Congenital Toxoplasmosis Prenatal Aspect of Toxoplasma Gondii Infection

Reproductive Toxicology 21 (2006) 458472
Review
Congenital toxoplasmosisprenatal 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 Ofce, 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 5080% in Europe). Vertical transmission from a recently infected pregnant woman to her fetus may lead to congenital toxoplasmosis. The risk of such transmission increases as primary maternal infection occurs later in pregnancy. However, consequences for the fetus are more severe with transmission closer to conception. The timing of maternal primary infection is, therefore, critically 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, specic 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. 2. Case report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The parasite . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Life cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Mechanism of infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Virulence of T. gondii strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Congenital toxoplasmosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Incidence and prevalence in pregnant women and infants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Diagnostic evaluation, manifestation and consequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Prenatal laboratory diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.1. Sabin Feldman dye test (SFDT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.2. Enzyme immunoassays (EIA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.3. Immunosorbent agglutination assay test (IAAT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.4. Indirect uorescent assay (IFA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.5. Avidity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.6. Animal and cell culture inoculation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 459 459 459 460 460 460 461 461 461 463 463 465 465 465 465 466
3. 4.
Corresponding author. Tel.: +972 50 6242904; fax: +972 3 6826996. E-mail address: efrat.rorman@phlta.health.gov.il (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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5. 6.
4.3.7. Molecular diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Laboratory diagnosis of infants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4.1. Western blots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Treatment of congenital toxoplasmosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Primary prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.1. Vaccine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Secondary prevention screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4.
466 467 467 467 468 468 468 468 468 469
1. Case report A 26-year-old woman from a rural village in northern Israel presented with cervical lymphadenopathy during the 13th week of her rst pregnancy. The woman was otherwise healthy and without any symptoms. She was followed up by her primary care physician and as the lymphadenopathy did not resolve, was sent for surgical consultation during the 26th week of pregnancy. The surgeon referred her to laboratory tests for Toxoplasma gondii-specic antibodies and for various other infections. The results obtained from testing a serum sample from the 26th week of gestation, performed at the Israel National Toxoplasmosis Reference Center were: positive for total T. gondii-specic immunoglobulins (Ig) (250 IU/ml by Sabin Feldman Dye Test) and for T. gondii-specic IgM antibodies (by ELFA, Enzyme-Linked Fluorescent immuno-Assay) with low IgG avidity (0.027). These results were reported and an additional serum sample, as well as an earlier sample (whether available) were requested. Blood samples were subsequently delivered to our laboratory; from the 12th (sample drawn as part of the routine pregnancy follow-up) and from the 34th weeks of pregnancy. The results of the earlier sample were negative for both total Ig and IgM antibodies. The results from the 34th week were positive for total T. gondii-specic immunoglobulins (250 IU/ml by Sabin Feldman Dye Test) and negative for T. gondii-specic IgM antibodies (by ELFA, Enzyme-Linked Fluorescent immuno-Assay) with low IgG avidity (0.055). The sum interpretation of the three above tests results of the 12th, 26th and 34th week of pregnancy was consistent with denite recent T. gondii infection (seroconversion, constant high T. gondii-specic immunoglobulins, emergence and disappearance of IgM, low avidity and cervical lymphadenopathy). Amniocentesis was performed during the 35th week of pregnancy and PCR result for T. gondii DNA in the amniotic uid was positive. The woman was referred for follow-up at a high risk pregnancy clinic in a tertiary medical center. Anti-T. gondii therapy including Pyrimethamine, Sulfadiazine and folinic acid was started and continued until birth. The pregnancy course was otherwise uneventful and fetal growth assessment through ultrasound follow-up did not reveal any abnormality. During the 38th week of pregnancy a female infant was born by spontaneous delivery. Birth weight was 2830 g and head circumference was 33 cm. Physical examination was normal. Laboratory tests including complete blood count, glucose, electrolytes, liver function tests and cerebro-spinal uid (CSF) tests were all
normal. Cranial ultrasonography, brain stem evoked response (BERA), audiometry and eye examination were all normal. Tests for T. gondii in the infant included: PCR of CSFnegative, immuno-sorbent agglutination assays (IgM-ISAGA)negative and Sabin Feldman Dye Test (SFDT)positive (250 IU/ml), probably reecting maternal antibodies transfer. Despite the serological indicators of maternal infection (most probably towards the end of the rst trimester) and positive PCR of the amniotic uid, there was no evidence of congenital toxoplasmosis in the neonate. The infant was treated with the same therapeutic protocol as the mother planned to be continued until the age of 1 year. Medical evaluation, auditory and ophthalmic tests at the age of 4 and 8 months revealed normal physical growth and development and intensive follow-up continues (at the age of 6 months laboratory analysis was reported to be normal). This case demonstrates the complexity of establishing clinical diagnosis and interpretation of laboratory results in regard to T. gondii infection in pregnancy. The favourable outcome despite the timing of infection may be attributed to providing anti parasitic therapy, although the specic role of therapy or other unknown variables is unclear. Since many T. gondii infections are sub-clinical or present with non-specic signs, physicians should be able to integrate clinical and laboratory data in order to make diagnostic and therapeutic decisions. 2. The parasite T. gondii is a member of the phylum Apicomplexa, order Coccidia, which are all obligate intracellular protozoan parasites. Other members of this phylum include known human pathogens such as Plasmodium (malaria) and Cryptosporidium. 2.1. Life cycle The life cycle of T. gondii consists of two stagesasexual and sexual: the asexual stage takes place in the intermediate hosts, which are mammals or birds. During this phase rapid intracellular growth of the parasite as tachyzoite takes place (generation time in vitro is 68 h). The oval or crescent-shaped tachyzoites can infect and multiply in almost any nucleated mammalian or avian cell [1]. Following accumulation (64128), tachyzoites are secreted into the blood stream [2] and spread in the body, leading to development of an acute disease (parasitemia). The normal immune response and transformation of the tachyzoite into cystforming bradyzoites limit the acute stage and establish a chronic infection. Bradyzoites differ from tachyzoites mainly in their
