Seroprevalence: Lisbon (Portugal) and Luanda (Angola)

A wide variability in the prevalence of T. gondii infection in pregnant/childbearing-age female populations has been reported worldwide. As observed for the seroprevalence in the general population, these dissimilarities are associated with several distinct factors related to each region and population-specific characteristic. Furthermore, when comparing seroprevalence data, the different laboratory methods used, which are not yet standardized, should be taken into account.

Before 2000, seroprevalence of T. gondii infection, in European countries has been estimated to range between 23% and 73% in women of childbearing age. Lower seroprevalence rates have been observed in similar populations in cold countries such as Finland (20%), Sweden (20%) and Norway (11%) [Reference Tenter, Heckeroth and Weiss2]. Although more recently published reports indicate a decrease in seroprevalence in some European countries, the range of values are not much different to the earlier reports [Reference Tenter, Heckeroth and Weiss2, Reference Pappas, Roussos and Falagas3].

In Portugal, a middle-income country located in southwestern Europe, [9], the 21·94% T. gondii antibody prevalence in pregnant women observed in this study is much lower than the prevalence reported more than 20 years ago in Portugal which showed a value of 64·3% in pregnant women from the Lisbon region (1984) [Reference Antunes10]; 30% and 60% in women of childbearing age, respectively, from the south and north of Portugal [Reference Ângelo11]. The results above suggest that seroprevalence of T. gondii infection has considerably decreased in the last three decades in Portugal. Unlike previous studies that indicate a gradual decline from north to south of Portugal in prevalence values between the populations analysed, the seroprevalence in this study is similar to results reported in 2012 for women of childbearing age from the north of Portugal (24·4%) [Reference Lopes12, Reference Lopes13] and also in other Southern European countries such as Spain (21–44%), Greece (21–36%) and Italy (17–34%), in pregnant/childbearing age women populations [Reference Tenter, Heckeroth and Weiss2, Reference Pappas, Roussos and Falagas3].

Angola is a low-income country located in Southern Africa [9]. In addition to the official language, the people from Angola retain much of the Portuguese influence, including dietary and other cultural habits. In Angola, only two reports on T. gondii seroprevalence in humans were found in literature [Reference Martins and Abranches14, Reference López15]. To the best of our knowledge this is the first study of T. gondii infection in pregnant women in Angola, and more specifically in the capital, Luanda. Concerning T. gondii seroprevalence in the pregnant/childbearing age female population, the majority of studies performed in the early 1990s in the African continent were conducted in countries from the West coast, and usually showed high rates (54–78%) [Reference Tenter, Heckeroth and Weiss2]. In contrast, during that period there were fewer reports on the North and East African coasts, with data mostly showing lower T. gondii seroprevalence rates (i.e. Ethiopia 20%, Tanzania 35%) [Reference Tenter, Heckeroth and Weiss2]. Although some of the recent data contradict the initial trend observed in countries located on both African coasts, some studies were performed after 2000 in Morocco (50·6%), Benin 54%, Ivory Coast (60·0%), São Tomé and Principe (75·2%) and Ethiopia (76·4% and 88·6%) [Reference Pappas, Roussos and Falagas3, Reference Endris16–Reference Hung18]. With the exception of the Republic of Congo [Reference Tenter, Heckeroth and Weiss2], which is a neighbour of Angola to the north, no data on T. gondii seroprevalence in these particular female populations were available from other countries neighbouring Angola. The prevalence of antibodies to T. gondii in pregnant women from Luanda region found in this study (27%) is lower than most of those reported from Western African countries, with the exception of the rate found in Burkina Faso (25·3% and 34·7%) [Reference Pappas, Roussos and Falagas3, Reference Bamba19], and is nearer to the prevalence found in East African coast countries, Sudan (34·1%) [Reference Elnahas20] and Mozambique (18·7%) [Reference Sitoe21], which is also another country with influence of Portuguese culture and dietary habits. It is also quite similar to the prevalence found in some European countries such as Portugal (21·9%, this study), Spain (21%) [Reference Pappas, Roussos and Falagas3], Italy (22·7%) [Reference Pappas, Roussos and Falagas3], Greece (20·1%) [Reference Pappas, Roussos and Falagas3], Slovakia (22·1%) [Reference Pappas, Roussos and Falagas3] or Ireland (24·6%) [Reference Pappas, Roussos and Falagas3]. Since this study was also conducted in a developing region (Luanda, Angola), where 93·3% of the studied population was unaware of the disease and of the potential risk factors associated with the transmission of infection, and with absence of adequate sanitation among other deficiencies, it was expected to find a higher prevalence of antibodies to T. gondii. The similarity with the seroprevalence reported in European countries is indeed an interesting fact. It should be noted that the Angolan people assimilated multiple cultural and gastronomic habits during the presence of the Portuguese in this African region, which is probably one of the reasons for the similar prevalence rates between the two cities. In addition, the urban nature of the region where this study was conducted in Luanda, and the fact that the pregnant women involved were participating in prenatal care, may have contributed, among other possible factors, to the lower than expected infection rates observed in Luanda. This relatively low seroprevalence is in contrast with values observed in several similar low- to middle-income regions outside Africa, e.g. Brazil (40–80%) [Reference Tenter, Heckeroth and Weiss2, Reference Pappas, Roussos and Falagas3]. The difference in rates observed may be associated with distinct dietary habits, socioeconomic features, hygiene and health conditions of the populations enrolled, as well as with the circulating parasite strains, among other possible reasons.

