Immunocompetent and immunocompromised women contracting T. gondii infection for the first time during pregnancy run a risk of transmitting the infection to the foetus because of the parasite crossing the placenta from the maternal blood. A host that is immunocompetent acquires life-long immunity against toxoplasmosis when infected with T. gondii. A new T. gondii infection contracted 4–6 months before conception will usually not result in mother-to-child transmission of toxoplasmosis on subsequent exposures. In immunocompromised women who are positive for human immunodeficiency virus (HIV), T. gondii infection may reactivate with resulting vertical transmission.^3,4 In a study in Brazil, 28% of T. gondii Immunoglobulin M (IgM)-positive pregnant women with unknown HIV status transmitted the infection vertically.²

Only about 30% of infants born to mothers who had seroconversion in pregnancy acquire prenatal infection. If the infection in a pregnant woman is not treated, the risk of intrauterine infection increases with gestational age, that is, from 14% for primary maternal infection in the first trimester to 59% for primary maternal infection in the last trimester.³ When the primary infection occurs during advanced gestation, the risk of transmission to the foetus is higher. However, when transmission occurs early in gestation, the risk of symptomatic infection and severe disease is high.⁵ Clinical signs of toxoplasmosis in neonates include the classical triad, namely, intracranial calcification, hydrocephalus and retinochoroiditis. About 10% of prenatal infections result in abortion or neonatal death. The surviving infants suffer from progressive neurologic complications like mental retardation, learning problems, hearing and visual impairment or seizures and have special care and education needs. Children who have been infected before birth might be asymptomatic at birth but may develop complications associated with toxoplasmosis later in life.^3,6,7 Clinical trials indicate that early treatment of these children can decrease manifestations of disease and improve the quality of life.³

Congenital toxoplasmosis can best be prevented by ensuring that pregnant women who are seronegative do not get infected, or by treatment of pregnant women who are diagnosed with a primary infection, as evident from seroconversion, T. gondii Immunoglobulin M (IgM) positivity and low Immunoglobulin G (IgG) avidity. Screening programmes for toxoplasmosis have been introduced in many European countries and in the United States. These include either diagnosis of primary maternal infection with T. gondii during pregnancy or detection of congenital infections in neonates at birth.³ No routine screening for T. gondii is done in Namibia, and only two previous studies were conducted to determine the prevalence of toxoplasmosis among blood donors.^8,9 The aim of this study was to determine the prevalence of antibodies to T. gondii among urban pregnant women attending public antenatal care in Windhoek, Namibia, from September to October 2016.

This was a descriptive study aiming to describe the seroprevalence of T. gondii among pregnant women in Windhoek.

At an expected prevalence of 6% of T. gondii infection, using Cochran’s formula, a sample size of 542 would provide a precision (error margin, above and below the prevalence estimate) of 2%. Estimated prevalence was based on two previous publications in Namibia and the seroprevalence in neighbouring South Africa. Funding was available to enrol 344 participants in the study.

All pregnant women who attended public antenatal care in Windhoek, from 12 September to 11 October 2016, were informed about the study, and all women who were identified by the clinic to have their blood drawn for routine antenatal testing were eligible to participate. Five participants were excluded because of not signing the consent form by omission and one Ovahimba woman was excluded because of not understanding English or Afrikaans.

Participation in the study was voluntary, and written consent was obtained from each participant and guardian, where applicable. A venous blood sample was collected from each consenting participant and transported immediately to the testing laboratory. A questionnaire was administered to collect information on demographic characteristics and exposure to risk factors, and was filled by consenting participants. Questions were derived from the literature.

Blood samples were transported to the laboratory within an hour. Serum was stored for not more than a week at 2ºC – 8ºC and then at -80ºC until testing.

An automated chemiluminescent microparticle immunoassay was performed in an ISO 15189 accredited laboratory to determine seroprevalence of IgG antibodies to T. gondii (access immunoassay systems, Beckman Coulter Eurocenter SA, 22 rue Juste-Olivier, Case Postale 1044, CH – 1260 Nyon 1, Switzerland). The presence of anti-T. gondii IgM antibodies was determined in samples testing positive for specific IgG using the ELISA method (Euroimmun Medizinische Labordiagnostika, D-23560 Lubeck, Seekamp 31, Germany). Cut-off points were calculated by using the ratio of the extinction of the control or patient sample to the extinction of the calibrator. The extinction value of the calibrator defined the cut-off value as recommended by the manufacturer. IgG avidity was determined in samples testing positive for specific IgG by performing a manual ELISA test with urea neutralisation (Euroimmun).

