Since the discovery of Toxoplasma gondii (T.gondii) by Alfonso Splendore in 1908, many studies have attempted to explain its mechanisms of transmission and the host immune response to the parasite, to discover drugs to inhibit its proliferation, and to develop excellent diagnostic techniques using highly sensitive and specific immune and bio-molecular methods [1–6]. The rates of transmission and clinical manifestation of congenital toxoplasmosis vary sharply in newborns (NB, and depend on the immune response of the fetus to the parasite, the gestational age when infection took place, the parasite load in the fetal circulation at the time of infection, the genotype of the parasite, and the specific treatment administered to the pregnant patient [1, 2]. Clinical follow-up of cases of suspected congenital transmission is very difficult once the pregnant woman has been treated with spiramycin, which affects the identification of the parasite in the NB [1, 2]. In addition, tests for anti-T.gondii IgM and IgA have poor sensitivity in fetuses and NB [6–9]. Most infected NB (60%) are asymptomatic [1, 2]; however, they may develop severe sequelae such as blindness or mental retardation if not treated [1, 2, 10–12].

It has not yet been conclusively proven that maternal treatment reduces the risk of fetal infection [3, 13–19], although some studies have demonstrated a beneficial effect of treatment [20–27]. Moreover, treatment with a combination of pyrimethamine and sulphadiazine is thought to be more effective than spiramycin treatment for reducing the risk of clinical manifestations in infected children [1, 2, 20–27]. There are conflicting views in the literature regarding the effectiveness of spiramycin treatment when the fetus has already been infected [1, 2]. However, many authors believe that spiramycin is able to reduce the severity of fetal infection by delaying the onset of fetal disease and thus allowing greater maturation of the fetal immune system [28–30].

A number of studies have evaluated the performance of polymerase chain reaction (PCR) using amniotic fluid samples for the diagnosis of fetal infection [31–35]. In addition, in some countries cerebrospinal fluid (CSF) is the most frequently used diagnostic sample [1], while in others placental samples are used [36]. However, few studies have attempted to evaluate the sensitivity of PCR testing of the peripheral blood of NB congenitally infected with T.gondii[37]. Considering the difficulty of diagnosing congenital toxoplasmosis and the low frequency of clinical manifestations in infected NB and infants, the purpose of this study was to evaluate the influence of prenatal spiramycin treatment on the sensitivity and specificity of diagnostic tests, and its effects on clinical manifestations of the disease in NB.

Congenital toxoplasmosis was confirmed in 61.4% (27/44) of the NB in group 1 and 79.2% (19/24) of the NB in group 2. Women from group 1 had received prenatal treatment for toxoplasmosis and almost all of these women had undergone serological testing during the first trimester because of suspicion of acute toxoplasmosis. The women from group 2 who had been seronegative for toxoplasmosis during first trimester screening had probably seroconverted during the 2nd or 3rd trimester. Of the mothers in group 2 who gave birth to infected NB, 89.5% (17/19) had been seronegative for T. gondii during the first 3 months of pregnancy and 10.5% (2/19) had not undergone prenatal toxoplasmosis testing. None of these seronegative pregnant women were screened for seroconversion during pregnancy; and the patients reported that they had not been informed of prophylactic measures to prevent infection by T.gondii.

The presence of serological markers for toxoplasmosis was not associated with greater severity of congenital infection.

In Goiânia (2003), Avelino et al. [40] found one of the highest rates of toxoplasmosis seroconversion during pregnancy described in the literature (8.6%). This result led to the introduction of a public program to control toxoplasmosis during pregnancy in the state of Goiás. Although the treatment of toxoplasmosis in pregnancy is not followed by a reduction in its transmission to the fetus [13–16], it has been shown that treatment can reduce the severity of fetal infection [17–27] as we found in this study. However, in our study as in others [11, 14, 18], some children born to treated mothers developed ocular or neurological sequelae to congenital infection. However, severe clinical manifestations of congenital infection were found only among children born to untreated women (Table 2).

Our results highlight flaws in the implementation of primary prophylactic measures for seronegative women at risk of becoming infected by T. gondii. Of the mothers in group 2, 89.5% gave birth to infected children. Moreover, they were not informed of possible prophylactic measures. In France, implementation of a prevention program and screening of pregnant women led to a reduction of symptomatic neonates [24]. Preventive measures should be taken throughout prenatal care (by doctors or the healthcare professional team). Additional information on how to prevent the occurrence of acute infection during pregnancy should be provided. Simple measures such as avoiding undercooked meat, eggs and raw vegetables; not drinking untreated water or unpasteurized milk; using hygienic gardening practices; and limiting interaction with cats can help to prevent infection [41]. Failure to provide information on primary prophylaxis measures, and the lack of seroconversion surveillance during prenatal care, were responsible for the high incidence of congenital infection among NB born to the initially seronegative pregnant women of group 2. There is a high possibility of contact between pregnant women and the sources of the parasite in our environment [40]. In addition to failure of primary prophylaxis, the initially seronegative women in this study did not undergo the seroconversion surveillance by monthly serologic screening that is indicated in regions with a high prevalence of toxoplasmosis [1, 2, 42, 43]. Thus, many women were acutely infected but not properly diagnosed and treated.

