humans and otherintermediate hosts. Oocysts can spread in the environment andcontaminate water, soil, fruits, vegetables and herbivores fol-lowing consumption of infected plant material. Investigation ofoutbreaks of toxoplasmosis has led to recovery of oocytes fromsoil [7] but not from water [8–10]. Oocytes have been found tobe very stable, especially in warm and humid environments, andresistant to many disinfecting agents [11], but survive poorly inarid, cold climates [12].

2.2. Mechanism of infection

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genome of T. gondii, consisting of 14 chromosomes, is currentlybeing investigated and sequenced [26] (http://ToxoDB.org).

2.3. Virulence of T. gondii strains

Clinical manifestations and severity of illness followinginfection are affected by features of the interaction between theparasite and the host and include strain virulence, inoculum size,route of infection, competence of the host’s immune response(both cellular and humoral), integrity of the host’s mucosal andepithelial barriers, host’s age and genetic background [27]. Var-ious strains of T. gondii have long been known to differ invirulence and pathogenicity [28,29]. These strains can be classi-fied by immunologic assays, isoenzyme analysis and molecularanalysis [30–33]. There are three T. gondii clonal lineages, ofthem one carries conserved genetic loci, suspected of codingfor the virulence trait [24]. Grigg et al. [34] demonstrated that asexual recombination, performed in vitro, between the two rela-tively avirulent strains can give rise to the virulent strain. This isin accordance with polymorphism analysis of the three T. gondiistrains, which indicated that they emerged within the last 10,000years, following a single genetic cross [34,35]. Acquisition ofan efficient mechanism to spread by direct oral transmission,bypassing a sexual phase, leads to successful clonal expansionof this virulent lineage [35,36].

Genetic background plays a significant role in increased sus-cat

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T. gondii has been shown to migrate over long distances in theost’s body; crossing biological barriers, actively enter the bloodtream, invade cells and cross substrates and non-permissiveiological sites such as the blood-brain-barrier, the placenta andhe intestinal wall. At the same time, the parasite minimizesxposure to the host’s immune response, by rapidly entering andxiting cells. These two functions share common mechanismshich depend on Ca2+ regulation [13].Unlike many bacteria and viruses, T. gondii actively enters

he cell, in a mechanism which is mediated by the para-ites’ cytoskeleton and regulated by a parasite-specific calcium-epended secretion pathway [2,14]. The first step of cell invasiony T. gondii is recognition of an attachment point. The twopecial organelles involved in this invasion process, rhoptriesnd micronemes, each discharging proteins during the process5,15]. Following the rapid cellular invasion the parasite residesithin a vacuole, derived primarily from the host cell’s plasmaembrane [2,16]. The active motion of T. gondii, called “glid-

ng”, occurs with no major changes in cell shape. It is fast (about0 times faster than the “crawling” rate of amoeboid cells), andonsists of both circular gliding in a counter-clockwise directionnd clockwise helical gliding [17–21]. As an obligatory parasite,t’s invasive capabilities play an important role in virulence andathogenicity, since it can only survive intracellularly where itets nutrients and escapes from the host’s immune response [22].he most virulent T. gondii strain has been shown to exhibit supe-

ior migratory capacity [23] and a subpopulation of this strainisplays a special, long distance migration phenotype [14]. Thebility to cross biological barriers is associated with acute vir-lence and is linked to genes on chromosome VII [24,25]. The

eptibility to T. gondi in humans; HLA-DQ3 appears to begenetic marker associated with susceptibility to developing

oxoplasma-dependent encephalitis [37,38].

. Epidemiology

T. gondii infection is most frequently caused by ingestionf row or undercooked meat, which carries tissue cysts, byonsuming infected water or food or by accidental intake ofontaminated soil. Toxoplasmosis is also an occupational haz-rd for laboratory workers. A total of 47 laboratory-acquiredases have been reported, 81% of them were symptomatic cases39].

Tender et al. [40] collected data of nation-wide T. gondii sero-revalence in women at child-bearing age (1990–2000). Theates of positive sero-prevalence, were 58% in Central Euro-ean countries, 51–72% in several Latin-American countriesnd 54–77% in West African countries. Low seroprevalence,–39%, was reported in southwest Asia, China and Korea asell as in cold climate areas such as Scandinavian countries

11–28%). In the US 15% of females at childbearing age wereound to be seropositive [41]. It should be noted that seropositiverevalence in the same country may differ among populationsr geographical regions and world-wide prevalence is higher inlder populations.

In a limited case–control study that included six large Euro-ean centers it was shown that the consumption of undercookedeat was the major risk factor for toxoplasmosis infection [42].nother study aimed to determine the prevalence of T. gondii

n edible meat tested 71 meat samples from commercial sourcesn the UK for the parasite—positive results were found in 27

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E. Rorman et al. / Reproductive Toxicology 21 (2006) 458–472 461

samples. Twenty-one of these contaminated meat samples car-ried the virulent T. gondii type I [43]. Although cats play adefinite role in the epidemiology of toxoplasmosis, no signif-icant correlation between human toxoplasmosis infection andcat ownership could be proven [44]. Furthermore, the oocytesare not found on cat fur but rather are buried in the soil as theyare shed with cat faeces [45–47].

Data regarding seroprevalence of specific T. gondii anti-bodies in the Israeli population are based on several regionalsurveys performed in collaboration with the Israeli NationalToxoplasmosis Reference Center. The prevalence in certain sub-populations of pregnant women in northern Israel had beenreported to be 21% on average and the incidence rate of infectionacquired during pregnancy estimated as 1.4% [48].

Human contact with infected oocyst from contaminated soil[7,49,50] and water [8–10] were associated with several reportedepidemics caused by T. gondii. Only in one case were T.gondii oocysts recovered from the soil—the suspected sourceof 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 ofsample is required for isolation of the organism. In addition,there is a lag period between the time of infection and the timethat the contaminated source is tested, further reducing the like-lihood of recovery of oocytes from the suspected environmentd

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disease, resulting in brain lesions or diffuse encephalitis. Otherorgans, such as the heart, lung, liver, and retina may also beinvolved. Most of these cases result from reactivation of latentinfection [54] although re-infection with a different T. gondiistrain in the transplanted organs may also occur.

4.1. Incidence and prevalence in pregnant women andinfants

The disease is caused by vertical transmission of T. gondiifrom a seronegative pregnant woman, who is acutely infectedwith T. gondii to her fetus.

The prevalence of T. gondii and its incidence of human infec-tion vary widely amongst various countries. Worldwide, 3–8infants per 1000 live births are infected in utero [55]. Multiplefactors are associated with the occurrence of congenital toxo-plasmosis infection, including route of transmission, climate,cultural behaviour, eating habits and hygienic standards. Thiscombination leads to marked differences even among developednations. For example, the incidence of congenital infection inBelgium and France is 2–3 cases per 1000 live births—markedlyhigher than the US incidence, which is between 1 in 10,000 to1 in 1000 live births [47,56].

In a research conducted in Goiania, Brazil, a region witha relatively high seroconversion rate, pregnant women werefound to have a 2.2 times higher risk for seroconversion thannahtms

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

llnesses attributed to a known pathogen, and 20.7% of the totalood-borne mortality caused by a known pathogen, in the Unitedtates in 1996–1997. Many of these cases involved HIV-infectedatients [53].

The largest reported toxoplasmosis outbreak resulting fromontaminated water occurred in British Columbia and causedcute infection in 100 people; 19 with retinitis and 51 with lym-hadenopathy. The likely source was a municipal water systemhat used unfiltered, chloraminated surface water [10]. There waslso a seasonal correlation to rainfall and turbidity in this watereservoir. In another small outbreak North of Rio de Janeiro,razil, the source of the parasite was traced to an unfiltered water

ource. It was also linked to high prevalence of seropositivity inhis region of low socio-economic background [8].

. Congenital toxoplasmosis

Most cases of acquired toxoplasma infection are asymp-omatic and self-limited; hence many cases remain undiagnosed.he incubation period of acquired infection is estimated to beithin a range of 4–21days (7 days on average) [10]. When

ymptomatic infection does occur the only clinical findings maye focal lymphadenopathy, most often involving a single siteround the head and neck. Less commonly, acute infection isccompanied by a mononucleosis-like syndrome characterizedy fever, malaise, sore throat, headache and an atypical lympho-ytosis on peripheral blood smear [54]. In immunocompromisedatients, most commonly HIV infected and organ transplantecipients, T. gondii may cause a severe central nervous system

on-pregnant women of equivalent characteristics. In addition,mongst pregnant women, adolescents were shown to have theighest risk for seroconversion [57]. The authors hypothesizedhat higher vulnerability to T. gondii infection during pregnancy

ay be due to a combination of pregnancy associated immuno-uppression as well as hormonal changes.

Only a few cases of congenital toxoplasmosis transmittedy mothers who were infected prior to conception have actu-lly been reported [58–60]. One such case published recentlynvolved a woman who had ocular toxoplasmosis 20 years prioro giving birth to a newborn, who suffered from congenital tox-plasmosis. The mother had a “toxoplasmic scar” in the retinand was tested positive for specific toxoplasma IgG antibodies.he newborn was found to be positive for both IgG and IgMntibodies and had a macular scar on the retina, typical to tox-plasmosis, as well as a calcified brain granuloma. [59]. Suchcase could be attributed to re-infection with a different, moreirulent strain or by reactivation of a chronic disease[58].

