Incidence of congenital toxoplasmosis estimated by neonatal screening: relevance of diagnostic confirmation in asymptomatic newborn infants

Incidence of congenital toxoplasmosis estimated by neonatal

screening: relevance of diagnostic conﬁrmation in asymptomatic

newborn infants

C. G. CARV A L HEIRO, M. M. M U SSI-PI N H ATA*, A. Y. Y AMAM O TO,

C. B. S. D

E SOUZA AND L. M. Z. M ACIE L

Department of Pediatrics, Faculty of Medicine of Ribeira

o Preto, University of Sa

o Paulo, Sa

o Paulo, Brasil

(Accepted 12 November 2004)

SUMMARY

Congenital toxoplasmosis is rarely identiﬁed by routine clinical examination. The aim of this

study was to estimate the incidence of the disease in the region of Ribeira

o Preto, south-eastern

Brazil. A deﬁnitive diagnosis was made on the basis of the persistence of anti-Toxoplasma IgG

antibodies beyond 1 year of age. Blood samples obtained from 15 162 neonates and adsorbed

onto ﬁlter paper were tested for anti-Toxoplasma IgM antibodies. Fifteen samples gave positive

results. A deﬁnitive diagnosis was conﬁrmed in ﬁve of the 13 infants (38

5%) who completed

follow-up. These ﬁve infants presented with serum IgM and/or IgA antibodies, and clinical

abnormalities. Disease incidence was estimated to be 3

3/10 000 (95 % CI 1

0–7

7), indicating the

need for preventive measures. Neonatal screeni ng is feasible, but screening tests with a better

performance are required ; positive screening results must be carefully conﬁrmed.

INTRODUCTION

Congenital toxoplasmosis (CT) is characterized by a

broad spectrum of clinical manifestations at birth,

including varying degrees of neurological, ophthal-

mological and systemic involvement [1]. Although

most newborns are asymptomatic [2], up to 85% may

develop visual disturbances within the ﬁrst two

decades of life, and up to 55 % will show neurological

abnormalities [3, 4]. Several lines of evidence suggest

that the early institution of appropriate treatment

may reduce the frequency of late sequelae [1, 5].

The seroprevalence of toxoplasmosis in pregnant

women and the incidence of CT are well known in

various regions, particular ly in European countries

[6–11]. This knowledge has allowed adequate assess-

ment of the burden of the disease and the planning of

preventive measures.

In Brazil, a large country characterized by hetero-

genous socio-economic conditions and cultural and

hygiene-nutritional habits, studies involving a limited

number of pregnant women from diﬀerent com-

munities have reported high rates of toxoplasmosis

seroprevalence, ranging from 59

8to74

5% [12–16].

However, there have been few investigations that

identify the real incidence of CT in diﬀerent regions of

Brazil. Recent reports have esti mated that the inci-

dence of CT ranges from 3

3to19

6/10 000 live new-

borns [17–19]. In these investigations, conﬁrmation of

the disease was obtained by the detection of anti-

Toxoplasma IgM antibodies in the newborn.

Although this detection has been used for deﬁnitive

diagnosis [1, 5, 20], its use generally requires per-

forming conﬁrmatory testing. A deﬁnitive diagnosi s

would also be possible by the direct detection of the

parasite in clinical samples, by demonstrating the

persistence of anti-Toxoplasma IgG antibodies

beyond 1 year of age and/or by the detection of anti-

Toxoplasma IgA in the ﬁrst 6 months of life [1, 5, 20].

* Author for correspondence : Dr M. M. Mussi-Pinhata, Av.

Bandeirantes 3900, 14049-900, Ribeira

o Preto, Sa

o Paulo, Brasil.

(Email : [email protected])

Epidemiol. Infect. (2005), 133, 485–491. f 2005 Cambridge University Press

doi :10.1017/S095026880400353X Printed in the United Kingdom

The region of Ribeira

o Preto, located in the state of

o Paulo, south-eastern Brazil, is characterized by a

hot climate, favouring the survival of Toxoplasma

oocytes in the soil. The seroprevalence of toxo-

plasmosis in pregnant women in this region was esti-

mated to be 61% in 1999 [12]. These data include

the population of this area in the group at high

risk for CT according to the model proposed by

Frenkel [21].