Of the children in group 2 (untreated mothers), 68.4% (13/19) were born with severe clinical conditions (Table 2). In contrast, only 29.6% of the NB in group 1 were symptomatic and none had severe clinical disease. Thirteen children required tertiary prophylactic measures as their mothers had been untreated. They needed to be followed by a multidisciplinary team and the cost was estimated to be exorbitant for the government program. According to Remington et al. [1], such costs could reach a million US dollars during the lifetime of a patient. This could have been alleviated if those women had followed prophylactic measures against T.gondii and had also undergone seroconversion surveillance. Only 10.5% (2/19) of the untreated women had not undergone prenatal care.

The higher percentage of infants without clinical symptoms in group 1 (70.3%) compared to group 2 (31.5%) suggests that maternal treatment with spiramycin reduced the fetal load of T.gondii, thus minimizing the sequelae of congenital toxoplasmosis infection [28–30]. Research conducted by Andrade et al. [44] showed that 60% of infants born to treated mothers were asymptomatic, which is similar to our results. However, NB outcomes after maternal spiramycin treatment are variable within the literature, with results ranging from no benefit [13–15] to reduced severity of congenital infection [17–27]. A European multicenter study [14] concluded that spiramycin was of no benefit in reducing the severity of fetal infection. However, recent studies have shown that early treatment can interfere with the transmission of infection, and decrease the severity of congenitally acquired toxoplasmosis [23–25]. A review by Wallon et al. [3] reported conflicting results within the literature; five studies showed that maternal treatment reduced the severity of vertical transmission, but this was not confirmed in another four studies. Still other studies, such as that by Foulon et al. [13], have shown that early treatment was able to reduce the severity of the clinical consequences of congenital infection, but not vertical transmission to the fetus. Hohlfeld et al. [12] demonstrated that treatment slightly reduced the severity of congenital infection.

For the infected children whose mothers were not treated it was not possible to determine when maternal infection took place. Severe neurological lesions were observed only among the children of untreated mothers, suggesting that maternal treatment contributed to a decrease in the development of severe neurological lesions. The same result was shown by the European Multicenter Study [45]. However, Cortina-Borja et al. [46], found no reduction in the severe neurological sequelae affecting children of acutely infected mothers, regardless of whether maternal treatment consisted of spiramycin or the combination of pyrimethamine and sulfadiazine. Those authors report that 31% of the children born to mothers acutely infected by T. gondii during pregnancy had severe neurological sequelae. This is a higher percentage than reported in regions that do not have preventive government programs, indicating a more aggressive strain of toxoplasmosis circulating in this environment [46].

The severity of the infection was greatest in NB from group 2; 68.4% were symptomatic at birth and developed severe manifestations of congenital toxoplasmosis: 4 (30.8%) had neuro-optical lesions, 2 (15.4%) blindness, 5 (38.5%) hydrocephalus, 2 (15.4%) microcephaly, and 1 (7.7%) systemic toxoplasmosis. Gilbert et al. [26], showed that the risk of being born with clinical signs of congenital infection, or developing signs until the age of three years, is lower in countries in which intensive treatment of pregnant women is performed, such as in Austria.

Olariu et al. [47] reported a clinical picture as severe as that found in Goiania for untreated children. One or more severe clinical manifestations of congenital toxoplasmosis were reported in 84% of the affected infants. These manifestations included eye lesions (92.2%), brain calcifications (79.6%), and hydrocephalus (67.7%). In 61.6% of the infants, eye lesions, brain calcifications, and hydrocephalus were present simultaneously. Soares et al. [48] found that 72.4% of patients appeared asymptomatic at birth, but 34.5% had chorioretinitis, 32.3% had intracranial calcification, and 42.9% had neuromotor delay.

Hearing dysfunction was observed in five NB in our study, 4 (14.8%) from group 1 and one (5.3%) from group 2 (Table 2). Andrade et al. [44] found auditory dysfunction in six children with congenital toxoplasmosis, two of whom were born to mothers who were properly treated for toxoplasmosis during pregnancy. These data suggest that congenital toxoplasmosis, even in the absence of other clinical manifestations, should be considered in the evaluation of children with hearing loss, and that maternal treatment does not exclude the possibility of developing this dysfunction. Brown et al. [49] reported hearing impairment in 28% of children born to untreated mothers; however, in our study there was a higher incidence of hearing loss in the treated group.

