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Transactions of the Royal Society of Tropical Medicine and Hygiene xxx (2010) xxx–xxx

Contents lists available at ScienceDirect

Transactions of the Royal Society ofTropical Medicine and Hygiene

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prospective study of Toxoplasma-positive pregnant women inouthern Brazil: a health alert

ourenco T. Higaa, Silvana M. Araújob, Luiza Tsunetob, Marcela Castilho-Pellosoc,oão L. Garciad, Rosangela G. Santanae, Ana L. Falavigna-Guilhermeb,∗

Department of Medicine/University Hospital, Universidade Estadual de Maringá, 1590 Mandacaru Avenue, CEP 87083-240 Maringá, Paraná, BrazilDepartment of Clinical Analyses, Universidade Estadual de Maringá, Bloco I-90, 5790 Colombo Avenue, CEP 87020-900 Maringá, Paraná, BrazilEpidemiology Sector of the 15th Health District of Paraná, 50 Demétrio Ribeiro Street, CEP 87020-110 Maringá, Paraná, BrazilDepartment of Veterinary Medicine, Universidade Estadual de Londrina, 445 Celso Garcia Cid High Road, CEP 86051-990 Londrina, Paraná, BrazilDepartment of Statistics, Universidade Estadual de Maringá, Bloco 38, 5790 Colombo Avenue, CEP 87020-900 Maringá, Paraná, Brazil

r t i c l e i n f o

rticle history:eceived 16 June 2009eceived in revised form 7 January 2010ccepted 7 January 2010vailable online xxx

eywords:oxoplasmosisoxoplasma gondiiregnant womeniagnosis

a b s t r a c t

We evaluated anti-Toxoplasma gondii IgM-reactive pregnant women seen at a high-riskpregnancy outpatient clinic. From March 2005 to January 2008 in Paraná, Brazil, pregnantwomen seen by the Brazilian Public Health System, in any gestational period, who were anti-T. gondii IgM-positive, were followed. Clinical symptoms were noted, and tests performedincluding IgA, IgG avidity, ultrasonogram, and amniocentesis (PCR/inoculation in mice). Of75 patients, 8 showed low, 3 intermediate and 31 high IgG avidity. Of those who underwentthe avidity test, 31 (70.5%) were in the second trimester of pregnancy. Thirty-two (42.7%)pregnant women received specific treatment. Six received triple combination treatment; inthree, tachyzoites were isolated, although only one was PCR-positive, showing changes inthe cerebral sonogram, borderline IgA, and the Sabin tetrad. One fetus died, and one non-

isk factorsrazil

reactive IgM pregnant woman showed ocular recurrence. The municipality of residence,contact with cats during adulthood, and ingestion of unpasteurized milk were shown to beimportant risk factors. Congenital toxoplasmosis was observed in a pregnancy referred latefor treatment. Follow-up of children born to mothers with diagnosed or suspected acutetoxoplasmosis is crucial in the management of the changes that toxoplasmosis may cause.

Society

© 2010 Royal

. Introduction

In Brazil, the highest rates of toxoplasmosis are found inhe southern and northern states.1,2 In the state of Paraná,he most recent data indicate a prevalence of 66% in adults.3

Severe forms of toxoplasmosis can be observed in new-

Please cite this article in press as: Higa LT, et al. A prospective stBrazil: a health alert. Trans R Soc Trop Med Hyg (2010), doi:10.1

orns with congenital infections.4,5 Fetal lesions can leado subclinical forms of infection, development of chori-retinitis, microphthalmus with strabismus, damage tohe central nervous system or intrauterine death.6 These

∗ Corresponding author. Tel.: +55 44 3261 8987; fax: +55 44 32614860.E-mail address: alfguilherme@uem.br (A.L. Falavigna-Guilherme).