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extremely slow multiplication rate (their name reects this slow process) and in the distinct set of proteins they express [1,35]. The cysts are formed mainly in neural and muscular tissues, especially brain, skeletal and cardiac muscles, and can persist, inactivated, in the body for a very long time. In the immunocompromised patient the release of bradyzoites from the cyst may cause acute encephalitis. The sexual stage takes place in the intestine of the denitive host. Known denitive hosts are members of the feline family, predominantly domestic cats. When bradyzoites or oocytes are ingested by a feline, formation of oocytes proceeds in the epithelium of the small intestine. Several million unsporulated oocytes may be released in the feces of a single cat over a period 318 days, depending on the stage of T. gondii ingested [1]. Under mild environmental conditions oocytes may sporulate within a 3-week period [6], then infecting humans and other intermediate hosts. Oocysts can spread in the environment and contaminate water, soil, fruits, vegetables and herbivores following consumption of infected plant material. Investigation of outbreaks of toxoplasmosis has led to recovery of oocytes from soil [7] but not from water [810]. Oocytes have been found to be very stable, especially in warm and humid environments, and resistant to many disinfecting agents [11], but survive poorly in arid, cold climates [12]. 2.2. Mechanism of infection T. gondii has been shown to migrate over long distances in the hosts body; crossing biological barriers, actively enter the blood stream, invade cells and cross substrates and non-permissive biological sites such as the blood-brain-barrier, the placenta and the intestinal wall. At the same time, the parasite minimizes exposure to the hosts immune response, by rapidly entering and exiting cells. These two functions share common mechanisms which depend on Ca2+ regulation [13]. Unlike many bacteria and viruses, T. gondii actively enters the cell, in a mechanism which is mediated by the parasites cytoskeleton and regulated by a parasite-specic calciumdepended secretion pathway [2,14]. The rst step of cell invasion by T. gondii is recognition of an attachment point. The two special organelles involved in this invasion process, rhoptries and micronemes, each discharging proteins during the process [5,15]. Following the rapid cellular invasion the parasite resides within a vacuole, derived primarily from the host cells plasma membrane [2,16]. The active motion of T. gondii, called gliding, occurs with no major changes in cell shape. It is fast (about 10 times faster than the crawling rate of amoeboid cells), and consists of both circular gliding in a counter-clockwise direction and clockwise helical gliding [1721]. As an obligatory parasite, its invasive capabilities play an important role in virulence and pathogenicity, since it can only survive intracellularly where it gets nutrients and escapes from the hosts immune response [22]. The most virulent T. gondii strain has been shown to exhibit superior migratory capacity [23] and a subpopulation of this strain displays a special, long distance migration phenotype [14]. The ability to cross biological barriers is associated with acute virulence and is linked to genes on chromosome VII [24,25]. The
genome of T. gondii, consisting of 14 chromosomes, is currently being investigated and sequenced [26] (http://ToxoDB.org). 2.3. Virulence of T. gondii strains Clinical manifestations and severity of illness following infection are affected by features of the interaction between the parasite and the host and include strain virulence, inoculum size, route of infection, competence of the hosts immune response (both cellular and humoral), integrity of the hosts mucosal and epithelial barriers, hosts age and genetic background [27]. Various strains of T. gondii have long been known to differ in virulence and pathogenicity [28,29]. These strains can be classied by immunologic assays, isoenzyme analysis and molecular analysis [3033]. There are three T. gondii clonal lineages, of them one carries conserved genetic loci, suspected of coding for the virulence trait [24]. Grigg et al. [34] demonstrated that a sexual recombination, performed in vitro, between the two relatively avirulent strains can give rise to the virulent strain. This is in accordance with polymorphism analysis of the three T. gondii strains, which indicated that they emerged within the last 10,000 years, following a single genetic cross [34,35]. Acquisition of an efcient mechanism to spread by direct oral transmission, bypassing a sexual phase, leads to successful clonal expansion of this virulent lineage [35,36]. Genetic background plays a signicant role in increased susceptibility to T. gondi in humans; HLA-DQ3 appears to be a genetic marker associated with susceptibility to developing toxoplasma-dependent encephalitis [37,38]. 3. Epidemiology T. gondii infection is most frequently caused by ingestion of row or undercooked meat, which carries tissue cysts, by consuming infected water or food or by accidental intake of contaminated soil. Toxoplasmosis is also an occupational hazard for laboratory workers. A total of 47 laboratory-acquired cases have been reported, 81% of them were symptomatic cases [39]. Tender et al. [40] collected data of nation-wide T. gondii seroprevalence in women at child-bearing age (19902000). The rates of positive sero-prevalence, were 58% in Central European countries, 5172% in several Latin-American countries and 5477% in West African countries. Low seroprevalence, 439%, was reported in southwest Asia, China and Korea as well as in cold climate areas such as Scandinavian countries (1128%). In the US 15% of females at childbearing age were found to be seropositive [41]. It should be noted that seropositive prevalence in the same country may differ among populations or geographical regions and world-wide prevalence is higher in older populations. In a limited casecontrol study that included six large European centers it was shown that the consumption of undercooked meat was the major risk factor for toxoplasmosis infection [42]. Another study aimed to determine the prevalence of T. gondii in edible meat tested 71 meat samples from commercial sources in the UK for the parasitepositive results were found in 27
