Congenital toxoplasmosis: Current status of diagnosis, treatment, and prevention

Congenital Toxoplasmosis: Current Status of Diagnosis, Treatment, and Prevention Kenneth M. Boyer, MD Congenital toxoplasmosis, like congenital syphilis and perinatally acquired human immunodeficiency virus infection, usually is not apparent clinically in the newborn, and, because screening is seldom performed, the condition seldom is entertained as a diagnosis. Early recognition and treatment of Toxoplasma infection in pregnant women may prevent transplacental infection and avoid its clinical expression when the infant reaches young adulthood. New programs for screening and early diagnosis have awakened interest among pediatricians in early treatment of this infection. This article describes the parasite that causes the infection, Toxoplasma gondii; its epidemiology; the clinical manifestations of infection; diagnostic tests; treatment; and prevention. Copyright 娀 2000 by W.B. Saunders Company

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ike congenital syphilis and perinatally acquired human immunodeficiency virus (HIV) infection, congenital toxoplasmosis is a condition that usually is not apparent clinically in the newborn baby.1 However, unlike syphilis and HIV infection, screening of mothers for Toxoplasma infection in the United States seldom is done, and, for this reason, the condition seldom is entertained as a diagnosis by obstetricians or pediatricians. Although 70 to 90 percent of infants with congenital toxoplasmosis have no abnormalities on a routine newborn physical examination, most, if left untreated, will develop significant clinical expression of their infection by young adulthood. An emerging body of evidence from this country and abroad indicates that early recognition and treatment of Toxoplasma infection in the pregnant woman can prevent transplacental infection. Treatment of the infected fetus or newborn also can improve outcome.2,3 Thus, interest in developing new programs for screening and early diagnosis of this important congenital infection is awakening.

The Parasite Toxoplasma gondii is an intracellular parasite that is a member of the apicomplexan phylum of protozoans. It is taxonomically related to Plasmodium (the cause of malaria) and Pneumocystis (the cause of Pneumocystis pneumonia). The organism is an obligate intracellular parasite and for many years has been the subject of research as a model of intracellular parasitism in humans. Because of its relationship to Plasmodium, Pneumocystis, and other similar apicomplexan parasites, new therapeutic

From the Section of Pediatric Infectious Diseases, Rush Children’s Hospital, Chicago, IL. Address correspondence to Kenneth M. Boyer, MD, Rush Children’s Hospital, 1653 W Congress Parkway, Chicago, IL 60612. Copyright 娀 2000 by W.B. Saunders Company 1045-1870/00/1103-0004$10.00/0 doi:10.1053/pi.2000.6226

approaches to one may have profound implications for treatment of related organisms. The structure of T gondii is schematized in Figure 1. This figure depicts the tachyzoite phase of the organism. The apical complex of microtubules, polar rings, and rhoptries is apparent and is the basis for its taxonomy. Numerous outer membrane antigens have been characterized and have known gene sequences. Organelles such as the nucleus, Golgi apparatus, and mitochondrion are typical of eukaryotic cells. Dense granules contain intracellular enzymes, important for metabolism. Of particular current interest is the presence of a plastid in the cytoplasm. It has been considered to be an evolutionary remnant of uncertain significance. Recently, this plastid was found to be most similar to the chloroplasts of green plants. Enzyme systems typical of plant metabolic pathways (eg, the shikimic acid pathway) have been found within T gondii and have opened the possibility of using new approaches to chemotherapy for diseases caused by T gondii and related organisms.4,5 The life cycle of T gondii is well-known.6 The definitive host is the cat, a broad designation that includes essentially all known feline species. The sexual reproduction of this parasite, which takes place only in the intestinal tract of felines, leads to the production of oocysts that are shed in cat excrement. Ingestion of sporulated oocysts by other mammalian species, as well as birds, leads to bloodborne invasion of many body organs, where the mature tachyzoite phase of the organism can damage tissue and also establish parasitism. Included in the list of possible intermediate hosts for the organism are human beings, including pregnant women. Infection of mammalian and avian cells also can lead to persistent latent infection. This type of infection occurs because, after invasion of skeletal muscles and other organs of an infected animal, the parasite forms bradyzoites, which encyst and can remain dormant in the tissue for the life of the animal. Such muscle tissue, if it is ingested as meat, also can lead to human infection. T gondii infection also can occur by laboratory accident, blood transfusion, and organ transplantation, but these modes of transmission are extremely rare

