Petersen 2001

International Journal for Parasitology 31 (2001) 115144
www.parasitology-online.com
Invited review
Recent trends in research on congenital toxoplasmosis q

Eskild Petersen*, Arnold Pollak, Ingrid Reiter-Owona
a
Department of Mycobacteria and Parasitic Infections Statens Serum Institut, Copenhagen, Denmark
Received 11 September 2000; received in revised form 12 September 2000; accepted 12 September 2000
Keywords: Toxoplasma gondii; Treatment; Diagnosis; Prevalence; Neonates; HIV
1. The european research network on congenital tries was made (Ho-Yen et al., 1995). From the start the
toxoplasmosis working group on epidemiology concentrated on studies
of risk factors of infection with a study from Naples, Italy
Eskild Petersen: (Buffolano et al., 1996). Later a European multicentre study
Department of Mycobacteria and Parasitic Infections, showed that risk factors vary from area to area, but that
Statens Serum Institut, Copenhagen, Denmark overall, infection from meat can explain approximately
two-thirds of all infections (Gilbert et al., 2000). This was
The start of the European Research Network on Conge- the rst study from Europe that actually provided sound data
nital Toxoplasmosis was a seminar in Copenhagen on 17 on the proportion of infections transmitted through meat and
January 1992, with more than 100 participants from Europe is important when future strategies for prevention are
and the United States (Lebech and Petersen, 1992). It was decided.
realised that there were many aspects of congenital infection The serological working group started with an ambitious
with Toxoplasma gondii that were not known, or where the project comparing 17 different diagnostic assays for T.
existing data dated back more than 30 years. The seminar gondii-specic IgG-, IgM- and IgA-antibodies. The working
resulted in an application to the European Union for fund- group collected sera from the centres where the time of
ing, and the network received funding for a concerted action seroconversion and thus the time of infection was known
in the autumn of 1992, ofcially commencing operation on and was able to establish a bank of sera where the date of
January 1st 1993. From the beginning the idea was to create infection was known within weeks. Using these well-char-
a network open to individuals interested in all aspects of acterised sera, it was possible to show that no method, either
congenital toxoplasmosis, who were interested in contribut- single or in combination, could predict the time of infection
ing to research projects which would create good quality within the rst year after infection. The study is in still in the
data, on the basis of which, sound decisions on case deni- process of being published.
tion, prevention, screening, diagnosis and treatment could A second study compared Western blot with ELIFA for
be taken. the diagnosis of prenatal infection in newborns without
The rst meeting took place in Grenoble and working detectable T. gondii-specic IgM- or IgA-antibodies at
groups were established on serological and non-serological birth. The study again took advantage of collecting well-
diagnostic methods, ophthalmology, epidemiology, quality characterised sera from newborns who were born to mothers
control and quality assurance, treatment, and health educa- with Toxoplasma-infection during pregnancy and who were
tion. Later meetings took place in Bonn, Warsaw and a nal followed-up to 1 year of age. The results of the study are still
open congress on congenital toxoplasmosis was held in in the process of being published. A retrospective multicen-
Vienna in June 2000. The submissions making up this tre study in newborns born to mothers treated during preg-
review are the highlights of the Vienna meeting. nancy showed that the diagnostic sensitivity of T. gondii-
Some working groups have been more successful than specic IgM- and IgA-antibodies varied between 41 and
others. It proved particularly difcult to provide common 66%. However, it should be noted that the results can not
guidelines on health education, for instance, although a be extended to children born to mothers not treated during
review of preventive measures in different European coun- pregnancy (Naessens et al., 1999).
The working group on standardisation and quality control
q
Selected reviews from the European Congress on Congenital Toxoplas- was very concerned with the quality of different diagnostic
mosis, Vienna, June 2000. tests and performed two large quality assurance studies
* Corresponding author. Fax: 1 45-3268-3033. where test sera kindly provided by the French National
E-mail address: ep@ssi.dk (E. Petersen).
0020-7519/01/$20.00 q 2001 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.
PII: S 0020-751 9(00)00140-5
116 E. Petersen et al. / International Journal for Parasitology 31 (2001) 115144
Quality Assurance Programme were sent to more than 200 with the planning of the European Multicentre Study on
European laboratories (Petithory et al., 1996). Another Congenital Toxoplasmosis (EMSCOT), coordinated from
study looked at the SabinFeldmann dye test (SFDT), the Institute of Child Health in London. The study started
which is still performed in a few laboratories. The SFDT in 1997 and recruitment ended in 2000 when more than
still has its place in acute diagnosis as a supplement to other 1300 infected women and their newborns from more than
tests (Reiter-Owona et al., 1999). 14 European centres had been recruited into the study. The
The working group on non-serological diagnostic meth- study now focuses on the follow-up of 3-year-old children in
ods rapidly decided to try and evaluate the newly developed particular with regard to new cases of retinochoroiditis
direct detection of T. gondii DNA by the PCR. This new despite treatment and neurological symptoms. A further
method was performed in many laboratories but no external follow-up at 6 years of age is also being prepared based
quality control scheme existed, protocols varied widely, and on the hypothesis that slight damage in infected children
there was great uncertainty on how reliable and reproducible may not be apparent before school age.
the results were. The working group managed to organise
two European multicentre studies which beyond doubt
showed a wide variation in the quality of different PCR 2. Neonatal screening for congenital toxoplasmosis
assays and helped focus on the quality and the need to
2.1. Early evaluation of the postnatal treatment efcacy in
have a certain number of tests in the laboratory to maintain
infants with congenital toxoplasmosis from West Poland
expertise (Guy et al., 1996; Pelloux et al., 1998).
Another group discussed a common case denition and M. Paul a,*, A. Jaworska b, E. Petersen c, J. Szczapa b, H.
agreed on guidelines classifying congenital infections into
Twardosz-Pawlik b:
certain, probable, possible and no infection. The guidelines a
Department of Medical Microbiology, Institute of
provide case denitions which could be used to standardise
Microbiology and Infectious Diseases; bDepartment of
data from different studies to allow comparisons (Lebech et
Neonatal Infectious Diseases, Hospital of Gynaecology
al., 1996).
and Obstetrics; cDepartment of Ophthalmology, Karol
The ophthalmology working group started working on the
Marcinkowski University of Medical Sciences, Poznan,
protocol for a blinded study of retinal changes with the aim
Poland; dDepartment of Mycobacteria and Parasitic Infec-
of providing data on the prociency of clinical diagnosis. tions, Statens Serum Institut, Copenhagen, Denmark
However, the protocol proved very difcult in practical
terms and the study was eventually abandoned. A planned 2.1.1. Introduction
randomised study comparing traditional daily treatment
with sulfadiazine and pyrimethamine with weekly sulfadox- Infection with T. gondii acquired during pregnancy may
ine and pyrimethamine (Fansidar w) also had to be aban- cause intrauterine damage and sequelae in the newborn. Up
doned due to lack of support from the manufacturers of to 87% of congenitally infected infants are asymptomatic at
the drugs. birth (Foulon et al., 1999), and the infection may pass unno-
Through the years it became more and more apparent that ticed until childhood or adolescence, when ocular lesions
the data from the 1950s and 1960s on transmission and and neurodevelopmental disorders may develop (Foulon et
clinical severity may not be valid any longer. A retrospec- al., 1999; Dunn et al., 1999; Wilson et al., 1980; Koppe et
tive pilot study had shown that treatment during pregnancy al., 1986). Anti-parasitic chemotherapy is believed to reduce
may reduce clinical symptoms in congenitally infected chil- the risk of sequelae and should be initiated as soon as the
dren (Foulon et al., 1999), but it was realised that new data infection is diagnosed, but the impact of treatment during
from a prospective study on the natural history of infection pregnancy on materno-foetal transmission is still controver-
and the long term consequences in congenitally infected sial (Foulon et al., 1999; Wallon et al., 1999; Couvreur et al.,
children were much needed. 1993; Stray-Pedersen, 1992).
The working group on the epidemiology of congenital In Poland, like in most European centres, we currently
toxoplasmosis has performed several projects looking at use a combination of pyrimethamine with sulfadiazine alter-
existing databases in European centres. One particular nating with spiramycin for 12 months. Other centres, parti-
large study was a retrospective evaluation of all infected cularly in France, prefer a continuous therapy combining
women from Lyon, France, which provided precise data pyrimethamine and sulphadoxine (Fansidar w) for 2 years
on transmission and clinical symptoms in children with (Villena et al., 1998). In Poland, in the absence of nation-
congenital toxoplasmosis related to gestational age at infec- wide screening of pregnant women, the early postnatal treat-
tion (Dunn et al., 1999). A collaborative study with the ment of all newborns with congenital toxoplasmosis was
European study group working with HIV infection in preg- performed in Poznan since 1996, when the regional screen-
nancy showed that congenital toxoplasmosis was not a ing programme detecting Toxoplasma-specic IgM anti-
problem in HIV-positive pregnant mothers (Dunn et al., body at birth was introduced (Paul et al., 2000).
1996). The aim of the present study has been to evaluate the
The most ambitious project of the network started in 1996 effectiveness of serological screening in newborns followed
E. Petersen et al. / International Journal for Parasitology 31 (2001) 115144 117
by postnatal anti-parasitic therapy for the prevention of clin- estimated on the basis of the kinetics of specic IgG, IgM
ical and immunological recurrences in patients with and IgA antibodies by different serological techniques
conrmed congenital T. gondii infection in early infancy. performed in the post partum period (Guerina et al., 1994).
Treatment management: Children with subclinical infec-
2.1.2. Materials and methods tion or non-specic signs at birth were treated with pyri-
methamine (1 mg/kg body weight per day) plus sulfadiazine
Patients: Fifteen infected infants (eight males and seven (100 mg/kg body weight per day) completed with folinic
females, all singletons) born before November 1998 were acid (5 mg three times per week) for 4 weeks with spiramy-
included. Thirteen of 15 patients were diagnosed during the cin (375 000 IU/kg body weight per day) for 4 weeks. For
rst days of life by detection of anti-Toxoplasma IgM from infants with clinical toxoplasmosis, the continuous therapy
Guthrie cards eluates; two remaining infants from the study with pyrimethamine, sulfadiazine and folinic acid (Lederfo-
area, with no positive specic IgM ELISA at birth were lat) was prescribed for 1224 months, according to the
recognised only by the typical triad of clinical signs: hydro- severity of infection and immunological status of the
cephalus, intracranial calcications, chorioretinitis, and patient. Leucocyte and thrombocyte counts were controlled
later conrmed serologically. The study was approved by twice per month during the periods of the pyrimethamine/
the University of Medical Sciences Ethical Council sulfadiazine administration. Infants with neurological
(Poznan, Poland). abnormalities were managed by regular rehabilitation ther-
Clinical investigations: The children were systematically apy using Voyta's or Balott's methods.
evaluated by the multidisciplinary group consisting of a
neonatologist, an infectious disease specialist, an ophthal- 2.1.3. Results
mologist, a paediatric radiologist and an immunologist.
Symptomatic cases were additionally surveyed by a paedia- In June 1996, the regional screening programme for
tric neurologist, managed under the rigorous care of a physi- congenital T. gondii infection was developed for all
cal therapist and/or an audiologist. The paediatric follow- neonates from the Grand Poland Province. During the rst
up, including an assessment of psychomotor and growth 28 months of the study period, 27 516 liveborn infants were
development with a routine neurological examination was tested for anti-Toxoplasma IgM using peripheral blood
performed soon after birth and again every 2 months during spotted on neonatal Guthrie cards (Lebech et al., 1999).
the rst year of life, and then every 4 months after anti- Fifteen children born with congenital toxoplasmosis in the
parasitic therapy was discontinued. study area, gave an incidence of one per 1834 live births
At each paediatric visit, a blood sample was taken for (0.55 per 1000), which is relatively higher than other coun-
specic antibody measurement. Transfontanel ultrasound tries conducting similar preventive management of
of the brain was repeated, when feasible, every 2 months newborns (Guerina et al., 1994; Malm et al., 1999;
for 1 year. The general ophthalmic assessment, followed by Pawlowski et al., 1994). A seroprevalence of Toxoplasma
direct and indirect ophthalmoscopy after pupil dilatation, antibodies in pregnant women in Poland of 58.9% in the
was usually done before 4 weeks of age and repeated early 1990s (Couvreur et al., 1984) and 44% in 1998
every 3 months, and at each immunological rebound. Hear- 2000 is also elevated. Therefore, passively transmitted anti-
ing investigation was performed during the neonatal period bodies make the conrmation of neonatal screening results
by the Echo-Screen apparatus (FischerZoth GmbH) as well more difcult.
as cranial radiography done in the lateral and sagittal projec- Clinical pattern of infection: Eleven of 15 children with
tions. The imaging investigation was completed by congenital T. gondii infection were followed-up clinically
computed tomography scan near 12 months of age. and serologically between 25 and 47 months (mean
Laboratory diagnosis: The non-commercial immunocap- 34.6 ^ 8.4 months). The remaining four children were lost
ture ELISA, adapted for testing of specic IgM in dried for follow-up between the age of 3 and 16 months. Two
blood neonatal samples collected at birth has been families (patient nos. 3 and 5) refused treatment and/or
previously described (Lebech et al., 1999; Cazenave and further follow-up. Two postnatal deaths occurred at 3 and
Bessieres, 1992). When the screening result was positive, 12 months, respectively; one of severe staphylococcal sepsis
the suspected infection was veried by a mother and child (patient no. 13), and one (patient no. 6) from respiratory
comparative immunological prole analysis (CIP-WB) insufciency (Table 1).
using the Western blot assay (LDBIO Diagnostics) and There were 11 children born at term (73.3%), and four
conventional serology by ELISA for anti-Toxoplasma premature babies delivered between the 32nd and 35th week
IgG, IgM and IgA antibodies (PLATELIA TOXO, Sano of pregnancy (patients nos. 3, 6, 14 and 15). The birth-
Diagnostics Pasteur, VIDAS TOXO, bioMerieux), and weight ranged from 1560 to 3920 g (mean 2983 ^ 776 g),
Immunosorbent Agglutination Assay (ISAGA) for IgM and the head circumference of neonates measured from 29
and IgA (ISAGA PLUS IgA/IgM, bioMerieux), according to 38 cm (mean 32.9 ^ 3.0 cm) (Table 2).
to the manufacturer's standard procedures. One of 13 infected newborns (patient no. 6) identied by
Age of maternal primary infections during pregnancy was serological screening developed hydrocephalus, brain calci-
Table 1
Clinical expression of congenital Toxoplasma gondii infection in 15 infants during the follow-up period a
Patient no. Age of the last Signs or symptoms recognised after the neonatal period
follow-up months
1 47 Transitory muscular hypertonia by 3 months