035-9203/$ – see front matter © 2010 Royal Society of Tropical Medicine and Hoi:10.1016/j.trstmh.2010.01.006

of Tropical Medicine and Hygiene. Published by Elsevier Ltd. Allrights reserved.

manifestations are directly related to the virulence of theparasite, and to the mother’s immune response and stageof pregnancy.7 The vertical transmission rate is directlyproportional to the stage of pregnancy when the motheracquired the primary infection.8 The chances of complica-tions and death decrease as the gestational age at initialinfection advances.9

The primary infection in pregnant women is usuallyasymptomatic, and the mother’s diagnosis in the acute

udy of Toxoplasma-positive pregnant women in southern016/j.trstmh.2010.01.006

stage depends on laboratory tests.5 It is widely agreedthat anti-Toxoplasma gondii immunoglobin M (IgM) is anexcellent marker for the initial evaluation of expectantmothers at public health services.10 However, this shouldnot be employed as the sole diagnostic tool, because

ygiene. Published by Elsevier Ltd. All rights reserved.

INy of Trop

ARTICLEG ModelTRSTMH-1335; No. of Pages 6

2 L.T. Higa et al. / Transactions of the Royal Societ

individuals can remain positive for months or even yearsafter infection.5,11 The combination of serological, parasito-logical and molecular techniques is important for a definitediagnosis of acute infection and/or fetal transmission.12–14

Although there is no consensus as to itsdesirability,9,15–16 anti-parasite treatment of the expectantmother and early diagnosis of congenital toxoplasmosiscan mitigate the risk of transmission or sequelae in thefetus, if initiated early,17 even considering the adverseeffects of the therapy and the risk that the mother willdiscontinue the treatment.

In Brazil, the combination of different techniques isseldom applied, except in laboratories associated with aca-demic or research institutions.18 Furthermore, expectantmothers with presumed T. gondii infections are not alwaysadequately diagnosed and monitored.18 It is necessary toemphasize the statistics regarding the consequences anddamage that the pregnant women and their conceptuseswith suspected toxoplasmosis have suffered, even in thegreat regional centers of Brazil. This study aims to alertthe scientific community that generates the knowledgebase for decision-making to the problematic situation ofthe majority of health services in the country, where preg-nant women and their conceptuses with suspected T. gondiiinfection do not receive systematic care, principally withregard to diagnosis, follow-up and treatment. We also aimto alert the community to the risk to pregnant women whoare seronegative for toxoplasmosis and are seldom moni-tored. In this study, we aim to prospectively evaluate anti-T.gondii IgM-reactive pregnant women who were seen at ahigh-risk pregnancy outpatient clinic at a teaching hospitalin southern Brazil.

2. Materials and methods

2.1. Study patients

The study included expectant mothers attended by thepublic health services, in any trimester of pregnancy, withanti-T. gondii IgM reactive status, who were referred bythe municipal health authorities of the 30 cities that makeup the 15th Health District of the State of Paraná, to theHigh-Risk Pregnancy Outpatient Clinic at the Regional Uni-versity Hospital of Maringá (HURM/UEM), Maringá, Paraná,Brazil, between March 2005 and January 2008. All expec-tant mothers signed the consent form, giving their free andinformed consent.

2.2. Anti-Toxoplasma gondii antibody research

The measurement of IgM and IgG antibodies wasrepeated at the UEM Teaching and Research Laboratory orby the Paraná State Central Laboratory for Public Health, inorder to standardize the result according to the diagnos-tic kit and the technical execution, using the microparticleenzyme-linked immunoassay (MEIA) method in an auto-

Please cite this article in press as: Higa LT, et al. A prospective stBrazil: a health alert. Trans R Soc Trop Med Hyg (2010), doi:10.1

mated Axsym system (Abbott Laboratório do Brasil Ltda,São Paulo, Brazil). The results were interpreted accord-ing to the criteria established by the manufacturer, whichconsiders an IgM index ≥0.600 as reactive, 0.500–0.599 asundetermined, and ≤0.499 as non-reactive. Values for IgG

PRESSical Medicine and Hygiene xxx (2010) xxx–xxx

≥3 IU/ml were considered as positive, between 2 and 3IU/ml as undetermined, and ≤2 IU/ml as negative.