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Figure 1. Schematic diagram of localization of intracellular organelles and antigens of Toxoplasma gondii. (Reprinted with permission.32) compared with transmission as oocysts or as parasites encysted in meat. The infectious forms of the parasite are of interest because, in the case of oocysts, they are capable of persisting in the environment for months to years, particularly in warmer climates where humidity is high.7 Bradyzoites remain within cysts in food animals or in infected humans essentially for life. Because the metabolic rate of the parasites is very slow at this stage, they are extremely difficult to eradicate with chemotherapy. At present, no safe and effective chemotherapeutic agent is available for eliminating the bradyzoite stage of the parasite.

Epidemiology Most infected cats acquire T gondii soon after being weaned, a reflection of carnivorism. For this reason, infection is more prevalent in feral cats than in domestic cats. Shedding of oocysts by cats is relatively brief, approximately 7 to 20 days after primary infection. Although reactivation of feline infection can occur, the number of oocysts shed is much less. At any given time, fewer than 1 percent of cats in the United States are shedding oocyts. Approximately 30 percent of cats in the United States are seropositive. Transmission of T gondii to humans, particularly pregnant women, most commonly is attributed to handling kitty litter. Numerous more subtle means of accidental ingestion, including gardening, playing with children in sandboxes, and frequenting riding stables where cats are used to suppress the mouse population, also are thought to occur.8 A

recent epidemic in Victoria, British Columbia, was attributed to inadequate purification in a municipal water system.9 Among the mothers of children with congenital toxoplasmosis interviewed in our Chicago Collaborative Trial between 1981 and 1998, most indicated that they had some cat exposure during pregnancy. Interestingly, however, only a minority owned a cat and only 15 percent recalled emptying a litter pan. Thus, at present, exposure to cats accounts for only a fraction of congenitally infected infants. The exposure of humans to T gondii in infected meat probably is now comparable in frequency to cat exposure as a means of disease transmission in the United States. Analysis of grocery store meats indicates that 10 to 80 percent of beef, 20 percent of pork, and 30 percent of lamb is infected.10 Detection of infection of meat is difficult because it requires feeding to susceptible laboratory animals and because it is difficult to differentiate between viable and nonviable parasites by using laboratory procedures (eg, polymerase chain reaction [PCR]) other than feeding to susceptible animals. An important factor in the transmission of T gondii by meats is that tissue cysts are killed by holding temperatures below 0°F for more than 24 hours. Thus, meat processing or storage in a home freezer may render much potentially infectious meat noninfectious. Transmission of T gondii by meat typically is thought to occur through ingestion of ‘‘gourmet’’ dishes such as steak tartare. However, inadvertent exposure to parasites can occur in numerous other ways, such as by sampling uncooked hamburger during preparation, ingesting rare or undercooked rather than completely cooked meat, and by butchering wild animals such as deer, raccoons, or opossums. Of the mothers with congenitally infected infants in our Collaborative Trial, approximately one-half indicated some possibility of raw meat exposure during pregnancy, although only 12 percent had a recollection of actually eating a raw meat dish per se. The global epidemiology of T gondii infection varies widely. High prevalences are found in such diverse settings as France, numerous Pacific islands, and Central American countries such as Panama and Costa Rica. Low prevalences are found in several Scandinavian countries, Japan, and Iceland. In the United States, the overall prevalence of infection ranges from less than 5 percent in the Rocky Mountain states to 15 to 25 percent in the southeastern states.11 In Massachusetts, where neonatal blood samples have been screened systematically, 17 percent of pregnant women are IgG antibody positive and approximately 1 in 10,000 newborns are positive for IgM-specific antibody.12 Most IgM-positive infants have proven to be congenitally infected. Thus, currently an estimated 400 to 4,000 congenital infections occur per year in the United States. The incidence of congenital infection is related in an interesting manner to the prevalence of infection in women of childbearing age. An insightful mathematical model of congenital toxoplasmosis infection was developed by Frenkel13 (Fig 2). In this model, a very high prevalence of antibody in women of childbearing age would be associated with a very low incidence of congenital infection because, in such a population, very few women would be expected to be susceptible to Toxoplasma infection during the 9 months of gestation. A very low prevalence of infection in women of childbearing age also would be associated with a low incidence of congenital infection because,