2 44 Asymptomatic
3 13 Moderate ventricular enlargement without neurodevelopmental complications
4 41 Anaemia, slight dilatation of the both lateral ventricles without neurological impairment
5 16 Asymptomatic
6 12 Progressing hydrocephalus, ventriculoperitoneal shunt, cerebral atrophy, multiple intracranial calcications,
seizures, muscular hypotonia, unilateral microphthalmia and cataract with blindness (left eye), severe
developmental delay, death from respiratory insufciency at 1 year b
7 32 Asymptomatic
8 28 Asymptomatic
9 28 Asymptomatic
10 27 Asymptomatic
11 26 Asymptomatic
12 25 Asymptomatic
13 3 Dilatation of sagittal suture and anterior fontanel, death of severe staphylococcal sepsis at 3 months
14 40 Severe hydrocephalus at the 6th week of life, ventriculoperitoneal shunt, multiple intracranial calcications,
unilateral and peripheral retinochoroidal scar (right eye), strabismus convergent of the right eye, slight
asymmetry of eyes size, suspicious changes in EEG
15 43 Unilateral and peripheral retinochoroidal scar (left eye), multiple intracranial calcications, moderate
dilatation of the both lateral ventricles, anaemia, no neurological impairment
a
Cases 113: Identied solely by the neonatal screening programme detecting Toxoplasma-specic IgM in lter-paper blood specimens; cases 14 and 15:
recognised from clinical signs and conrmed by serology.
b
This patient had abnormal results of foetal ultrasonography at 27 weeks of gestation (intracranial calcications, hypotrophy) but was not treated in utero.
cations, microphthalmia and hepatosplenomegaly and ve hydrocephalus and cerebral atrophy, blindness in one eye,
neonates had non-specic clinical abnormalities at birth, multiple intracranial calcications and profound neurologi-
undetectable later in life, resulting from prematurity or cal disorders, leading to death at the age of 1 year; (ii) one
adaptative respiratory disorders. In one infant (patient no. case of hydrocephalus, brain calcications, unilateral chor-
4) previously classied as asymptomatic at birth, some addi- ioretinal scar without visual impairment; (iii) one case of
tional clinical signs for intrauterine infection became moderate dilatation of ventricular system without hydroce-
obvious after the neonatal period (anaemia at 2 months phalus, brain calcications, unilateral chorioretinal scar
and slight dilatation of lateral ventricles at 12 months). without vision loss; (iv) two cases of isolated slight enlarge-
The remaining six IgM-positive children had no signs or ment of lateral ventricles without any symptoms of hydro-
symptoms of Toxoplasma infection, especially no signs of cephalus or neurological dysfunction (Table 3).
vision impairment due to retinochoroiditis. Four patients exhibited some congenital malformations
Two infants (patient nos. 14 and 15) with a lack of speci- such as unilateral microphthalmia with cataract (patient
c IgM ELISA at birth and probably infected in the early no. 6), slight asymmetry in eye size, bilateral inguinal hernia
intrauterine period demonstrated a typical triad of clinical with hydrocele (patient no. 14), ependymal cyst of the brain
signs recognised during the rst year of life. One of them (patient no. 12), and disturbed skull sutures development
necessitated an introduction of ventriculoperitoneal shunt (patient no. 13).
due to a suddenly increasing ventricular size at 6 weeks Specic antibodies monitoring: The sensitivity of the
(normal at birth) and its revision at 11 months of age. detection of specic IgG, IgM and IgA from lter-paper
Although only two episodes of febrile seizures occurred, eluates at birth has been previously described (Lebech et
the infant received periodic cover of anti-convulsion drugs al., 1999). Paired sera were available for a conrmatory
because of suspicious changes in the EEG. His psychomotor analysis between the 2nd day and 11 weeks after birth
development investigated with the Infants Developmental (mean 4.6 ^ 3.4 weeks). Specic IgM and IgA were less
Scale of the National Institute of Mother and Child was frequently detected after the rst week of life; there were no
normal at 3 years of age. Generally the children demon- differences in sensitivity of ISAGA and ELISA (nine of 13
strated substantial symptoms or signs, if the maternal infec- were positive). During the rst weeks of life before the
tion took place in the rst half of pregnancy (patients no. 6, treatment, seven infants were positive for IgM and IgA
14 and 15). During the follow-up period, no clinical relapses antibodies (53.8%), two children had only positive values
were reported through detailed paediatric, ophthalmic and of IgM (15.4%), one patient was only positive for IgA
imaging surveillance (Table 1). Signs of the classic triad (7.7%), and two cases had borderline levels of IgM or IgA
were found in ve cases (33.3%): (i) one case of progressing (15.4%).
Table 2
Newborns maturity parameters and estimated age of maternal infection in relation to individual treatment recommendations for 15 congenitally infected infants a
Patient Sex Gestational age Birth weight Head Apgar score Delivery Estimated maternal Treatment regimen Age when treatment Duration of the Side effects
no. at delivery (g) circumference mode infection (weeks) started (days) treatment (months)
(weeks) (cm)
1 M 37 3300 32 10 C 3536 P 1 S/R 23 14 None

2 F 40 3240 35 10 S 3739 P 1 S/R 37 13 None
3 F 35 1560 28 9 C 2832 P/S 89 b 10 b None
4 M 42 3050 33 6/8/9 S 2832 P 1 S/R 50 15 None
5 F 40 2700 32 9 S 3640 P 1 S/R 81 b 14 None
6 F 32 1800 28 7/7/8 S 1220 P/S 1c 12 None
7 F 38 3150 34 9 S 2125 P 1 S/R 23 13 None
8 M 40 3700 34 10 S Periconceptional P 1 S/R 17 13 None
9 M 41 3820 36 10 S 2832 P 1 S/R 23 14 None
10 F 40 3150 32 10 S 3236 P 1 S/R 28 12 None
11 M 41 3920 37 7 S 2530 P 1 S/R 26 12 Skin rash
12 M 37 3880 38 4/9/10 S 2630 P 1 S/R 35 12 Crystalluria
13 M 40 3260 34 6/7/7 C Periconceptional P 1 S/R 20 3d None
14 M 32 1790 29 2/5/7 S ,8 P/S 61 24 Crystalluria
15 F 34 2430 31 9 C Unknown P/S 360 24 None
a
Presumptive dates of infections estimated on maternal serology (Foulon et al., 1999).M, male; F, female; S, spontaneous; C, Caesarean section; P/S, pyrimethamine and sulfadiazine; P 1 S/R: pyrimethamine
and sulfadiazine alternated with spiramycin.
b
Treatment and follow-up refusal by parents.
c
Death of severe staphylococcal sepsis.
d
E. Petersen et al. / International Journal for Parasitology 31 (2001) 115144
Cover with spiramycin during the 1st day of life.

119
Table 3 observed in one infant, and transitory crystalluria occurred

Incidence of clinical ndings typical for congenital T. gondii infection in two cases; one of them (patient no. 14) had hyperuricaemia
among 15 infected infants untreated prenatally
and a family predisposition to renal lithiasis (Table 2).
Clinical sign Number of affected children
1. Unilateral retinchoroiditis N2 2.1.4. Discussion