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

ccurrence is difficult to quantify, but it is probably low. Latent. gondii infection may be reactivated in immunodeficient indi-iduals (such as HIV-infected women) and result in congenitalransmission of the parasite [61].

.2. Diagnostic evaluation, manifestation andonsequences

The diagnostic evaluation of T. gondii is part of routine preg-ancy follow-up and differential diagnosis of intrauterine infec-ion. Intrauterine ultrasonographic findings of T. gondii infection

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462 E. Rorman et al. / Reproductive Toxicology 21 (2006) 458–472

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 intrauterineinfection in which the diagnosis was based upon hydrocephalusin fetal ultrasound, even though PCR of amniotic fluid wasnegative 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 withinseveral months post-infection in cases of intrauterine infectionof T. gondii. Additional ultrasonographic findings may includehepatomegaly, splenomegaly, ascitic fluid, cardiomegaly andplacental abnormalities [55,64]. Safadi et al. [65] followed 43children with congenital toxoplasmosis for a period of at least5 years. Most of them (88%) had sub-clinical presentationat birth. The most common neurological manifestation was adelay in neuro-psychomotor development. Half of the childrendeveloped neurological manifestations, 7 children had neuro-radiologic alterations in skull radiography, and 33 children intomography. Notably, cerebral calcifications were not associatedwith an increased incidence of neurological sequelae. Choriore-tinitis was the main ocular sequelae, found in almost all childrenand noted years after birth, despite specific therapy in the firstyear of life.

An important step in the diagnosis of congenital toxoplasmo-stiotabt1b

ties. They concluded that positive screening results must be care-fully confirmed [66]. Laboratory methods and their implicationsin supporting evidence-based diagnoses are discussed below.

The risk of fetal infection is multifactorial, depending on thetime of maternal infection, immunological competence of themother during parasitemia, parasite load and strain’s virulence[40]. The probability of fetal infection is only 1% when pri-mary maternal infection occurs during the preconception periodbut increases as pregnancy progresses; infection acquired dur-ing the first trimester by women not treated with anti-T. gondiidrugs 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 30–54% and60–65%, respectively [54].

The consequences are more severe when fetal infectionoccurs in early stages of pregnancy, when it can cause miscar-riage (natural abortion or death occurs in 10% of pregnanciesinfected with T. gondii [67]), severe disease, intra-uterine growthretardation or premature birth. A multi-centre prospective cohortstudy evaluated the association between congenital toxoplas-mosis and preterm birth, low birth weight, and small size forgestational age [68]. Freeman et al. reported that infected babieswere born earlier than uninfected babies and that congenitalinfection 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 weightoiaili

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is and evaluation of time of infection is achieved by laboratoryechniques, monitoring the immune response: titer and affin-ty of specific antibodies (Fig. 1). Other laboratory tools focusn direct detection of the parasite by animal or tissue inocula-ion or more commonly, by molecular techniques. Carvalheiro etl. studied the incidence of congenital toxoplasmosis in Brazil,ased on persistence of anti-Toxoplasma IgG antibodies beyondhe age of 1year. Disease incidence was estimated to be 3.3 per0,000. A definitive diagnosis was confirmed in five infants withoth serum IgM and/or IgA antibodies, and clinical abnormali-

Fig. 1. Laboratory diagnosis of congenital toxoplasmosis.

r small size for gestational age. The cause for shorter gestations not yet known. The highest frequency of severe abnormalitiest birth is seen in children whose mothers acquired a primarynfection between the 10th and 24th week of gestation [67]. Theikelihood of clinical symptoms in the newborn is reduced whennfection occurs later.

Clinical manifestations in newborns with congenital toxo-lasmosis vary and can develop at different times before andfter birth. Most newborns infected with T. gondii are asymp-omatic at birth (70–90%) [61]. When clinical manifestations areresent they are mainly non-specific and may include: a mac-lopapular rash, generalized lymphadenopathy, hepatomegaly,plenomegaly, hyperbilirubinemia, anemia and thrombocytope-ia [69]. The classic triad of chorioretinitis, intracranial calcifi-ations and hydrocephalus is found in fewer than 10% of infectednfants [47]. Hydrocephalus and/or microcephaly may develophen intra-uterine infection results in meningo-encepahlitis

69]. All these signs and symptoms are included in the generalork-up of suspected congenital TORCH infections: toxoplas-osis, other (syphilis, varicella-zoster, parvovirus B19), rubella,

ytomegalovirus (CMV) and herpes infections. Cerebral cal-ifications can be demonstrated by cranial radiography, ultra-onography or computerized tomography. Neurologic impair-ent may initially present as seizures, necessitating specific

valuation and treatment.The most prevalent consequence of congenital toxoplasmosis

s chorioretinitis.Chorioretinitis is diagnosed based on characteristic retinal

nfiltrates. Vutova et al. [70] investigated eye manifestations ofongenital toxoplasmosis in 38 infants and children. The mostrequent finding was chorioretinitis (92%), together with other

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E. Rorman et al. / Reproductive Toxicology 21 (2006) 458–472 463

ocular lesions in 71% of cases, and the second most commonfinding was microphthalmia with strabismus. Lesions of theanterior segment of the eye included iridocyclitis, cataracts andglaucoma. Other uncommon findings were diminished visualacuity and neurological sequelae such as hydrocephalus, calci-fication in the brain, paresis, and epilepsy.

Wallon et al. [71] reported the clinical evolution of ocularlesions and final visual function, in a prospective cohort of 327congenitally infected children in France. The children were iden-tified by maternal prenatal screening and monitored for up to14 years. After 6 years, 79 (24%) children had at least oneretinochoroidal 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 withlesions in one eye, half the children with lesions in both eyesand none had bilateral visual impairment. Most of the mothers(84%) had been treated. A combination of pyrimethamine andsulfadiazine had been prescribed in all the children (38% beforeand 72% after birth). Late-onset retinal lesions and relapse canoccur many years after birth, but the overall ocular prognosis ofcongenital toxoplasmosis seems satisfactory, when infection isidentified early and appropriately treated. Early diagnosis andtreatment 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 testsas described in details below. If T. gondii infection is suspectedaac

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sue inoculation or more commonly, by molecular techniques. Itis important to combine all available clinical and laboratory dataduring the evaluation of toxoplasmosis diagnosis and providingtreatment recommendations.

Infection during gestation may cause serious damage tothe fetus and hence, a major objective of the diagnosis is toestimate the time of maternal infection. IgG antibodies usu-ally appear within two weeks of infection, peak within 6–8weeks and persist in the body indefinitely [67]. IgM antibod-ies are considered the indicators of recent infection and canbe detected by enzyme immunoassay (EIA) or immunosor-bent agglutination assay test (IAAT) relatively early—within2 weeks of infection. Uncertainty may arise as IgM may per-sist for years following primary infection [73]. IgA antibodiesmay also persist for more than a year [67] and their detectionis informative mainly for the diagnosis of congenital toxoplas-mosis. The level of specific IgE antibodies increases rapidlyand 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 fortoxoplasma infection should be sought. Both the laboratoryperforming the tests and the referring physician should beaware of the limitations and select the best combination oftests available to timely evaluate the stage of toxoplasmaiT

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t the time of birth, diagnostic work-up includes ophthalmic,uditory and neurological examinations, lumbar puncture andranial imaging [69].

In a large percentage of children the disease sequelae mayecome apparent and present with visual impairment, mental andognitive abnormalities of variable severity, seizures or learningisabilities only after several months or years [55].

Infants born to women infected simultaneously with HIVnd T. gondii should be evaluated for congenital toxoplasmosis,onsidering the increased risk of reactivation of parasitemia andisease in these mothers.

In a case–control study in Israel, Potasman et al. tested 95hildren with variable neurological disorders: cerebral palsy,pilepsy and nerve deafness compared with a control group of09 healthy children, for the presence of T. gondii-specific anti-odies in the serum. They found that children with any of theeurological disorders were significantly more likely to have. gondii specific IgG antibodies, especially those with nerveeafness (relative risk 2.5 and 7.1, respectively) [72].

A definite diagnosis cannot be made in the following situa-ions: (1) the infant is older than one year of age and was notested for toxoplasmosis previously, (2) either the child or theother is seronegative, or (3) the mother was known to be sero-

ositive prior to conception.

.3. Prenatal laboratory diagnosis

The principle method used to diagnose and evaluate tim-ng of congenital infection relies on indirect evidence, and isased on detection of specific antibodies, by monitoring themmune response. Direct evidence is obtained by animal or tis-

nfection [75]. Laboratory tests available are summarized inable 1.

.3.1. Sabin Feldman dye test (SFDT)This is the first test developed for the laboratory diagnosis of

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

es (total Ig) and is performed only in reference centers. Thehange in antibody titer as determined in SFDT in consecu-ive serum samples taken at least 3 weeks apart is importantor the evaluation of infection during pregnancy. A “significant”hange is considered to be at least a four-fold difference. Thebsolute antibody titer is also important—values over 250 IU/mlre considered “high” suggestive of recent infection. The testedera are serially diluted and incubated with live tachyzoitescarrying toxoplasma-specific antigens) in the presence of sep-rated human plasma from “sero-negative” donors (providingomplement components). The antigen–antibody–complementomplexes formed are subsequently lysed in the presence ofhe dye methylene blue. End-point titer is established by count-ng the numbers of dead (unstained) and live (stained) para-ites. The reported titer is that producing lysis of 50% of therganisms. End-point titer can be converted to internationalnits (IU): additional standardization is achieved by prepara-ion of a standardised control serum (consisting of a pool ofera), tested by numerous reference centers, and adjusted sohat the SFDT value of this control serum is set at 1000 IU/ml77]. Recently, the WHO recognized the first international stan-ard for human anti-toxoplasma IgG, with an assigned potencyf 20 IU per ampoule of total anti-toxoplasma antibodies78].