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samples. Twenty-one of these contaminated meat samples carried the virulent T. gondii type I [43]. Although cats play a denite role in the epidemiology of toxoplasmosis, no significant correlation between human toxoplasmosis infection and cat ownership could be proven [44]. Furthermore, the oocytes are not found on cat fur but rather are buried in the soil as they are shed with cat faeces [4547]. Data regarding seroprevalence of specic T. gondii antibodies in the Israeli population are based on several regional surveys performed in collaboration with the Israeli National Toxoplasmosis Reference Center. The prevalence in certain subpopulations of pregnant women in northern Israel had been reported to be 21% on average and the incidence rate of infection acquired during pregnancy estimated as 1.4% [48]. Human contact with infected oocyst from contaminated soil [7,49,50] and water [810] were associated with several reported epidemics caused by T. gondii. Only in one case were T. gondii oocysts recovered from the soilthe suspected source of infection [7]. There are ongoing efforts to develop sensitive detection techniques for environmental samples [11,51,52]. Unfortunately, isolation of oocytes from such samples is difcult, since infectious doses are small while large volume of sample is required for isolation of the organism. In addition, there is a lag period between the time of infection and the time that the contaminated source is tested, further reducing the likelihood of recovery of oocytes from the suspected environment during epidemiological investigation. T. gondii was reported to cause 0.8% of the total food-borne illnesses attributed to a known pathogen, and 20.7% of the total food-borne mortality caused by a known pathogen, in the United States in 19961997. Many of these cases involved HIV-infected patients [53]. The largest reported toxoplasmosis outbreak resulting from contaminated water occurred in British Columbia and caused acute infection in 100 people; 19 with retinitis and 51 with lymphadenopathy. The likely source was a municipal water system that used unltered, chloraminated surface water [10]. There was also a seasonal correlation to rainfall and turbidity in this water reservoir. In another small outbreak North of Rio de Janeiro, Brazil, the source of the parasite was traced to an unltered water source. It was also linked to high prevalence of seropositivity in this region of low socio-economic background [8]. 4. Congenital toxoplasmosis Most cases of acquired toxoplasma infection are asymptomatic and self-limited; hence many cases remain undiagnosed. The incubation period of acquired infection is estimated to be within a range of 421days (7 days on average) [10]. When symptomatic infection does occur the only clinical ndings may be focal lymphadenopathy, most often involving a single site around the head and neck. Less commonly, acute infection is accompanied by a mononucleosis-like syndrome characterized by fever, malaise, sore throat, headache and an atypical lymphocytosis on peripheral blood smear [54]. In immunocompromised patients, most commonly HIV infected and organ transplant recipients, T. gondii may cause a severe central nervous system
disease, resulting in brain lesions or diffuse encephalitis. Other organs, such as the heart, lung, liver, and retina may also be involved. Most of these cases result from reactivation of latent infection [54] although re-infection with a different T. gondii strain in the transplanted organs may also occur. 4.1. Incidence and prevalence in pregnant women and infants The disease is caused by vertical transmission of T. gondii from a seronegative pregnant woman, who is acutely infected with T. gondii to her fetus. The prevalence of T. gondii and its incidence of human infection vary widely amongst various countries. Worldwide, 38 infants per 1000 live births are infected in utero [55]. Multiple factors are associated with the occurrence of congenital toxoplasmosis infection, including route of transmission, climate, cultural behaviour, eating habits and hygienic standards. This combination leads to marked differences even among developed nations. For example, the incidence of congenital infection in Belgium and France is 23 cases per 1000 live birthsmarkedly higher than the US incidence, which is between 1 in 10,000 to 1 in 1000 live births [47,56]. In a research conducted in Goiania, Brazil, a region with a relatively high seroconversion rate, pregnant women were found to have a 2.2 times higher risk for seroconversion than non-pregnant women of equivalent characteristics. In addition, amongst pregnant women, adolescents were shown to have the highest risk for seroconversion [57]. The authors hypothesized that higher vulnerability to T. gondii infection during pregnancy may be due to a combination of pregnancy associated immunosuppression as well as hormonal changes. Only a few cases of congenital toxoplasmosis transmitted by mothers who were infected prior to conception have actually been reported [5860]. One such case published recently involved a woman who had ocular toxoplasmosis 20 years prior to giving birth to a newborn, who suffered from congenital toxoplasmosis. The mother had a toxoplasmic scar in the retina and was tested positive for specic toxoplasma IgG antibodies. The newborn was found to be positive for both IgG and IgM antibodies and had a macular scar on the retina, typical to toxoplasmosis, as well as a calcied brain granuloma. [59]. Such a case could be attributed to re-infection with a different, more virulent strain or by reactivation of a chronic disease[58]. Chronically infected women, who are immunodecienct, may also transmit the infection to their fetus; the risk of this occurrence is difcult to quantify, but it is probably low. Latent T. gondii infection may be reactivated in immunodecient individuals (such as HIV-infected women) and result in congenital transmission of the parasite [61]. 4.2. Diagnostic evaluation, manifestation and consequences The diagnostic evaluation of T. gondii is part of routine pregnancy follow-up and differential diagnosis of intrauterine infection. Intrauterine ultrasonographic ndings of T. gondii infection
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are usually non-specic and in most cases no pathological evidences are revealed. In certain cases the ultrasonographic ndings may include: intracranial calcications, echogenic streaks, microcephalus, ventricular dilatation and hydrocephalus [62]. Gay-Andrieu et al. [63] described two cases of intrauterine infection in which the diagnosis was based upon hydrocephalus in fetal ultrasound, even though PCR of amniotic uid was negative in both cases. The authors emphasized that hydrocephalus is the most frequent lesion detected by fetal ultrasound, reecting the pathological process taking place within several months post-infection in cases of intrauterine infection of T. gondii. Additional ultrasonographic ndings may include hepatomegaly, splenomegaly, ascitic uid, cardiomegaly and placental abnormalities [55,64]. Safadi et al. [65] followed 43 children with congenital toxoplasmosis for a period of at least 5 years. Most of them (88%) had sub-clinical presentation at birth. The most common neurological manifestation was a delay in neuro-psychomotor development. Half of the children developed neurological manifestations, 7 children had neuroradiologic alterations in skull radiography, and 33 children in tomography. Notably, cerebral calcications were not associated with an increased incidence of neurological sequelae. Chorioretinitis was the main ocular sequelae, found in almost all children and noted years after birth, despite specic therapy in the rst year of life. An important step in the diagnosis of congenital toxoplasmosis and evaluation of time of infection is achieved by laboratory techniques, monitoring the immune response: titer and afnity of specic antibodies (Fig. 1). Other laboratory tools focus on direct detection of the parasite by animal or tissue inoculation or more commonly, by molecular techniques. Carvalheiro et al. studied the incidence of congenital toxoplasmosis in Brazil, based on persistence of anti-Toxoplasma IgG antibodies beyond the age of 1year. Disease incidence was estimated to be 3.3 per 10,000. A denitive diagnosis was conrmed in ve infants with both serum IgM and/or IgA antibodies, and clinical abnormali-
Fig. 1. Laboratory diagnosis of congenital toxoplasmosis.