Pigmented scar 2
Fresh lesion 0 Thirteen of 15 neonates with proven congenital toxoplas-
2. Intracranial calcications N3 mosis were identied solely by serological screening, and
Perivendular region alone 0
appeared healthy at birth. Guerina et al. (1994) observed
Cerebral cortex and 3
periventricular that all infants diagnosed by serological screening were
3. Dilatation of ventricualr N5 judged by neonatologists on routine clinical examination
system to have no clinical symptoms, but careful investigations
Severe with hydrocephalus 2 showed neurological and/or ocular abnormalities in about
Mild or moderate 3
40% (19 of 48) of the newborns. Couvreur et al. (1984)
4. More than one clinical sign 3
found neurological abnormalities in 33% of infants diag-
nosed as asymptomatic by their primary physicians.
The prevalence of typical clinical signs or symptoms in
The comparative mother and child Western blot assay our patients was 33% (ve of 15) and rather exceeds the data
allowed the detection of actively synthesised IgM in 14 of reported by previous studies, where 8087% of newborns
15 (93.3%) cases examined at admission; 13 children were free of symptoms (Foulon et al., 1999; Villena et al.,
produced IgG of different antigenic specicity but the differ- 1998; Lebech et al., 1999; Gratzl et al., 1998; Pratlong et al.,
ence was not statistically signicant. 1994). Such discrepancies are difcult to explain but may be
During the rst year of life, a signicant increase of speci- due to a limited number of cases, to the observation period,
c IgG was observed in three cases (patient nos. 1, 9 and or possibly to the Toxoplasma strain or inoculum size.
11), all treated and a short reappearance of IgM and IgA was Except for one case with severe neurological sequela and
documented in another patient (no. 4). Toxoplasma-specic a poor prognosis at birth, the clinical pattern was stable or
IgG showed decreasing titres in the infants when treated. In improved through intensive treatment. In the New England
three cases, mostly with severe clinical damage, specic screening trial (Guerina et al., 1994), new retinochoroidal
antibodies were temporarily undetectable. Specic IgG anti- lesions causing vision impairment occurred in 10% of cases
bodies reappeared at the age of 10 months (patient no. 8) or in early infancy (four patients) and a severe persistent
5 months after the cessation of treatment (patient no. 14). neurological decit was observed in one patient.
The third patient (patient no. 6) was IgM- and IgA-positive
for 4 weeks after birth and then synthesised IgG of different
antigenic specicity to the mother.
Nine of 11 children (81.8%) with complete serological
follow-ups showed serological rebounds of specic IgG
(two cases) or IgG and IgA (seven cases) between 3 and
10 months after the cessation of treatment (mean 6.6 ^ 2.1
months) at the age of 16.5 to 34 months (mean 24.1 ^ 5.4
months) without any signs of clinical reactivation (Fig. 1).
Anti-parasitic treatment regimen and its tolerability: for
13 IgM-positive neonates the anti-parasitic chemotherapy
was initiated between the rst day to 11 weeks of life
(mean 4 weeks); for two other children (patients nos. 14
and 15) not identied by the screening programme, the
treatment was administered at the age of 7 weeks and near
12 months, respectively, after recognition of clinical symp-
toms and serological conrmation.
Considering the estimated date of maternal infection, the
postnatal treatment of IgMpositive infants was initiated
between 4.5 and 42 weeks after the presumed T. gondii infec-
tion of the mothers (mean 18.0 ^ 11.5 weeks). The duration
of the specic anti-Toxoplasma therapy ranged from 3 to 24
months (mean 13.7 ^ 5.0 months). No important side-effects
giving indications for the interruption of treatment were seen. Fig. 1. Occurrence of immunological rebounds in relation to (A) the time
Mild skin rash, probably related to the treatment, was lapse after stopping the treatment and (B) age of infected children.
In contrast, in the Chicago collaborative study following specic treatment given to children with a postnatal diag-
44 congenitally infected infants, there were few asympto- nosis of Toxoplasma infection soon after birth. Although the
matic patients with biological signs of infection only and no long-term follow-up of the patients is necessary to make an
systematic prenatal or neonatal screening (McAuley et al., accurate evaluation of the benet within adolescence and
1994). A higher risk for clinical abnormalities in early adult life, the actual prognosis of infected patients remains
infancy due to Toxoplasma was also mentioned by Berrebi satisfactory.
et al. (1993), who found 41% (nine of 22) of symptomatic Acknowledgements: We are indebted to Emeritus Profes-
children during a mean observation period of 32 months, sor Zbigniew S. Pawlowski from the University of Medical
although half of them were treated prenatally. The incidence Sciences in Poznan, Poland for his early encouragement and
of typical clinical ndings was also high in a total group of advice, Ewa Gajewska and Iwona Napierala from the Unit
103 newborns (56 symptomatic cases) born to mothers trea- of Developmental Pediatry and Early Infants' Diagnosis
ted during pregnancy (Couvreur et al., 1993). (University Hospital of Gynaecology and Obstetrics) for
Sequelae were diagnosed in two children with a treatment their experience with rehabilitation management, as well
delay, but probably existed already prenatally without being as all the nurses from the Department of Neonatal Infectious
diagnosed at birth. In only two children, progressing or Diseases for their generous co-operation and skilful care for
spontaneous hydrocephalus needed neurosurgical interven- the children. We thank Dr. Jadwiga Winnicka and all the
tion. Three infants identied by regional screening only had team of the University Pharmacy in Poznan for importing
typical clinical ndings but no retinochoroidal lesions. The drugs not available in Poland and the laborious preparation
incidence of Toxoplasma retinochoroiditis in newborns of therapeutic doses. This work was supported by the Polish
varies between studies. Villena et al. (1998) reported four Research Committee in Warsaw, Poland (grant nos. 4PO5E
cases with inactive eye lesions at birth of 78 newborns 08413 and 4PO5E 10017).
(5.1%), whose mothers were screened during pregnancy,
and some of them treated in utero. The new ocular sequelae 2.2. Screening for Congenital Toxoplasmosis in Turkey
developed in 12 infants (15.4%) during the rst year of life
N. Altintas a,*, S. Ertug a, A. Yolasigmaz a, G. Sonmez a, M.
or in early post-treatment period. Couvreur et al. (1985) ztekin c, F.
Uysalci a, N. Altintas d N. Kultursay b, K. O
observed retinal lesions in 24% (26 of 108) of newborns c a e
Sendag , Y. Guruz , E. Petersen :
at initial examination after birth with a secondary apparition a
Department of Parasitology; bDepartment of Pediatrics;
of new ocular complications between 4 months and 7.5 c
Department of Obstetrics and Gynaecology; dDepartment
years in 8% of them who remained untreated, but not in
of Medical Biology, Ege University, Medical Faculty,
patients receiving intensive postnatal chemotherapy.
Bornova-Izmir, Turkey; eDepartment of Mycobacteria and
The signicant diminution or resolution of intracranial
Parasitic Infections, Statens Seruminstitut, Copenhagen S,
calcications during intensive therapy as well as an
Denmark
improvement of psychomotor development in symptomatic
cases is now unquestionable (Roizen et al., 1995). 2.2.1. Introduction
The serological rebounds following treatment occurred
frequently (81.8%) in our patients, but never had clinical Toxoplasmosis is usually asymptomatic in immunocom-
implications. Among 63 infants followed by Fortier et al. petent subjects. However, T. gondii infection acquired
(1997) during the rst 2 years of age, clinical recurrences during pregnancy can lead to infection of the foetus,
were noted in ve patients, and four of them were preceded which may result in foetal loss or lesions that usually
by a serological rebound. So far, a mechanism of this involve the brain and the eyes (Naessens et al., 1999; Roizen
phenomenon is not known. Some authors recommend et al., 1995; Lyneld and Guerina, 1997).
Fansidar w treatment for 46 months following an initial The major inuences on the spread of Toxoplasma to
treatment of pyrimethamine/sulfadiazine (Couvreur, humans include hygienic routes, contamination of vegeta-
1993), but we believe that only clinical relapses justify a bles, fruit, and unfrozen meat, and association with cats, but
further treatment (Stray-Pedersen, 1992; Villena et al., handwashing, rinsing of vegetables and fruit, and the hand-
1998). Garin (1988) reported that secondary synthesis of ling of cats are unlikely to have altered very much over the
antibodies occurred frequently 34 months after therapy years.
withdrawal, usually close to the end of the 2nd year, and Stray cats are one of the major problems that face the
he proposed a treatment prolongation until this age. Turkish community in this regard. Tradition is another chal-
Negative serology especially in immature infants with lenge for the Turkish people who like consuming raw meat
small birth weight or hypotrophy is not surprising and has balls or consuming uncooked salami or sucuk (which is a
already been reported by others (Villena et al., 1998; meat lling with spices). On a large scale and as a develop-
Fricker-Hidalgo et al., 1996). Some authors observed symp- ing country, most people consume fresh meat which is not
tomatic infants or infants with parasitological proven infec- kept in the freezer. Meat frozen at 2208C for 23 days kills
tion without specic IgG (McAuley et al., 1994). most of the T. gondii bradyzoites, so this does not occur.
The study provides more information on the outcome of The prevalance of antibodies against T. gondii in fertile
women differs widely with geographic location. Risk factors substrate was added. The enzymic hydrolysis of substrate
for vertical transmission from an infected mother to her was stopped after 30 min by the addition of 100 ml NaOH.
children also differ in different studies. The estimated inci- Samples with absorbance values greater than two or usually
dence of congenital Toxoplasma infection is as high as 14 three times of the absorbance values of negative control are
per 1000 births in certain European countries. In Turkey considered as positive. Values between two and three times
there is no obligation for TORCH screening before marriage are considered as suspected positive and further investi-
except for AIDS recently. The serological screening of gated.
pregnant women for toxoplasmosis and the follow-up until Indirect immunouorescence antibody (IFA) test: IFAT
delivery, is not a routine procedure and there is no obliga- was used in the detection of T. gondii IgG and IgM in our
tion to report veried cases of toxoplasmosis in Turkey. laboratory. Briey, multiwell slides were coated with a
Accurate serological tests for screening of Toxoplasma suspension of T. gondii, adjusted to contain 100 organisms
infections can only be done in the big cities (such as Istan- per 5 ml, air dried and stored at 2708C until used. For the
bul, Ankara, Izmir, etc.) and in certain laboratories (mostly test, parasite smears were incubated at 378C for 30 min with
parasitology laboratories of the universities). In most of the serial dilutions of the sera to be tested, washed three times
private laboratories different commercial kits are used and with PBS, and air dried. The slides were then incubated with
of course the results obtained are difcult to compare. Many a 1/300 dilution in PBS of uorescein-labelled goat anti-
gynaecologists do not realise the importance of Toxoplasma human immunoglobulin IgG or IgM (bioMerieux 75692,
infection and hence they usually do not recommend testing bioMerieux 75672) at 378C for 30 min. After three washes
for toxoplasmosis during pregnancy. Due to geographic and with PBS, the slides were mounted with glycerol. The test
nancial problems no programme has ever been planned for slides were viewed with a uorescence microscope. Reac-
this purpose. As a result there are no good data concerning tions were considered positive when .50% of the organ-
the prevalence of parasitic diseases, as well as toxoplasmo- isms showed complete peripheral uorescence. Dilutions of
sis, in Turkey. Some local studies have been performed but 1:16 and higher were evaluated as positive (Ertug et al.,
these can not be applied all over the country and so far the 2000).
incidence of congenital toxoplasmosis in Izmir is unknown. IgM Immunocapture: Microtitre plates were coated with
Because antenatal screening was not available, the aim of 100 ml rabbit anti-human IgM (DAKO A 0425) and kept at
this study was to examine the children and mothers who 48C overnight, washed three times with PBS (pH 7.2) with
were affected by prenatal toxoplasmosis after delivery. 0.0005% Triton X-100. Serum samples (100 ml) diluted
1:100 were tested in duplicate. The microtitre plates were
2.2.2. Materials and methods incubated at 378C for 1 h, washed three times. T. gondii
antigen (100 ml) was applied and incubated at 378C for 1
Samples: Between 1998 and 1999, altogether 953 cord h, washed and 100 ml mAb (S13) was added per well and
blood samples were collected from all pregnant women incubated at 378C for 1 h. Rabbit anti-mouse immunoglo-
who delivered at Ege University Medical Faculty Hospital. bulins (100 ml) (DAKO D 0314) with conjugated alkaline
The serological proceedings (ELISA, IFA) were performed phosphatase was applied and incubated at 378C for 1 h and
after all cord bloods were collected. IgM immunocapture washed. Enzyme substrate [p-nitrophenyl phosphate] (100
and IgM ISAGA were performed on sera of suspected ml) were applied and incubated for 30 min before the reac-
patients. The mean age of the study population was 26.3 tion was stopped. Greater than 8 IU were regarded as posi-
years (range 1642). tive. WHO International standard sera (Statens
Antigens: Antigen preparations were made from tachy- Seruminstitut, Denmark) were used as negative and positive
zoites of the TRH strain (RH like strain; personal informa- controls (Lebech et al., 1993).
tion from Dr. Sibley) of T. gondii. Tachyzoites were IgM immunosorbent agglutination assay (ISAGA): IgM
obtained from the peritoneal exudates of mice infected 2 ISAGA (bioMerieux) was used. The ISAGA index was
days earlier. These antigens have been routinely used for interpreted as follows: 05 negative reaction; .5 positive
the serological Toxoplasma tests in our laboratory (Ertug et reaction (Duffy et al., 1989).
al., 2000; Kuman et al., 1999).
Enzyme Linked Immunosorbant Assay (ELISA). The
wells in microtiter plates were sensitised with the Toxo- 2.2.3. Results
plasma antigen overnight at 48C. The plates were then
given three washes each of 3 min in PBS containing Nine hundred and fty-three cord bloods were analysed
0.05% Tween 20 and 100 ml diluted serum was added to for IgG and IgM anti-Toxoplasma antibodies by ELISA and
each well and incubated for 1 h at 378C. The plates were IFAT. Specic IgG anti-Toxoplasma antibodies were found
washed as before in PBS/Tween 20. One hundred microli- in 375 (39.34%) cord bloods. Five cord bloods were eval-
tres anti-human IgG or IgM labelled with alkaline phospha- uated positive by IgM-ELISA and -IFAT. ISAGA and
tase conjugate (Sigma A3187, Sigma A-3437) was added to Immunocapture were performed on these ve sera and one
each well and incubated for 1 h at 378C and 100 ml enzyme of them was found positive by these tests. This child was
followed up until 2 years of age but no signs of sequelae, tive women and 1.5 million pregnancies in Turkey. Accord-
either neurological or ophthalmological were found. ing to our laboratory records the seropositivity of
toxoplasmosis is 55% in pregnant women but in this study
2.2.4. Discussion we could not follow up the pregnant women and so we do
not have a serologic prole for them. We can only say that
The reasons for differences in the prevalence of Toxo- the seropositivity found was 39.34% in pregnant women and
plasma infections in different countries or regions, as seroconversion was 0.1%.
noted also in Turkey, have been attributed to variations in, The data from the present study in the Izmir area conrms
for example, human behaviour such as cooking habits and that seroconversion to T. gondii during pregnancy is not a
the number of cats living outdoors, but these are still not rare event. In order to determine the incidence of congenital
fully understood (Gilbert et al., 2000; Frenkel et al., 1995; toxoplasmosis in children in Izmir a study will be
Rey and Ramalho, 1999). performed, but a Turkish nationwide screening program
The relationship of toxoplasmosis with feeding habits, should be set up to identify women at risk of Toxoplasma
domestic animals, and urban and rural settlements have infection during pregnancy. The risk to the foetus does not
been studied and higher Toxoplasma-seropositivity has correlate with whether the infection in the mother was
been found in people living in urban areas. But there was symptomatic or asymptomatic during gestation. Therefore,
no signicant difference between their culinary habits neonatal screening for toxoplasmosis is important to iden-
(eating raw meat or rare cooked meat, uncooked vegetables, tify infection and start early treatment of congenitally Toxo-
drinking raw or pasteurised milk) as well as their relations plasma-infected children.
with domestic animals (cats, dogs, birds, etc.) (Kuman et al.,
1996).
No programme has ever been planned for detection of
congenital toxoplasmosis in Turkey and only local studies 3. Toxoplasmosis in central Europe
have been performed. According to these local serological
3.1. Toxoplasmosis Prevalence in Eastern Romania
studies the seroprevalence found is between 17.3 and 78%
(Table 4) (Altintas et al., 1997; Altintas et al., 1998; Altin-
Elena Crucerescu:
tas, 1996)
Laboratory of Parasitology, Institute of Public Health, 14
A seroconversion rate between 0.2 and 1% was found
Victor Babes street, Iassy 6600, Romania
(Table 5) (Dilmen et al., 1990; Dabakoglu et al., 1995)
In another study in Izmir in 1990, the sera of 300 mothers
were tested with ELISA and 136 (45.33%) and ve (1.66%) 3.1.1. Introductionn
were found positive for Toxoplasma-IgG and IgM, respec-
tively, and the rate of prenatal toxoplasmosis was deter- Toxoplasma gondii is common in Romania, but there is
mined to be three in the 300 cord blood samples by no systematic serological screening in pregnant women so
testing for IgM antibodies (1%) (Table 6) (Sasmaz et al., toxoplasmosis diagnosis is often a problem of physician
1990). inspiration.
It has been suggested that there are 13 million reproduc- The aim of this study was to estimate toxoplasmosis
Table 4
Results of T. gondii IgG and IgM antibodies in various cities of Turkey (seroprevalence)
Year of survey Province Number of patients Name of the test Positive (%)
IgG IgM
1954 Ankara 687 Skin test 40.3

1954 Istanbul 687 Skin test 31.27
1971 Adiyaman 500 Skin test 60.6
1980 Ankara 550 SabinFeldman 51.8
1981 Kayseri 1232 IHA, ELISA 25
1985 Izmir 18233 IFAT 24.12
1985 Kayseri 4603 IHA 17.3
1988 Adana 4200 IFAT 48.88
1989 Ankara 1772 IHA, ELISA 65
1993 Konya 1423 ELISA 39 13.4
1995 Istanbul 110 ELISA 34 0.9
1995 Malatya 510 ELISA, SF 22.6 1.1
1995 Izmir 9410 IFAT, ELISA 49.4
1998 Izmir 1865 IFAT, ELISA 23.1
Table 5
T. gondii IgG antibodies and seroconversion rates obtained in different studies
Year of survey Number of pregnant women IgG positive (%) Seroconversion (%)
1980 1772 65.0 1.00

January 1988 July 1995 11170 41.5 0.20
prevalence among the human population in eastern Roma- 3.1.3. Results