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464 E. Rorman et al. / Reproductive Toxicology 21 (2006) 458–472

Table 1Laboratory diagnostic tests for congenital toxoplasmosis

Test Matrix Results Interpretation Time Degree ofexpertiseequired

Other remarks Suggested use

Sabin Feldman DyeTest (SFDT)

Serum Titer in internationalunits (IU) of totalspecific Ig

Quantitative data:detection of high(≥250 IU) antibodiestiters and significantchanges (≥×4) intiter in consecutivesamples – importantfor evaluation ofrecent infection

Routine = ∼2–4× week

High, referencecenter only

Gold standard Confirmation ofinfection

“handson” = severalhours

Live parasitesand animalinjection → riskto lab employee

Standardizedassay(internationaleffort)

Follow-upchange in titer

EIAa/total Igb Serum Positive/negative fortotal specific Ig

Exposure to T. gondii Several hours Low = simpleautomated test

Possible falsenegative veryearly infection

Screening test

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

Exposure to T. gondii Several hours High, referencecenter only

Verysubjective anddifficult tostandardize

When SFDT isunavailable

IgG by EIA Serum Positive/negative forspecific IgG Abs

Exposure to T. gondii Several hours Low = simpleautomated test

Partial results(combine withIgM detection)

Screening test

IgM/IgA or IgE byEIA

Serum Positive/negative forspecific IgM, IgA orIgE Abs

Possible recentinfection with T.gondii

Several hours Low = simpleautomated test

Requiresfurther testing,IgE – not innewborn

IgM –Screening

IgM/IgA –NewbornIgE – Earlier

IgM/IgA or IgE byIAAT

Serum Positive/negative forspecific IgM, IgA orIgE Abs

Possible recentInfection with T.gondii

Several hours Relatively high Most sensitiveand specifictest

IgM/IgA –Newborn

Western blotshould beconsidered ifcontaminationwith maternalblood issuspected

IgM/IgA or IgE byIFA

Serum Positive/negative forspecific IgM, IgA orIgE Abs

Possible recentInfection with T.gondii

Several hours High, referencecenter only

Verysubjective anddifficult tostandardize

When ISAGA isunavailable

IgG avidity Serum Avidity = functionalaffinity

High avidity supports“past infection” (≥4months)

Several hours Relativelysimple

Supportiveevidence

When only asingle serumsample isavailable, in thebeginning ofpregnancy

Mice Body flu-ids/tissue

Positive/negative Presence of parasite 3–6 weeks High, referencecenter only

Lowsensitivity

Strain isolation

Live parasitesand animalinjection → riskto lab employee

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E. Rorman et al. / Reproductive Toxicology 21 (2006) 458–472 465

Table 1 (Continued)

Test Matrix Results Interpretation Time Degree ofexpertiseequired

Other remarks Suggested use

Cells Body flu-ids/tissue

Positive/negative Presence of parasite 3–6 days Very highreference centeronly

Lowsensitivity

When availablefor a direct proofof infection

Live parasitesPCR Body flu-

ids/tissuePositive/negative Presence of parasite’s

DNASeveral hours High High

sensitivityAmniotic fluid

Western blot IgG,IgM

Serum Identical/unidenticalto maternal Ig

Fetal/newborninfection

1 day High, referencecenter

Infrequentavailability

Confirmatorytest orfetal/newborninfection

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-linkedimmunosorbent assay (ELISA) and enzyme linked fluorescentimmuno-assay (ELFA) which test for the presence of IgG and/orIgM antibodies specific for the parasite in human sera. EIA areuseful as fast, low-cost screening tests and have been improvedover the years to avoid false positive results due to non-specificdetection of interfering factors such as rheumatoid factor andantinuclear antibodies.

There is no standardization of these tests, which causes highvariability in results obtained with different kits and/or in differ-ent laboratories. Consequently, and also as a result of the highincidence of false-positive results even in reference centers, theUS Food and Drug Administration (FDA) issued a health advi-sory to physicians on July, 1997. The FDA recommends avoidingreliance of results obtained with any single commercial kit forthe detection of toxoplasma-specific IgM, as the sole deter-minant of recent toxoplasma infection in pregnant women. Inour experience at the Israeli National Toxoplasmosis ReferenceCenter, during the years 1997–2002, in an average of 747 sam-ples (range: 652–816) received annually for confirmation, only17% ± 2.6% were indeed positive for T. gondii-specific IgM. Itis therefore recommended that patient follow-up would be per-formed by a reference center, and that commercial kits would belr

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genital infection (where the expected levels of antibodies arevery low) [74,83].

Toxoplasma-specific IgE antibodies can be detected by EIAor IAAT in sera of recently infected adults, congenitally infectedinfants, and children with congenital toxoplasmic chorioretinitis[84]. IgE detection is, however, ineffective in evaluating fetal ornewborn 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 withlive, 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 then detected using fluoresceinisothiocyanate-labeled anti-human Ig (or anti-IgG or anti-IgM).IFA is safer to perform and more economical than the SFDT. Itappears to measure the same antibodies as the dye test, and itstiters tend to parallel dye test titers [47,85]. However, the IFAinterpretation is subjective and time consuming. False positiveresults may occur with sera containing antinuclear antibodiesand rheumatoid factor [86], and false negative results of IFA forIgM may occur due to blockage by T. gondii-specific IgG [87].

4.3.5. AvidityIgG avidity testing was developed by Hedman et al. and is

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ocally evaluated to achieve the highest degree of accuracy andepeatability possible for screening tests.

In general, when toxoplasma infection is suspected based onetection of specific IgM antibodies specimens are referred foronfirmation by a reference center where SFDT, PCR and otherdvanced assays can be performed.

.3.3. Immunosorbent agglutination assay test (IAAT)IAAT is highly specific in detection of anti-T. gondii IgM,

gA or IgE antibodies [79]. This assay utilizes the entire tachy-oite and is the most sensitive commercially available method80–82]. Unfortunately, it is expensive, requires a high degree ofxpertise and is not automated. It is consequently seldom used ineference centers, usually in neonates suspected of having con-

ased on the increase in functional affinity (avidity) between. gondii-specific IgG and the antigen over time, as the hostmmune response (and specific B cell selection) evolves [88].issociation of the antigen–antibody complexes reflects the

ower avidity closer to primary infection. Pregnant women withigh avidity antibodies are those who have been infected at least–5 months earlier, which makes the avidity test most useful andeliable in the first trimester when high-avidity is detected [89].n one study, 35 out of 63 patients (55%) who were classifiedy toxoplasma-specific serology as having recent or border-ine infection showed high avidity-antibodies and were thereforereated as chronic patients [90]. Lappaplainen et al. [91] wereble to follow 13 women who showed high-avidity antibodies inhe first trimester and confirmed that none of the born infants wasound to be infected with T. gondii (as determined serologically

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466 E. Rorman et al. / Reproductive Toxicology 21 (2006) 458–472

after birth). The avidity test is most important when only a sin-gle serum sample is available at the time when critical decisionsmust be made. To the best of our knowledge, commercial IgGavidity kits have been licensed in Europe but not in the US [92].When avidity is low or borderline it may be misleading anda more careful interpretation of all laboratory tests results inconjunction with other clinical findings, should then be under-taken. Several studies have shown that this test is reliable andvaluable in diagnosis of recent infection during early pregnancy[88,93–98].

Accurate and definitive serologic diagnosis of recentlyacquired toxoplasma infection is still difficult and depends ontesting of more than one sample. Efforts to develop betterdiagnostic approaches continue based on antigens specificallyexpressed either during the primary phase (i.e. GRA7, GRA4)or the latent phase (i.e. GRA1) of infection. These antigens canbe produced by recombinant DNA technologies and may lead toa more informative serologic diagnosis, based on a single serumsample [47,99,100].

4.3.6. Animal and cell culture inoculationA definite laboratory confirmation of active toxoplasmosis

infection (especially in immunocompromised patients and preg-nant women) can be established by inoculation of body fluids ortissue into mice or cell culture [47].

Mice are injected intraperitoneally or subcutaneously with1TAp

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fii

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As in all diagnostic tests based on amplification of DNA,a few technical aspects are of crucial importance in achievingreliable results. Therefore, PCR based test should be carefullydesigned to include negative, positive and internal control, targetDNA for amplification should be specific, sample preparationtechniques should be perfected to extract minute parasite DNA[105] and to prevent cross contamination.

In a small (5 laboratories) inter-laboratories comparativework [106] followed by a larger study (15 laboratories) [104] sig-nificant differences in test performances were obtained, includ-ing false negatives and false positives. These results shoulddefinitely urge optimization and standardization of the test. Morerecently, 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 observedbetween the results of the three protocols [107].