ties. They concluded that positive screening results must be carefully conrmed [66]. Laboratory methods and their implications in supporting evidence-based diagnoses are discussed below. The risk of fetal infection is multifactorial, depending on the time of maternal infection, immunological competence of the mother during parasitemia, parasite load and strains virulence [40]. The probability of fetal infection is only 1% when primary maternal infection occurs during the preconception period but increases as pregnancy progresses; infection acquired during the rst trimester by women not treated with anti-T. gondii drugs results in congenital infection in 10 to 25% of cases. For infections occurring during the second and third trimesters, the incidence of fetal infection ranges between 3054% and 6065%, respectively [54]. The consequences are more severe when fetal infection occurs in early stages of pregnancy, when it can cause miscarriage (natural abortion or death occurs in 10% of pregnancies infected with T. gondii [67]), severe disease, intra-uterine growth retardation or premature birth. A multi-centre prospective cohort study evaluated the association between congenital toxoplasmosis and preterm birth, low birth weight, and small size for gestational age [68]. Freeman et al. reported that infected babies were born earlier than uninfected babies and that congenital infection was associated with an increased risk of preterm delivery when seroconversion occurred before 20 weeks of gestation. Congenital infection was not associated with low birth weight or small size for gestational age. The cause for shorter gestation is not yet known. The highest frequency of severe abnormalities at birth is seen in children whose mothers acquired a primary infection between the 10th and 24th week of gestation [67]. The likelihood of clinical symptoms in the newborn is reduced when infection occurs later. Clinical manifestations in newborns with congenital toxoplasmosis vary and can develop at different times before and after birth. Most newborns infected with T. gondii are asymptomatic at birth (7090%) [61]. When clinical manifestations are present they are mainly non-specic and may include: a maculopapular rash, generalized lymphadenopathy, hepatomegaly, splenomegaly, hyperbilirubinemia, anemia and thrombocytopenia [69]. The classic triad of chorioretinitis, intracranial calcications and hydrocephalus is found in fewer than 10% of infected infants [47]. Hydrocephalus and/or microcephaly may develop when intra-uterine infection results in meningo-encepahlitis [69]. All these signs and symptoms are included in the general work-up of suspected congenital TORCH infections: toxoplasmosis, other (syphilis, varicella-zoster, parvovirus B19), rubella, cytomegalovirus (CMV) and herpes infections. Cerebral calcications can be demonstrated by cranial radiography, ultrasonography or computerized tomography. Neurologic impairment may initially present as seizures, necessitating specic evaluation and treatment. The most prevalent consequence of congenital toxoplasmosis is chorioretinitis. Chorioretinitis is diagnosed based on characteristic retinal inltrates. Vutova et al. [70] investigated eye manifestations of congenital toxoplasmosis in 38 infants and children. The most frequent nding was chorioretinitis (92%), together with other
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ocular lesions in 71% of cases, and the second most common nding was microphthalmia with strabismus. Lesions of the anterior segment of the eye included iridocyclitis, cataracts and glaucoma. Other uncommon ndings were diminished visual acuity and neurological sequelae such as hydrocephalus, calcication in the brain, paresis, and epilepsy. Wallon et al. [71] reported the clinical evolution of ocular lesions and nal visual function, in a prospective cohort of 327 congenitally infected children in France. The children were identied by maternal prenatal screening and monitored for up to 14 years. After 6 years, 79 (24%) children had at least one retinochoroidal lesion. In 23 of them a new lesion was diagnosed within10 years, mainly in a previously healthy location. Normal vision was found in about two thirds of children with lesions in one eye, half the children with lesions in both eyes and none had bilateral visual impairment. Most of the mothers (84%) had been treated. A combination of pyrimethamine and sulfadiazine had been prescribed in all the children (38% before and 72% after birth). Late-onset retinal lesions and relapse can occur many years after birth, but the overall ocular prognosis of congenital toxoplasmosis seems satisfactory, when infection is identied early and appropriately treated. Early diagnosis and treatment are believed to reduce the risk of visual impairment. Relevant laboratory tests include complete blood count (CBC), liver function tests and specic T. gondii diagnostic tests as described in details below. If T. gondii infection is suspected at the time of birth, diagnostic work-up includes ophthalmic, auditory and neurological examinations, lumbar puncture and cranial imaging [69]. In a large percentage of children the disease sequelae may become apparent and present with visual impairment, mental and cognitive abnormalities of variable severity, seizures or learning disabilities only after several months or years [55]. Infants born to women infected simultaneously with HIV and T. gondii should be evaluated for congenital toxoplasmosis, considering the increased risk of reactivation of parasitemia and disease in these mothers. In a casecontrol study in Israel, Potasman et al. tested 95 children with variable neurological disorders: cerebral palsy, epilepsy and nerve deafness compared with a control group of 109 healthy children, for the presence of T. gondii-specic antibodies in the serum. They found that children with any of the neurological disorders were signicantly more likely to have T. gondii specic IgG antibodies, especially those with nerve deafness (relative risk 2.5 and 7.1, respectively) [72]. A denite diagnosis cannot be made in the following situations: (1) the infant is older than one year of age and was not tested for toxoplasmosis previously, (2) either the child or the mother is seronegative, or (3) the mother was known to be seropositive prior to conception. 4.3. Prenatal laboratory diagnosis The principle method used to diagnose and evaluate timing of congenital infection relies on indirect evidence, and is based on detection of specic antibodies, by monitoring the immune response. Direct evidence is obtained by animal or tis-
sue inoculation or more commonly, by molecular techniques. It is important to combine all available clinical and laboratory data during the evaluation of toxoplasmosis diagnosis and providing treatment recommendations. Infection during gestation may cause serious damage to the fetus and hence, a major objective of the diagnosis is to estimate the time of maternal infection. IgG antibodies usually appear within two weeks of infection, peak within 68 weeks and persist in the body indenitely [67]. IgM antibodies are considered the indicators of recent infection and can be detected by enzyme immunoassay (EIA) or immunosorbent agglutination assay test (IAAT) relatively earlywithin 2 weeks of infection. Uncertainty may arise as IgM may persist for years following primary infection [73]. IgA antibodies may also persist for more than a year [67] and their detection is informative mainly for the diagnosis of congenital toxoplasmosis. The level of specic IgE antibodies increases rapidly and remains detectable for less than 4 months after infection, which leaves a very short time to be used for diagnostic purposes [74]. However, IgE serology is not useful in samples from newborns. When serology alone is insufcient direct evidence for toxoplasma infection should be sought. Both the laboratory performing the tests and the referring physician should be aware of the limitations and select the best combination of tests available to timely evaluate the stage of toxoplasma infection [75]. Laboratory tests available are summarized in Table 1. 