nia and to study its consequences.
Pregnant women: The prevalence of Toxoplasma-speciifc
3.1.2. Materials and methods IgG-antibodies in toxoplasmosis in pregnant women was
41%. There were no signicant variation between the clini-
Study population: The study was performed in 1996 cally healthy women group and the group suffering abortion
1999 in Eastern Romania. The study population included (Table 7). The serologic screening of the apparently healthy
pregnant women, newborns, and HIV infected patients women revealed 15 cases (1.9%) of asymptomatic, IgM-
and patients with eye disease and lymphadenopathy. positive women who could have acquired the infection
The pregnant women included 810 healthy women and during pregnancy, compared with 16 cases (4.2%) with
378 women with spontaneous abortion in the rst trimester IgM-antibodies among the women with spontaneous abor-
of pregnancy. Sero-negative healthy women were consid- tion.
ered at risk of acute acquired toxoplasmosis and were sero- Newborns: Toxoplasma gondii antibodies were found in
logically followed up until birth. 44.5%. The majority of cases (43.8%) were uninfected
Cord blood from 1226 newborns that were analysed for newborns with maternal transplacental antibodies. Nine
specic Toxoplasma IgM- and IgG-antibodies and newborns (0.7%) with Toxoplasma-specic IgM- and or
newborns with suspected infection were followed until IgA-antibodies were followed up during the rst year of
they became seronegative or the diagnosis was conrmed. life and one infant was conrmed as having congenital toxo-
In addition we studied the seroprevalence of antibodies plasmosis by persistent IgG-antibodies at 1 year of age.
against T. gondii in 102 HIV infected children (mean age Toxoplasma prevalence among HIV infected patients was
14.5 years), 50 chorioretinitis patients, 17 uveitis patients, 34.3% and no patient showed either specic IgM- or IgA-
and 43 patients with other ocular pathology (strabismus, antibodies. Toxoplasma-specic IgG-antibodies varied
cataract, microphthalmia, etc) and 343 children (mean age between 4 u.i./ml and 6400 u.i./ml.
15.8 years) with lymphadenopathy. In patients with chorioretinitis 68% had specic Toxo-
Antibody analysis: The following tests were used for plasma antibodies and patients with other eye pathology had
serologic diagnosis of T. gondii infection: IFAT, 2- a seroprevalence of 37.3%.
mercapto-ethanol agglutination test for Toxoplasma IgG, The seroprevalence in patients with lymphadenopathy was
and ISAGA for Toxoplasma IgM and IgA (Thulliez et al., 34.1% and acute infection was conrmed in 44 cases
1986; Dannemann et al., 1990; Desmonts et al., 1981). (12.8%).
Positive diagnosis of acute acquired toxoplasmosis was
considered when all of the following criteria were fullled:
positive IgG, IgM, and IgA, high level or signicant 3.1.4. Discussion
increase of specic IgG-antibodies. Chronic toxoplasmosis
was considered when IgG only was positive. Equivocal The results show that T. gondii is common in Eastern
serologic pictures were cleared up by repeatedly testing at Romania. The seroprevalence of 41% in pregnant women
23 week intervals. with an average age of 26 years shows that Toxoplasma
A risk of congenital toxoplasmosis in newborns were seroprevalence is at the same level as in France and Austria
considered if we found a high level of IgG, positive/equi- Table 7
vocal IgM and/or IgA. A child that remained seropositive Toxoplasma seroprevalence among pregnant women
after rst year of life, with or without signs and symptoms of
disease was considered congenitally infected. T. gondii serologic Healthy pregnant women Women with abortion
status (810 cases) (378 cases)
Table 6 No. % No. %
Results of sera from 300 mothers and the corresponding cord blood samples
Seropositive 335 41.4 155 41.0
Working group Toxo-IgM Toxo-IgG Number of total patients Toxoplasma IgG 320 39.5 139 36.8
alone
1 % 1 % Suspected 15 1.9 16 4.2
infection during
Mothers 5 1.67 136 45.3 300 (100 %) pregnancy
Cord bloods 3 1.0 136 45.3 300 (100 %) Seronegative 475 58.6 223 59.0
and thus that the situation in Romania is comparable with on the 1994 epidemic is based on epidemiological examina-
other central European countries. tions conducted in hygiene stations Brno and Olomouc and
The level of IgM-antibodies do not necessarily show that the Mikrochem Laboratory in Olomouc. Demographic data
these women were infected during pregnancy as IgM-anti- concerning population gures or toxoplasmosis-associated
bodies may persist for years. mortality are provided by the Czech Statistical Ofce and
One conrmed case of congenital toxoplasmosis in the Institute of Health Information and Statistics of the
Romania was found in 1226 newborns which show that Czech Republic.
the birth prevalence of congenital toxoplasmosis is at the Serological surveys: The programme for monitoring sero-
same level as that in Poland (Paul et al., 2000). prevalence of selected infections (Multi-purpose immunolo-
The level of 68% toxoplasmosis seroprevalence among gical surveys of infection in the Czech Republic) includes
chorioretinitis patients compared to the pregancy cases, monitoring of the prevalence of toxoplasmosis. Samples of
HIV-positive children and patients with other eye diseases sera for the purposes of this monitoring were collected in
may indicate that infection with T. gondii may be an impor- each case by district and paediatric practitioners from 20 to
tant risk factor for chorioretinitis. However, the seropreva- 30 randomly-selected medical districts in selected regions.
lence need to be adjusted for age before a direct comparison Each participating medical practitioner was specically
can be made. Patient and physician restraint regarding instructed as to which age-group, category and number of
ocular uid testing made it difcult to denitively conrm samples were to be collected (always from subjects without
toxoplasmosis as a cause for all tested chorioretinitis. acute clinical symptoms). A random selection of samples
from the complete collection of sera was examined, encom-
3.2. Toxoplasma in the Czech Republic 19231999: rst passing in equal measures gender and number of subjects in
case to widespread outbreak all nine age-groups.
In 1971, 1977, 1983, 1985, 1987, 1990 and 1996 the
P. Kodym a,*, M. Maly a, E. Svandova a, H. Lezatkova b, M. NRLT examined a total of 5431 samples of sera taken
Bazoutova c, J. Vickova d, C. Benes a, M. Zastera a: from 21 regions of the Czech Republic (Table 8). A total
a
National Institute of Public Health, Srobarova 48, CZ- of 1775 samples were collected in towns with a population
100 42 Praha 10; bMikrochem Laboratories, Wolkerova 6, exceeding 100 000 inhabitants (urban areas) and 3656 in
CZ-779 00 Olomouc; cCity Hygiene Station, Jerabkova 4, predominantly rural regions. Data associated with the
CZ-602 00 Brno; dDistrict Hygiene Station, Wolkerova 6, samples contain the district of domicile, gender, age,
CZ-779 00 Olomouc, Czech Republic month and year of birth and the results of serological exam-
inations for other specic infectious diseases. None of the
3.2.1. Introduction regions were examined repeatedly with the exception of
Research into human toxoplasmosis originated in Bohe- various Prague districts.
mia: T. gondii was identied as a serious human pathogen Surveillance of toxoplasmosis: Data concerning cases of
by Janku (1923) in Prague. Subsequently it became clear acute toxoplasmosis were taken from centralised databases
that toxoplasmosis constitutes the most signicant parasito- (systems ISPO, Epidat) and reviewed at NRLT. Cases of
sis in this region and the disease was subjected to very close serologically conrmed clinical toxoplasmosis are reported
attention (Kouba et al., 1974; Jra and Rosicky, 1983). Since by the practitioner concerned. Reports include place of resi-
1955, cases of the disease are subject to mandatory reporting dence, gender and age, date of rst symptoms, clinical form
and its prevalence is repeatedly monitored by means of and epidemiological case history. HIV-positive patients are
serological surveys. Although a great amount of data have not included in the survey.
been gathered, the majority were published for internal use The 1994 epidemic: Data include reported cases and the
only, or not at all. The extensive 1994 epidemic of toxoplas- results of epidemiological surveillance. All patients with
mosis in the Czech Republic resulted in further information, conrmed toxoplasmosis in Brno (257) and Olomouc
as collected by the existing reporting system. The aim of this (148) during the period December 1993 to April 1994
report is to evaluate all hitherto unused data and further responded to a questionnaire detailing clinical complaints,
existing knowledge of the epidemiological aspects of toxo- contact with animals and soil and dietary habits. The
plasmosis in central Europe. percentage of positive answers to individual questions was
calculated. The number of persons with acute toxoplasmosis
3.2.2. Materials and methods among members of one family was also followed up.
Serological tests: Sera were examined by complement-
Data sources: The National Reference Laboratory for xation test - CFT (Pokorny et al., 1972b) using antigens
Toxoplasmosis (NRLT) continuously gathers and evaluates from tween-ether extraction (Pokorny et al., 1972a). Titres
data from two major sources: data concerning seropreva- of 8 and higher were evaluated as positive. Reaction using
lence of toxoplasmosis gained from irregular serological the antigen supplied since 1972 by SEVAC (now SEVA-
surveys and incidence, gained from the database of reported PHARMA) Prague, managed by the same procedure
cases of clinical toxoplasmosis. Supplementary information throughout the monitoring period, is still the most wide-
Table 8
Results of the Multipurpose serological surveys of infection in the Czech Republic 19711996: Seroprevalence of toxoplasmosis in selected districts with
predominatly urban or rural character of residence
Year District Character Men Women Total
Tested Positive % Tested Positive % Tested Positive %
1971 Prague 1, 4, 5, 7, 8, 9 Urban 148 6 4.1 160 16 10.0 308 22 7.1

Benesov Rural 29 2 6.9 27 3 11.1 56 5 8.9
Pelhrimov Rural 61 14 23.0 59 11 18.6 120 25 20.8
Tabor Rural 73 13 17.8 85 18 21.2 158 31 19.6
Southern Moravia a Rural 66 63 95.4 87 77 88.5 153 140 91.5
1977 Beroun Rural 125 45 36.0 126 67 53.2 251 112 44.6
Strakonice Rural 131 68 51.9 131 90 68.7 262 158 60.3
Semily Rural 132 48 36.4 128 71 55.5 260 119 45.8
Brno Urban 125 34 27.2 120 57 47.5 245 91 37.1
1983 Teplice Rural 133 38 28.6 132 43 32.6 265 81 30.6
Gottwaldov Rural 129 54 41.9 134 69 51.5 263 123 46.8
Prague 3, 7 Urban 130 41 31.5 124 49 39.5 254 90 35.4
1985 Trutnov Rural 99 56 45.5 108 67 62.0 207 123 59.4
1987 Psek Rural 150 30 20.0 148 32 21.6 298 62 20.8
Cheb Rural 148 16 10.8 150 17 11.3 298 33 11.1
1990 Prague 1, 2 Urban 110 19 17.3 110 21 19.1 220 40 18.2
Jindrichuv Hradec Rural 127 34 26.8 128 45 35.2 255 79 31.0
Trebic Rural 133 35 26.3 131 39 29.8 264 74 28.0
1996 Prague 2, 4, 7, 8, 9 Urban 311 37 11.9 291 45 15.5 602 82 13.6
Hradec Kralove Urban 57 6 10.5 69 13 18.8 126 19 15.1
Chrudim Rural 73 14 19.2 66 17 25.8 139 31 22.3
Jicin Rural 75 12 16.0 74 20 27.0 149 32 21.5
Nachod Rural 78 11 14.1 68 20 29.4 146 31 21.2
Svitavy Rural 62 16 25.8 70 23 32.9 132 39 29.5
Total 2705 712 26.3 2726 930 34.1 5431 1642 30.2
a
Southern Moravia: samples collected from neighbouring districts Breclav, Gottwaldov, Hodonin, Znojmo.
spread screening system in use and form the basis of immu- 5431 persons examined as part of the immunological
nological surveys. In the 1980s, the NRLT began using the survey, 30.2% were CFT-positive. Seroprevalence in
ELISA test for IgG (Pokorny et al., 1989) and double-sand- women (34.1%) was signicantly higher than in men
wich ELISA for specic IgM (Pokorny et al., 1990). Since (26.3%). The ratio of seropositive persons has a marked
the early 1990s, both NRLT and regional diagnostic labora- uctuation from 7.1% (Prague) to 91.5% (southern Mora-
tories also use commercial immunoenzyme tests on anti- via) (Table 8).
toxoplasma IgG, IgM and IgA for conrmation of results The percentage of positivity correlates with the type of
and determination of the phase of infection (Sano Diag- environment: In urban regions an average of 19.6% persons
nostics Pasteur, Captia Centocor, Abbott IMX and Test- (16.2% men, 23% women) from our sample were positive,
Line Brno). while in rural areas average positivity was 35.3% (31.2%
Data processing: All available data from serological men, 39.4% women). These differences are statistically
surveys were combined for analysis. The Poisson regression signicant.
model (for categorical variables) with multiplicative risk, The seroprevalence of toxoplasmosis increases with age
which links a count and a rate multiplier with a set of xed (Table 9). Increasing seropositivity is marked in children of
covariate values, was used for analysis of the count of toxo- both sexes up to 4 years of age and continues until age 9. In
positive subjects from immunological surveys by gender, higher age-groups the increase is less marked. For women,
domicile (rural/urban) and age-group in the EPICURE soft- seroprevalence generally increases in a linear manner up to
ware package. Chi-square test was used for comparison of age 50 when peak values are reached; in higher age-groups
percentages in the contingency table in Epi-Info programme. positivity declines. For men, seroprevalence begins to
From the data on reported clinical cases, morbidity per increase in the over 20 years age-groups, while in persons
100 000 population and month was calculated. All statistical older than 60 years it is lower than in previous age-groups.
tests were carried on 5% signicance level. Analysis of the effects of domicile and gender on seropre-
valence in the various age-groups: Seroprevalence in the
3.2.3. Results individual age-groups is strongly inuenced by place of
residence (Fig. 2). While in urban inhabitants of both
Prevalence of toxoplasmosis in the Czech Republic: Of sexes it increases at a more or less even rate, in the rural
Table 9
Seroprevalence of toxoplasmosis by age-group and gender in the Czech
Republic
Age-group (years) Men Women
Tested Prevalence (%) Tested Prevalence (%)
0 23 0.0 23 0.0
1 70 11.4 80 10.0
2 102 13.7 84 27.4
3 108 11.1 109 25.7
4 125 24.8 129 24.8
04 428 15.2 425 21.4
59 416 27.9 418 30.1
1014 299 20.4 294 27.9 Fig. 2. Comparison of seroprevalence by age and gender in urban and rural
1519 282 25.5 325 31.1
districts of the Czech Republic. Solid square: males; Cross: females; Solid
2029 401 28.7 351 38.5 line: urban; Dotted line: rural.
3039 215 27.4 240 46.3
4049 224 34.4 214 46.7
5059 211 34.6 234 41.0
601 229 32.3 225 39.1 and only reaches 5.24%, which is signicantly lower.
Total 2705 26.3 2726 34.1 However, in the higher age-groups the differences between
urban and rural areas diminish. In age-groups over 50 years
the multiplication factor reaches about 0.8 and the differ-
ences are no longer statistically signicant (the condence
population there is a very high seroprevalence from age 9 interval covers the value 1).
onwards. In men, seroprevalence remains practically Antibody titres of examined sera were generally low and
unchanged in the higher age-groups while in women there diagnosed toxoplasmosis can be evaluated as latent or
is a slight increase up to age 4049 years (52.3% positive); chronic. For all sera with a CFT titre of 64 and higher,
seroprevalence decreases in subsequent age-groups. The collected in 1990 (N 23) and 1996 (N 23), specic
resultant Poisson regression model for assessment of toxo- IgM antibodies were determined. Two samples from 1990
plasmosis-positivity prevalence in the monitored sub- were positive and the infection was conrmed as acute.
groups expresses the inuence of residence character Reported cases of acute toxoplasmosis: Prior to 1945,
(urban/rural) as dependent on age. For the effects of gender, only one case of toxoplasmosis is on record (Janku, 1923),
uniform coefcient is sufcient (Table 10). The effect of and until the mid-1960s the disease was diagnosed sporadi-
gender is similar in all age-groups (no statistically signi- cally (Table 11).
cant differences); prevalence in women in a given sub-group The creation of new diagnostic centres (from three prior
is therefore calculated as 1.293-fold the prevalence in the to 1966 to 82 in 1999) contributed to the increase in
corresponding male sub-group. This increase is statistically numbers of conrmed cases. The number of reported
signicant. For instance, in the up to 4 years age-group, the cases rose slightly during the periods 19711975, 1979
estimated prevalence in men from rural areas exceeds 20 % 1983 and 19871993, with a decrease in the interim periods.
(0.212 100), while in the city it is 0.247-fold this gure Average morbidity during 19821991 was 0.538 per
Table 10
Inuence of urban and rural environments on prevalence of toxoplasmosis by age-group and gender
Age-group Baseline relative prevalence (males in rural regions) Effect of urban area
Estimate 95% CI a Estimate 95% CI a
04 0.212 (0.178, 0.253) 0.247 (0.149, 0.409)