To assess the anti-T. gondii IgA antibodies, the ETI-TOXOK-reverse PLUS manual capture immunometric ELISA(Diasorin, Saluggia, Italy) was used. Results >2.0 were con-sidered as reactive, between 0.5 and 2.0 as borderline, and<0.5 as non-reactive.

The IgG avidity test was performed by the Paraná StateCentral Laboratory, using the enzyme linked fluorescentassay (ELFA; Vidas/Biomerieux, Marcy l’Etoile, France). Theinterpretation of the results was as follows: <20.0%, lowavidity; between 20.0 and 29.9%, intermediate avidity; and≥30.0%, high avidity.

2.3. Toxoplasma gondii in amniotic fluid

Ten millilitres of amniotic fluid (AF) was collected underultrasonography from IgM-reactive pregnant women withlow IgG avidity, between the second and third trimestersof pregnancy. In order to eliminate the possibility of con-tamination of the amniotic fluid by tachyzoites from theplacenta, two syringes were used: the first collected 2 mlof AF to be discarded, and the second removed 10 ml to beused. Of this volume, approximately 5 ml was centrifugedfor 10 min at 3000 rpm. The remainder was discarded,except for 2 ml. Of this, 0.3 ml was used for fresh exami-nation and Giemsa staining. The remainder was inoculatedintraperitoneally into five male Swiss mice, 6–8 weeksold, which were periodically examined for the presenceof tachyzoites. In negative groups, the mice were killedby ether after 30 days, and parts of the lung, brain, liverand peritoneal fluid were removed. The organ fragmentswere macerated, resuspended in saline, added to strepto-mycin and penicillin, and inoculated into five other mice, toconfirm the results. This process was repeated five times.On the second pass, indirect immunofluorescence was per-formed on serum from each mouse, for the anti-T. gondii IgGassay.

The remaining amniotic fluid was used for moleculardiagnosis (PCR), according to Fuentes et al. (1996).19 ForDNA extraction, the QiAamp DNA Kit (Sample & Assay Tec-nologies, Qiagen, Valencia, CA, USA) was used, accordingto the manufacturer’s recommendations. Oligonucleotideprimers 694–715, 5′-GGAACTGCATCCGTTCATGAG-3′ and887–868, 5′-TCTTTAAAGCGTTCGTGGTC-3′, which amplifythe fragments of the B1 gene in T. gondii, were used.20

A single T. gondii parasite can be directly detected fromcell lysate materials by PCR using the 35-fold-repetitive B1gene as a target for amplification. With 100 000 humanleukocytes and DNA extraction, as few as 10 organismswere detected by using the B1 gene as a target for amplifi-cation.

2.4. Ultrasonograms and treatment

The expectant mothers were monitored monthly

udy of Toxoplasma-positive pregnant women in southern016/j.trstmh.2010.01.006

through an obstetric ultrasound examination (Philips EnVi-sor, Riverside, CA, USA); if they showed low or intermediateIgG avidity, they were monitored every other week. Dila-tion of lateral brain ventricles, intracranial calcification,placenta thickness, hepatosplenomegaly, and restricted

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ARTICLEG ModelRSTMH-1335; No. of Pages 6

L.T. Higa et al. / Transactions of the Royal Societ

ntrauterine growth were used as suggestive markers forongenital T. gondii infection.6

Women with positive amniocentesis results for T.ondii (by inoculation in mice and/or PCR) and who wereetween the 14th and 34th week of gestation were treatedith the triple combination of sulphadiazine (3.0 g/day),yrimethamine (100 and 50 mg/day), and folinic acid15 mg/day), alternating with spiramycin (3.0 g/day). Fromhe 35th week on, they received spiramycin (3.0 g/day).regnant women with IgM reactivity, high and low IgGvidity received spiramycin (3.0 g/day) until the end ofestation.

.5. Clinical, laboratory and ophthalmologicalxaminations of the newborns

At birth, babies from mothers who had received theriple combination treatment were clinically examined andiven an ophthalmological examination by fundoscopy,nd an IgM/IgG serological investigation, using ELISA MEIA.