Chabbert et al. [108] used two different primer sets of the B1gene to compare PCR performance followed by Southern blot,on various sample types (including amniotic fluid, blood andtissues). For amniotic fluid both PCR conditions produced sim-ilar results. The fragments produced by one of the primer setshad to be confirmed by specific hybridization, otherwise non-specific results were obtained. The PCR product of the sameamplification procedure was sequenced by Kompalic-Cristo andsuspected of originating from human DNA, as predicted bybioinformatics analysis [109].

oPHbraooIatnnedPo

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0–30 ml of sediment from amniotic fluid or whole fetal blood.he mice are bled prior and 3–6 weeks following inoculation.ntibody detection by SFDT establishes infection and finalroof is obtained by staining to demonstrate brain cysts [101].

Cell culture inoculation with amniotic fluid or blood usesndirect IFA to detect the parasite in monolayers within 3–6ays following inoculation [102]. When compared, inoculationf both blood and amniotic fluid from an infected fetus resultedn toxoplasma isolation from both cultures in 70% of cases.owever, in 40% of the cases T. gondii isolation is successful innly one of the samples [100]. Derouin et al. [100] demonstratedimilar sensitivities comparing cell culture and mice inoculation.hulliez et al. [102] reported that the sensitivity of amnioticuid cell culture inoculation is only 53% compared with 73%ensitivity in mice inoculation.

Currently, the principle role for these methods may be con-rmation of PCR as they are complex, expensive and relatively

nsensitive. [103].

.3.7. Molecular diagnosisReplacing fetal blood analysis, which is a high risk proce-

ure for the fetus, with molecular evaluation of amniotic fluidas provided a low risk diagnosis of congenital toxoplasmosis.olymerase chain reaction (PCR) is currently the most commonolecular technique routinely used for diagnosis of toxoplasmo-

is, although, it has not yet been standardized. No attempts haveeen made to standardize either the sample preparation processr the PCR amplification itself, and numerous laboratories useultiple “in-house” methods of varying sensitivities and relia-

ility [104,105]. Recently, a commercial PCR proficiency testecame available.

Different protocols influence the sensitivity and specificityf PCR assays. The specificity and positive predictive value ofCR tests on amniotic fluid samples is close to 100% [110,111].owever, the sensitivity of these PCR tests varies and estimated,ased on a large number of studies, to be 70–80% [105]. Oneeport showed that the sensitivity of PCR from amniotic fluid isffected by the stage of pregnancy in which maternal infectionccurs: best sensitivity was detected when maternal infectionccurred between 17 and 21 weeks of pregnancy [89,111,112].n addition, treatment with anti-toxoplasma drugs may alsoffect the sensitivity [89,112]. However, the reliability of a PCRest performed on amniotic fluid prior to the 18th week of preg-ancy requires further evaluation [110,111]. It should also beoted, that testing amniotic fluid for T. gondii was found to beffective about 4 weeks following infection, which is alreadyuring the parasitemic stage in the infected mother. Therefore,CR test should not be performed in the absence of serologic orther clinical/sonographic data indicative of infection.

In the last 4 years there have been reports on the use of Realime PCR, a sensitive and specific technique, which enablesapid detection of amplification products as well as hybridizationf amplicon-specific probes, similar to PCR followed by South-rn blot analysis. The method, which will ultimately replaceraditional PCR, enables an overall time for amplification andetection of less than two hours. In addition, cross contamina-ion is prevented by elimination of the need to handle amplifiedmplicons. In Real Time PCR it is possible to perform a quanti-ative study and follow the parasite load, allowing determinationf parasite count and its correlation with clinical symptoms andmpact of treatment. The technique permits linear range over 6ogs of DNA concentrations [113,114].

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E. Rorman et al. / Reproductive Toxicology 21 (2006) 458–472 467

The most popular target gene for PCR diagnosis of T. gondiiis the 35-fold repetitive gene B1. A variety of primers have beenused for amplification, some of which include nested primers.The second common locus is the single copy gene P30 alsoknown as SAG1, which encodes for a surface antigen. AnotherPCR target is the 18S ribosomal DNA. As reviewed by Bastien[105], two other target loci have been examined but are currentlynot used by most laboratories. Recently, some laboratories haveshown success in amplification of a DNA fragment, AF146527,which is repeated 200–300 times [89,113,114].

4.4. Laboratory diagnosis of infants

Laboratory diagnosis of Toxoplasma infection in infants isbased 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 directevidence as described above should be repeated as well duringthis period.

Serologic tests should follow total (or IgG) T. gondii specificantibodies titer (taking into account that closely after birth theseare maternal in origin, transferred through the placenta), IgMand IgA titers. Though passively transferred maternal IgG has ahalf life of approximately 1 month, it can still be detected in thenewborn for several months, generally disappearing completelywipdm

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drugs such as spiramycin (adult dosage 3–4 g/d × 3–4 weeks)and sometimes clindamycin are recommended in certain circum-stances. Spiramycin is used to prevent placental infection; it isused in many European countries especially France, Asia andSouth America. In the US, spiramycin is currently not approvedby the FDA but, available as an investigational drug, requiringspecial approval. Treatment with pyrimethamine and sulfadi-azine to prevent fetal infection is contraindicated during the firsttrimester of pregnancy due to concerns regarding teratogenicity,except when the mother’s health is seriously endangered. Duringthe first trimester sulfadiazine can be used alone.

As recently reviewed by Montoya and Liesenfeld [112], treat-ment protocols vary among different centers. The effectivity ofanti-T. gondii treatment is evaluated based on two criteria: rateof mother to child transmission and prevalence and severity ofsequelae. The majority of the studies are retrospective or cohortstudies of various populations and case definitions. The differ-ence in study patterns and methodologies affects the reliabilityand validity of the results and thus prevents issuing further rec-ommendations.

Wallon et al. [120] reviewed studies comparing treated anduntreated concurrent groups of pregnant women with provedor likely acute toxoplasma infection. Outcomes data of theoffspring were reported. The results showed treatment to beeffective in five studies but ineffective in four. Gras et al. [121]reported that the effect of prenatal pyrimethamine–sulfadiazinecicpp(sidaaAipatttdimArnos1ntww

ithin one year [112]. Appearance of autonomous IgG antibod-es in a congenitally infected newborn begins, in an untreatedatient, about 3 months after birth. Anti-parasitic therapy mayelay antibody production for about 6 months and, occasionally,ay completely prevent antibodies production [86].

.4.1. Western blotsRemington et al. introduced Western blots (using T. gondii-

pecific labeled antigens to detect antibodies, separated by elec-rophoresis and transferred to a membrane) to compare newbornersus maternal antibodies [115–117]. Western blotting couldotentially separate maternal from fetal/newborn antibodies.he test is not widely used mainly because of its technical com-lexity and high price.

. Treatment of congenital toxoplasmosis

Anti T. gondii treatment initiation generally requires con-rmatory laboratory tests in a reference center, followed byonsultation with experts. Treatment is indicated in the fol-owing conditions: infection during pregnancy and congenitalnfection as well as infection of an immunocompromised hoste.g. HIV/AIDS) and in case of an invasive disease. In pregnantomen and infected neonates, both symptomatic and asymp-

omatic, specific treatment of T. gondii infection is indicatedmmediately following established diagnosis. The combinationf pyrimethamine, (adult dosage 25–100 mg/d × 3–4 weeks),ulfadiazine adult dosage 1–1.5 g qid × 3–4 weeks) and foliniccid (leucovorin, 10–25 mg with each dose of pyrimethamine,o avoid bone marrow suppression) is the basic treatment pro-ocol recommended by the WHO [118] and CDC [119]. Other

ombination treatment on the cerebral and ocular sequelae ofntrauterine infection with T. gondii was not beneficial in 181hildren of infected mothers. Neto reported the outcome ofatients with congenital toxoplasmosis who were all treated withyrimethamine, sulfadiazine and folinic acid; of 195 patients 13871%) were asymptomatic until the age of 2 years. The authorsuggest that for six patients with sequelae because of the delayn anti-toxoplasma treatment (6–14 months post diagnosis) theisease was not prevented [122]. Gratzl et al. [123] reported vari-ble concentrations of spiramycin and its metabolites in serumnd amniotic fluid of 18 pregnant women following treatment.ll the drug concentrations were below the level reported to

nhibit parasite growth in vitro. The authors suggested that theossible reasons being individual pharmatokinetic variabilitynd patients’ treatment compliance. Gilbert et al. [124] reportedhe effect of prenatal treatment in 554 infected women andheir offspring. In this study comparison of early versus latereatment and of combination treatment (pyrimethamine, sulfa-iazine) with spiramycin or no-treatment, were all statisticallynsignificant. The possible interpretation is that delayed treat-

ent initiation led to failure to prevent parasite transmission.nother European multicenter study comparing transmission

ates and clinical outcomes in 856 mother–infant pairs, foundo significant association between the outcome and the intensityf treatment protocol in pregnancy [125]. Bessieres et al. [126]tudied the effect of treatment during pregnancy in a cohort of65 women and found that cases could be identified during preg-ancy as well as during the neonatal period. They also notedhat T. gondii was less frequently isolated in women treatedith pyrimethamine and sulfadoxine than in women treatedith spiramycin only. Foulon et al. [127] reviewed the measures

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468 E. Rorman et al. / Reproductive Toxicology 21 (2006) 458–472

of prevention of congenital toxoplasmosis and concluded thattreatment during pregnancy significantly reduces sequelae andtreatment 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 effectof treatment when comparing the different studies because of:different treatment regimes and timing (for small groups ofpatients), the pharmacokinetics patterns of drugs (concentra-tion in amniotic fluid and fetal CSF), patient (none) compliancewith treatment and different methodologies of follow-up in eachstudy.