Some variances in seroprevalence have been observed along ethnic lines in some studies in the United States and Europe [Reference Pappas, Roussos and Falagas3, Reference Jones22]. Flatt & Shetty [Reference Flatt and Shetty23] observed, in a study performed on pregnant women in London that the risk factors for antibodies to T. gondii presence were associated with foreign-born ethnic origin, particularly for African/Afro-Caribbean, Middle Eastern and mixed origins.

Molecular diagnosis using PCR can be a useful tool for diagnosis of T. gondii. However, the utilization of PCR using peripheral blood samples has shown varying results (from 25% to 77%) [Reference Franzen24]. Some studies have reported low levels of sensitivity (25–35%), mainly in peripheral blood [Reference Franzen24–Reference Joseph26]. PCR is recommended to be used as a confirmatory assay in addition to serological methods to detect recent infection. Some authors suggest that in acute acquired infections in healthy individuals, there is only a transient parasitaemia [Reference Ho-Yen27]. This fact may justify the absence of T. gondii DNA amplification in blood samples collected on filter paper from IgM antibodies to T. gondii-seropositive Angolan women. Blood samples from Portuguese pregnant women were not tested by PCR as no cases of active infection were detected.

Risk factors evaluation: Lisbon (Portugal) and Luanda (Angola)

In the statistical analysis between risk factors and infection by T. gondii in pregnant women from the Lisbon area using a single variable analysis, we found a significant association between the number of births, contact with cats belonging to others (family or friends), gardening practices and the consumption of meat from hunting sources (i.e. birds, rabbits, boars) (Table 2). However, the binary logistic regression analysis performed with a backward stepwise algorithm, just confirmed contact with cats belonging to others, significant differences between the >2 births and the one-birth classes, as effective risk factors for T. gondii infection (Table 3).

In the pregnant women from Luanda, the main risk factors among the variables studied were contact with cats belonging to family/friends and the consumption of pasteurized milk and dairy products (Table 2). The logistic regression analysis for risk factors assessment indicated that none of the variables analysed were identified as statistically significant risk factors effectively associated to T. gondii infection in Angolan pregnant women (Table 3).

In the statistical analysis between risk factors and infection by T. gondii a significant association between the number of births and T. gondii-seropositive Portuguese pregnant women were verified, but were not confirmed by binary logistic regression analysis. The pregnant women with >2 births had increased chance of a seropositive diagnosis compared to those with only one birth (Table 3). Although, it was expected that multiparous women would have knowledge of toxoplasmosis prevention measures, in a study by Ramsewak et al. [Reference Ramsewak28] in the region of Trinidad and Tobago, it was found that there was an increase in prevalence of IgG/IgM antibodies to T. gondii in pregnant women who had more than three children than in the other groups. However, Porto et al. [Reference Porto29] did not corroborate these findings in pregnant women from Brazil. The higher percentage of T. gondii infection found in multiparous women from Portugal compared to nulliparous women may simply reflect the fact that multiparous women are normally older than nulliparous women, and were exposed to potential sources of Toxoplasma for a longer time.

The potential risk factor of contact with cats of family/friends was found to be associated with IgM/IgG antibodies to T. gondii seroprevalence in both groups of women studied (Table 2). However, when using logistic regression analysis, this variable was only confirmed to be an effective risk factor for the Portuguese pregnant women (Table 3).

In the Portuguese pregnant women sample, an association at the limits of significance (Table 2) was observed between activities and practices that promote contact with soil (gardening) and the presence of antibodies to T. gondii. However, no association between these two variables was verified in pregnant women from Angola. In addition, the above factors were not confirmed to be associated with T. gondii seropositivity in the two populations studied by logistic regression analysis. The absence of association at the limit of significance between T. gondii seropositivity with activities and practices that promote contact with soil (gardening) may have been influenced by the fact that most of the pregnant women studied originate from urban areas in both countries. Studies conducted in China describe a seroprevalence variation with location (rural vs. urban) with a higher prevalence in the countryside (12·7% vs. 7·5%) [Reference Liu30]. In contrast, Ramsewak et al. [Reference Ramsewak28] could not corroborate these findings.