.6. Risk factors associated with Toxoplasma gondiinfection in pregnant women

Data were recorded for age, educational level, place ofesidence (rural or urban), contact with cats, exposure toarbage and waste, management of soil, consumption ofntreated water, ingestion of unpasteurized milk, freshheeses, raw eggs, poorly cooked meat, or fresh vegetablesnd fruit.

.7. Statistical analysis

The statistical comparison of epidemiological factorsas conducted between two groups of pregnant women:

he non-reactive IgM and IgG anti-T.gondii group and thegM-reactive pregnant women treated with spiramycin orhe triple scheme. Fisher’s exact test and logistic regressionere used, with a significance level of 5%.

. Results

Of the 30 municipalities that compose the 15th Healthistrict of Paraná, 17 referred pregnant women suspectedf toxoplasmosis to the HURM/UEM High-Risk Pregnancyutpatient Clinic.

.1. Seroprevalence of Toxoplasma gondii in pregnantomen

During the study period, 420 women were seen athe HURM/UEM High-Risk Pregnancy Outpatient Clinic. Ofhese women, 81 were referred with suspected acute toxo-lasmosis and 75 were confirmed as IgM-reactive. From the

atter group, 42 (56.0%) underwent the IgG avidity test: twoere in the first trimester and showed low avidity; 29 in the

Please cite this article in press as: Higa LT, et al. A prospective stBrazil: a health alert. Trans R Soc Trop Med Hyg (2010), doi:10.1

econd trimester (six with low, two with intermediate and1 with high avidity) and 11 in the third trimester (two with

ow, one with intermediate and eight with high avidity).Of 42 pregnant women who were T. gondii IgM-reactive

nd had the IgG avidity test, the anti-T. gondii IgA assay

PRESSical Medicine and Hygiene xxx (2010) xxx–xxx 3

was carried out in 31. Of these, 10 showed low avidity andreceived chemotherapy (eight IgA-reactive, two border-line), three expectant woman showed intermediate avidity(one IgA reactive, two borderline). The IgA-reactive womenand one IgA-borderline woman received chemotherapy.

Of the 31 pregnant women with high avidity (one IgA-reactive, one borderline for IgA, 16 non-reactive for IgA, 13not tested for IgA), eight were treated, one of whom wasIgA-reactive, five were not reactive and two had no IgA test.

Four categories of pregnant women received treatment:(a) seven of the eight expectant mothers with low or inter-mediate avidity and IgA-reactive; (b) three of the fourpregnant women who did not undergo the avidity test andhad reactive or borderline IgA; (c) eight women who didnot participate in the avidity survey and with non-reactiveIgA; and (d) seven expectant mothers with high avidity,five of them with non-reactive IgA and two without theIgA test.

3.2. Laboratory data and treatment of pregnant women

Appropriate treatment was administered to 32 expec-tant mothers (42.7% of the IgM-reactive women). Theprescription and adoption of the treatment did not fol-low a standard protocol. For example, some pregnantwomen arrived at the High-Risk Pregnancy OutpatientClinic already taking spiramycin.

Six women were treated with the triple combination(Table 1). Of these, three were diagnosed with low avidityand reactive IgA; one was a late arrival into the High-Risk Pregnancy Outpatient Clinic and progressed to fetaldeath (case 5), and in another it was possible to diagnosethe parasite by inoculation of mice. This woman (case 2)showed high avidity and was IgA-reactive, and her testfor Toxoplasma was positive in mice (Table 1). In case 1(Table 1), the avidity test was not performed, but the IgAwas borderline, PCR and mice tests were positive, andthe infant showed the Sabin tetrad. Among the expectantmothers treated with the triple combination, there was onesevere case of acute exacerbation of ocular toxoplasmo-sis.