Considering the lack of information regarding the

incidence of CT in the Ribeira

o Preto region, the

present study aims to estimate the incidence of the

disease through neonatal screening, using as con-

ﬁrmatory criteria the persistence of anti-Toxoplasma

IgG antibodies beyo nd 1 year of age and/or the pres-

ence of elevated titres after termination of treatment.

In addition, clinical and laboratory ﬁndings for the

identiﬁed infants are described.

METHODS

Study population

As part of the obligatory neonatal screening pro-

gramme for phenylketonuria, congenital hypothy-

roidism and haemoglobinopathies, the Screening

Laboratory of the University Hospital of the Faculty

of Medicine of Ribeira

o Preto of the University of

o Paulo (HCFMRP–USP) receives all blood

samples adsorbed onto ﬁlter paper cards collected

within the public health system of the 25 towns in the

region of Ribeira

o Preto. These samples accou nt for

y90% of the 17 000 live newborns delivered per year

in the region. All 15 162 children whose ﬁlter-paper

samples were sent to this laboratory in 2001 were

included in the present study.

The study was approved by the Research Ethics

Committee of HCFMRP–USP. Prior to the study, the

team responsible for sample collection in the 25 towns

in the region received instruction regarding the

characteristics of the investigation. Information con-

cerning the study was provided through informative

posters and leaﬂets distributed to the families at the

time of sample collection. Written, informed consent

for additional assessment and follow-up was obtained

from the guardians responsible for those children who

tested positive on neonatal screening.

Neonatal screening

Blood samples adsorbed onto ﬁlter paper were

collected in hospitals and health centres throughout

the region by trained personnel, using the standard

technique of skin puncture in the calcaneal area.

Samples were generally obtained from children aged

up to 16 days, and stored for a maximum period of 10

days at room temperature. A 3-mm diameter disc was

punched from each ﬁlter paper card, eluted and ana-

lysed for anti-Toxoplasma IgM by a commercially

available, ﬂuorometric, enzyme immunocapture

assay (Neonatal Toxoplasma gondii IgM FEIA

Labsystems, Helsinki, Finland) according to the

manufacturer’s instructions [22]. Samples showing an

optical density o80% of the mean obtained for

borderline controls were considered positive. Positive

results wer e conﬁrmed by repeating the assay using

two aliquots from the same sample.

Complementary assessment of mot her and infant

Children who had conﬁrmed positive results on neo-

natal screening for CT were invited for diagnostic

conﬁrmation. Peripheral blo od was collected from the

mothers and their infants for conﬁrmatory serological

tests. Serum obtained from the child was tested using

a double-sandwich, enzyme immunocapture assay

for the qualitative detection of anti- Toxoplasma IgM

and IgA antibodies (Platelia Toxo

, Sanoﬁ Pasteur,

Marnes-la-Coquette, France) according to the

manufacturer’s instructions. Samples showi ng optical

densities o80% of the control cut-oﬀ provided by

the manufacturer were considered positive. Maternal

and infant serum samples were also submitted to

an indirect ﬂuorescent antibody test (IFA ) for the

quantitative detection of anti-Toxoplasma IgG and

IgM. Brieﬂy, serum was diluted successively up to a

maximum dilution of 1 in 4000 and added to slides

containing ﬁxed Toxoplasma antigens. The slides

were incubated with ﬂuorescein-conjugated human

immunoglobulin anti-IgG or IgM. The antibody titre

was deﬁned as the highest serum dilution at which

ﬂuorescence was observed.

In addition, these children were submitted to a

complete clinical examination, ophthalmological

assessment and cranial ultrasonography and/or com-

puterized tomography. Consequent to any alteration

identiﬁed during the initial clinical assessment, the

children were tested for liver function, complete blood

count, cerebrospinal ﬂuid analysis and instrumental

auditory evaluation (auditory screening). The children

were also screened for congenital cytomegalovirus

infection (dete ction of viral DNA by polyme rase

chain reaction) [23], syphilis (VDRL), and rubella

486 C. G. Carvalheiro and others

(haemagglutination inhibition test). None were in-

fected by these agents.

Follow-up and diagnostic criteria

All infants identiﬁed by neonatal screening were

followed up until their infection status had been

established. Assessments included the detection of

anti-Toxoplasma IgG antibodies by IFA every 2–4

months. A diagnosis of CT was conﬁrmed on the

basis of persistence of speciﬁc IgG antibodies beyond

12 months of age, or the presence of elevated titres

after termination of the antiparasite treatment. Chil-

dren with decreasing IgG titres and who became

anti-Toxoplasma IgG negative were considered to

be non-infected. Treatment was pro vided based on

current recommendations [1, 5] with sulphadiazine,

pyrimethamine, folinic acid and spiramycin being

provided free of charge.