In Portugal, hunting practices have been decreasing in the last few decades. In this study, the prevalence of Portuguese pregnant women consuming hunting meat was low (5·4%). However, a statistically significant association between consumption of game and T. gondii seropositivity was observed (Table 2). Due to the low percentage of women consuming game, binary logistic regression analysis was unable to test this variable as an effective risk factor for T. gondii infection in the Portuguese pregnant female population. Wild animals’ meat from hunting activities, including rabbit, wild boar, deer or poultry are potential sources of T. gondii transmission to humans, since their feeding habits are based on other vertebrates/bushes/insects that could be contaminated by Toxoplasma and have wide soil contact. Cook et al. [Reference Cook31] showed a significant relationship between the consumption of hunting meat and seropositivity for T. gondii in pregnant women from six European countries, corroborating our findings. In other studies the results are discrepant, Ertug et al. [Reference Ertug32], studying pregnant women from Turkey, found no relationship between consumption of game meat species and seropositivity for T. gondii. Moreover, consumption of unpasteurized milk/dairy products may pose a risk of human infection by T. gondii [Reference Moura33]. In our study, there was a low percentage (5·7%) of Portuguese pregnant women reporting the habit of consuming milk or unpasteurized dairy products. The lack of association between T. gondii seropositivity and this variable was also in agreement with the study of Ertug et al. [Reference Ertug32]. However, other authors found that the consumption of unpasteurized milk was associated with T. gondii infection in some European countries, concluding that this dietary habit may pose a significant risk factor for T. gondii infection [Reference Cook31, Reference Findal34]. Interestingly, and unexpectedly, in the Angolan pregnant women studied an association was observed between T. gondii seropositivity and consumption of pasteurized milk/dairy products with the single variable analysis (Table 2), but this was not confirmed by binary logistic regression analysis. Of the total pregnant women studied the highest seropositivity value for the specific antibodies (57·3%) (Table 2) was observed in the group that claimed not consuming unpasteurized milk/dairy products, which contradicts the expected results and described data in the literature [Reference Galisteu35, Reference Heukelbach36]. This can be explained by several reasons: (i) pregnant women consume pasteurized dairy products purchased in the markets, or on the street (milk, ice cream, yogurt, cheese). These products are usually prepared from powdered milk. These powdered milk products need to be reconstituted with water locally, so if there are no adequate hygienic measures available, leading to the use of unsafe water to prepare the products, women may believe they are consuming safe pasteurized milk, when in reality it is contaminated through the addition of unsafe water. (ii) Unawareness of the origin of goods consumed as supposedly pasteurized products. (iii) Inappropriate handling of the products consumed on the street. The sale of drinking water and milk-derived products on the street is a very common practice in Luanda. (iv) Misinterpretation by the pregnant women, of the concept of pasteurized products. Data obtained in this region, highlight the importance of following proper hygienic practices and consuming reliable pasteurized milk/dairy products for the prevention of infection by T. gondii.

In this study, no significant association was observed between some variables that are reported in literature [Reference Tenter, Heckeroth and Weiss2, Reference Pappas, Roussos and Falagas3], as potential risk factors for T. gondii infection, such as awareness of toxoplasmosis, access to basic sanitation, contact with own cats, or consumption of raw or undercooked meat, in both groups of the pregnant women studied. It must be taken into account that some of the results may have been influenced by misinterpretation of some questions by the women monitored. Further studies are needed to clarify the real importance of these variables as a risk factor in T. gondii epidemiology in these regions, especially in the Angolan population.

Based on the results obtained in this study, showing quite similar results in pregnant women from both Lisbon and Luanda regions, it was concluded that the prevalence of antibodies to T. gondii is still significant enough to alert the health authorities of the epidemiological importance of preventing this infection.

This study verified that a high percentage of all pregnant women analysed were non-immune to T. gondii infection, especially in Angola with a high risk of acquiring the infection during pregnancy. Thus, a routine serological surveillance for T. gondii infection with standardized techniques, particularly focused on women of childbearing age and pregnant women, especially for those in early pregnancy, is strongly encouraged for monitoring and preventive purposes. Health education on toxoplasmosis and its risk factors is required to increase the awareness about this disease and to minimize the effects of Toxoplasma infection in the general population, and particularly in pregnant women. The absence of a statistically significant relationship between the prevalence of Toxoplasma infection in the two pregnant women populations and several of the risk factors investigated in the study and cited before in this report does not rule out that these factors have some effect on the transmission of toxoplasmosis. Such factors may play a limited role in these regions, possibly due to cultural, religious and dietary behavioural characteristics.

Data obtained in this study highlights the importance of conducting further large-scale epidemiological studies in Portugal, and particularly in Angola where this is the first attempt to study the subject, and to take into account the diversity of climate, geographical and population features which characterize this vast African country.