As concluded by Peyron et al. [128] and others, further largescale, carefully controlled studies are necessary in order to clar-ify this controversial issue. At present the anti-parasite treatmentrecommended for toxoplasmosis as outlined above, should beconsidered 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 the parasite (hygienicand culinary practice during pregnancy). In Poland, an extensiveha4idcasop

6

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6

w

State of Goias, Brazil as recommended by experts [127]. Screen-ing of women should begin prior to conception with follow-upmonthly tests during pregnancy to detect seroconverion. This isthe basis for the French [138] screening program and the Aus-trian Toxoplasmosis Prevention Programs, both recommend rou-tine serologic testing, in Austria three times during pregnancy:in the first, second and third trimesters and in France six timesfollowing the initial finding [139]. Treatment is recommendedif one of the tests suggests definite or probable primary mater-nal infection [140]. In Massachusetts, USA, where there is lowseroprevalence in the population, only newborns are screenedfor the presence of T. gondii-specific IgM [141]. IgM detec-tion is followed by an extensive clinical evaluation and a oneyear treatment regimen combination of pyrimethamine and sul-fadiazine [140]. A recent study screened 364,130 neonates inthe United States for T. gondii specific IgM and confirmed 195cases of congenital toxoplasmosis (1 in 1867). Moreover, a 7-year follow-up of the treated patients revealed no symptoms orat least no progress of the disease. Based on these findings, theauthors suggest including toxoplasmosis in neonatal screeningprograms [122].

In the United Kingdom a national committee concluded thatno 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 pregnantwomen with primary T. gondii infection and recommendedmsrH(Tapa

peo

stcsira

lpaa

A

f

ealth education campaign, increased toxoplasmosis awarenessnd knowledge of preventive behaviour significantly within theyears of the reported study [129]. Many other countries have

ntroduced educational programs aimed at reducing the inci-ence of congenital toxoplasmosis. Such programs depend onareful identification of unique target-populations and tailoringppropriate approaches of education. To evaluate the success ofuch programs it is important to measure incidence rate beforenset and at pre-determined intervals after introducing the cam-aign.

.1.1. VaccineDevelopment of a vaccine for toxoplasmosis can prevent

uman disease by immunization of human as well as animalsthe source of infection). Both attenuated parasite and immuno-enic antigens are considered as potential agents for vaccination.ive attenuated S48 strain is in use for vaccination of sheep inurope and New Zealand but is unsuitable for human use due to

ts expense, short shelf life and most importantly, to the ability ofhe attenuated parasite to revert to a pathogenic strain [130–133].

uch of the work has been focused on SAG1, a surface anti-en expressed on tachyzoites, in attempts to induce protectivemmune response (mainly T-helper response) when introducedo the host with various adjuvants [134–136]. Development ofaccine using antigens expressed by bradyzoites and oocytes islso under investigation[134,137].

.2. Secondary prevention – screening

Routine toxoplasmosis screening programs for pregnantomen have been established in France, in Austria and in the

aternal screening during pregnancy rather than neonatalcreening [143]. In Norway, screening of pregnant women wasecommended until 1977 when the National Institute of Publicealth discouraged it, following a large study that showed low

0.17 %) incidence of primary infection during pregnancy [144].wo years following this change in policy, a study by Eskild etl. [145] showed that despite the recommendations, 81% of theregnant women were still routinely tested for T. gondii-specificntibodies.

In Finland, a cost-benefit analyses of screening programs forregnant women as well as education programs revealed the ben-ficial effect of such programs in both low and high incidencesf toxoplasmosis [146].

Cost-effectiveness of optional screening programs (nocreening, pre-conception or neonates screening, frequency ofests during pregnancy) depends on local factors: incidence ofongenital toxoplasmosis, available diagnostic and therapeuticervices, and the population compliance with screening. It ismportant to promote public, as well as professional, knowledgeegarding the disease, in order to effectively prevent, diagnosend treat congenital toxoplasmosis.

In conclusion, it is highly recommended to educate the pub-ic and professionals to minimize risk of infection. Screeningrograms of women at childbearing age and upon gestation ort least newborn screening is highly effective for early treatmentnd prevention of sequelae.

cknowledgments

Dr. Irena Volovik Sub-district Health Officer, Hadera, Israel,or providing data of the presented case.

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E. Rorman et al. / Reproductive Toxicology 21 (2006) 458–472 469

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

References

[1] Dubey JP, Lindsay DS, Speer CA. Structures of Toxoplasma gondiitachyzoites, bradyzoites, and sporozoites and biology and developmentof tissue cysts. Clin Microbiol Rev 1998;11:267–99.

[2] Black MW, Boothroyd JC. Lytic cycle of Toxoplasma gondii. MicrobiolMol Biol Rev 2000;64:607–23.

[3] Lekutis C, Ferguson DJ, Boothroyd JC. Toxoplasma gondii: identifica-tion of a developmentally regulated family of genes related to SAG2.Exp Parasitol 2000;96:89–96.

[4] Lyons RE, McLeod R, Roberts CW. Toxoplasma gondii tachyzoite-bradyzoite interconversion. Trends Parasitol 2002;18:198–201.

[5] Manger ID, Hehl AB, Boothroyd JC. The surface of Toxoplasmatachyzoites is dominated by a family of glycosylphosphatidylinositol-anchored antigens related to SAG1. Infect Immun 1998;66:2237–44.

[6] Dubey JP, Miller NL, Frenkel JK. The Toxoplasma gondii oocyst fromcat feces. J Exp Med 1970;132:636–62.

[7] Coutinho SG, Lobo R, Dutra G. Isolation of Toxoplasma from thesoil during an outbreak of toxoplasmosis in a rural area in Brazil. JParasitol 1982;68:866–8.

[8] Bahia-Oliveira LM, Jones JL, Azevedo-Silva J, Alves CC, Orefice F,Addiss DG. Highly endemic, waterborne toxoplasmosis in north Riode Janeiro state, Brazil. Emerg Infect Dis 2003;9:55–62.

[9] Benenson MW, Takafuji ET, Lemon SM, Greenup RL, Sulzer AJ.Oocyst-transmitted toxoplasmosis associated with ingestion of contam-inated water. N Engl J Med 1982;307:666–9.

[10] Bowie WR, King AS, Werker DH, Isaac-Renton JL, Bell A, EngSB, et al. Outbreak of toxoplasmosis associated with municipaldrinking water. The BC Toxoplasma Investigation Team. Lancet1997;350:173–7.

[11] Dumetre A, Darde ML. How to detect Toxoplasma gondii oocysts inenvironmental samples? FEMS Microbiol Rev 2003;27:651–61.

[12] Jones JL, Lopez A, Wilson M, Schulkin J, Gibbs R. Congenital toxo-plasmosis: a review. Obstet Gynecol Surv 2001;56:296–305.

[13] Hoff EF, Carruthers VB. Is Toxoplasma egress the first step in inva-sion? Trends Parasitol 2002;18:251–5.

[14] Barragan A, Sibley LD. Migration of Toxoplasma gondii across bio-logical barriers. Trends Microbiol 2003;11:426–30.

[15] Kasper LH, Mineo JR. Attachment and invension of host cell by Tox-oplasma gondii. Parasitol Today 1994;10:82–5.

[16] Suss-Toby E, Zimmerberg J, Ward GE. Toxoplasma invasion: theparasitophorous vacuole is formed from host cell plasma mem-brane and pinches off via a fission pore. Proc Natl Acad Sci USA1996;93:8413–8.

[17] Hakansson S, Morisaki H, Heuser J, Sibley LD. Time-lapse videomicroscopy of gliding motility in Toxoplasma gondii reveals anovel, biphasic mechanism of cell locomotion. Mol Biol Cell1999;10:3539–47.

[18] Mordue DG, Desai N, Dustin M, Sibley LD. Invasion by Toxoplasmagondii establishes a moving junction that selectively excludes host cellplasma membrane proteins on the basis of their membrane anchoring.J Exp Med 1999;190:1783–92.

[19] Mordue DG, Hakansson S, Niesman I, Sibley LD. Toxoplasma gondiiresides in a vacuole that avoids fusion with host cell endocytic andexocytic vesicular trafficking pathways. Exp Parasitol 1999;92:87–99.

[20] Lecordier L, Mercier C, Sibley LD, Cesbron-Delauw MF. Transmem-brane insertion of the Toxoplasma gondii GRA5 protein occurs aftersoluble secretion into the host cell. Mol Biol Cell 1999;10:1277–87.

[21] Opitz C, Soldati D. ’The glideosome’: a dynamic complex power-ing gliding motion and host cell invasion by Toxoplasma gondii. MolMicrobiol 2002;45:597–604.

[22] Sibley LD. Intracellular parasite invasion strategies. Science2004;304:248–53.

[23] Barragan A, Sibley LD. Transepithelial migration of Toxoplasmagondii is linked to parasite motility and virulence. J Exp Med2002;195:1625–33.

[24] Su C, Howe DK, Dubey JP, Ajioka JW, Sibley LD. Identification ofquantitative trait loci controlling acute virulence in Toxoplasma gondii.Proc Natl Acad Sci USA 2002;99:10753–8.