3.3. Risk factors associated with Toxoplasma gondii

A comparison of epidemiological factors was conductedamong 19 pregnant women who were non-reactive to IgMand IgG and 32 who were IgM-reactive and undergoingtreatment. All the pregnant women were living in urbanareas. Those not residing in the municipality of Maringáwere 5.8 times more likely to be IgM-reactive (P = 0.008).Those who had contact with cats during adulthood were19 times more likely to be IgM-reactive (P = 0.003). Preg-nant women who consumed unpasteurized milk were 9.5times more likely to be IgM-reactive (P = 0.027). The inges-tion of cheese (P = 0.534), unfiltered water (P = 1.000), eggs(P = 0.673), or raw or poorly cooked meat (P = 0.234); age

udy of Toxoplasma-positive pregnant women in southern016/j.trstmh.2010.01.006

(P = 0.7419); educational level (P = 0.650); management ofsoil (P = 1.000); exposure to garbage and waste (P = 1.000);contact with soil (P = 1.000); or ingestion of fresh vegeta-bles and fruit (P = 0.376) showed no statistical differencebetween the two groups.

Please cite this article in press as: Higa LT, et al. A prospective stBrazil: a health alert. Trans R Soc Trop Med Hyg (2010), doi:10.1

ARTICLE ING ModelTRSTMH-1335; No. of Pages 6

4 L.T. Higa et al. / Transactions of the Royal Society of Trop

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PRESSical Medicine and Hygiene xxx (2010) xxx–xxx

4. Discussion

The difficulty in obtaining definite diagnostic and treat-ment tools, together with the decision-making delays in thepublic health system, were all apparent in this investiga-tion as aggravating factors in the outcome for IgM-reactivewomen. A previous retrospective study in the same regionwarned of the risk to expectant mothers with serologysuggestive of acute toxoplasmosis, which is not alwaysadequately monitored.18 During the period of the presentstudy, the different health departments were periodicallyalerted to refer anti-T. gondii IgM-reactive expectant moth-ers to the clinic. However, only 56.3% of municipalitiescomplied with this request.

Most expectant mothers were referred to the High-Risk Pregnancy Outpatient Clinic starting in the secondtrimester, i.e. after the 16th week of the pregnancy,which is considered very late for diagnostic evaluationand intervention in pregnant women at risk for verticaltransmission.6

Considering the result of the avidity test for IgG, IgAassay, PCR or parasite isolation, the specific treatment wascontinued for those women who were already taking themedications, and was prescribed for those whose diagno-sis of the acute phase was confirmed. The administrationof spiramycin was continued because the mother or fam-ily did not agree to interrupt a treatment prescribed by atrusted family doctor. These situations illustrate the irreg-ularity of the preventive treatment with spiramycin. Thisstudy documented the unnecessary treatment of pregnantwomen who showed high avidity and non-reactive IgA,burdening the public health system and greatly inconve-niencing the patient, and once again revealed the needto formulate guidelines and disseminate scientific knowl-edge on the subject. The establishment of reference centersfor diagnosis, follow-up and treatment of toxoplasmosis inexpectant mothers, such as those in France,6 is one wayto improve the process. However, Brazil’s vast territorymust be taken into account in this process, which requirescommunication and prompt decisions beginning from thetime of the first consultation at basic healthcare units, withdirect and interconnected contact with the referring ser-vice.

Almost all the expectant mothers with low or interme-diate avidity and reactive or borderline IgA underwent thetreatment. This decision was prudent, because these anti-bodies can persist for months after an acute infection.21

IgA has more dynamic formation and disappearance kinet-ics than IgM, and its presence indicates that the infectionwas acquired less than 8 months prior to detection.6,22

The inoculation in mice, despite the delay in the result,was fundamental to inform the conduct and prognosisin some cases. Nevertheless, it must be considered thatthis is an academic method that involves the creation andmaintenance of a dedicated facility, with operational costsincluding specially qualified technicians, which would

udy of Toxoplasma-positive pregnant women in southern016/j.trstmh.2010.01.006

limit its viability under current conditions in Brazil.Amniocentesis was generally refused by the expectant

mothers because of the associated risks, which explains thesmall number of PCR results and inoculation in mice. ThePCR from amniotic fluid was negative for two expectant