Congenital Toxoplasmosis

Figure 2. Cumulative antibody prevalences in human populations and the fetal risk of congenital toxoplasmosis in pregnancies of women aged 20 to 30 years. (Reprinted with permission from Frenkel JK: BioScience 23:350, 1973.33 娀1973 American Institute of Biological Sciences.) in such a population, very few women would be expected to acquire Toxoplasma infection during the 9 months of gestation. At prevalence rates of approximately 50 to 70 percent, which correspond to a 3 percent acquisition rate per year, one would expect to see the highest incidence of congenital Toxoplasma infection. Such prevalence rates occur in France, for example, where the highest incidence of congenital infection occurs (approximately 1 per 200 births). Transplacental transmission of toxoplasma varies greatly according to the trimester of maternal acquisition of infection. Transmission occurs in 17 percent of first-trimester acquisitions, 25 percent of second-trimester acquisitions, and 65 percent of third-trimester acquisitions.14 In contrast, the severity of disease is greatest in the fetus that acquires infection in the first trimester, and generally is mild or asymptomatic in the fetus that acquires infection in the third trimester. This pattern of transmission and severity of infection has important implications for programs involving maternal screening for acquisition of infection.

Clinical Manifestations Most infants with congenital toxoplasmosis are asymptomatic as newborns. The minority of infants who receive a diagnosis of congenital toxoplasmosis on the basis of clinical findings have either ‘‘generalized’’ or ‘‘neurologic’’ disease. These two catego-

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ries of disease were defined initially by Eichenwald15 in the early 1960s. A generalized presentation occurs in approximately one-third of symptomatic patients. Prominent features are hepatosplenomegaly, petechiae, thrombocytopenia, jaundice, and a septic appearance. Children with this form of presentation may appear very similar to the ‘‘blueberry muffin’’ presentation of congenital rubella and congenital cytomegalovirus infection. This form of the disease is clinically obvious in earliest infancy. There is some clinical overlap with the ‘‘neurologic’’ form of disease, which generally manifests later in infancy or childhood. The classic triad of features are chorioretinitis, intracranial calcifications, and hydrocephalus. Intracranial calcifications in congenital toxoplasmosis are visualizable by ultrasound, but the most sensitive technique for detecting them is computed tomography (CT). Lesions may be multiple or isolated, tiny or coalescing and large. Usually, they are located in the basal ganglia, but they may be present anywhere in the brain substance.16 Cerebrospinal fluid abnormalities are common. A lymphocyctic pleocytosis is seen most commonly, usually accompanied by remarkable elevations of cerebrospinal fluid (CSF) protein as high as 1 to 2 g/dL. The elevated levels of CSF protein serve to differentiate this condition from viral aseptic meningitis. Hydrocephalus may be detected in utero by ultrasound or may be present at birth. More commonly, hydrocephalus is a late-appearing manifestation, with the peak age at presentation being approximately 6 months of age. In general, the earlier hydrocephalus is detected, the more likely permanent neurologic sequalae will occur.17 Eye involvement by T gondii is the most characteristic feature of all forms of congenital infection.18 The macula most often is involved, with or without peripheral lesions. Active disease is associated with vitreous haze, a reflection of lymphocytic inflammation. Quiescent disease may be in the form of large pigmented scars that extend through the retina to the sclera, or it may simply involve tiny discrete hyperpigmented areas in the retina that can be confused with other conditions. Symptoms of toxoplasmic choriorentinitis generally are subtle in infancy and early childhood. The most common presentation is strabismus. Later on, as a child’s ability to describe visual effects improves, blurred vision or the presence of ‘‘floaters’’ may be symptoms that will lead to ophthalmologic evaluation. Because of the common involvement of the macula, most toxoplasmic chorioretinitis is threatening to vision. Exacerbations of eye disease may occur multiple times over many years and lead to ultimate loss of vision, even when the earlier stages in a particular patient were relatively mild. In a study from the Netherlands, 82 percent of a group of asymptomatic children known to be infected in infancy had developed retinal lesions by the age of 20 years.19 Onset of disease leading to blindness developed as late as 18 years of age.