For interpretation of IgG results, recommendations from the manufacturer were followed. Specimens with specific IgG concentration of > 10.5 IU/mL were considered positive for T. gondii past or current infection. Acute infection with T. gondii was determined by the presence of anti-T. gondii IgM antibodies, and specific IgG antibodies with low avidity. Recommendations from the manufacturer were followed for interpretation of results. For interpretation of IgM results, an extinction ratio (sample relative to calibrator) of <0.8 was considered negative, ≥0.8 to <1.1 was borderline and ≥1.1 was considered positive. For interpretation of avidity results, the ratio of the enzyme immunoassay optical density when urea-treated relative to untreated was used. Low avidity was defined as a ratio of <40%, which was indicative of acute T. gondii infection. A ratio of 40% – 60% was considered equivocal, while a ratio above 60% was an indication of high avidity antibodies.

Association of demographic characteristics (maternal age, gestational age and parity) with seroprevalence of T. gondii infection among pregnant women was investigated. Association of risk factors (water source, living with cats or dogs, blood transfusion, as well as consumption of untreated water, unpasteurised cow’s milk, goat’s milk, raw or undercooked meat or unwashed fruit/vegetables) with seroprevalence of T. gondii infection was investigated. A case-based description of anti-T. gondii IgG and IgM seropositivity and IgG avidity was provided for nine participants who tested IgG positive. Seropositivity among five age groups was evaluated with the assumption that all age groups had had similar exposure to T. gondii. All data were entered into an Excel spreadsheet (Microsoft Corporation, One Microsoft Way, Redmond, WA 98052-7329) and analysed using Statistical Package for the Social Sciences statistical software (version 24, IBM North America, 590 Madison Avenue, New York, NY 10022) and R version 3.2.2.¹⁰ Chi-square test and Fisher’s exact test (when the expected frequency in any cell of the two-by-two table was less than 5) were used to assess associations between population characteristics provided in the surveys and seropositivity results. Multivariate analysis was performed with generalised linear models using binary logistic regression. Beta values were used to calculate odds ratios of variables, while the Wald 95% confidence intervals gave an indication of the width of the odds ratios; p-values less than 0.05 were considered statistically significant.

Ethical approval to conduct the study was obtained from the School of Health and Applied Sciences, Polytechnic of Namibia; the Ministry of Health and Social Services in Namibia; and the Health Research Ethics Committee at Stellenbosch University, Cape Town, South Africa.

Very low seroprevalence of anti-T. gondii IgG (2.61%) was found among urban pregnant women attending public antenatal care in Windhoek, in 2016. This is comparable to a previous study in Namibia which found 1% seroprevalence of anti-T. gondii among blood donors.⁹ Possible reasons for this low seroprevalence could be the arid climate and the high altitude of the capital city, Windhoek.¹¹ Oocysts cannot withstand dry conditions that prevail during extended periods of drought in central Namibia. The worst drought, since recording of rainfall had started, occurred in Namibia in 2016. The seroprevalence of anti-T. gondii found in 2016 was lower than the 12.1% recorded among blood donors in Windhoek in 1978.⁸ This finding is in line with the worldwide trend of decreasing seroprevalence of anti-T. gondii over the previous decades.¹² Factors that could contribute to the decreasing trend are urbanisation, which is accompanied by better access to safe drinking water and abattoirs, as well as increased use of pasteurised cow’s milk.