59 0.320 (0.277, 0.370) 0.312 (0.211, 0.459)
1014 0.246 (0.202, 0.300) 0.607 (0.418, 0.883)
1519 0.305 (0.256, 0.365) 0.464 (0.321, 0.670)
2029 0.332 (0.285, 0.386) 0.646 (0.484, 0.862)
3039 0.346 (0.288, 0.414) 0.780 (0.549, 1.108)
4049 0.393 (0.331, 0.468) 0.643 (0.446, 0.927)
5059 0.347 (0.289, 0.418) 0.826 (0.586, 1.164)
601 0.333 (0.276, 0.401) 0.798 (0.563, 1.131)
Effect of female gender 1.293 (1.173, 1.426)
a
CI, condence interval.
Table 11
Cases of clinical toxoplasmosis reported in the Czech Republic 19231999 including the three most commonly specied clinical forms and mortality
Year No. of Men Women Proportion of Morbidity per No. of Clinical form
cases men (%) 100 000 pop. deaths
and month
Lymphadenopathy Congenital Ocular
1923 1 1
19451950 4
19511955 45
19561960 40
19611965 61
19661968 486
1969 100 0.084 3
1970 101 0.086 4
1971 103 0.087 3
1972 155 0.131 2
1973 212 0.178 3 61 7 3
1974 342 0.285 3 96 10 11
1975 431 0.357 3 100 7 27
1976 402 0.331 1 149 11 19
1977 368 0.301 0 167 8 22
1978 388 0.316 2 198 3 17
1979 291 0.236 0 155 9 12
1980 346 114 232 32.9 0.279 0 231 12 20
1981 476 150 326 31.5 0.385 0 313 9 19
1982 578 215 363 37.2 0.467 0 444 10 18
1983 747 273 474 36.5 0.603 0 560 13 22
1984 635 230 405 36.2 0.512 0 481 14 18
1985 706 266 440 37.7 0.569 0 536 12 39
1986 667 232 435 34.8 0.538 0 528 19 17
1987 513 193 320 37.6 0.413 1 412 5 16
1988 601 240 361 39.9 0.484 0 515 4 13
1989 560 220 340 39.3 0.450 0 458 4 16
1990 746 267 479 35.8 0.600 0 609 10 18
1991 706 255 451 36.1 0.571 1 563 10 14
1992 823 0.665 0
1993 860 275 585 32.0 0.694 0 596 3 22
1994 2056 716 1340 34.8 1.658 0 1558 12 26
1995 1514 475 1039 31.4 1.221 0 1065 8 44
1996 1217 413 804 33.9 0.983 0 952 7 30
1997 952 328 624 34.5 0.770 0 696 11 37
1998 777 258 519 33.2 0.629 0 595 2 30
1999 857 291 566 34.0 0.695 0 664 3 41
100 000 inhabitants per month. In 1994, certain regions epidemic did not spread to other parts of the Czech Republic
were struck by a widespread epidemic of toxoplasmosis or neighbouring Slovakia and Austria.
resulting in a threefold increase of average nationwide The outbreak had a focal character: whereas certain
morbidity against the previous average. districts reported an abnormal incidence of toxoplasmosis
The 1994 Toxoplasmosis epidemic started simultaneously in 1994, neighbouring districts had no such increase. Preva-
in all districts. Most of the patients reported rst symptoms lence of the disease varied widely within the epidemic area
in January 1994 (in some areas at the end of December (Fig. 3).
1993), a smaller proportion in February to March 1994 The number of aficted persons is unknown. The ten most
(Fig. 4). From April onwards the number of new cases heavily affected districts had 722 reported cases (Table 12)
declined rapidly. Nevertheless, the number of cases within the rst 3 months of 1994 (in contrast to 91 cases
remained above average for some time and pre-epidemic during the same period of the previous year). As a result of
status was regained in 1997 (Table 11). the epidemic, the total number of cases throughout the
A transitional increase in numbers of cases was recorded Czech Republic in 1994 rose by 1196 cases as compared
in many areas of the Czech Republic, particularly in the with the previous year an increase of 2.4-fold. The major-
eastern regions. The greatest increase was in ten Moravian ity of cases were amongst inhabitants of large cities such as
districts, where monthly morbidity exceeded ten times the Brno and Olomouc.
19821991 national average (Table 12, Fig. 4). The In Brno and Olomouc, less than 25% of patients queried
Fig. 3. The geographical distribution of the monthly morbidity from Toxoplasma gondii-infection per 100 000 population and month during the peak epidemic
period JanuaryMarch, 1994, Czech Republic.
consumed raw meat or cereals, kept pet cats, ate fast-food or male patients 50% were under 16 years of age and 75%
visited a rural location regularly. Less than 50% consumed under 25 years. Amongst the female patients 50% were
the products of hog-killing or had contact with sand or soil. under 23 years and 75% under 32 years and 66% were
The following foods were frequently consumed: raisins 1845 years of age (data from the period 1995 to 1999).
(80%), peanuts (85%), mayonnaise, ham (93%), milk A similar trend was recorded during the epidemic.
(pasteurised) and milk products (95%), oranges and apples Of all cases reported in the years 1993 and 19951999,
(99.2%). Vegetarians constituted 2.2%, three-quarters of from 5% (1999) to 7.8% (1993) are recorded as familial
who eat ham occasionally. Drinking water for individual incidence, e.g. associated with families where at least two
areas is locally supplied. cases of acute toxoplasmosis were recorded simultaneously.
Prole of persons with clinical toxoplasmosis: The major- In 1994, the nationwide percentage of familial incidence
ity of all patients infected during 19801999 were women rose to 13.2%. In certain areas, over 60% of cases during the
(Table 11), the proportion of men uctuated from 31.4% epidemic qualify as familial (Table 13). The infection was
(1995) to 39.9% (1988). Acute toxoplasmosis was recorded detected among male and female patients, both children and
mainly amongst the younger population: of all reported adults. An unusual case was that of a family of eight in
Table 12
Ten districts in Moravia (Czech Republic) where monthly morbidity exceeded 5.52 per 100 000 population (ten times the nationwide average for 19831993)
during the peak of the epidemic (JanMar 1994). The urban character of the epidemic is highlighted by comparison of morbidity data for Brno-city and Brno-
rural districts
District No. of inhabitants Cases JanMar 1994 Morbidity per 100000

population and month
JanMar 1994 Mean 19821991
Brno-city 390112 250 21.36 0.26

Olomouc 226091 133 19.61 0.83
Opava 181976 85 15.57 1.61
Blansko 107885 40 12.36 0.23
Prostejov 111490 37 11.06 0.25
Karvina 285903 65 7.58 2.36
Prerov 138472 30 7.22 0.98
Novy Jicn 160526 34 7.06 1.07
Kromerz 108917 20 6.12 0.35
Brnorural 155208 28 6.01 0.30
Total 1866580 722 12.89 0.91

individuals in each group for accurate analysis when distri-

buting by age, gender or residence. In view of the fact that
the main screening test is still the CFT, which remains a
satisfactory test with no changes in technique, and because
there is no reason to suppose marked changes of toxoplas-
mosis prevalence in the Czech Republic during the moni-
tored period, we have combined the results of all seven
serological surveys. This yielded a group of 5431 examined
persons and enabled in-depth analysis.
Of the examined group, 30.2% were positive. This does
not signicantly differ from older available data. Jrovec
(1971) detected 37.7% positivity in 15 972 persons in the
former Czechoslovakia by intradermal testing (IDT). Simi-
Fig. 4. Morbidity per month and 100 000 in ten Moravian districts (Czech
Republic) with most apparent outbreak of toxoplasmosis, 19931995.
lar seroprevalence was detected in the Czech Republic in
HIV-positive patients (27.5%) (Pospslova et al., 1997);
(29.8%) (Sykora et al., 1992). However, average counts
Olomouc, of which six members suffered from lymphade- tell us little about the pattern of toxoplasmosis seropreva-
nopathy and one child from subclinical toxoplasmosis; the lence in the population, which varies widely from region to
father was uninfected. region. For instance, in southern Moravia seroprevalence
Clinical presentation: The most frequent symptom of was recorded as 91.5 against 7.1% in Prague. Local differ-
toxoplasmosis is lymphadenopathy; in the past 15 years ences in prevalence are quoted by a number of authors who
7688% of cases reported were the lymphatic form. The examined pregnant women (Prague: 24.8%, Jindrick uv
number of reported cases of congenital toxoplasmosis Hradec: 44.8% (Ziteck, 1998), Ceske Budejovice district:
continues to be low. Cases of the ocular form multiplied 37% (Hejlzek et al., 1999); Karvina district: 40% (Palicka
following the 1994 epidemic (Table 11). Lymphadenopathy et al., 1998) and blood donors (Strakonice district: 24.6%
was also prevalent amongst epidemic patients (86% Brno, (Hejlcek and Literak, 1993a) Novy Jicn region: 32.1%
89.9% Olomouc). Two thirds of patients reported `protracted (Svobodova and Literak, 1998). The great differences
angina' with subfebrility and lassitude. A total of 212 chil- between individual regions are due to a number of factors:
dren were hospitalised in Brno clinics for infectious diseases. Prevalence of toxoplasmosis may be inuenced by climate
Laboratory tests revealed elevated gamma globulin and and height above sea-level (Shevkunova, 1980) and the
slight increase in ALT in 7% of child and 12% of adult epidemiological situation in previous years. The governing
patients. Drug therapy was successful in all but two cases factor is, in all likelihood, whether the area is urban or
where the therapy had to be repeated (Cerny et al., 1995). rural. In the Czech Republic, prevalence in the rural popu-
Mortality: In the Czech Republic 19691999, 26 deaths lation is signicantly (1.8 times on average) higher than in
from toxoplasmosis are recorded (Table 11). Child mortality the urban population. These differences are far more
(boys/girls) was as follows: up to 1 year age (10/9), age 1 marked in the lower age-groups. For instance, seropreva-
year (2/2), age 3 years (1/0) and 59 years (2/0). The last lence amongst male children up to 4 years of age in rural
reported mortality was in May 1991, a 2-month old male areas is approximately four-fold higher than in their urban
with generalised toxoplasmosis and hydrocephalus. NRLT counterparts. Rural children have earlier contact with the
isolated a cyst-forming strain from the tissue. infection and their prevalence increases at a more rapid
rate than in the city. In the over 10 years age-group, the
3.2.4. Discussion prevalence in rural-dwelling boys practically tails off, and
rises only slightly in girls of the same age. In contrast,
Since 1971, NRLT has examined sera from a randomly urban children who have less opportunity to become
selected cross-section of the population as part of a program infected exhibit a less rapid rate of seroprevalence for a
of immunological surveys. Although large-scale population longer period. In the over 50 age-groups the difference
groups were examined annually, there were not enough between urban and rural populations is practically nil.
Table 13
Familial cases of toxoplasmosis in Brno (reported) and in Olomouc (diagnosed at Mikrochem laboratory) 1994
City Cases in 1 family Familial occurrence Families Total cases Percentage of family cases
1 2 3 4 5 6 7
Brno 183 24 13 1 1 0 0 96 39 279 34.4%

Olomouc 50 14 6 5 3 0 1 88 29 138 63.8%
The above data show that the risk of acquiring toxoplas- be about 100, if estimating from the frequency of 1.2 cases
mosis in pregnancy is paradoxically higher for the urban per 1000 in Karvina district (Palicka et al., 1998) and approx
population of women who have a sharp rise of serprevalence 90 000 births annually. In the Czech Republic there is no
at a fertile age, than in rural women who, for the most part, screening for toxoplasmosis apart from several districts
were infected at an earlier age. Moreover, in young mothers (Karvina and western Bohemia) and many cases are unrec-
the disease causes psychological changes (Flegr and Havl- ognised; the problem is also due to lack of discipline in the
cek, 1999). reporting of cases.
The source of infection, apart from contaminated soil or Up to 1976, 14 deaths from toxoplasmosis were reported
sand, can be certain types of meat. For instance, 14% of pigs annually. The virulent P-Cz strain was isolated from the
(Hejlzek and Literak, 1993b) and 4484% of rabbits (Hejl- brain of a girl aged 2 months who died of congenital toxo-
cek and Literak, 1994) from domestic breeding are Toxo- plasmosis in 1963 (Kouba et al., 1974; Literak et al., 1998).
plasma positive. Contact with raw meat, but not cats, in the It is difcult to ascertain whether there are any long-term
family appears to be a major risk factor (Flegr et al., 1998). changes of incidence and prevalence of toxoplasmosis in
Women appear to be in contact with these sources to a the Czech Republic. The increasing trend of reported cases
greater extent with a prevalence 1.3 times higher than is probably due to greater access to improved diagnostic
men. This ratio is independent of age, being practically methods. Figures for prevalence recorded in serological
identical in all age-groups. surveys from past years are inuenced more by actual
The majority of Toxoplasma-positive persons in the site of collection and locality (urban versus rural) than by
Czech Republic become infected at age 9 years or younger. the time factor. Hejlcek and Literak, 1993a) found no
If we compare prevalence year by year we can see that all changes among blood donors in the Strakonice district
examined children under 1 year of age were negative. In 14 over the period 19801990. Repeated examinations were
year olds, particularly from rural regions, there is a sharp performed only on the population of Prague, showing low
rise. A further increase in prevalence, particularly in prevalence and no dramatic changes between the rst CFT
women, is seen between the ages of 15 and 40 during examinations in 1960 (14.2%) (Seeman, 1960) and the
peak fertility. serological survey in 1996 (13.6%).
Toxoplasma survives in the infected host until the death The epidemic started at the end of December 1993. The
of the host (Krahenbuhl and Remington, 1982). Hence the greatest increase in morbidity (14 times the long-term aver-
increase of prevalence is cumulative. However, in our age) was recorded in ten Moravian districts containing 18%
group there was a decline in seroprevalence in the over of the Czech Republic population. At the peak of the
50 age-groups, more marked in women, particularly those epidemic (JanuaryMarch 1994) the disease was conrmed
from rural areas. Decreased prevalence in the highest age- in these districts in a total of 722 patients.
groups (unfortunatelly uncommented) is apparent from The largest local outbreak of toxoplasmosis was docu-
IDT data by Jrovec (1971) and Shevkunova (1980), but mented by Bowie et al. (1997) in 1995 in Victoria, Canada,
not by Sandow et al. (1989) who noted a continual increase where 29007700 cases were estimated; outbreaks,
leading to 100% prevalence in persons over 67 years using described by Radulovic et al. (1990): (78 persons); Bene-
the IFAT. nson et al. (1982): (38 persons) and Teutsch et al. (1979):
A possible explanation for this decline is that the general (37 persons), were much smaller. Other epidemics
rule of life-long seropositivity is not absolute and that in mentioned in the literature involved four to ten persons,
certain individuals immune response may fade after a long on a local level. An epidemic of toxoplasmosis affecting
period p.i. NRLT has recorded two cases: a man and a hundreds of people over such an extensive area is a unique
woman who were seronegative 20 and 15 years following phenomenon.
hospitalisation for conrmed acute toxoplasmosis (Kodym- Morbidity declined after the epidemic subsided but
unpublished data). Another explanation would be the lower remained at elevated levels for a period of 3 years. Aside
seroprevalence in the past, although this variant does not t from increased circulation of the infectious agent this fact
with established data. The third explanation for the may be due to increased diagnostic attention and reporting
decrease of seropositive individuals in higher age-groups- around the critical period.
that they have died-is adamantly rejected by the authors, Sources of the infection are given as oocysts excreted by
some of whom are seropositive themselves. cats contaminating water (Bowie et al., 1997; Benenson et
Clinical toxoplasmosis in the Czech Republic manifests al., 1982), food from army kitchens (Radulovic et al., 1990),
itself as a disease affecting children and persons (mainly riding school environments (Teutsch et al., 1979), sandpits
women) of fertile age. The most frequent symptom is (Stagno et al., 1980) or farm soil (Coutinho et al., 1982); or
lymphadenopathy, and following the outbreak in 1993/ bradyzoites/tachyzoites from animal food products, unpas-
1994 there was an increased incidence of ocular toxoplas- teurised goat's milk (Sacks et al., 1982), uncooked meat
mosis. There is an unexpectedly low incidence of reported from arctic mammals (McDonald et al., 1990), traditional
congenital toxoplasmosis (no increase was detected even kibbi from raw mutton (DeSilva et al., 1984) or hamburgers
during the epidemic). The expected annual number should (Kean et al., 1969).
Eight cases of toxoplasmosis have been reported in number of deaths (Clumeck, 1991). However, since autop-
genetic laboratory personnel infected by tissue cultures sies are not regularly performed in Africa and facilities for
(Markvart et al., 1978). Epidemiological investigations the accurate diagnosis of these opportunistic pathogens are
have eliminated all of the above as sources of the epidemic only rarely available, the denite cause of death in AIDS
described in this paper. patients often is unclear.
In view of the fact that we have eliminated contact with In order to determine whether toxoplasmosis might be
soil (winter epidemic), cats or other animals and common important in the progression of AIDS in Africa as well,
water supplies as causative factors; the urban character of we analysed in this study 668 serum samples from Nigerian
the epidemic (occurring simultaneously in isolated foci individuals.
during the Christmas period) and with a high familial inci-
dence, denotes an alimentary route of infection.
4.1.2. Materials and methods
The majority of patients reported consumption of food-
stuffs that may potentially have contained tissue cysts (ham) The samples were collected between January and June
or may have been contaminated during manufacture or 1994 and consisted of 380 sera from female individuals
storage by oocysts from cat faeces (nuts, fruit). Infected (average age 30.1 years) and 288 sera from male individuals
foodstuffs, imported or domestic, were probably distributed (average age 32.6 years). The majority (365) of serum
to shops in various towns prior to the Christmas period with samples were from outpatients with various ailments, such
resulting infection of the local population. Isolation of T. as typhoid fever, diabetes, and malaria fever. Additional
gondii developmental stages in foods or the environment samples (137) were from antenatal patients and 166 sera
during the epidemic was unsuccessful. As such, this has were from apparently healthy individuals, 65 of whom
occurred in only three of the 11 above-mentioned were blood donors.
epidemics: from mice and cats captured in the infested The serum samples were examined for T. gondii-specic
riding school (Dubey et al., 1981), farm soil (Coutinho et antibodies by the direct agglutination test (DA, Toxo-
al., 1982a,b) and drinking water (Isaac-Renton et al., 1998). Screen), the CFT, and the SFDT, and for antibodies to
The exact route of transmission of the Moravian epidemic in HIV I and II by an enzyme immunoassay (EIA, Enzygnost
1993/1994 will probably remain unsolved. Anti-HIV 1/2 plus, Behring). EIA HIV-positive and border-
Acknowledgements: The authors acknowledge the line results were retested by an immunoblot (New LAV
considerable contribution of late Jan Pokorny to Toxo- BLOT I&II, Sano Diagnostics Pasteur, Freiburg,
plasma research in the Czech Republic. They are grateful Germany). A serum was considered positive for T. gondii
to Stanislava Polednakova and Vera Tomaskova for antibodies if it was reactive in at least both DA and SFDT;
performing serological tests, to Radek Chodera for language and for HIV antibodies if it was reactive in both EIA and
cooperation and to Ladislav Tomasek for computational immunoblot.
support. The research was supported by the Grant Agency
of the Czech Republic, No. 524/98/0111.
4.1.3. Results
4. Coinfection between HIV and Toxoplasma gondii The overall prevalence of 25.9% for T. gondii antibodies
in the general population recorded in this preliminary study
4.1. Coinfection between HIV and Toxoplasma gondii in an is surprisingly low (Table 14), compared with studies in
african population an underestimated threat? other parts of Nigeria, which demonstrated a seroprevalence
of 72.2% (Olurin et al., 1971), especially as the dietary habit
T.A. Olusi a, B. Weissbrich b, J.A. Ajayi c, U. Gross d: of the sampled population (Olusi et al., 1994) and the abun-
a
Department of Biological Sciences, University of Agri- dance of stray cats particularly expose the population to
culture, Makurdi, Nigeria; bInstitute of Virology, University infection with T. gondii. The HIV infection rate of 5.7%
of Wurzburg, Wurzburg, Germany; cDepartment of Zool- was expected since promiscuity is common in Nigeria.
ogy, University of Jos, Jos, Nigeria; dDepartment of Bacter- Interestingly, the seroprevalence of HIV antibodies in
iology, University of Gottingen, Gottingen, Germany Nigeria, all of them specic for HIV I, is signicantly
4.1.1. Introduction Table 14