Data analysis

Assuming that the incidence of CT would be 1/3000

births, the sample size necessary to estimate the inci-

dence of infection with a 95% conﬁdence level and a

precision of 0

03% would be 12 639. The incidence

was estimated based on the number of children

showing CT, using the binomial exact 95 % conﬁ-

dence interval (CI) for descriptive studies employing

random samples. Calculations were performed

using the Epi-Info 6 program (CDC, Atlanta, GA,

USA, November 1993). The age at which infant s

became anti-Toxoplasma IgG negative was estimated

as the midpoint of the interval between the last

positive anti-Toxoplasma IgG test and the ﬁrst nega-

tive test.

RESULTS

Anti-Toxoplasma IgM was initially detected in

samples from 101 (0

67%) of the 15 162 infants in-

cluded in the study. This resul t was conﬁrmed in 15

infants (14

85%), 10 (66

7%) being males. The Figure

summarizes the results of the initial evaluation and

the follow-up.

Most infants (73

3%) were from urban areas. Birth

weight ranged from 2060 to 3790 g (median 3120 g),

and gestational age ranged from 32 to 41 weeks

(median 39

5 weeks). The media n age of the 14 infants

evaluated during complementary assessment was 94

days (2–338). All mothers reported regular prenatal

follow-up. However, only two mothers (of infant

nos. 6 and 12) were diagnosed with probable asymp-

tomatic gestational toxoplasmosis due to detection

of anti-Toxoplasma IgM in the serum at 26 weeks

gestation, and to seroconversion at 31 weeks respect-

ively. In the ﬁrst mother, detection of genomic

fractions of the parasite in the amniotic ﬂuid by

polymerase chain reaction (PCR) gave a negative

result. The use of spiramycin until the end of preg-

nancy was prescribed in both cases, but only the

second mother used the medication regularly.

With respect to the initial complementary labora-

tory evaluation, anti-Toxoplasma IgG at titres o1in

4000 were detected in serum samples from all 14

mothers by IFA, with no identiﬁcation of IgM.

Except for one child, initially evaluated at 11 months

of age, IFA detected IgG antibodies in the remaining

13 infants, titres ranging from 1 in 256 to >1in

4000; IgM was not detected. Enzyme immunoassays

for the detection of serum anti-Toxoplasma IgA

and IgM were positive in six of the 14 infants

(42

9%), ﬁve infants being positive for IgM and IgA,

and one infant being positive for IgA and negative

for IgM.

Routine clinical examination showed signs and

symptoms suggestive of co ngenital infection in two of

the 14 infants (14

3%). One infant was not evaluated

further. Complementary asses sment revealed clinical

abnormalities in ﬁve of the 13 remaining infants

(Table). None of the 13 infants was diagnosed with

auditory deﬁciency. All ﬁve symptomatic infants were

15 162

newborns

Neonatal screening

IgM negative

15 147 (99·9%)

Complementary clinical and laboratory assessments

Not performed

1/15 (6·7%)

Not performed

1/14 (7·1%)

Congenital

toxoplasmosis excluded

8/13 (61·5%)

Status of infection

Follow-up

IgM positive

15 (0·1%)

Performed

14/15 (93·3%)

Performed

13/14 (92·9%)

Confirmed congenital

toxoplasmosis

5/13 (38·5%)

Fig. Neonatal screening for toxoplasmosis in children from

the region of Ribeira

o Preto, Sa

o Paulo, south-eastern

Brazil.

Neonatal screening for congenital toxoplasmosis 487

treated for 1 year. Four of the eight asymptomatic

infants received treatment for periods ranging from

15 days to 2 months, followed by withdrawal of

the medication after a documented decline greater

than 10 dilutions in anti-Toxoplasma IgG antibody

titres.

The 13 infants were followed up for a mean period

of 15

9 months (12

6–19

2 months). Five infants ful-

ﬁlled the conﬁrmatory criteria for CT : four of them

(infant nos. 2, 3, 7, 8) maint ained positive IgG titres at

1 year of age, and the other (infant no. 12) was sero-

negative at 12 months of age, but had a sudden rise in

anti-Toxoplasma IgG titres after cessation of therapy.