[25] Weiss LM, Kim K. The International Congress on Toxoplasmosis. IntJ Parasitol 2004;34:249–52.

[26] Kissinger JC, Gajria B, Li L, Paulsen IT, Roos DS. ToxoDB: accessingthe Toxoplasma gondii genome. Nucleic Acids Res 2003;31:234–6.

[27] Bhopale GM. Pathogenesis of toxoplasmosis. Comp Immunol Micro-biol Infect Dis 2003;26:213–22.

[28] Sibley LD, Boothroyd JC. Virulent strains of Toxoplasma gondii com-prise a single clonal lineage. Nature 1992;359:82–5.

[29] Howe DK, Sibley LD. Toxoplasma gondii comprises three clonal lin-eages: correlation of parasite genotype with human disease. J InfectDis 1995;172:1561–6.

[30] Ware PL, Kasper LH. Strain-specific antigens of Toxoplasma gondii.Infect Immun 1987;55:778–83.

[31] Kong JT, Grigg ME, Uyetake L, Parmley S, Boothroyd JC. Serotypingof Toxoplasma gondii infections in humans using synthetic peptides. JInfect Dis 2003;187:1484–95.

[32] Ajzenberg D, Cogne N, Paris L, Bessieres MH, Thulliez P, FilisettiD, et al. Genotype of 86 Toxoplasma gondii isolates associated withhuman congenital toxoplasmosis, and correlation with clinical findings.J Infect Dis 2002;186:684–9.

[33] Ferguson DJ, Pittilo RM. Toxoplasma gondii and the professor. Para-sitol Today 1999;15:301–2.

[34] Grigg ME, Bonnefoy S, Hehl AB, Suzuki Y, Boothroyd JC. Successand virulence in Toxoplasma as the result of sexual recombinationbetween two distinct ancestries. Science 2001;294:161–5.

[35] Su C, Evans D, Cole RH, Kissinger JC, Ajioka JW, Sibley LD. Recentexpansion of Toxoplasma through enhanced oral transmission. Science2003;299:414–6.

[36] Volkman SK, Hartl DL, Parasitology. A game of cat and mouth. Sci-ence 2003;299:353–4.

[37] Suzuki Y, Wong SY, Grumet FC, Fessel J, Montoya JG, Zolopa AR,et al. Evidence for genetic regulation of susceptibility to toxoplasmicencephalitis in AIDS patients. J Infect Dis 1996;173:265–8.

[38] Mack DG, Johnson JJ, Roberts F, Roberts CW, Estes RG, David C, etal. HLA-class II genes modify outcome of Toxoplasma gondii infec-tion. Int J Parasitol 1999;29:1351–8.

[39] Herwaldt BL. Laboratory-acquired parasitic infections from accidentalexposures. Clin Microbiol Rev 2001;14:659–88, table of contents.

[40] Tenter AM, Heckeroth AR, Weiss LM. Toxoplasma gondii: from ani-mals to humans. Int J Parasitol 2000;30:1217–58.

[41] Jones JL, Kruszon-Moran D, Wilson M, McQuillan G, Navin T,McAuley JB. Toxoplasma gondii infection in the United States: sero-prevalence and risk factors. Am J Epidemiol 2001;154:357–65.

[42] Cook AJ, Gilbert RE, Buffolano W, Zufferey J, Petersen E, JenumPA, et al. Sources of toxoplasma infection in pregnant women: Euro-pean multicentre case–control study. European Research Network onCongenital Toxoplasmosis. BMJ 2000;321:142–7.

[43] Aspinall TV, Marlee D, Hyde JE, Sims PF. Prevalence of Toxoplasmagondii in commercial meat products as monitored by polymerase chainreaction—food for thought? Int J Parasitol 2002;32:1193–9.

[44] Lappin MR, Burney DP, Hill SA, Chavkin MJ. Detection of Toxo-plasma gondii-specific IgA in the aqueous humor of cats. Am J VetRes 1995;56:774–8.

[45] Dubey JP. Duration of immunity to shedding of Toxoplasma gondiioocysts by cats. J Parasitol 1995;81:410–5.

[46] Dubey JP. Sources of Toxoplasma gondii infection in pregnancy. Untilrates of congenital toxoplasmosis fall, control measures are essential.BMJ 2000;321:127–8.

[47] Dubey JP, Beattie CP. Toxoplasmosis of animals and man. Boca Raton:CRC Press; 1988.

[48] Franklin DM, Dror Z, Nishri Z. The prevalence and incidence of Tox-oplasma antibodies in pregnant women. Isr J Med Sci 1993;29:285–6.

Page 13: Congenital toxoplasmosis—prenatal aspects of Toxoplasma gondii infection

470 E. Rorman et al. / Reproductive Toxicology 21 (2006) 458–472

[49] Teutsch SM, Juranek DD, Sulzer A, Dubey JP, Sikes RK. Epi-demic toxoplasmosis associated with infected cats. N Engl J Med1979;300:695–9.

[50] Stagno S, Dykes AC, Amos CS, Head RA, Juranek DD, Walls K.An outbreak of toxoplasmosis linked to cats. Pediatrics 1980;65:706–12.

[51] Isaac-Renton J, Bowie WR, King A, Irwin GS, Ong CS, Fung CP, etal. Detection of Toxoplasma gondii oocysts in drinking water. ApplEnviron Microbiol 1998;64:2278–80.

[52] Kourenti C, Heckeroth A, Tenter A, Karanis P. Development and appli-cation of different methods for the detection of Toxoplasma gondii inwater. Appl Environ Microbiol 2003;69:102–6.

[53] Mead PS, Slutsker L, Dietz V, McCaig LF, Bresee JS, Shapiro C, etal. Food-related illness and death in the United States. Emerg InfectDis 1999;5:607–25.

[54] Lynfield R, Guerina NG. Toxoplasmosis. Pediatr Rev 1997;18:75–83.[55] Feigen RD, Adcock LM, Edwards MS. Fungel and protozoal infec-

tions (Toxoplasmosis). In: Fanaroff AA, Martin RJ, editors. Neonatalperinatal medicine disease of the fetus and infant. 5th ed. Mosby YearBook; 1992. p. 688–90.

[56] Guerina NG. Congenital infection with Toxoplasma gondii. PediatrAnn 1994;23:138–42, 47–51.

[57] Avelino MM, Campos Jr D, do Carmo Barbosa de Parada J, de CastroAM. Pregnancy as a risk factor for acute toxoplasmosis seroconversion.Eur J Obstet Gynecol Reprod Biol 2003;108:19–24.

[58] Dollfus H, Dureau P, Hennequin C, Uteza Y, Bron A, Dufier JL.Congenital toxoplasma chorioretinitis transmitted by preconceptionallyimmune women. Br J Ophthalmol 1998;82:1444–5.

[59] Silveira C, Ferreira R, Muccioli C, Nussenblatt R, Belfort Jr R. Toxo-plasmosis transmitted to a newborn from the mother infected 20 yearsearlier. Am J Ophthalmol 2003;136:370–1.

[71] Wallon M, Kodjikian L, Binquet C, Garweg J, Fleury J, Quantin C, etal. Long-term ocular prognosis in 327 children with congenital toxo-plasmosis. Pediatrics 2004;113:1567–72.

[72] Potasman I, Davidovitch M, Tal Y, Tal J, Zelnik N, Jaffe M. Congenitaltoxoplasmosis: a significant cause of neurological morbidity in Israel?Clin Infect Dis 1995;20:259–62.

[73] Bobic B, Sibalic D, Djurkovic-Djakovic O. High levels of IgM anti-bodies specific for Toxoplasma gondii in pregnancy 12 years afterprimary toxoplasma infection. Case report. Gynecol Obstet Invest1991;31:182–4.

[74] Pinon JM, Toubas D, Marx C, Mougeot G, Bonnin A, Bonhomme A,et al. Detection of specific immunoglobulin E in patients with toxo-plasmosis. J Clin Microbiol 1990;28:1739–43.

[75] Roberts A, Hedman K, Luyasu V, Zufferey J, Bessieres MH, BlatzRM, et al. Multicenter evaluation of strategies for serodiagnosis ofprimary infection with Toxoplasma gondii. Eur J Clin Microbiol InfectDis 2001;20:467–74.

[76] Sabin AB, Feldman HA. Dyes as microchemical indicators of a newimmunity phenomenon affecting a protozon parasite (Toxoplasma). Sci-ence 1948;108:660–3.

[77] Reiter-Owona I, Petersen E, Joynson D, Aspock H, Darde ML,Disko R, et al. The past and present role of the Sabin-Feldman dyetest in the serodiagnosis of toxoplasmosis. Bull World Health Org1999;77:929–35.

[78] Rigsby P, Rijpkema S, Guy EC, Francis J, Das RG. Evaluationof a candidate international standard preparation for human anti-Toxoplasma immunoglobulin G. J Clin Microbiol 2004;42:5133–8.

[79] Stepick-Biek P, Thulliez P, Araujo FG, Remington JS. IgA antibodiesfor diagnosis of acute congenital and acquired toxoplasmosis. J InfectDis 1990;162:270–3.

[80] Duffy KT, Wharton PJ, Johnson JD, New L, Holliman RE. Assessment

[60] Chemla C, Villena I, Aubert D, Hornoy P, Dupouy D, Leroux B, et al.