The sensitivity of serological diagnostic markers of congenital toxoplasmosis in NB (T. gondii-specific IgM and IgA antibodies and PCR evidence of T. gondii DNA) reported in the literature is low, with independent studies reporting different results (1,2,6–9,32–36). Diagnosis seems to be adversely affected by maternal treatment as reported by Gilbert et al. [9]. In our study, Toxoplasma gondii was not detected by PCR in 96.3% (26/27) of samples from NB born to treated mothers, indicating that spiramycin treatment reduced the sensitivity of this technique. A similar phenomenon was reported by Rodrigues et al. [50], Fricker Hidalgo et al. [51] and Bessières et al. [8]. There was also low PCR sensitivity (31.6%) in group 2 NB, suggesting that peripheral blood may not be the best biological sample to use for the diagnosis of congenital toxoplasmosis. Sterkers et al. found an even lower percentage (21.2%) of PCR-positive congenitally infected NB in 2012 [31], and in 2001 Bessières et al. reported levels similar to those we found in this study [8]. The identification of the parasite in the amniotic fluid is easier than its detection in the NB, thus the sensitivity of the diagnostic technique (either PCR or mouse inoculation) is greater when there is intrauterine parasitemia. Spalding et al. [52] suggest that the low sensitivity of PCR for the diagnosis of NB with congenital T. gondii infection relative to its high sensitivity when used to test amniotic fluid is due to the transient persistence of T.gondii in peripheral blood [9, 33–35]. However, according to Okay et al. [53], the amniotic fluid from only 40.47% of congenitally infected NB was PCR-positive [53]. However, despite these difficulties, PCR can be highly sensitivity (around 90%) if it is used to test amniotic fluid collected close to the time that the pregnant woman seroconverted, which is the time of acute fetal infection [1, 2, 9, 32–35]. Bessières et al. [8] found a PCR sensitivity of 43% when using umbilical cord blood, which is higher than the sensitivity that we report for peripheral blood.

The sensitivity of specific anti-T.gondii IgM and IgA antibodies in NB of both groups (43.5 and 21.7%, respectively) was similar to that observed in other studies. Pinon et al. [7] found the sensitivity of anti-T.gondii IgM and IgA in NB samples to be approximately 25%. Naessens et al. [54] reported a sensitivity of 40% for IgM. Bessières et al. [8] found greater sensitivities for IgA (60%) and IgM levels (50%) than those found in this study. Foulon et al. [13] found positive results for IgA (58%) and IgM (54%) using the enzyme-linked immunosorbent assay (ISAGA). In a study involving 14 laboratories supported by the European Community Biomed 2 program, Pinon et al. [55] evaluated immunologic methods for the postnatal diagnosis of congenital toxoplasmosis and compared ELFA with a commercial enzyme immunoassay (EIA) or in-house immunosorbent agglutination assay (ISAGA) for the detection of IgM or IgA. The results were highly sensitive when the techniques were combined.

Treatment of pregnant women with spiramycin did not interfere, from a statistical viewpoint, with the sensitivity of specific anti-T.gondii IgM and IgA antibodies in NB within the 2 groups (Table 1). This is similar to the findings of other studies [8]. However, the sensitivity was higher (68.4% for IgM and 36.8% for IgA) in infants from group 2, similar to the findings of Lebech et al. [56]. It is likely that maternal treatment reduced the parasite load transmitted to the fetus, consequently reducing antigenic stimulation and the fetal humoral immune response. This, along with delayed fetal disease, may be one of the reasons for the reduced incidence of neuro-optical lesions and hydrocephalus in babies born to treated mothers.

Detection of specific anti-T.gondii IgA did not improve early diagnosis of congenital toxoplasmosis in the study group, because in 90% (9/10) of the infants with anti-T. gondii IgA, IgM was also present. However, IgA seemed to be the marker with the poorest prognosis for the congenital infection, as it was present in all NB who developed the neuro-optical form of toxoplasmosis (8.7% or 4/46). The presence of other serological markers for toxoplasmosis was not associated with greater severity of congenital infection.

The high specificity of the laboratory markers for congenital toxoplasmosis (PCR, specific IgM and IgA) observed in this study reinforces the need for such procedures to be performed in the routine diagnosis of congenital infection. They add parameters to confirm the diagnosis and need for subsequent treatment, which uses toxic drugs in patients suspected of congenital infection.

The effectiveness of treating pregnant women in order to prevent fetal infection is highly controversial [12–27]; however, our results suggest that treatment of the mother reduces the severity of fetal infection. The poor prognosis observed in infected group 2 NB suggests that the guidelines of the Program for Congenital Infection Control from France [57, 58] should be followed, including monthly serologic testing throughout pregnancy for seronegative patients and a focus on earlier medical treatment using sulfadiazine, pyrimethamine and folinic acid instead of spiramycin [1, 2, 12, 18–27] in case of proven fetal infection. Earlier diagnosis and treatment may prevent vertical transmission and the treatment of pregnant women identified as having recent toxoplasmosis can reduce the severity of the fetal infection [24, 25].

Spiramycin treatment of women with acute toxoplasmosis did not interfere significantly with the detection of laboratory markers of congenital toxoplasmosis. Treatment did reduce the frequency of the clinical manifestations in the NB, minimizing the severity of the congenital infection.

The detection of laboratory markers for congenital infection was positively correlated with the clinical diagnosis of toxoplasmosis in NB from both groups. However, due to the low sensitivity of the laboratory markers, negative results do not exclude the possibility of congenital infection. These results emphasize the importance of clinical follow-up of NB suspected to have congenital toxoplasmosis until at least 12 months of age.

This article in the field of immunology shows the effectiveness of maternal treatment in reducing the clinical manifestations of congenital toxoplasmosis.