4.3.1. Sabin Feldman dye test (SFDT) This is the rst test developed for the laboratory diagnosis of T. gondii infection [76], it is still considered the gold standard. SFDT detects the presence of anti-T. gondii specic antibodies (total Ig) and is performed only in reference centers. The change in antibody titer as determined in SFDT in consecutive serum samples taken at least 3 weeks apart is important for the evaluation of infection during pregnancy. A signicant change is considered to be at least a four-fold difference. The absolute antibody titer is also importantvalues over 250 IU/ml are considered high suggestive of recent infection. The tested sera are serially diluted and incubated with live tachyzoites (carrying toxoplasma-specic antigens) in the presence of separated human plasma from sero-negative donors (providing complement components). The antigenantibodycomplement complexes formed are subsequently lysed in the presence of the dye methylene blue. End-point titer is established by counting the numbers of dead (unstained) and live (stained) parasites. The reported titer is that producing lysis of 50% of the organisms. End-point titer can be converted to international units (IU): additional standardization is achieved by preparation of a standardised control serum (consisting of a pool of sera), tested by numerous reference centers, and adjusted so that the SFDT value of this control serum is set at 1000 IU/ml [77]. Recently, the WHO recognized the rst international standard for human anti-toxoplasma IgG, with an assigned potency of 20 IU per ampoule of total anti-toxoplasma antibodies [78].
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Table 1 Laboratory diagnostic tests for congenital toxoplasmosis Test Matrix Results Interpretation Time Degree of expertise equired High, reference center only Other remarks Suggested use
Sabin Feldman Dye Test (SFDT)
Serum
Titer in international units (IU) of total specic Ig
Quantitative data: detection of high (250 IU) antibodies titers and signicant changes (4) in titer in consecutive samples important for evaluation of recent infection
Routine = 24 week
Gold standard
Conrmation of infection
hands on = several hours
Live parasites and animal injection risk to lab employee Standardized assay (international effort) Possible false negative very early infection Very subjective and difcult to standardize Partial results (combine with IgM detection) Requires further testing, IgE not in newborn Follow-up change in titer
EIAa /total Igb
Serum
Positive/negative for total specic Ig Titer (in IU) of total specic Ig
Exposure to T. gondii
Several hours
Low = simple automated test High, reference center only
Screening test
IFAc -total Ig
Serum
Several hours
When SFDT is unavailable
IgG by EIA
Serum
Positive/negative for specic IgG Abs Positive/negative for specic IgM, IgA or IgE Abs
Several hours
Low = simple automated test Low = simple automated test
Screening test
IgM/IgA or IgE by EIA
Serum
Possible recent infection with T. gondii
Several hours
IgM Screening
IgM/IgA or IgE by IAAT
Serum
Positive/negative for specic IgM, IgA or IgE Abs
Possible recent Infection with T. gondii
Several hours
Relatively high
Most sensitive and specic test
IgM/IgA Newborn IgE Earlier IgM/IgA Newborn Western blot should be considered if contamination with maternal blood is suspected When ISAGA is unavailable
IgM/IgA or IgE by IFA
Serum
Positive/negative for specic IgM, IgA or IgE Abs Avidity = functional afnity
Possible recent Infection with T. gondii High avidity supports past infection (4 months)
Several hours
High, reference center only
IgG avidity
Serum
Several hours
Relatively simple
Very subjective and difcult to standardize Supportive evidence
Mice
Body uids/tissue
Positive/negative
Presence of parasite
36 weeks
High, reference center only Live parasites and animal injection risk to lab employee
Low sensitivity
When only a single serum sample is available, in the beginning of pregnancy Strain isolation
E. Rorman et al. / Reproductive Toxicology 21 (2006) 458472 Table 1 (Continued) Test Matrix Results Interpretation Time Degree of expertise equired Very high reference center only Live parasites High High, reference center Other remarks
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Suggested use
Cells
Body uids/tissue
Positive/negative
Presence of parasite
36 days
Low sensitivity
When available for a direct proof of infection Amniotic uid Conrmatory test or fetal/newborn infection
PCR Western blot IgG, IgM
Body uids/tissue Serum
Positive/negative Identical/unidentical to maternal Ig
Presence of parasites DNA Fetal/newborn infection
Several hours 1 day
High sensitivity Infrequent availability
a b c
EIA: enzyme immunoassay. Ig: immunoglobulin. IFA: indirect uorescent assay.
4.3.2. Enzyme immunoassays (EIA) The most common laboratory tests for toxoplasmosis infection, also available as commercial kits and/or automated platforms, are EIA. These tests include: enzyme-linked immunosorbent assay (ELISA) and enzyme linked uorescent immuno-assay (ELFA) which test for the presence of IgG and/or IgM antibodies specic for the parasite in human sera. EIA are useful as fast, low-cost screening tests and have been improved over the years to avoid false positive results due to non-specic detection of interfering factors such as rheumatoid factor and antinuclear antibodies. There is no standardization of these tests, which causes high variability in results obtained with different kits and/or in different laboratories. Consequently, and also as a result of the high incidence of false-positive results even in reference centers, the US Food and Drug Administration (FDA) issued a health advisory to physicians on July, 1997. The FDA recommends avoiding reliance of results obtained with any single commercial kit for the detection of toxoplasma-specic IgM, as the sole determinant of recent toxoplasma infection in pregnant women. In our experience at the Israeli National Toxoplasmosis Reference Center, during the years 19972002, in an average of 747 samples (range: 652816) received annually for conrmation, only 17% 2.6% were indeed positive for T. gondii-specic IgM. It is therefore recommended that patient follow-up would be performed by a reference center, and that commercial kits would be locally evaluated to achieve the highest degree of accuracy and repeatability possible for screening tests. In general, when toxoplasma infection is suspected based on detection of specic IgM antibodies specimens are referred for conrmation by a reference center where SFDT, PCR and other advanced assays can be performed. 4.3.3. Immunosorbent agglutination assay test (IAAT) IAAT is highly specic in detection of anti-T. gondii IgM, IgA or IgE antibodies [79]. This assay utilizes the entire tachyzoite and is the most sensitive commercially available method [8082]. Unfortunately, it is expensive, requires a high degree of expertise and is not automated. It is consequently seldom used in reference centers, usually in neonates suspected of having con-