Preconception seroconversion and maternal seronegativity at deliverydo not rule out the risk of congenital toxoplasmosis. Clin Diagn LabImmunol 2002;9:489–90.

[61] Lebech M, Joynson DH, Seitz HM, Thulliez P, Gilbert RE, DuttonGN, et al. Classification system and case definitions of Toxoplasmagondii infection in immunocompetent pregnant women and their con-genitally infected offspring. European Research Network on Congen-ital Toxoplasmosis. Eur J Clin Microbiol Infect Dis 1996;15:799–805.

[62] El Ayoubi M, de Bethmann O, Monset-Couchard M. Lenticulostriateechogenic vessels: clinical and sonographic study of 70 neonatal cases.Pediatr Radiol 2003;33:697–703.

[63] Gay-Andrieu F, Marty P, Pialat J, Sournies G, Drier de Laforte T,Peyron F. Fetal toxoplasmosis and negative amniocentesis: necessityof an ultrasound follow-up. Prenat Diagn 2003;23:558–60.

[64] Crino JP. Ultrasound and fetal diagnosis of perinatal infection. ClinObstet Gynecol 1999;42:71–80, quiz 174–5.

[65] Safadi MA, Berezin EN, Farhat CK, Carvalho ES. Clinical presentationand follow up of children with congenital toxoplasmosis in Brazil. BrazJ Infect Dis 2003;7:325–31.

[66] Carvalheiro CG, Mussi-Pinhata MM, Yamamoto AY, De Souza CB,Maciel LM. Incidence of congenital toxoplasmosis estimated by neona-tal screening: relevance of diagnostic confirmation in asymptomaticnewborn infants. Epidemiol Infect 2005;133:485–91.

[67] Remington JS, Desmonts G. Infectious Diseases of the Fetus and New-born Infant. In: Remington JS, Klein JD, editors. Toxoplasmosis. 3rded. Philadelphia: WB Saunders; 1990. p. 89–195.

[68] Freeman K, Oakley L, Pollak A, Buffolano W, Petersen E, Sem-prini AE, et al. Association between congenital toxoplasmosis andpreterm birth, low birthweight and small for gestational age birth.BJOG 2005;112:31–7.

[69] Swisher CN, Boyer K, McLeod R. Congenital toxoplasmosis. The Tox-oplasmosis Study Group. Semin Pediatr Neurol 1994;1:4–25.

[70] Vutova K, Peicheva Z, Popova A, Markova V, Mincheva N, Todorov T.Congenital toxoplasmosis: eye manifestations in infants and children.Ann Trop Paediatr 2002;22:213–8.

of immunoglobulin-M immunosorbent agglutination assay (ISAGA) fordetecting toxoplasma specific IgM. J Clin Pathol 1989;42:1291–5.

[81] Ashburn D, Joss AW, Pennington TH, Ho-Yen DO. Specificity andusefulness of an IgE immunosorbent agglutination assay for toxoplas-mosis. J Clin Pathol 1995;48:64–9.

[82] Desmonts G, Naot Y, Remington JS, Immunoglobulin. M-immunosorbent agglutination assay for diagnosis of infectious diseases:diagnosis of acute congenital and acquired Toxoplasma infections. JClin Microbiol 1981;14:486–91.

[83] Wong SY, Hajdu MP, Ramirez R, Thulliez P, McLeod R, RemingtonJS. Role of specific immunoglobulin E in diagnosis of acute toxo-plasma infection and toxoplasmosis. J Clin Microbiol 1993;31:2952–9.

[84] Remington PL, Anderson DE, Manering MC, Peterson EA, AndersonH. The PRECEDES Project: background and methods. Wis Med J1990;89:695–6.

[85] Araujo FG, Handman E, Remington JS. Use of monoclonal antibodiesto detect antigens of Toxoplasma gondii in serum and other body fluids.Infect Immun 1980;30:12–6.

[86] Wilson M, Jones JL, McAuley JB. Toxoplasma. In: Murray PR, BaronEJ, Jorgensen JH, Pfaller MA, Yolken RH, editors. Manuel of clinicalmicrobiology, Vol. 2, 8th ed. ASM Press; 2003. p. 1970–80.

[87] Remington JS, Araujo FG, Desmonts G. Recognition of different Tox-oplasma antigens by IgM and IgG antibodies in mothers and theircongenitally infected newborns. J Infect Dis 1985;152:1020–4.

[88] Montoya JG, Liesenfeld O, Kinney S, Press C, Remington JS. VIDAStest for avidity of Toxoplasma-specific immunoglobulin G for confir-matory testing of pregnant women. J Clin Microbiol 2002;40:2504–8.

[89] Remington JS, Thulliez P, Montoya JG. Recent developments for diag-nosis of toxoplasmosis. J Clin Microbiol 2004;42:941–5.

[90] Montoya JG. Laboratory diagnosis of Toxoplasma gondii infection andtoxoplasmosis. J Infect Dis 2002;185(Suppl. 1):S73–82.

[91] Lappalainen M, Koskiniemi M, Hiilesmaa V, Ammala P, Teramo K,Koskela P, et al. Outcome of children after maternal primary Toxo-plasma infection during pregnancy with emphasis on avidity of specificIgG. The Study Group. Pediatr Infect Dis J 1995;14:354–61.

[92] Alvarado-Esquivel C, Sethi S, Janitschke K, Hahn H, Liesen-feld O. Comparison of two commercially available avidity tests

Page 14: Congenital toxoplasmosis—prenatal aspects of Toxoplasma gondii infection

E. Rorman et al. / Reproductive Toxicology 21 (2006) 458–472 471

for toxoplasma-specific IgG antibodies. Arch Med Res 2002;33:520–3.

[93] Liesenfeld O, Montoya JG, Kinney S, Press C, Remington JS. Effect oftesting for IgG avidity in the diagnosis of Toxoplasma gondii infectionin pregnant women: experience in a US reference laboratory. J InfectDis 2001;183:1248–53.

[94] Pietkiewicz H, Hiszczynska-Sawicka E, Kur J, Petersen E, NielsenHV, Stankiewicz M, et al. Usefulness of Toxoplasma gondii-specificrecombinant antigens in serodiagnosis of human toxoplasmosis. J ClinMicrobiol 2004;42:1779–81.

[95] Li S, Galvan G, Araujo FG, Suzuki Y, Remington JS, Parmley S.Serodiagnosis of recently acquired Toxoplasma gondii infection usingan enzyme-linked immunosorbent assay with a combination of recom-binant antigens. Clin Diagn Lab Immunol 2000;7:781–7.

[96] Aubert D, Maine GT, Villena I, Hunt JC, Howard L, Sheu M,et al. Recombinant antigens to detect Toxoplasma gondii-specificimmunoglobulin G and immunoglobulin M in human sera by enzymeimmunoassay. J Clin Microbiol 2000;38:1144–50.

[97] Li S, Maine G, Suzuki Y, Araujo FG, Galvan G, Remington JS, et al.Serodiagnosis of recently acquired Toxoplasma gondii infection witha recombinant antigen. J Clin Microbiol 2000;38:179–84.

[98] Nigro M, Gutierrez A, Hoffer AM, Clemente M, Kaufer F, Carral L,et al. Evaluation of Toxoplasma gondii recombinant proteins for thediagnosis of recently acquired toxoplasmosis by an immunoglobulin Ganalysis. Diagn Microbiol Infect Dis 2003;47:609–13.

[99] Daffos F, Forestier F, Capella-Pavlovsky M, Thulliez P, Aufrant C,Valenti D, et al. Prenatal management of 746 pregnancies at risk forcongenital toxoplasmosis. N Engl J Med 1988;318:271–5.

[100] Derouin F, Mazeron MC, Garin YJ. Comparative study of tissue cul-ture and mouse inoculation methods for demonstration of Toxoplasmagondii. J Clin Microbiol 1987;25:1597–600.

[113] Homan WL, Vercammen M, De Braekeleer J, Verschueren H. Iden-tification of a 200- to 300-fold repetitive 529 bp DNA fragment inToxoplasma gondii, and its use for diagnostic and quantitative PCR.Int J Parasitol 2000;30:69–75.

[114] Reischl U, Bretagne S, Kruger D, Ernault P, Costa JM. Comparisonof two DNA targets for the diagnosis of Toxoplasmosis by real-timePCR using fluorescence resonance energy transfer hybridization probes.BMC Infect Dis 2003;3:7.

[115] Robert-Gangneux F, Commerce V, Tourte-Schaefer C, Dupouy-CametJ. Performance of a Western blot assay to compare mother and newbornanti-Toxoplasma antibodies for the early neonatal diagnosis of con-genital toxoplasmosis. Eur J Clin Microbiol Infect Dis 1999;18:648–54.

[116] Pinon JM, Dumon H, Chemla C, Franck J, Petersen E, Lebech M, etal. Strategy for diagnosis of congenital toxoplasmosis: evaluation ofmethods comparing mothers and newborns and standard methods forpostnatal detection of immunoglobulin G, M, and A antibodies. J ClinMicrobiol 2001;39:2267–71.

[117] Tissot Dupont D, Fricker-Hidalgo H, Brenier-Pinchart MP, Bost-BruC, Ambroise-Thomas P, Pelloux H. Usefulness of Western blot in sero-logical follow-up of newborns suspected of congenital toxoplasmosis.Eur J Clin Microbiol Infect Dis 2003;22:122–5.