Diagnostic Tests The major diagnostic issues related to congenital toxoplasmosis are (1) identification and establishment of the time of infection in a pregnant woman; (2) determination of whether fetal infection has taken place prior to birth; (3) proof of congenital infection after birth; and (4) adaptation of testing procedures to

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enable cost-effective mass screening of either pregnant women or newborn infants. A major point concerning diagnostic testing is that the tests available in many hospital-based and commercial laboratories frequently are misinterpreted or inaccurate. This concern is particularly true of indirect fluorescence tests for IgG and IgM antibodies and of enzyme-linked immunosorbent assay (ELISA) systems for quantitation of IgM-specific antibodies. A Food and Drug Administration (FDA) warning has been issued recently about misinterpretation of IgM serologies.20 The recommendation is that all suspected infections be confirmed in a reference laboratory setting, such as the Palo Alto Medical Foundation (phone number: [650] 853-4828). The problem of diagnosis of toxoplasmosis in a pregnant woman is complex because most pregnant women who acquire toxoplasmosis are asymptomatic. Detection of IgG antibody then creates the problem for the obstetrician of differentiating acute primary infection from long-standing latent infection. Current thought is that only primary infection occurring during gestation or periconceptionally can lead to fetal infection. Screening for IgG antibodies generally is performed using indirect immunofluorescence or ELISA systems. Confirmation should be determined with the Sabin-Feldman dye test. Several tests are available to determine the acuity of maternal infection and include testing for toxoplasma antibodies of IgM, IgA, or IgE classes. Acuity can be determined also by the use of the ‘‘differential agglutination’’ test or with recently developed tests for antibody avidity.21 If a panel of tests is used, generally the timing of maternal infection can be established with relative certainty. If recent acute infection is established in a mother during pregnancy, fetal infection is determined using PCR amplification of the B1 gene of T gondii in a sample of amniotic fluid.22 This confirmatory approach has replaced diagnostic cordocentesis. Whether the fetus actually has acquired organ damage as a result of fetal infection is determined using serial ultrasound examinations. Obviously, diagnosis of congenital infection in the newborn infant is aided by recognition that maternal infection has occurred. In circumstances of suspected congenital infection, a thorough evaluation of the newborn infant is carried out as summarized in Table 1. Infection can be established most definitively by actual cultivation of Toxoplasma organisms in placental samples, which is performed by inoculating placental tissues into laboratory mice. The presence of specific IgA, IgM, or IgE antibody also is strong evidence for the diagnosis. Unfortunately, approximately 30 percent of truly infected infants lack IgM antibodies, a problem that occurs in both seriously affected infants and apparently normal babies. Blast transformation of lymphocytes in response to Toxoplasma lysate antigens also can be used diagnostically, but again it is negative in approximately one-half of infected babies at the time of birth.23 Recognition of maternal or infant infection with mass screening has engendered considerable interest. In several European countries (eg, France, Austria), serial monthly testing of pregnant women is considered a standard element of good prenatal care. Such serial testing enables establishment of the time of maternal infection by relatively simple laboratory means but obviously requires a complex system of data management and serum collection. In the United States, a universal screening