Seroprevalence of HIV/Toxoplasma gondii co-infections in Benue state of
Although AIDS is widespread in several African coun- Nigeria.
tries, the inuence of opportunistic infections for the Infection Number Number Percent
outcome of the disease is unknown. Tuberculosis is thought tested positive positive
to be one of the most important causes of death in HIV-
infected patients in Africa (Lucas et al., 1993), whereas in HIV I 668 38 5.7
Toxoplasma gondii 668 173 25.9
Europe other opportunistic pathogens such as Pneumocystis
HIV/Toxoplasma gondii 668 10 1.5
carinii or T. gondii have been shown to account for a large
lower than found in neighboring Cameroon and ve other acquired toxoplasmic uveitis and some peculiarities of
central African countries (Kanki et al., 1987). the ocular disease caused by T. gondii infection in the
Rio Grande do Sul state (Melamed, 1989). However, an
4.1.4. Discussion unusually high prevalence of ocular toxoplasmosis in
southern Brazil that was reported at the beginning of the
No signicant difference was demonstrated for the sero- 1990's has been the classical reference citation of acquired
prevalence of T. gondii-specic antibodies between HIV- ocular toxoplasmosis all over the world (Glasner et al.,
positive and -negative individuals. This is perhaps not 1992).
surprising because HIV infection has not been shown to
be a risk factor for infection with T. gondii. However, the 5.1.2. Epidemiology of toxoplasmosis in Campos dos
number of HIV-positive individuals chronically infected Goytacazes: a cross-sectional study
with T. gondii is signicantly higher in Nigeria compared
with Europe and thus might be important to know in view of In Brazil, serologic prevalence of T. gondii infection
the fact that reactivation of chronic Toxoplasma infection ranges from 50 to 80% of the healthy adult population,
might cause fatal toxoplasmic encephalitis. the highest values being found in some northern and south-
Acknowledgements: Supported by the European Union ern states. The lowest values tend to occur in some south-
(BMH1-CT92-1535 to UG), the Nigerian National Univer- eastern states (Sao Paulo and Minas Gerais) (Orece and
sities Commission World Bank-assisted staff development Bonoli, 2000). The highest incidence of posterior uveitis
programme, and the University of Agriculture, Makurdi, seems to occur in southern of Brazil where it can reach a
Benue State. prevalence of 95% (Melamed, 1989). In Rio de Janeiro state
there is a paucity of information on the epidemiology of
toxoplasmosis. In 1987 a survey in the capital of the state
5. Toxoplasmosis in southeastern Brazil: an alarming (Rio de Janeiro city) showed the following prevalence: 30%
situation of highly endemic acquired and congenital for children younger than 5 years, 59% in the age range from
infection 6 to 10 years, 69% for those between 11 and 15 years and
71% for persons ranging in age from 16 to 20 years (Souza
Llian Maria Garcia Bahia-Oliveira a,*, Annelise Maria et al., 1987).
Wilken de Abreu a, Juliana Azevedo-Silva a, Fernando Ore- Campos dos Goytacazes, which is located at north of Rio
ce b: de Janeiro is the third city in terms of importance in the
a
Universidade Estadual do Norte Fluminense (UENF), state. Enormous social disparities are seen in the region
Centro de Biociencias e Biotecnologias (CBB), Laboratorio where about 70% of the population depends on the public
de Biologia do Reconhecer (LBR). Av. Alberto Lamego health system, which cannot provide for even the basic
2000, Horto, Campos dos Goytacazes, RJ Brazil 28015-620; necessities for the entire population. Local infectious
b
Departamento de Oftalmologia e Otorrinolaringologia, disease specialists and ophthalmologists have expressed
Escola de Medicina, Universidade Federal de Minas Gerais their concern regarding the great number of cases of acute
(UFMG). Av. Alfredo Balena 190 Belo Horizonte MG toxoplasmosis in children as well as posterior uveitis caused
Brazil 30130-100 by toxoplasmic infection. This concern led to an epidemio-
logic survey on toxoplasmosis, in Campos dos Goytacazes
5.1.1. Introduction the results of which we present herein, as an initiative of a
new University (the University of the North of Rio de
The organisms belonging to the Toxoplasma genus were
Janeiro State-UENF).
described independently in 1908 in Brazil by Splendore
(1908) in rabbits, which he named T. cuniculi, and by 5.1.3. Material and methods
Nicolle and Manceaux, 1908, 1909) in Tunisia in gondii
(Ctenodactylus gondii) a South African rodent, which Study population: The study population consisted of (A)
they named T. gondii. Although toxoplasmosis is undoubt- individuals from schools and slums or poor communities
edly an ancient disease it was recognised as a specic participating in a survey for toxoplasmosis; (B) children
entity only after the parasite was discovered. The rst assisted on a outpatient basis by a county health public
epidemiologic studies in Brazil came after the introduction programme for assistance to children and teens for the
of the SFDT in 1948. Busacca et al. (1953) reported that pilot study of seroprevalence of toxoplasmosis in neonates
23% of posterior uveitis had T. gondii as an etiological and young children; (C) neonates screened for toxoplasmo-
agent. Subsequently Abreu Fialho (1953), Pereira et al. sis based on specic IgM testing of lter paper blood
(1965) and Martins et al. (1969) reported seropositivity samples, as described in detail below.
rates of 38, 30 and 68%, respectively, in patients with (A) The individuals participating in this survey belong to
posterior uveitis in Brazil (Orece and Bonoli, 2000). In three different economic and social strata dened as follows:
1988 it was shown in the World uveitis symposium in Sao persons living in slums or very poor communities under
Paulo, Brazil, that there existed a high prevalence of precarious sanitary and poor social conditions (N 316),
population 1 P1; lower middle-class children attending Neonatal screening assay: All assays were performed by
public school and the adult school's staff (N 564), popu- one laboratory with expertise in toxoplasmosis neonatal
lation 2 P2; and upper middle-class children attending screening (Centro de Triagem Neonatal in Porto Alegre
private schools and the adult school's staff (N 337), popu- RS) using a commercial capture T. gondii IgM uorometric
lation 3 P3. The schools and slums or poor communities are enzyme immunoassay (FEIA) (Labsystems)
located in four different geographic regions of the city, with Ophthalmic examinations: after pupil dilatation indivi-
urban, suburban or rural characteristics predominating. duals were examined by a team of nine ophthalmologists
Only those who provided written consent were included in from the UFMG, Federal Fluminense University in Niteroi
this study. (UFF) and Campos dos Goytacazes. Indirect ophthalmo-
(B) Children who received assistance on a outpatient scopy was used to investigate the presence of posterior
basis from a county public health program for assistance uveitis. At the time of the examination the ophthalmologists
to children and teens were included in the pilot study of were unaware of the serologic status of the individuals.
seroprevalence of toxoplasmosis in neonates and young Active inammatory retinal lesions as well as pigmented
children: a total of 100 neonates (from 7 to 28 days) and retinal or retinochoroidal lesions consistent with healed reti-
children (from 1 month to 6 years) who were randomly nochoroiditis were observed. Every retinochoroidal lesion
selected during 1998 for a pilot study of seroprevalence of suggestive of an uveitic process, active or not, was notied.
toxoplasmosis in neonates and young children were The size, number, and location of lesions, and inammatory
included. Blood samples from babies and children were and vascular ocular disease were documented in a standar-
collected for routine examination as part of the protocol of dised protocol. All the healed and active lesions were clas-
the program for assistance to children and teens from the sied independently of the serological results. According to
county public health service. their morphological characteristics, the retinal lesions were
(C) Neonates screened for toxoplasmosis based on speci- classied as type A, B and C, type A and B being those with
c-IgM testing of lter paper blood samples. A total of 2550 the greatest probability of being caused by toxoplasmic
neonates aged 320 days during April 1999 to June 2000 infection. The signs taken into account for the classication
were tested for specic IgM in lter paper blood samples were the degree of involvement of retina and choroid,
which were collected for evaluation of phenylketonuria and judged mainly by spatial features of pigment mobilisation,
hypothyroidism in a county-funded program. The babies gliosis and tissue atrophy. For babies with congenital toxo-
participating in the screening were randomly selected plasmosis ocular examination was carried out under anaes-
using days of the week as criteria for selection. Written thesia by two ophthalmologists from Campos participating
informed consent was obtained from patients or their guar- in this study.
dians according to the guidelines of the Ethical Committee Neonatal examinations took place according to a protocol
from the FIOCRUZ (Ministry of Health, Brazil) for serolo- which includes: routine general physical examination,
gical and lter paper testing, ocular examinations and inter- neurological and ophthalmologic evaluations.
view surveys. Analysis of data: The data were entered into the computer
Serological assays: For the survey from P1, P2 and P3 and and analysed using Epi-info.
for the pilot study to investigate seroprevalence in neonates
and young children the sera were tested for toxoplasmosis
using a commercial ultramicro ELISA for specic IgG with 5.1.4. Results
uorometric detection (UMELISA Toxoplasma, Havana,
Cuba) at the Nucleo de Apoio Diagnostico (NUPAD) from We observed an increase in the seroprevalence by age for
the Federal University of Minas Gerais (UFMG). The corre- all population groups as shown in Fig. 5. The prevalence
lation between the UMELISA and the SFDT, as well as the levels for P1 and P2 in the age range of 625 years is
quality control of the UMELISA's system were evaluated by substantially higher in comparison with P3. The seropreva-
exchanging blind sera samples between NUPAD and the lence and the prevalence of ocular disease for the three
Serology Laboratory, Department of Immunology and Infec- populations (without considering age of individuals) are
tious Diseases, Research Institute, Palo Alto Medical Foun- shown in Fig. 6. The overall prevalence of ocular disease
dation. All the samples exchanged agreed 100% in terms of for the three populations ranges from 10 to 12%, however,
the value of titre and with regard to positivity or negativity for some communities within each strata that dened a popula-
toxoplasmosis. tion (see Section 5.1.2) showed differing prevalence of
IgG and IgM measurements for conrmatory serological ocular disease which ranged from 8 to 14%. For instance,
tests which are done for positive lter paper blood samples the lowest prevalence of ocular disease (8%) occurred in an
were performed using the kit VIDAS-TOXO-IgM (ELFA- urban slum community (with 110 invididuals) in which
Enzyme Linked Fluorecent Assay, Biolab-Merieux). The seroprevalence for toxoplasmosis is higher than 90% for
mothers of IgM positive babies were also tested for IgM individuals older than 25 years. On the other hand the high-
and IgG. These tests were done in a private laboratory in est prevalence of ocular disease (14%) was observed for a
Campos. poor community (with 104 individuals) living in a predomi-
Fig. 7. Seroprevalence in neonates (328 days) and children in the age