These ﬁve infants presented abnormal clinical ﬁndings

on complementary assessment, and the presence of

IgA and/or IgM in peripheral blood on ﬁrst evalu-

ation. The remaining eight infants were considered to

be non-infected. In these infants, median age of

becoming anti-Toxoplasma IgG antibody negative

was 10

2 months (5

8–12

8 months), and no signs or

symptoms attributable to CT or positive serological

IgA or IgM tests were observed.

Estimation of the incidence of CT

Considering that ﬁve infants with conﬁrmed CT were

identiﬁed from a total of 15 162 infants submitted to

neonatal screening, the incidence of the disease in the

population studied was estimated to be 3

3/10 000

infants (95% CI 1

0–7

7). Assuming that the two

infants who did not complete the study were also in-

fected (both showed elevated quantitative results on

neonatal screening, and one exhibited IgA and IgM

antibodies in the peripheral blood), the incidence

would be 4

6/10 000 infants (95% CI 1

9–9

5).

DISCUSSION

Based on the results of the present study, the inci-

dence of CT in the Ribeira

o Preto region is estimated

to be 3

3 (95% CI 1–7

7) cases per 10 000 births.

While this rate is similar to that reported in various

Brazilian regions (3

3, 95% CI 2

5–4

4) [17] and in

southern Brazil (8, 95 % CI 0

2–44

5) [19] and to that

found in Norway (3, 95% CI 1

5–5

5) [7], Denmark

(3, 95 % CI 2–4

4) [9], Slovenia (5

1, 95 % CI 2

6–9

[11] and in Poland (5

5, 95% CI 3–9) [24], it is ap-

parently lower than in Rio de Janeiro (19

6, 95% CI

4–45

7) [18], Switzerland (7

2, 95 % CI 4

5–11

[25], Belgium (9

6, 95% CI 5

8–14

8) [26] and France

(y10) [8]. However, the incidence of CT reported for

the North-American region of New England [2],

Sweden [10] and Hungary [6] is <1 case per 10 000

infants. These ﬁndings perhaps suggest epidemiologi-

cal diﬀerences in infection in distinct localities, but the

methodological peculiarities of the diﬀerent study

designs should be considered.

One advantage of the present study design is that

the occurrence of CT in infants identiﬁed by neonatal

screening was conﬁrmed using a criterion considered

to be a true indicator of the presence or absence of

the disease [1, 20], but that has been applied in

only a few previous studies [9, 10, 25]. Studies em-

ploying serological evidence of gestational maternal

Table. Complementary clinical and radiological evaluations of ﬁve children

Child (age in days

at ﬁrst evaluation) Routine clinical examination Positive ﬁndings on complementary assessment

No. 2 (110) Normal Macular scars due to chorioretinitis

No. 3 (104) Normal Temporal scars due to chorioretinitis, strabismus.

Compensated moderate hydrocephalus. Elevated AST

and ALT

No. 7 (98) Hepatosplenomegaly, petechiae,

ascites, jaundice

Turbid vitreous humor with active chorioretinitis in the

left eye, strabismus. Parenchymatous calciﬁcations

No. 8 (67) Normal Macular scars due to chorioretinitis, strabismus. Cranial

US: images suggestive of previous haemorrhage. Cranial

CT: prominence of the supratentorial ventricles. Elevated

CSF proteins

No. 12 (2) Hepatosplenomegaly, jaundice,

microcephaly

Turbid vitreous humor with active chorioretinitis.

Cerebral and cerebellar calciﬁcations. Elevated CSF

proteins. Elevated AST

US, Ultrasound ; CT, computerized tomography ; CSF, cerebrospinal ﬂuid ; ALT, alanine aminotransferase ; AST, aspartate

aminotransferase.

488 C. G. Carvalheiro and others

seroconversion [9, 1 0], in addition to the detection of

IgM and/or IgA in newborns, are potentially

more likely to estimate the true incidence of CT

because they are not limited by the performance of

neonatal screening, which may not detect some of the

aﬀected newborns [9].

In the present study, the use of a commercially

available, ﬂuorometric, IgM enzyme immunocapture

assay developed for the detection of anti-Toxoplasma

IgM in blood samples adsorbed onto ﬁlter paper was

chosen because more sensitive tests were not avaliable

[27]. The retest rate of 0

67% (101/15 162) obtained

after the initial test for detection of anti-Toxoplasma

IgM in dried blood samples was similar to the

frequency of 0

5% reported by others [17]. This ﬁgure

may have been elevated by the low cut-oﬀ level used

in the screening tests, established to assure a high

detection rate.