Table 1. Recommended Studies and Laboratory Evaluation for the Infant With Suspected Congenital Toxoplasmosis Clinical Evaluation and Nonspecific Tests By a pediatric ophthalmologist By a pediatric neurologist CT scan of the brain Blood tests Complete blood cell count with differential and platelet counts Serum IgM, IgG, IgA, and albumin Serum alanine aminotransferase, total and direct bilirubin Cerebrospinal fluid: cell count, glucose, protein, and total IgG T gondii–Specific Tests Serum Sabin-Feldman dye test, IgM ELISA, IgM ISAGA, IgA ELISA, IgE ELISA/ISAGA (0.5 mL serum, sent to Serology Laboratory, Palo Alto Medical Foundation, 860 Bryant St, Palo Alto, CA 94301) Lumbar puncture: 0.5 mL CSF sent to Serology Laboratory (see above address) for dye test and IgM ELISA Sterile placental tissue (100 g in saline, no formalin from near insertion of cord from the fetal side) and newborn blood obtained for inoculation into mice (2 mL clotted whole blood in redtopped tube) Maternal serum analyzed for antibody detected by dye test, IgM ELISA, IgE ELISA/ISAGA, and AC/HS Abbreviations: CT, computed tomography; ELISA, enzyme-linked immunosorbent assay; ISAGA, immunosorbent agglutination assay; CSF, cerebrospinal fluid; AC/HS, differential agglutination of acetone- and formalin-fixed organisms.

program has been established in Massachusetts and New Hampshire, using IgM ELISA testing on newborn heel-stick blood samples. This system appears to have good, although not complete, diagnostic accuracy. However, it is a less desirable approach than is prenatal screening because irreversible eye or brain damage may already have occurred in some babies by the time infection is recognized at or soon after birth. A recent conference at the Centers for Disease Control and Prevention has identified the issue of developing simple screening techniques for mothers or infants that would be widely adaptable in the United States as a public health priority for this disease.

Treatment Congenital toxoplasmosis is a treatable infection, although at present it is not a curable one. Available therapeutic agents are effective in killing the tachyzoite (pathogenic) phase of the parasite in man but are not capable of eradicating encysted bradyzoites. Development of drugs that would have this capability is a research priority. The treatment of choice for toxoplasmosis is the synergistic combination of the drugs pyrimethamine and sulfadiazine. A less toxic but probably less efficacious alternative medication is the macrolide drug spiramycin. Pyrimethamine and sulfadiazine are available in the United States routinely. Spiramycin

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Congenital Toxoplasmosis may be obtained only as an investigational drug through its manufacturer, Aventis (phone number: [301] 827-2127). Numerous other available drugs that are known to have activity against Toxoplasma include clindamycin, azithromycin, and atovaquone. These medications have been used as alternative drugs. Trimethoprim/sulfamethoxazole has equivalent potential for toxicity to pyrimethamine/sulfadiazine but is believed to have somewhat less efficacy. Folinic acid (leukovorin), although not a therapeutic agent against Toxoplasma, blocks the marrow-suppressive effects of pyrimethamine/sulfadiazine and should be used in any therapeutic regimen in combination with it. Prednisone may be used as an anti-inflammatory adjunctive therapy in severe eye or central nervous system disease. Guidelines for dosage of the available agents are found in most pediatric infectious disease textbooks and are summarized in Table 2.24-26 Although rigorous, randomized, clinical trials with untreated controls are not ethically possible, experience developed over the last 15 years in the Chicago Collaborative Treatment Trial has shown dramatically improved clinical outcomes in symptomatically treated infants compared with untreated historical controls. Remaining issues are the optimal duration of treatment of a congenitally infected infant, the intensity of treatment that is necessary to balance efficacy and potential toxicity, and

the degree to which early treatment results in a reduced risk of recurrent disease in older children and adults. In the Chicago trial, all enrolled infants received treatment of a year’s duration, although with varying intensity in different regimens. Experience with the long-term follow-up of these children is accumulating but is not complete. The Chicago studies have enabled development of data regarding the pharmacokinetics of pyrimethamine in infancy.27 The drug has a prolonged half-life of approximately 64 hours. CSF levels of drug are approximately 10 to 25 percent of concomitant serum levels. Coadministration of phenobarbital reduces the half-life of pyrimethamine by approximately onehalf (33 hours). If marrow suppression is observed, an increased dose of folinic acid generally results in improvement. In vitro studies show that folinic acid does not protect the parasite from the therapeutic effect of the drug. The development of ‘‘serologic rebound’’ after the completion of a year of treatment shows that encysted organisms are not completely eradicated by treatment. Although children with congenital toxoplasmosis average lower scores on rigorous psychological intelligence tests than do their siblings, outcomes in treated children are markedly better than in untreated historical controls, as described by Eichen-