genital infection (where the expected levels of antibodies are very low) [74,83]. Toxoplasma-specic IgE antibodies can be detected by EIA or IAAT in sera of recently infected adults, congenitally infected infants, and children with congenital toxoplasmic chorioretinitis [84]. IgE detection is, however, ineffective in evaluating fetal or newborn samples where IgA tests are most informative. 4.3.4. Indirect uorescent assay (IFA) The IFA was widely used to demonstrate T. gondii-specic antibodies: serially diluted serum samples are incubated with live, inactivated toxoplasma xed to a glass slide. T. gondiispecic antibodies present in the serum would bind to the inactivated parasite, and the complex is then detected using uorescein isothiocyanate-labeled anti-human Ig (or anti-IgG or anti-IgM). IFA is safer to perform and more economical than the SFDT. It appears to measure the same antibodies as the dye test, and its titers tend to parallel dye test titers [47,85]. However, the IFA interpretation is subjective and time consuming. False positive results may occur with sera containing antinuclear antibodies and rheumatoid factor [86], and false negative results of IFA for IgM may occur due to blockage by T. gondii-specic IgG [87]. 4.3.5. Avidity IgG avidity testing was developed by Hedman et al. and is based on the increase in functional afnity (avidity) between T. gondii-specic IgG and the antigen over time, as the host immune response (and specic B cell selection) evolves [88]. Dissociation of the antigenantibody complexes reects the lower avidity closer to primary infection. Pregnant women with high avidity antibodies are those who have been infected at least 35 months earlier, which makes the avidity test most useful and reliable in the rst trimester when high-avidity is detected [89]. In one study, 35 out of 63 patients (55%) who were classied by toxoplasma-specic serology as having recent or borderline infection showed high avidity-antibodies and were therefore treated as chronic patients [90]. Lappaplainen et al. [91] were able to follow 13 women who showed high-avidity antibodies in the rst trimester and conrmed that none of the born infants was found to be infected with T. gondii (as determined serologically
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after birth). The avidity test is most important when only a single serum sample is available at the time when critical decisions must be made. To the best of our knowledge, commercial IgG avidity kits have been licensed in Europe but not in the US [92]. When avidity is low or borderline it may be misleading and a more careful interpretation of all laboratory tests results in conjunction with other clinical ndings, should then be undertaken. Several studies have shown that this test is reliable and valuable in diagnosis of recent infection during early pregnancy [88,9398]. Accurate and denitive serologic diagnosis of recently acquired toxoplasma infection is still difcult and depends on testing of more than one sample. Efforts to develop better diagnostic approaches continue based on antigens specically expressed either during the primary phase (i.e. GRA7, GRA4) or the latent phase (i.e. GRA1) of infection. These antigens can be produced by recombinant DNA technologies and may lead to a more informative serologic diagnosis, based on a single serum sample [47,99,100]. 4.3.6. Animal and cell culture inoculation A denite laboratory conrmation of active toxoplasmosis infection (especially in immunocompromised patients and pregnant women) can be established by inoculation of body uids or tissue into mice or cell culture [47]. Mice are injected intraperitoneally or subcutaneously with 1030 ml of sediment from amniotic uid or whole fetal blood. The mice are bled prior and 36 weeks following inoculation. Antibody detection by SFDT establishes infection and nal proof is obtained by staining to demonstrate brain cysts [101]. Cell culture inoculation with amniotic uid or blood uses indirect IFA to detect the parasite in monolayers within 36 days following inoculation [102]. When compared, inoculation of both blood and amniotic uid from an infected fetus resulted in toxoplasma isolation from both cultures in 70% of cases. However, in 40% of the cases T. gondii isolation is successful in only one of the samples [100]. Derouin et al. [100] demonstrated similar sensitivities comparing cell culture and mice inoculation. Thulliez et al. [102] reported that the sensitivity of amniotic uid cell culture inoculation is only 53% compared with 73% sensitivity in mice inoculation. Currently, the principle role for these methods may be conrmation of PCR as they are complex, expensive and relatively insensitive. [103]. 4.3.7. Molecular diagnosis Replacing fetal blood analysis, which is a high risk procedure for the fetus, with molecular evaluation of amniotic uid has provided a low risk diagnosis of congenital toxoplasmosis. Polymerase chain reaction (PCR) is currently the most common molecular technique routinely used for diagnosis of toxoplasmosis, although, it has not yet been standardized. No attempts have been made to standardize either the sample preparation process or the PCR amplication itself, and numerous laboratories use multiple in-house methods of varying sensitivities and reliability [104,105]. Recently, a commercial PCR prociency test became available.
As in all diagnostic tests based on amplication of DNA, a few technical aspects are of crucial importance in achieving reliable results. Therefore, PCR based test should be carefully designed to include negative, positive and internal control, target DNA for amplication should be specic, sample preparation techniques should be perfected to extract minute parasite DNA [105] and to prevent cross contamination. In a small (5 laboratories) inter-laboratories comparative work [106] followed by a larger study (15 laboratories) [104] signicant differences in test performances were obtained, including false negatives and false positives. These results should denitely urge optimization and standardization of the test. More recently, three PCR protocols were optimized prior to a comparative study, using three different targets: 18S ribosomal DNA, B1 gene and AF146527. No signicant difference was observed between the results of the three protocols [107]. Chabbert et al. [108] used two different primer sets of the B1 gene to compare PCR performance followed by Southern blot, on various sample types (including amniotic uid, blood and tissues). For amniotic uid both PCR conditions produced similar results. The fragments produced by one of the primer sets had to be conrmed by specic hybridization, otherwise nonspecic results were obtained. The PCR product of the same amplication procedure was sequenced by Kompalic-Cristo and suspected of originating from human DNA, as predicted by bioinformatics analysis [109]. Different protocols inuence the sensitivity and specicity of PCR assays. The specicity and positive predictive value of PCR tests on amniotic uid samples is close to 100% [110,111]. However, the sensitivity of these PCR tests varies and estimated, based on a large number of studies, to be 7080% [105]. One report showed that the sensitivity of PCR from amniotic uid is affected by the stage of pregnancy in which maternal infection occurs: best sensitivity was detected when maternal infection occurred between 17 and 21 weeks of pregnancy [89,111,112]. In addition, treatment with anti-toxoplasma drugs may also affect the sensitivity [89,112]. However, the reliability of a PCR test performed on amniotic uid prior to the 18th week of pregnancy requires further evaluation [110,111]. It should also be noted, that testing amniotic uid for T. gondii was found to be effective about 4 weeks following infection, which is already during the parasitemic stage in the infected mother. Therefore, PCR test should not be performed in the absence of serologic or other clinical/sonographic data indicative of infection. In the last 4 years there have been reports on the use of Real Time PCR, a sensitive and specic technique, which enables rapid detection of amplication products as well as hybridization of amplicon-specic probes, similar to PCR followed by Southern blot analysis. The method, which will ultimately replace traditional PCR, enables an overall time for amplication and detection of less than two hours. In addition, cross contamination is prevented by elimination of the need to handle amplied amplicons. In Real Time PCR it is possible to perform a quantitative study and follow the parasite load, allowing determination of parasite count and its correlation with clinical symptoms and impact of treatment. The technique permits linear range over 6 logs of DNA concentrations [113,114].