Even though FEIA performs well compared to

similar assays conducted by experienced laboratories

[22], there are no studies designed to evaluate its

sensitivity and speciﬁcity properly, particularly when

a deﬁnite diagnosis of CT is considered. Previous

studies have reported sensitivities varying from 77

to 100% for IgM detection tests performed on neo-

natal dried blood samples [9, 10, 24], similarly to what

has been shown for assays performed on blood [27] .

In addition, IgM may not be present at birth in

infected children, due to various immunological

mechanisms [1, 28, 29]. Thus, supposing that the

sensitivity of the IgM test did not reach 100%, it is

possible that not all infected infants were identiﬁed,

and that the incidence of the disease may be under-

estimated.

On the other hand, in the present study, 61

5% of

the conﬁrmed positive neonatal screening results were

false-positive. Other investigators, using the same

assay and diagnostic criteria reported 78

6% [10] and

91% [17] false-positive results. A high proportion of

false-positive results is generally accepted for screen-

ing tests, especially in the case of populations with a

low prevalence of the disease [30]. However, an

elevated rate of false-positive results leads to a

marked increase in screening costs, and generates

anxiety and family issues due to the need for complex

investigation and medical follow-up. In addition,

emphasizing the need to follow IgG titres during

and after treatment and to take the occurrence of

serological rebounds into account for deﬁnitive diag-

nosis, it must be observed that infant no. 12, who was

seronegative at 12 months of age, showed a sudden

rise in anti-Toxoplasma IgG titres after cessation of

therapy.

All infants with conﬁrmed CT identiﬁed in this

study had ophthalmological impairment on initial

assessment, and 80% also presented neurological

abnormalities. These ﬁndings contrast with those

reported in other screening studies in which only

11–33% of infants presented such abnormalities, and

4–29%, neurological changes [2, 9, 10, 17, 24]. Even

in studies evaluating mostly symptomatic infants,

y60–80% presented with ophthalmological and/or

neurological impairment [31]. One fact that may

explain the higher percentage of symptomatic infant s

in the present study is the age at which the ﬁrst

complementary examination was performed, i.e. a

median age of 94

5 days. In contrast, other screening

studies examined infants within the ﬁrst 6 weeks of life

[2, 9, 10, 17, 24]. Since ophthalmological impairment

may not be clinically apparent for months or even

years, ocular lesions may have occurred during the

ﬁrst months of life. However, even studies which have

followed infants with CT up to 1–6 years of age have

not reported the presence of ocular lesions in all of

them [32]. Some investigators have tried to establish a

relationship between the Toxoplasma genotype and

the severity of clinical manifestations, with incon-

clusive results [33].

With respect to diagnostic conﬁrmation by sero-

logical test ing, it should be emphasized that, in con-

trast to non-infected infants, serum anti-Toxoplasma

IgA and/or IgM antibodies were detected in all

infants in whom infection was conﬁrmed. This ﬁnding

diverges from reports indicating that the sensitivity of

such antibody tests is lower when mothers are infected

at the beginning of pregnancy [28, 29]. Neonatal

screening, therefore, would not detect infants whose

mothers were infected during early pregnancy, when

the risk of fetal involvement is higher [28], a ﬁnding

not observed in the present study.

In conclusion, the demonstration of an elevated

incidence of CT in the region of Ribeira

o Preto,

Brazil, based on the analysis of a representative

population sample, reveals the need for the institution

of preventive measures, considering that 60% of

aﬀected infants may not have been identiﬁed by

routine clinical examination. The main measure

would be the implementation of informative cam-

paigns for the general population and health-care

professional, to promote the primary prevention of

gestational infection. Neonatal screening by sero-

logical testing of ﬁlter-paper sampl es is feasible and

Neonatal screening for congenital toxoplasmosis 489

may be widely available. However, tests with a better

performance are necessary. Positive screening results

must be carefully conﬁrmed by serological tests and

follow-up, particularly in asymptomatic children.

ACKNOWLEDGEMENTS

This research was supported by FAPESP (Fundac¸ a

de Amparo a

Pesquisa do Estado de Sa

o Paulo). We

are grateful to the staﬀ of the Neonatal Service of

Hospital das Clı

nicas da Faculdade de Medicina de

Ribeira

o Preto da Universidade de Sa

o Paulo.

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