Table 2. Treatment of Congenital Toxoplasmosis Manifestation of Disease Congenital toxoplasmosis*

Therapy

Dosage (oral unless specified)

Duration

Pyrimethamine*

Loading dose: 2 mg/kg/d for 2 1 yr days, then 1 mg/kg/d for 2 or 6 months, then this dose on and each Mon, Wed, and Fri Sulfadiazine* 100 mg/kg/d in 2 daily divided 1 yr and doses Leucovorin (folinic acid)* 5-10 mg 3 times weekly† 1 yr Spiramycin† 100 mg/kg/d in divided doses 1 yr used in alternate months in place of pyrimethamine/sulfadiazine/leucovorin in France Corticosteroids 1 mg/kg/d in 2 daily divided Until resolution of elevated (prednisone)§ doses (ⱖ 1 g/dL) CSF protein level or active chorioretinitis that threatens vision In pregnant women with acute Spiramycin† 1.5 g q 12 hr without food Until fetal infection documented toxoplasmosis first 21 wk of or excluded at 21 wk; if docugestation or until term if fetus mented, replace with pyrinot infected methamine, leucovorin, and sulfadiazine If fetal infection confirmed after Pyrimethamine Loading dose: 100 mg/d in Until delivery 17th wk of gestation or if divided doses for 2 days folinfection acquired in last few and lowed by 50 mg/d weeks of gestation Sulfadiazine Loading dose: 75 mg/kg/d in 2 Until delivery divided doses (maximum, 4 g/d) for 2 days, then 100 mg/kg/d in 2 divided doses and (maximum, 4 g/day) Leucovorin‡ 5-20 mg qd Until delivery

*Optimal dosage and feasibility currently being evaluated in ongoing National Collaborative Treatment Trial (773) 834-4152. †Available only on request from the Food and Drug Administration (301) 827-2127. ‡Monitor blood counts and platelets weekly, adjust for megaloblastic anemia, granulocytopenia, or thrombocytopenia. §Corticosteroids should be continued until signs of inflammation (high CSF protein ⱖ 1 g.dl) or active chorioretinitis that threatens vision have subsided, dosage then can be tapered and discontinued; use only in conjunction with pyrimethamine, sulfadiazine, and leucovorin. Modified and reprinted with permission.34

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wald15 and by Wilson et al.28 For example, of the 101 untreated children classified as having either generalized or neurologic disease by Eichenwald, 81 percent had epilepsy at subsequent evaluations, 70 percent had abnormal motor tone, and 86 percent had IQ scores lower than 70. The corresponding rates in the Chicago Collaborative Study were 11 percent for seizures, 24 percent for abnormal tone, and 32 percent for IQ scores lower than 70.29 An unexpected recent observation has been that intracranial calcifications in affected children, as detected by serial cranial CT scans, disappear or decrease in size in a high percentage of treated children.16 This observation appears to reflect the control of focal encephalitis, with an improved potential for brain growth and development.