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The most popular target gene for PCR diagnosis of T. gondii is the 35-fold repetitive gene B1. A variety of primers have been used for amplication, some of which include nested primers. The second common locus is the single copy gene P30 also known as SAG1, which encodes for a surface antigen. Another PCR target is the 18S ribosomal DNA. As reviewed by Bastien [105], two other target loci have been examined but are currently not used by most laboratories. Recently, some laboratories have shown success in amplication of a DNA fragment, AF146527, which is repeated 200300 times [89,113,114]. 4.4. Laboratory diagnosis of infants Laboratory diagnosis of Toxoplasma infection in infants is based on a combination of serologic tests, parasite isolation, and nonspecic ndings [112]. When suspected, serologic follow-up of the newborn is recommended for the rst year of life [90]. Evaluation for direct evidence as described above should be repeated as well during this period. Serologic tests should follow total (or IgG) T. gondii specic antibodies titer (taking into account that closely after birth these are maternal in origin, transferred through the placenta), IgM and IgA titers. Though passively transferred maternal IgG has a half life of approximately 1 month, it can still be detected in the newborn for several months, generally disappearing completely within one year [112]. Appearance of autonomous IgG antibodies in a congenitally infected newborn begins, in an untreated patient, about 3 months after birth. Anti-parasitic therapy may delay antibody production for about 6 months and, occasionally, may completely prevent antibodies production [86]. 4.4.1. Western blots Remington et al. introduced Western blots (using T. gondiispecic labeled antigens to detect antibodies, separated by electrophoresis and transferred to a membrane) to compare newborn versus maternal antibodies [115117]. Western blotting could potentially separate maternal from fetal/newborn antibodies. The test is not widely used mainly because of its technical complexity and high price. 5. Treatment of congenital toxoplasmosis Anti T. gondii treatment initiation generally requires conrmatory laboratory tests in a reference center, followed by consultation with experts. Treatment is indicated in the following conditions: infection during pregnancy and congenital infection as well as infection of an immunocompromised host (e.g. HIV/AIDS) and in case of an invasive disease. In pregnant women and infected neonates, both symptomatic and asymptomatic, specic treatment of T. gondii infection is indicated immediately following established diagnosis. The combination of pyrimethamine, (adult dosage 25100 mg/d 34 weeks), sulfadiazine adult dosage 11.5 g qid 34 weeks) and folinic acid (leucovorin, 1025 mg with each dose of pyrimethamine, to avoid bone marrow suppression) is the basic treatment protocol recommended by the WHO [118] and CDC [119]. Other
drugs such as spiramycin (adult dosage 34 g/d 34 weeks) and sometimes clindamycin are recommended in certain circumstances. Spiramycin is used to prevent placental infection; it is used in many European countries especially France, Asia and South America. In the US, spiramycin is currently not approved by the FDA but, available as an investigational drug, requiring special approval. Treatment with pyrimethamine and sulfadiazine to prevent fetal infection is contraindicated during the rst trimester of pregnancy due to concerns regarding teratogenicity, except when the mothers health is seriously endangered. During the rst trimester sulfadiazine can be used alone. As recently reviewed by Montoya and Liesenfeld [112], treatment protocols vary among different centers. The effectivity of anti-T. gondii treatment is evaluated based on two criteria: rate of mother to child transmission and prevalence and severity of sequelae. The majority of the studies are retrospective or cohort studies of various populations and case denitions. The difference in study patterns and methodologies affects the reliability and validity of the results and thus prevents issuing further recommendations. Wallon et al. [120] reviewed studies comparing treated and untreated concurrent groups of pregnant women with proved or likely acute toxoplasma infection. Outcomes data of the offspring were reported. The results showed treatment to be effective in ve studies but ineffective in four. Gras et al. [121] reported that the effect of prenatal pyrimethaminesulfadiazine combination treatment on the cerebral and ocular sequelae of intrauterine infection with T. gondii was not benecial in 181 children of infected mothers. Neto reported the outcome of patients with congenital toxoplasmosis who were all treated with pyrimethamine, sulfadiazine and folinic acid; of 195 patients 138 (71%) were asymptomatic until the age of 2 years. The authors suggest that for six patients with sequelae because of the delay in anti-toxoplasma treatment (614 months post diagnosis) the disease was not prevented [122]. Gratzl et al. [123] reported variable concentrations of spiramycin and its metabolites in serum and amniotic uid of 18 pregnant women following treatment. All the drug concentrations were below the level reported to inhibit parasite growth in vitro. The authors suggested that the possible reasons being individual pharmatokinetic variability and patients treatment compliance. Gilbert et al. [124] reported the effect of prenatal treatment in 554 infected women and their offspring. In this study comparison of early versus late treatment and of combination treatment (pyrimethamine, sulfadiazine) with spiramycin or no-treatment, were all statistically insignicant. The possible interpretation is that delayed treatment initiation led to failure to prevent parasite transmission. Another European multicenter study comparing transmission rates and clinical outcomes in 856 motherinfant pairs, found no signicant association between the outcome and the intensity of treatment protocol in pregnancy [125]. Bessieres et al. [126] studied the effect of treatment during pregnancy in a cohort of 165 women and found that cases could be identied during pregnancy as well as during the neonatal period. They also noted that T. gondii was less frequently isolated in women treated with pyrimethamine and sulfadoxine than in women treated with spiramycin only. Foulon et al. [127] reviewed the measures
468