[118] Drugs used in parasitic diseases, 2nd ed. Geneva: World Health Orga-nization; 1995.

[119] Chin J. Toxoplasmosis. In: Control of Communicable Disease Manual.17th ed. Washington DC: American Public Health Association; 2000.p. 500–3.

[120] Wallon M, Liou C, Garner P, Peyron F. Congenital toxoplasmosis:systematic review of evidence of efficacy of treatment in pregnancy.BMJ 1999;318:1511–4.

[121] Gras L, Gilbert RE, Ades AE, Dunn DT. Effect of prenatal treatment

[101] Derouin F, Thulliez P, Candolfi E, Daffos F, Forestier F. Early prenatal

diagnosis of congenital toxoplasmosis using amniotic fluid samples andtissue culture. Eur J Clin Microbiol Infect Dis 1988;7:423–5.

[102] Thulliez P, Daffos F, Forestier F. Diagnosis of Toxoplasma infectionin the pregnant woman and the unborn child: current problems. ScandJ Infect Dis Suppl 1992;84:18–22.

[103] Foulon W, Pinon JM, Stray-Pedersen B, Pollak A, Lappalainen M,Decoster A, et al. Prenatal diagnosis of congenital toxoplasmosis: amulticenter evaluation of different diagnostic parameters. Am J ObstetGynecol 1999;181:843–7.

[104] Pelloux H, Guy E, Angelici MC, Aspock H, Bessieres MH, BlatzR, et al. A second European collaborative study on polymerase chainreaction for Toxoplasma gondii, involving 15 teams. FEMS MicrobiolLett 1998;165:231–7.

[105] Bastien P. Molecular diagnosis of toxoplasmosis. Trans R Soc TropMed Hyg 2002;96(Suppl 1):S205–15.

[106] Guy EC, Pelloux H, Lappalainen M, Aspock H, Hassl A, Melby KK,et al. Interlaboratory comparison of polymerase chain reaction for thedetection of Toxoplasma gondii DNA added to samples of amnioticfluid. Eur J Clin Microbiol Infect Dis 1996;15:836–9.

[107] Filisetti D, Gorcii M, Pernot-Marino E, Villard O, Candolfi E. Diag-nosis of congenital toxoplasmosis: comparison of targets for detectionof Toxoplasma gondii by PCR. J Clin Microbiol 2003;41:4826–8.

[108] Chabbert E, Lachaud L, Crobu L, Bastien P. Comparison of two widelyused PCR primer systems for detection of toxoplasma in amniotic fluid,blood, and tissues. J Clin Microbiol 2004;42:1719–22.

[109] Kompalic-Cristo A, Nogueira SA, Guedes AL, Frota C, Gonzalez LF,Brandao A, et al. Lack of technical specificity in the molecular diag-nosis of toxoplasmosis. Trans R Soc Trop Med Hyg 2004;98:92–5.

[110] Hohlfeld P, Daffos F, Costa JM, Thulliez P, Forestier F, Vidaud M.Prenatal diagnosis of congenital toxoplasmosis with a polymerase chainreaction test on amniotic fluid. N Engl J Med 1994;331:695–9.

[111] Romand S, Wallon M, Franck J, Thulliez P, Peyron F, Dumon H.Prenatal diagnosis using polymerase chain reaction on amniotic fluidfor congenital toxoplasmosis. Obstet Gynecol 2001;97:296–300.

[112] Montoya JG, Liesenfeld O. Toxoplasmosis Lancet 2004;363:1965–76.

on the risk of intracranial and ocular lesions in children with congenitaltoxoplasmosis. Int J Epidemiol 2001;30:1309–13.

[122] Neto EC. Newborn screening for congenital infectious diseases. EmergInfect Dis 2004;10:1068–73.

[123] Gratzl R, Sodeck G, Platzer P, Jager W, Graf J, Pollak A, et al. Treat-ment of toxoplasmosis in pregnancy: concentrations of spiramycin andneospiramycin in maternal serum and amniotic fluid. Eur J Clin Micro-biol Infect Dis 2002;21:12–6.

[124] Gilbert RE, Gras L, Wallon M, Peyron F, Ades AE, Dunn DT. Effectof prenatal treatment on mother to child transmission of Toxoplasmagondii: retrospective cohort study of 554 mother-child pairs in Lyon,France. Int J Epidemiol 2001;30:1303–8.

[125] Gilbert R, Dunn D, Wallon M, Hayde M, Prusa A, Lebech M, etal. Ecological comparison of the risks of mother-to-child transmissionand clinical manifestations of congenital toxoplasmosis according toprenatal treatment protocol. Epidemiol Infect 2001;127:113–20.

[126] Bessieres MH, Berrebi A, Rolland M, Bloom MC, Roques C, CassaingS, et al. Neonatal screening for congenital toxoplasmosis in a cohortof 165 women infected during pregnancy and influence of in uterotreatment on the results of neonatal tests. Eur J Obstet Gynecol ReprodBiol 2001;94:37–45.

[127] Foulon W, Naessens A, Ho–Yen D. Prevention of congenital toxoplas-mosis. J Perinat Med 2000;28:337–45.

[128] Peyron F, Wallon M, Liou C, Garner P. Treatments for toxoplasmosisin pregnancy. Cochrane Database Syst Rev 2000:CD001684.

[129] Pawlowski ZS, Gromadecka-Sutkiewicz M, Skommer J, Paul M,Rokossowski H, Suchocka E, et al. Impact of health education onknowledge and prevention behavior for congenital toxoplasmosis: theexperience in Poznan, Poland. Health Educ Res 2001;16:493–502.

[130] Letscher-Bru V, Pfaff AW, Abou-Bacar A, Filisetti D, Antoni E, Vil-lard O, et al. Vaccination with Toxoplasma gondii SAG–1 protein isprotective against congenital toxoplasmosis in BALB/c mice but notin CBA/J mice. Infect Immun 2003;71:6615–9.

[131] Haumont M, Delhaye L, Garcia L, Jurado M, Mazzu P, DaminetV, et al. Protective immunity against congenital toxoplasmosis withrecombinant SAG1 protein in a guinea pig model. Infect Immun2000;68:4948–53.

Page 15: Congenital toxoplasmosis—prenatal aspects of Toxoplasma gondii infection

472 E. Rorman et al. / Reproductive Toxicology 21 (2006) 458–472

[132] Nielsen HV, Lauemoller SL, Christiansen L, Buus S, Fomsgaard A,Petersen E. Complete protection against lethal Toxoplasma gondiiinfection in mice immunized with a plasmid encoding the SAG1 gene.Infect Immun 1999;67:6358–63.

[133] Denkers EY, Gazzinelli RT. Regulation and function of T-cell-mediatedimmunity during Toxoplasma gondii infection. Clin Microbiol Rev1998;11:569–88.

[134] Bhopale GM. Development of a vaccine for toxoplasmosis: currentstatus. Microbes Infect 2003;5:457–62.

[135] Roque-Resendiz JL, Rosales R, Herion P. MVA ROP2 vaccinia virusrecombinant as a vaccine candidate for toxoplasmosis. Parasitology2004;128:397–405.

[136] Mohamed RM, Aosai F, Chen M, Mun HS, Norose K, Belal US, et al.Induction of protective immunity by DNA vaccination with Toxoplasmagondii HSP70, HSP30 and SAG1 genes. Vaccine 2003;21:2852–61.

[137] Buxton D, Innes EA. A commercial vaccine for ovine toxoplasmosis.Parasitology 1995;110(Suppl):S11–6.

[138] Thulliez P. Screening programme for congenital toxoplasmosis inFrance. Scand J Infect Dis Suppl 1992;84:43–5.

[139] Aspock H, Pollak A. Prevention of prenatal toxoplasmosis by serolog-ical screening of pregnant women in Austria. Scand J Infect Dis Suppl1992;84:32–7.

[140] Lopez A, Dietz VJ, Wilson M, Navin TR, Jones JL. Prevent-ing congenital toxoplasmosis. MMWR Recomm Rep 2000;49:59–68.

[141] Hsu HW, Grady GF, Maguire JH, Weiblen BJ, Hoff R. Newbornscreening for congenital Toxoplasma infection: five years experi-ence in Massachusetts USA. Scand J Infect Dis Suppl 1992;84:59–64.

[142] Gilbert RE, Peckham CS. Congenital toxoplasmosis in the UnitedKingdom: to screen or not to screen? J Med Screen 2002;9:135–41.

[143] Ricci M, Pentimalli H, Thaller R, Rava L, Di Ciommo V. Screeningand prevention of congenital toxoplasmosis: an effectiveness study ina population with a high infection rate. J Matern Fetal Neonatal Med2003;14:398–403.

[144] Eskild A, Magnus P. Commentary: Little evidence of effective pre-natal treatment against congenital toxoplasmosis––the implications fortesting in pregnancy. Int J Epidemiol 2001;30:1314–5.

[145] Eskild A, Fallas Dahl G, Melby KK, Nesheim BI. Testing for toxo-plasmosis in pregnancy: a study of the routines in primary antenatalcare. J Med Screen 2003;10:172–5.

[146] Lappalainen M, Sintonen H, Koskiniemi M, Hedman K, Hiilesmaa V,Ammala P, et al. Cost-benefit analysis of screening for toxoplasmosisduring pregnancy. Scand J Infect Dis 1995;27:265–72.