Prevention Congenital toxoplasmosis can be prevented or its consequences minimized by early treatment. It has an incidence comparable to other diseases for which screening is mandated by law (eg, phenylketonuria). Thus, interest in developing practical and cost-effective preventive programs has been renewed. There is no question that toxoplasmosis in a pregnant woman is preventable by not ingesting raw or undercooked meat and by avoiding exposure to cats and specifically cat excrement in the home or the environment. Programs of maternal education in several European countries where they have been aggressively pursued have resulted in reductions in the incidence of congenital infection and of maternal seroconversion.30 Measures a pregnant woman can take to avoid infection are described nicely in a brochure we developed in Chicago that is available from the March of Dimes. This brochure can be downloaded from http://www.iit.edu/⬃toxo9pamphlet (accessed 3/18/00). Primary prevention by means of maternal screening programs that detect seroconversion during pregnancy is mandated in several European countries, including France and Austria. Recognition of maternal seroconversion during pregnancy enables initial intervention with the drug spiramycin, which prevents transplacental infection.31 It also enables amniocentesis and detection of fetal infection by means of PCR. Documentation of fetal infection then permits a rational choice between pregnancy termination or carrying the pregnancy through to term, and it also permits more aggressive maternal/fetal therapy with pyrimethamine/sulfadiazine before delivery. Secondary prevention by means of neonatal screening with routine heel-stick blood samples has been studied in Massachusetts and is now performed as a routine in the state health department laboratories for the states of Massachusetts and New Hampshire.12 In these studies and their subsequent application, the incidence of congenital toxoplasmosis in Massachusetts (a state of relatively low incidence) has been approximately 1 in 10,000. Although most infected infants who have been detected in these programs were not recognized by their physicians during newborn and early infant examinations, detection of infection by this means has enabled early institution of therapy. Early experience seems to suggest that outcomes are improved. Although controversy over the choice of method remains, experts agree that instituting programs for more

widespread screening of mothers or infants for congenital toxoplasmosis in the United States would be desirable.

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Congenital Toxoplasmosis 22. Hohlfeld P, Daffos T, Costa JM, et al: Prenatal diagnosis of congenital toxoplasmosis with a polymerase-chain reaction test on amniotic fluid. N Engl J Med 331:695-699, 1994 23. McLeod R, Mack DG, Boyer KM, et al: Phenotypes and functions of lymphocytes in congenital toxoplasmosis. J Lab Clin Med 116:623635, 1990 24. Roberts F, Boyer K, McLeod R: Toxoplasmosis, in Gershon AA, Katz SL, Hotez PJ (eds): Infectious Diseases of Children (ed 10). St Louis, MO, Mosby-Year Book, 1998, pp 538-570 25. Boyer KM, McLeod R: Toxoplasma gondii (Toxoplasmosis), in Long SS, Pickering LK, Prober CG (eds): Principles and Practice of Pediatric Infectious Diseases. New York, NY, Churchill Livingstone, 1997, pp 1421-1448 26. Boyer KM, Remington JS, McLeod R: Toxoplasmosis, in Feigin RD, Cherry JD (eds): Textbook of Pediatric Infectious Diseases (ed 4). Philadelphia, PA, Saunders, 1998, pp 2473-2490 27. McLeod R, Mack D, Foss R, et al: Levels of pyrimethamine in sera and cerebrospinal and ventricular fluids from infants treated for congenital toxoplasmosis. Antimicrob Agents Chemother 36:10401048, 1992

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28. Wilson CB, Remington JS, Stagno S, et al: Development of adverse sequelae in children born with subclinical congenital Toxoplasma infection. Pediatrics 66:767, 1980 29. Roizen N, Swisher C, Stein MA, et al: Neurologic and developmental function in treated congenital toxoplasmosis. Pediatrics 95:1120, 1995 30. Carter AO, Gelmon SB, Wells GA, et al: The effectiveness of a prenatal education programme for the prevention of congenital toxoplasmosis. Epidemiol Infect 103:539-545, 1989 31. Couvreur J, Desmonts G, Thulliez P: Prophylaxis of congenital toxoplasmosis: Effect of spiramycin on placental infection. J Antimicrob Chemother 22:193-200, 1988 32. McLeod R, Mack D, Brown C: Toxoplasma gondii—New advances in cellular and molecular biology. Exp Parasitol 72:109-121, 1991 33. Frenkel JK: Toxoplasma in and around us. BioScience 23:343-352, 1973 34. McLeod R, Wisner J, Boyer K: Toxoplasmosis, in Krugman S, Katz SL, Gershon AA, et al (eds): Infectious Diseases of Children (ed 9). St Louis, MO, Mosby-Year Book, 1992, p 541