of prevention of congenital toxoplasmosis and concluded that treatment during pregnancy signicantly reduces sequelae and treatment of infected children has a benecial effect when therapy is begun soon after birth. In conclusion, the efcacy of anti-T. gondii treatment in pregnancy is still an unsettled matter. It is difcult to nd the effect of treatment when comparing the different studies because of: different treatment regimes and timing (for small groups of patients), the pharmacokinetics patterns of drugs (concentration in amniotic uid and fetal CSF), patient (none) compliance with treatment and different methodologies of follow-up in each study. As concluded by Peyron et al. [128] and others, further large scale, carefully controlled studies are necessary in order to clarify this controversial issue. At present the anti-parasite treatment recommended for toxoplasmosis as outlined above, should be considered as the guideline for good medical practice. 6. Prevention 6.1. Primary prevention In the United States efforts at prevention of congenital toxoplasmosis have been primarily directed towards health education, focused to avoid personal exposure to the parasite (hygienic and culinary practice during pregnancy). In Poland, an extensive health education campaign, increased toxoplasmosis awareness and knowledge of preventive behaviour signicantly within the 4 years of the reported study [129]. Many other countries have introduced educational programs aimed at reducing the incidence of congenital toxoplasmosis. Such programs depend on careful identication of unique target-populations and tailoring appropriate approaches of education. To evaluate the success of such programs it is important to measure incidence rate before onset and at pre-determined intervals after introducing the campaign. 6.1.1. Vaccine Development of a vaccine for toxoplasmosis can prevent human disease by immunization of human as well as animals (the source of infection). Both attenuated parasite and immunogenic antigens are considered as potential agents for vaccination. Live attenuated S48 strain is in use for vaccination of sheep in Europe and New Zealand but is unsuitable for human use due to its expense, short shelf life and most importantly, to the ability of the attenuated parasite to revert to a pathogenic strain [130133]. Much of the work has been focused on SAG1, a surface antigen expressed on tachyzoites, in attempts to induce protective immune response (mainly T-helper response) when introduced to the host with various adjuvants [134136]. Development of vaccine using antigens expressed by bradyzoites and oocytes is also under investigation[134,137]. 6.2. Secondary prevention screening Routine toxoplasmosis screening programs for pregnant women have been established in France, in Austria and in the
State of Goias, Brazil as recommended by experts [127]. Screening of women should begin prior to conception with follow-up monthly tests during pregnancy to detect seroconverion. This is the basis for the French [138] screening program and the Austrian Toxoplasmosis Prevention Programs, both recommend routine serologic testing, in Austria three times during pregnancy: in the rst, second and third trimesters and in France six times following the initial nding [139]. Treatment is recommended if one of the tests suggests denite or probable primary maternal infection [140]. In Massachusetts, USA, where there is low seroprevalence in the population, only newborns are screened for the presence of T. gondii-specic IgM [141]. IgM detection is followed by an extensive clinical evaluation and a one year treatment regimen combination of pyrimethamine and sulfadiazine [140]. A recent study screened 364,130 neonates in the United States for T. gondii specic IgM and conrmed 195 cases of congenital toxoplasmosis (1 in 1867). Moreover, a 7year follow-up of the treated patients revealed no symptoms or at least no progress of the disease. Based on these ndings, the authors suggest including toxoplasmosis in neonatal screening programs [122]. In the United Kingdom a national committee concluded that no prenatal or neonatal screening for T. gondii should be performed, which brought out controversy among specialists [142]. A survey conducted in Italy reported 35/1000 pregnant women with primary T. gondii infection and recommended maternal screening during pregnancy rather than neonatal screening [143]. In Norway, screening of pregnant women was recommended until 1977 when the National Institute of Public Health discouraged it, following a large study that showed low (0.17 %) incidence of primary infection during pregnancy [144]. Two years following this change in policy, a study by Eskild et al. [145] showed that despite the recommendations, 81% of the pregnant women were still routinely tested for T. gondii-specic antibodies. In Finland, a cost-benet analyses of screening programs for pregnant women as well as education programs revealed the benecial effect of such programs in both low and high incidences of toxoplasmosis [146]. Cost-effectiveness of optional screening programs (no screening, pre-conception or neonates screening, frequency of tests during pregnancy) depends on local factors: incidence of congenital toxoplasmosis, available diagnostic and therapeutic services, and the population compliance with screening. It is important to promote public, as well as professional, knowledge regarding the disease, in order to effectively prevent, diagnose and treat congenital toxoplasmosis. In conclusion, it is highly recommended to educate the public and professionals to minimize risk of infection. Screening programs of women at childbearing age and upon gestation or at least newborn screening is highly effective for early treatment and prevention of sequelae. Acknowledgments Dr. Irena Volovik Sub-district Health Ofcer, Hadera, Israel, for providing data of the presented case.
469
Mrs. R. Kaufman and Mrs. R. Avni for excellent laboratory work.
References
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Congenital Toxoplasmosis Prenatal Aspect of Toxoplasma Gondii Infection

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Reproductive Toxicology 21 (2006) 458472

Congenital toxoplasmosisprenatal aspects of Toxoplasma gondii infection

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

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

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

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

Fig. 1. Laboratory diagnosis of congenital toxoplasmosis.

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

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

Sabin Feldman Dye Test (SFDT)

Titer in international units (IU) of total specic Ig

hands on = several hours

EIAa /total Igb

Positive/negative for total specic Ig Titer (in IU) of total specic Ig

Low = simple automated test High, reference center only

When SFDT is unavailable

Low = simple automated test Low = simple automated test

IgM/IgA or IgE by EIA

Possible recent infection with T. gondii

IgM/IgA or IgE by IAAT

Positive/negative for specic IgM, IgA or IgE Abs

Possible recent Infection with T. gondii

Most sensitive and specic test

IgM/IgA or IgE by IFA

High, reference center only

Very subjective and difcult to standardize Supportive evidence

PCR Western blot IgG, IgM

Body uids/tissue Serum

Positive/negative Identical/unidentical to maternal Ig

Presence of parasites DNA Fetal/newborn infection

Several hours 1 day

High sensitivity Infrequent availability

EIA: enzyme immunoassay. Ig: immunoglobulin. IFA: indirect uorescent assay.

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

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

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

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

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

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