range of 15 months (30 individuals), 6 to 36 months (35 individuals)
and 3772 months (35 individuals). Neonates and children who have
Fig. 5. Toxoplasmosis serum prevalence according to the age of three been randomly selected during the year of 1998 for a pilot study of sero-
populations living under different sanitary and social conditions in Campos prevalence of toxoplasmosis (see Section 5.1.2).
dos Goytacazes RJ. Population 1 (P1) comprised individuals living under
precarious sanitary and poor social conditions (N 316); population 2 (P2)
comprised lower middle-class, individuals (N 564); population 3 (P3) the age of 6 years are seropositive for toxoplasmosis. In fact,
comprised individuals living under good sanitary and social conditions the children in the outpatient basis from the county public
(N 337). health program belong to the social strata P1 or P2. Two
babies with toxoplasmosis presenting with symptoms were
identied in the pilot study (Table 15) and during 1999, two
nantly rural area in which the seroprevalence is close to 90% neonates (not participating in the neonatal screening) were
for persons older than 25 years (not shown). referred to the paediatrician of our group for examination
In the preliminary data analysis of all three groups, for suspected suffering congenital toxoplasmosis; the sero-
factors associated with infection included (1) drinking unl- logical IgM tests conrmed the disease, however both died
tered water; (2) drinking water from wells; and (3) cat even before treatment was initiated. They had systemic
ownership. Drinking ltered or spring water was protective. illness. One of them presented splenomegaly and enlarged
Only within the lower-middle class population (P2) was lateral ventricles; the other had microcephaly and micro-
eating undercooked beef a risk factor (the very poor are phthalmy (not shown).
unable to afford meat). Among the upper class population, Screening of neonatal lter paper blood spots for Toxo-
no risk factors were identied. plasma-specic IgM followed by conrmatory testing
The IgG seroprevalence for 100 neonates and children during April 1999 to June 2000 showed that 5/2550 babies
who were randomly selected during the year of 1998 for a had congenital toxoplasmosis. This study is still ongoing
pilot study of seroprevalence of toxoplasmosis (see Section and the number of babies screened (2550) represents
5.1.2) is shown in Fig. 7. About 40% of the babies had anti- about 25% of the total births in Campos during that period.
Toxoplasma antibodies until the age of 5 months, from 6 to The clinical status of the babies is shown in Table 15. All the
36 months the prevalence of IgG anti-Toxoplasma decreases babies with congenital disease, independent of whether they
to 5%, which was expected because of the clearance of had symptoms, have been treated according to regimens
maternal IgG occurring by this age, and 22% of the babies described by Remington et al. (1995).
ranging in age from 37 to 72 months had IgG against T.
gondii antigens. This is in agreement with data for P1 and P2
5.1.5. Discussion
(Fig. 3) where respectively 40 and 30% of the children by
These data suggest that social and economic conditions
inuence risk factors for toxoplasmosis in the Rio de Janeiro
state. The contribution of congenital toxoplasmosis for the
total/global prevalence of toxoplasmosis in Campos is
ongoing and data have revealed that birth prevalence of
congenital toxoplasmosis is also elevated.
The lower seroprevalence of toxoplasmosis observed for
P3 (17%) and P2 (61%) in comparison with P1 (86%)
contrasts with the similar ocular prevalence found for the
three populations namely, P3 (12%) P2 (10%) and P1
(12%). The P1, P2 and P3 prevalence curves (Fig. 5) suggest
that the risk of women getting infected during pregnancy is
high for those under better economic and social conditions
Fig. 6. Seroprevalence and toxoplasmic ocular lesions in the three popula- (P3) in Campos. This gives rise to the question whether the
tions, P1, P2, and P3, without considering the age of individuals. ocular lesions found in the three populations are mainly
Table 15
Clinical status of babies with congenital toxoplasmosis in Campos
Baby Code Age therapy Age Evaluation Signs/symptoms observed a Treatment

begun (months) (months)
1b 1 1 No symptoms or sign PS
4b 1 1 Macular retinochoroidites (unilateral) PS
6c 4 4 Strabism, nistagmus, macular retinochoroidites (bilateral) cerebral calcications PS
7c 6 6 Abnormal cognitive development, mild motor development PS
a
Evaluation included general physical examination, neurologic (including computer tomography of the head) and ophthalmic evaluation. These children are
participating in a 5-year follow-up which started 1999.
b
Babies participating in the neonatal screening.
c
Babies assisted in the pilot study (see Section 5.1.2).P, Pyrimethamine; S, sulfadiazine.
caused by congenital or acquired infection. However it is the development of ocular disease. It might be possible
important to note that the prole of the P3 seroprevalence considering the fact that many variables such as host genet-
curve has to be interpreted carefully in terms of its value to ics, parasite strain, inoculum size, host age, sex hormones
estimate risk for persons living in Campos under good social and immune status clearly modulate and affect outcome of
and economic conditions to become infected with T. gondii, experimental infections (Melamed et al., 1989). In this sense
in particular for those older than 25 years. For the past 10 the chances of an individual to be infected with a large
years a great number of persons from different regions of oocyst inoculum (concentrated in faeces of felines spread
Brazil have migrated to Campos because of the oil explora- in soils of rural regions) is potentially higher in comparison
tion in the region. The demographic data of the P3 indivi- with those not exposed to constant contact with soil from
duals must be carefully taken into account to help to clarify urban regions.
the prole of P3 seroprevalence curve. This is not applicable The prevalence of congenital infection in Campos is
for P2 and P3 who are individuals not linked and not higher in comparison with that recently reported in Brazil
attracted to the oil exploration activities. estimated in a 3-year prospective neonatal screening study
It is interesting that drinking unltered water appears as in which the prevalence was 1 per 3000 live births (Bandoli
the rst risk factor when analysis is done for the three popu- and Basilio de Oliveira, 1977) and those reported previously
lations together as well as when it is done for P1 and P2 (Paul et al., 2000; Guerina et al., 1994; McLeod et al., 1996;
separately. Attempts to isolate parasites from water reser- Camargo-Neto et al., 2000). The long term follow-up of
voirs from Campos and surrounding have recently been babies participating in this study will help to clarify the
implemented. An interesting and curious report from 1977 impact of asymptomatic congenital infections in the life of
describing a case of a dolphin with disseminated toxoplas- individuals. The social and economic prole of women
mosis, which was found dead in the Guanabara bay, in Rio having infants with congenital toxoplasmosis in Campos
de Janeiro capital (Melamed et al., 1989), opens the question is still unknown, however it will be of vital importance
of contamination of water reservoirs in the Rio de Janeiro for the implementation of health public preventive
state which should be carefully pursued. The dolphin measures.
species, Sotalia guianensis lives in estuaries which put Acknowledgements: We express our gratitude to Mrs.
them in contact with sources of fresh water potentially Cindy Press, Drs. Fausto Araujo, Jack Remington, and
contaminated with oocysts of T. gondii. Jose Montoya from the Palo Alto Medical Foundation for
The high prevalence of ocular disease (14%) in a rural their valuable help on serologic questions and for advice on
poor community (with 104 individuals) as compared with treatment of congenital cases of toxoplasmosis. We thank
the prevalence observed for another community (with 110 also Mrs. Patricia Silva Santos, Cristiane Crespo, Thiago
individuals) living in an urban region in which 8% of ocular Tatagiba and Juliana Salgado Viana for their dedicated tech-
lesions was found, is also an interesting nding since both nical assistance, and the team of ophthalmologist: Drs.
communities belong to the same social and economic strata Acacio Muralha, Andre Curi, Dase Malheiros, Elisa
(P1). Anecdotal reports suggest higher prevalence of ocular Waked, Fernanda Porto, Lilia Muralha, Ricardo G. Peixe,
disease for individuals living in rural areas (without an asso- and Wesley Campos, for examining patients. Our gratitude
ciation with the habit of eating raw or undercooked meat) in also to Drs. David Addiss (from the Centers of Disease
comparison with their neighbors from urban regions in Control and Prevention, Atlanta) and Enrique Medina
south of Brazil (Melamed personal communication.). It Acosta (from the laboratory of Biotecnology of the Univer-
raises the question whether contact with soil contaminated sity of the Rio de Janeiro State in Campos dos Goytacazes)
with T. gondii oocysts would be an important risk factor for for the critical reading and suggestions on this manuscript.
This work has been supported by CNPq, PRONEX FAPERJ human isolate (1959) maintained in mice by continuous
and FENORTE. i.p. passage of brain cysts, was used for infection. According
to the classication of Gross et al. (1991) it is an avirulent
6.1. The activity of Fansidar w in chronic murine strain.
toxoplasmosis-a combined serological and morphological Mice: Thirty-eight female NMRI mice (own laboratory
study breeding), 6 weeks of age, were infected orally with brain
cysts from ve chronically infected mice. The brains were
Christiane Rock, Ingrid Reiter-Owona, Helge Kampen, dissected, ground with pestle and mortar and resuspended in
Hanns M. Seitz: a sterile solution of 0.9% NaCl. The mean value of cysts
Institute for Medical Parasitology, University of Bonn, detected in 10 10 ml of homogenate was used to calculate
Sigmund-Freud Strasse. 25, D-53105 Bonn, Germany the approximate total cyst number. The homogenate was
then diluted with 0.9% NaCl to get an infection dose of
6.1.1. Introduction seven cysts per 200 ml per mouse. Three months after Toxo-
Previous studies indicate that children with prenatal plasma infection all animals were tested for Toxoplasma
Toxoplasma infection benet from prolonged therapy antibodies by SFDT and proved to be positive with titres
(Roizen et al., 1995). The standard therapeutic regimen is ranging from 1:4000 to 1:64 000.
a combination of pyrimethamine and sulfadiazine given in Five female mice from the same age-group were kept
treatment courses of 3 or 4 weeks separated by spiramycine uninfected until treatment started (therapy control group).
treatment during the rst year of life. However, rising anti- Blood samples were collected in vivo by retro-orbital punc-
body titres (serological rebounds) which are frequently ture or from the heart at autopsy as indicated in Table 16.
observed in children after the end of a long-term therapy Treatment: Before treatment (3 months after Toxoplasma
signal that the antiparasitic effect is only limited and infection) the mice were randomly divided into four groups
temporary. Serological rebounds are rarely accompanied (Table 16). Groups AC received therapy for a period of
by clinical recurrences and do not occur during adequate maximum 30, 60 and 90 days, respectively. Five uninfected
therapy. There is evidence that in prenatally infected, post- mice were treated continuously for 90 days (therapy control
natally treated children the antibody decline is faster than in animals). The remaining ve Toxoplasma infected mice
untreated individuals (WHO, 1988). These ndings support (group 4) were kept untreated as Toxoplasma control
the probability that a release of drug pressure allows reacti- animals.
vation of the parasite. However, the ndings do not exclude Fansidar w (Roche) was diluted in a solution of 4% BSA in
that an immunosuppressive effect of the chemicals or the PBS, pH 7.2, to give a nal solution of 0.55 mg pyrimetha-
parasite itself could prevent antibody maturation during mine and 11 mg sulfadoxine per 100 ml. Every 3 days each
therapy. mouse received 100 ml of the solution s.c. corresponding to
In general, our knowledge of the efcacy of the standard an average dose of 18.3 mg of pyrimethamine and 367 mg of
chemotherapeutic agents on Toxoplasma cysts is limited. In sulfadoxine per kg of body weight. The mean body weight
vivo studies with different T. gondii/mouse models have of the mice was 30 g when treatment started.
demonstrated that the number of Toxoplasma brain cysts Parasite detection: Tissue and blood samples were recov-
decreases early after therapy but that cysts are not comple- ered from animals during (subgroup a) or after (subgroup b)
tely eliminated (Djurkovic-Djakovic et al., 2000; Couvreur treatment as indicated in Table 16. At autopsy the brain of
et al., 1991; Araujo et al., 1993). The survival of Toxo- each mouse was immediately removed for microscopic and
plasma parasites during therapy and the subsequent reacti- histologic studies. At rst, the weight of the whole brain was
vation may be due to either a limited anti-bradyzoite activity determined. Thereafter, the brain was cross-sectioned and
of the drugs or to the limited amount of chemicals which the caudal part cut longitudinally into two pieces. The
reach the brain. weight of the resulting two caudal pieces was recorded.
Only a few in vivo studies give information on the anti- Cyst count: The left caudal piece of each brain was
body response of chronically infected mice during Toxo- homogenised in 0.3 ml of 0.9% NaCl. The whole homo-
plasma therapy. To our knowledge long-term studies or genate was examined microscopically for the presence of
studies on the antibody response after cessation of therapy cysts. The right caudal piece was cut into two sections.
(serological rebound) are not available. The aim of our study Each section was squeezed between two slides, the number
was to demonstrate the effect of a long-term, pyrimetha- of tissue cysts was determined microscopically and the
mine/sulfadoxine (Fansidar w) application on brain cysts of result of both sections added. Finally, the total brain cyst
chronically infected mice and to study the immune response number of one animal was calculated in relation to the
during and after Toxoplasma therapy. weight of the respective caudal brain piece and the weight
of the total brain.
6.1.2. Materials and methods Histology, electron microscopy: the frontal brain part was
cut longitudinally. The left part was immediately xed in
Parasite: The cyst forming T. gondii strain `Gail', a 4% formaldehyde, the right part in 2% glutaraldehyde. The
Table 16 diaminobenzidine (Liquide-DAB). Slides were counter-