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ARTICLEG ModelRSTMH-1335; No. of Pages 6

L.T. Higa et al. / Transactions of the Royal Societ

others (one with high avidity and another with lowvidity) despite the isolation of T. gondii strains in mice.he sensitivity of PCR varies from 42.9% if the infectionccurs between the 4th and 16th week, 92.9% betweenhe 17th and 21st week, and 61.7% after the 22nd week ofregnancy.23 This sensitivity can be compromised depend-

ng on the primer used, considering the several genotypesf T. gondii and the time when the amniocentesis iserformed. The reference laboratories of the Europeannion have only recently adopted standardized PCR proto-ols, thus reducing false-negative/positive results.24 Evenhough the sensitivity of this technique varies over the ges-ational period, the accuracy of the result can be improvedy developing primers with T. gondii from each region.

Some expectant mothers were given triple combinationreatment, and even so there was one case of congenitaloxoplasmosis, severely affecting the neonate. This out-ome suggests that seroconversion must have occurred athe beginning of the pregnancy. The woman was referredo the High-Risk Pregnancy Outpatient Clinic during theast trimester of her pregnancy, making earlier diagnosisnd treatment impossible, which would have limited theequelae. This case again raises the issue, not only of aware-ess among expectant mothers who use the public healthystem, but also of the previously mentioned complexity ofoxoplasmosis and the slow response of the health services.

The remaining infants were born apparently normal,ith negative IgM for T. gondii. However, it is important toighlight the urgency of creating adequate follow-up pro-edures, because congenital infection can lead to sequelaeven years after exposure.6

All IgM-reactive mothers were asymptomatic, exceptor one non-IgM reactive with recurrence of ocular tox-plasmosis, in which the triple combination treatmentesulted in a significant improvement. Ocular toxoplasmo-is is usually the result of a recurring illness, regardless ofhether it is acquired or congenital.25

Different epidemiological factors may contribute to pri-ary infection of pregnant women in different regions.2,6

hose in this study had a greater chance of developingcute toxoplasmosis if they had contact with cats duringdulthood and/or consumed unpasteurized milk. The otherpidemiological factors investigated, such as ingestion ofnfiltered water or poorly cooked meat, did not signifi-antly affect the risk of contamination by T. gondii. Thoseregnant women living in the city of Maringá, the largestity in the region, were less likely to acquire toxoplasmosis,robably due to the greater access to goods and services,hich is typical of the planned cities of the country and the

dequate quality of life in these centers.The fragmentation of assistance, the damage to new-

orns which in many situations will only become apparentears later, the lack of mandatory notification and non-ompliance with some existing protocols all compromiseecision-making and lead to inadequate medication orxposure of the fetus. This adverse situation can become

Please cite this article in press as: Higa LT, et al. A prospective stBrazil: a health alert. Trans R Soc Trop Med Hyg (2010), doi:10.1

emedied through the creation of reference centers foriagnosis, follow-up and treatment of toxoplasmosis

n expectant mothers, and through the implementa-ion of efficient communication among public healthnits.

PRESSical Medicine and Hygiene xxx (2010) xxx–xxx 5

Authors’ contributions: ALFG designed the study; MCPrecruited the participants; LTH carried out the clinicalassessments and interviews; JLG analyzed the PCR; LT ana-lyzed the serological tests; RGS carried out the statisticalanalyses; SMA and ALFG analyzed and interpreted the data;LTH and ALFG drafted the manuscript. All authors revised,read and approved the final manuscript. LTH and ALFG areguarantors of the paper.

Acknowledgements: We thank all the expectant mothersand their children for participating in this study.

Funding: Fundacão Araucária, State of Paraná, Brazil.

Conflicts of interest: None declared.

Ethical approval: The study was approved by the Perma-nent Ethics Committee for Research Involving Humans,Universidade Estadual de Maringá (UEM), Paraná, Brazil(Protocol no. 159/2004-COPEP), and the UEM Ethics Com-mittee for Animal Experiments (decision no. 034/2004).

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