Being the driest African country south of the Sahara, Namibia shows much lower seroprevalence of T. gondii infection than in other developing countries (Table 4).^{8,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28} It is however comparable to seroprevalence in neighbouring South Africa, with which Namibia shares weather patterns.²³ The seroprevalence is the same as in Mexico, which has a similar arid climate.²⁹ A review of studies conducted in Africa found a median seroprevalence of T. gondii of 37%, with rates ranging from 6.4% to 74.5%.¹² Very little is known about the burden of CT in developing countries. In Brazil, Neto et al. found 0.06% of neonates being anti-T. gondii IgG seropositive.³⁰ It is postulated that the high burden of CT in South America can be attributed to more pathogenic genotypes that circulate in that part of the world.³¹

In Windhoek, 97.4% of pregnant women attending public antenatal care remain susceptible to T. gondii, posing a risk of vertical transmission and CT if they get infected while pregnant. Infection pressure is, however, low – a large population is at risk, but the probability of exposure is low. In hyperendemic settings, acquisition and immunity are likely before women get pregnant, whereas models suggest that when the incidence of T. gondii infection among pregnant women is 4% per annum, the risk of seroconversion during pregnancy and occurrence of CT would be the highest. This correlates with a susceptibility of 40% among pregnant women, which is much lower than the susceptibility found in this study.³¹

There was no significant difference in prevalence of anti-T. gondii IgG among the five different age groups of pregnant women. Seroprevalence linearly increased in the lowest three age groups (15–20 years, 21–25 years and 26–30 years), only to decrease in older women (31–47 years) (Table 1). Seroprevalence of anti-T. gondii IgG increased with gestational age although the difference was not statistically significant. Primigravidae showed the highest seroprevalence of anti-T. gondii IgG, which is in line with the finding of higher seroprevalence among younger women.

There was no significant association between risk factors and seroprevalence of T. gondii (Table 2). Surprisingly, participants with access to own tap showed the highest seroprevalence of T. gondii. However, this association was not significant. Another interesting finding was the lack of association between consumption of unwashed fruit or vegetables and seropositivity, while 3.45% of participants denied that this consumption showed seropositivity of T. gondii infection. Alvarado-Esquivel et al. reported a similar finding.²⁹ These exposures to T. gondii could possibly be because of other risk factors like working with raw meat, which were not assessed in our study.

Magwedere et al. included a discussion on T. gondii in their report on zoonoses posing a possible threat to the wildlife industry in Namibia.³² Working with raw game meat or consumption of undercooked game meat could possibly pose a risk of exposure to T. gondii. A previous study found presence of T. gondii in lions in Namibia. Among a pride of five lions in a private reserve, four showed evidence of T. gondii infection.³³ A high prevalence of T. gondii was also found in feral cats in the Western Cape Province in South Africa, providing a reservoir for the parasite and a potential source of contamination of the environment.³⁴ In the 1990s, a municipality in the Province of British Columbia in Western Canada experienced an outbreak of toxoplasmosis, with the municipal water supply implicated as the source of infection.^35,36

Very few pregnant women had possibly been infected during pregnancy; therefore, the risk of giving birth to an infant with CT was minimal. Anti-T. gondii IgM antibodies and specific IgG avidity were tested only in samples positive for anti-T. gondii IgG. Other studies found very low prevalence of samples positive for anti-T. gondii IgM only (0.3%,³⁷ 0.9%³⁸), so the possibility of finding IgM positive while IgG is negative could be negated.

It can thus be concluded that the pregnant women who presented with anti-T. gondii IgG antibodies were infected before they became pregnant, with low risk of vertical transmission. Avidity testing is widely used in Europe and increasingly in southern Africa. The French National Reference Center for Toxoplasmosis has proposed algorithms for interpretation of serologic testing (IgG, IgM and IgG avidity) for T. gondii.³⁹

The T. gondii IgG avidity test is useful to distinguish past infection from present infection in specific IgM-positive pregnant women. In a study in Brazil, the presence of anti-T. gondii IgM in an immune pregnant woman was often not associated with congenital infection. Twenty per cent of infants born to immune mothers with specific IgM antibodies and low avidity IgG antibodies were vertically infected, while 8% of infants born to mothers with specific IgM antibodies and intermediate or high IgG avidity were vertically infected.⁴⁰ Liesenfeld et al. evaluated the usefulness of testing for IgG avidity in association with T. gondii and found that among pregnant women with either positive or equivocal specific IgM, 55.9% had high avidity-specific IgG.

The high rate of T. gondii susceptibility among pregnant women could justify health education interventions. Women of childbearing potential found to be susceptible to T. gondii should be educated on behaviour to prevent infection.⁴¹ A health promotion strategy should be aimed at women of reproductive age, making them aware of preventive behaviour.³⁸