Treatment and observation periods of chronically T. gondii-infected NMRI stained with haemalaun.
mice a
Serology: Antibodies to T. gondii were determined by
Group Fansidar w Mouse Autopsy b Blood Sample b SFDT as described earlier (Reiter-Owona et al., 1998).
treatment no. Toxoplasma antibodies of the IgM and IgA class were
detected by an ISAGA described elsewhere (Saathoff and
Aa Up to 30 days 4 18 0, 18
Aa 9 27 0, 27 Seitz, 1991) with some variations. Plates were coated with a
Aa 15 18 0, 18 polyclonal sheep anti-mouse IgM or IgA antibody (Serotec
Aa 20 27 0, 12, 27 Ltd.) diluted 1:200 or 1:250 in 0.1 M carbonate buffer (pH
Ab 1 76 0, 38, 56, 68, 76 9.8). Toxoplasma antigen (Antigen Toxo AD, bioMerieux)
Ab 2 110 0, 38, 56, 68, 76, 110
was diluted 1:12 and 1:20, respectively. Antibodies to BSA
Ab 3 76 0, 38, 56, 68, 76
Ab 5 110 0, 38, 56, 68, 110 were detected by ELISA. Plates were coated with a 10%
Ab 6 55 0, 38, 55 BSA/carbonate buffer (pH 9.8) solution. Mouse serum
Ab 7 46 em 0, 38 samples (rst dilution 1:64) and goat anti-mouse HRP
Ab 8 46 0, 38 conjugate (1:1000, Southern Biotechnology) were diluted
Ab 10 55 0, 38, 55
in a solution of 10% human serum in PBS (pH 7.2). Visua-
Ab 11 140 0, 49, 140
Ba Up to 60 days 13 49 0, 49 lisation was by OPD at 450 nm after 10 min of incubation.
Ba 16 49 0, 49 PCR: DNA was extracted from 200 ml of mouse blood by
Ba 21 41 0, 38, 41 means of the QIAamp blood kit (QIAGEN) following the
Ba 22 41 0, 12, 41 manufacturer's instructions. In some cases the volume had
Bb 12 111 0, 49, 111
to be adjusted to 200 ml with physiological saline. Ampli-
Bb 14 111 0, 49, 111
Bb 17 140 0, 49, 140 cation was based on the 35-fold repetitive B1 gene of T.
Bb 18 75 0, 49, 75 gondii (Burg et al., 1989), making use of the primers
Bb 19 75 0, 49, 75 TOXOB22 and TOXOB23 as described (Bretagne et al.,
Ca Up to 90 days 29 c 88 0, 49, 70, 88 1993) as well as of the primers 1 and 2 (Eggers et al.,
Ca 30 c 88 0, 49, 70, 88
1995). With each DNA specimen two separate PCR assays
Ca 31 c 63 em 0, 12, 49, 63
Ca 32 c 77 em 0, 12, 49, 70, 77 were run with the different sets of primers, thereby one
Ca 33 c 63 0, 12, 49, 63 amplication result serving as a specicity control for the
Cb 23 c 161 em 0, 70, 90, 113, 138,161 other.
Cb 24 c 119 0, 49, 70, 90, 113, 119 Reaction mixtures consisted of 200 mM of dNTPs each,
Cb 25 c 182 em 0, 70, 90, 113, 138, 165, 182
10 pmol of each of the two primers (either TOXOB22/
Cb 26 c 103 em 0, 49, 70, 90, 103
Cb 27 c 103 0, 49, 70, 90, 103 TOXOB23 or 1/2), 10 mM KCl, 10 mM TrisHCl, pH
Cb 28 c 119 0, 49, 70, 90, 113, 119 8.3, 2 mM MgCl2, 1.25 units of AmpliTaq DNA-polymerase
4 No 34 c 85 0, 54 Stoffel-fragment (PerkinElmer), 10 ml of DNA solution,
4 35 161 em 0, 54, 113, 139, 161 and sterile water to give a nal volume of 50 ml. Negative
4 36 32 em 0, 32
controls (sterile distilled water instead of DNA solution as
4 37 c 139 0, 54, 113, 139
4 38 4 em 0, 4 well as QIAamp blood kit processed physiological saline)
and a positive control (QIAamp blood kit extracted Toxo-
a
em, brain tissue examined by electron microscopy; (a), mouse autopsied plasma DNA, strain BK, corresponding to three tachyzoite
during treatment; (b), mouse autopsied after treatment.
b DNA equivalents) were included.
Days after onset of therapy.
c
Parasite detection by PCR. Samples were cycled as described (Bretagne et al., 1993).
Amplication products were separated on a 1.8% agarose
gel, ethidium-bromide stained, and analysed under UV
material was then submitted to standard embedding as light.
described earlier (Reiter-Owona et al., 1996). Parafn
sections were cut 46 mm thick, stained by HE, or used
6.1.3. Results
for immunohistochemistry. Electron microscopic studies
were performed as indicated in Table 16. The Fansidar w application was well tolerated by all short-
Immunohistochemistry was performed on selected, serial and long-term treated animals without severe side effects.
parafn sections after clearance in xylene and acetone. A The total brain cyst number recorded was generally higher
rabbit hyperimmune anti-Toxoplasma serum (rabHS) in the tissue smear probes than in the tissue homogenate
diluted 1:300 in PBS (pH 7.6) was applied as primary anti- examined from each animal in parallel. Therefore, the
serum. The rabbit antibody was detected by an avidin-bioti- results are generally expressed as mean values of the results
nylated horseradish peroxidase (HRP) complex technique from both methods. A signicant reduction in the total brain
with the Vectastain w kit (Camon Labor-Service). Visualis- cyst numbers of treated animals was recorded early after
ing of the peroxidase-labelled secondary antibody was by therapy (Fig. 8). The concentration of Toxoplasma cysts
Toxoplasma-positive material (Fig. 9a) or degenerating

cysts with invading glia cells (Fig. 9b) were found. There
was no sign for cyst disruption or infection of the brain
tissue by extra-cystic parasites. The distribution of differ-
ently sized tissue cysts was similar in treated and non-trea-
ted animals.
The ultrastructural study of brain tissue from treated
animals during and after therapy was difcult due to the
low number of tissue cysts present. Bradyzoites in the
cysts of control animals were lying in close contact within
the electron-dense ground substance, whereas in cysts of
treated animals bradyzoites tended to be loosely packed
within a electron-lucent ground substance. Within the Toxo-
plasma cysts detected, degenerating bradyzoites (mice nos.
7, 26), chromatin aggregations at the border of the brady-
zoite nucleus (mouse no. 32) as well as alterations of the
cyst wall structure (mice nos. 26, 32) were seen (Fig. 10a,b).
In some cysts the wall appeared collapsed and thickened as
Fig. 8. Reduction in T. gondii brain cyst numbers during Fansidar w therapy. if several sections of the limiting membrane were lying on
Duration of therapy: (A) up to 30 days; (B) up to 60 days; (C) up to 90 days. top of each other. In mice nos. 25 and 26, autopsied 92 and
(a) Mice autopsied during therapy; (b) mice autopsied after therapy. 13 days after the end of a 90-day treatment, multiplying
bradyzoites (endodyogeny) were detected (Fig. 11).
detected in the brains of mice examined during Fansidar w The anti-Toxoplasma SFDT and IgM-/IgA-ISAGA anti-
treatment was 56% (group Aa), 48% (group Ba), and 43% body responses remained stable in the control group and in
(group Ca) in comparison with the control group (group 4). the short-to medium-term treatment groups A and B. In
The concentration of brain cysts in mice examined after group C, however, there was a slight decline in the SFDT
cessation of therapy was 52% (group Ab), 38% (group (Fig. 12) and IgM-/IgA-ISAGA antibody response in
Bb), and 39% (group Cb), respectively (Fig. 8). Histological animals treated more than 60 days. After cessation of the
examinations revealed little inammatory response in short- 90 day treatment course there was a slight increase of the
or long-term treated mice and very few signs of inamma- antibody response of one (mice nos. 23, 24, 26, 27, 28) or
tion in the control animals. In some of the immunolabelled two (mouse no. 25) dilution steps (Fig. 12, SFDT). In paral-
sections inammatory nodules surrounding amorphous lel, there was a steep rise of the anti-BSA antibody response
Fig. 9. Immunoreaction in brain tissue sections after labelling with a polyclonal Toxoplasma antibody (rabHS, brown label) and counterstaining with
haemalaun. (a) Inammatory nodules surrounding amorphous Toxoplasma-positive material (mouse no. 24, autopsy 29 days after a 90-day treatment
cycle; 462); (b) degenerating cyst with invading glia cells (mouse no. 20, autopsy 27 days after Fansidar w treatment; 693).
Fig. 10. Electron micrographs of T. gondii brain cysts in mice autopsied at different times during or after Fansidar w therapy (bars 1.1 mm). (a) Chromatin
aggregation at the border of the bradyzoite nucleus (Q) and multiple, deeply invaginated layers of the cyst membrane (CM). Mouse no. 32, autopsied at day 77
during Fansidar w treatment; (b) large defect (D) within the cyst matrix seen in mouse no. 26, autopsied 13 days after a complete 90-day Fansidar w treatment
(CM cyst membrane).
in all treated Toxoplasma- infected mice within the rst 40

days after Fansidar w application (Fig. 12b). Parasite detec-
tion by PCR was negative in all 13 blood samples examined
(Table 16).
6.1.4. Discussion
As presumed for the human host, an infection of NMRI

mice with the low-virulent `Gail' strain leads to a non-
progressive encephalitis during acute infection and a stable
brain cyst number at medium level during chronic infection
(personal observations). The results of our study demon-
strate a partial efcacy of a low-dose Fansidar w therapy
on the cyst stages of T. gondii which increases with
prolonged therapy. The ndings support the recommenda-
tions of different treatment trials (McAuley et al., 1994;
Roizen et al., 1995) that prenatally infected newborns prot
from a prolonged, continuous treatment.
The reduction of the brain cyst load seen in our study is
within the reported range of other in vivo models. Using a
similar parasite/mouse model but a high-dose, continuous
Fig. 11. Multiplying bradyzoites (endodyogeny, E) within a brain cyst of pyrimethamine treatment (3 150 mg/kg/day) Sarciron et
mouse no. 25, autopsied 92 days after a 90-day Fansidar w treatment period. al. (1998) observed an even more pronounced reduction of
(scale bar, 1.7 mm; CW, cyst wall). the brain cyst number (77%) within 5 days. Similar to our
observation), may vary in different mouse/parasite models.

Our histological studies did not evidence a signicant differ-
ence between treated and untreated animals. However, elec-
tron microscopy revealed degenerating and lysed
bradyzoites which, as suggested previously (Ferguson et
al., 1994), will nally be absorbed by the host cell and
cause a shrinkage of the cyst. Cyst shrinkage seems a possi-
ble explanation for the multimembraneous wall structure
seen after treatment.
In our study, the destruction of more than 50% of brain
cysts within the rst treatment cycle had no effect on the
specic antibody response of the animals. This is surprising
when we assume that extracerebrally located T. gondii cysts
will be destroyed to an even higher degree during Fansidar w
application. The fast anti-BSA antibody response of the
treated mice during the period of cyst destruction on the
one hand and the non-responding Toxoplasma-specic anti-
bodies together with a weak cellular reaction of the cerebral
tissue on the other hand, possibly indicate a specic, immu-
nosuppressive effect of parasitic antigens.
To summarise, a long-term anti-Toxoplasma therapy in
chronically infected mice is only partially efcacious. We
doubt whether a high-dose, more toxic therapy would result
in a complete destruction of cysts. As suggested by Werner
et al. (1979) it is not the cyst membrane (or the intracerebral
location) but the dormant bradyzoite metabolism of selected
cysts which limits the efcacy of the drugs applied.
Fig. 12. Antibody development in mice of group C during and after a 90-
day Fansidar w treatment period. (a) Individual dye test (DT) titers of mice
However, we could prove that cysts and their bradyzoites
nos. 2333. Dilution steps: 4-fold, beginning at 1:64 (0); (b) anti-BSA return to active metabolism when drug pressure is released.
antibody formation in mice nos. 2333. Endodyogeny, which is a rare event in mature cysts was
demonstrated in animals autopsied 13 and 92 days after
cessation of a 90-day Fansidar w treatment. The activation
histological ndings in short- and long-term treated mice, of bradyzoites, however, is limited to intracystic stages and
the cyst reduction was not correlated with an immunological does not result in a reactivation of the disease as demon-
reaction in the brain tissue. An inammatory response of the strated by the negative PCR results and the stable low cyst
cerebral tissue was reported when specic treatment was counts. In children, the rising antibody titres (serological
initiated shortly after Toxoplasma infection (Araujo et al., rebound) seen after cessation of long-term therapy are
1993) or before complete maturation of brain cysts (Fergu- also not correlated with clinical symptoms (Djurkovic-
son et al., 1994) when proliferative stages of the parasite are Djakovic et al., 2000; Coureur et al., 1991) or the presence
still present. of parasites in the peripheral blood (personal observation).
Cysts affected by therapy obviously disappear from brain Whether the intracystic reactivation of T. gondii alone may
tissue fast and absolutely. A previous report described that be responsible for serological rebounds in children could not
there is no difference in the number or the micromorphology be demonstrated by the mouse model due to the non signif-
of mouse brain cysts whether treatment (50 mg/kg pyri- icant changes in antibody titres.
methamine and 500 mg/kg sulfamethoxypyrazine) was Acknowledgements: We thank Gabriela Chioralia for
given for 10 days or 25 days (Werner et al., 1979). This dedicated assistance with electron microscopy. The study
conrms our ndings that it is difcult to demonstrate the was supported in part by a grant from the BMBF to I. R.-
self-destruction of the affected cysts by morphological O. (01KI 9456).
methods when the brain is examined only 34 weeks after
the onset of therapy.
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International Journal for Parasitology 31 (2001) 115144

Recent trends in research on congenital toxoplasmosis q

1 47 Transitory muscular hypertonia by 3 months

1 M 37 3300 32 10 C 3536 P 1 S/R 23 14 None

Cover with spiramycin during the 1st day of life.

Table 3 observed in one infant, and transitory crystalluria occurred

1. Unilateral retinchoroiditis N2 2.1.4. Discussion

1954 Ankara 687 Skin test 40.3

1980 1772 65.0 1.00

prevalence among the human population in eastern Roma- 3.1.3. Results

Year District Character Men Women Total

Tested Positive % Tested Positive % Tested Positive %

1971 Prague 1, 4, 5, 7, 8, 9 Urban 148 6 4.1 160 16 10.0 308 22 7.1

Age-group (years) Men Women

Tested Prevalence (%) Tested Prevalence (%)

Estimate 95% CI a Estimate 95% CI a

04 0.212 (0.178, 0.253) 0.247 (0.149, 0.409)

District No. of inhabitants Cases JanMar 1994 Morbidity per 100000

Brno-city 390112 250 21.36 0.26

Total 1866580 722 12.89 0.91

individuals in each group for accurate analysis when distri-

Brno 183 24 13 1 1 0 0 96 39 279 34.4%

4.1.1. Introduction Table 14

Fig. 7. Seroprevalence in neonates (328 days) and children in the age

Baby Code Age therapy Age Evaluation Signs/symptoms observed a Treatment

Table 16 diaminobenzidine (Liquide-DAB). Slides were counter-

Toxoplasma-positive material (Fig. 9a) or degenerating

in all treated Toxoplasma- infected mice within the rst 40

As presumed for the human host, an infection of NMRI

observation), may vary in different mouse/parasite models.

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