Toxoplasma gondii Seroprevalence in Mali

Abstract

The protozoan parasite Toxoplasma gondii is globally distributed, with considerable local variation in prevalence based on behavioral and environmental factors. To assess prevalence and estimate risk in Mali, we conducted a survey of 760 serum samples previously collected for malaria studies. A modified agglutination test detected antibodies in ~27% of the adult population, with no significant differences between men and women, or between urban and rural study sites. In the village of Kolle, seroprevalence rose from 0% in infants (<1 yr, but after weaning of maternal immunoglobulin G) to 0.8% (1–5 yr), 2.7% (6–10), 11.3% (11–15), and 26.8% (>15); differences between the <10-, 11–15-, and >15-yr age groups were highly significant (P ≤ 0.01). We also observed an increase in anti–T. gondii antibody titers with age. Modeling the observed age distribution suggests a seroconversion rate of ~1%/yr, indicating that congenital toxoplasmosis may be an under-appreciated public health concern in Mali.

The protozoan parasite Toxoplasma gondii is widely distributed throughout the world, and it is thought to be capable of infecting all warm-blooded animals, including humans. It is estimated that one third of the human population is chronically infected with T. gondii, although prevalence varies by locale based on climate, agricultural traditions, eating habits, feline population density, socio-economic conditions, and other factors (Feldman, 1968; Tenter et al., 2000). Seroprevalence has been estimated at 6–17% in the U.K., 50–60% in France, 10–25% in North America, 10–60% in Asia, 45–80% in South America, and 20–85% in Africa (Carme and Tirard-Fleury, 1996; Dromigny et al., 1996; Pal et al., 1996; Baril et al., 1999; Nash et al., 2005; Jones et al., 2007; Dubey and Jones, 2008; Rosso, Les et al., 2008; Fernandes et al., 2009; Pappas, Roussos et al., 2009; Sroka et al., 2010; Xiao et al., 2010).

Toxoplasma gondii may be transmitted by the ingestion of meat containing latent tissue cysts (bradyzoites), or food or water contaminated with oocysts shed in feline feces; cats are the definitive host for this parasite (Benenson et al., 1982). After passage through the acidic environment of the stomach, parasites excyst, invade the intestinal epithelium, and differentiate into the acutely lytic (tachyzoite) form. Tachyzoites divide rapidly within a specialized vacuole inside infected cells. The tachyzoites ultimately cause these cells to lyse, spreading infection to neighboring cells and tissues throughout the body. Continued cycles of infection in the absence of effective control can produce extensive tissue damage.

Toxoplasmosis is typically subclinical, as the infection is usually well controlled in immunocompetent adults even without treatment, through a combination of innate and acquired immune responses (Derouin, 1992). In parallel with the emergence of acquired immune responses, however, some parasites differentiate into latent bradyzoite tissue cysts, especially within the brain, establishing a life-long chronic infection in affected individuals. Although chemotherapy is available for acute infection, no drugs are known to be effective against these latent forms.

Primary, or recrudescent, infection can be fatal in immunocompromised individuals, and T. gondii is a well-known opportunistic pathogen in acquired immunodeficiency syndrome and patients immunosuppressed for cancer chemotherapy, transplantation, or other reasons (Clumeck et al., 1984; Zangerle et al., 1991; Luft and Remington, 1992; Weiss and Dubey, 2009). Toxoplasmosis is also a prominent source of congenital disease, as the highly promiscuous tachyzoite form is able to cross the placenta and infect the fetus. The severity of congenital toxoplasmosis is greatly influenced by the timing of maternal infection (Desmonts and Couvreur, 1974; Dunn et al., 1999; Nowakowska, Colon et al., 2006). Women infected before pregnancy rarely transmit to the fetus, except in immunodeficient patients (Dunn et al., 1999). Primary infection of the mother during the first trimester is typically controlled without transplacental transmission, but when transmission occurs, it is usually associated to a miscarriage or severe fetal lesions (e.g., intracranial calcification, hydrocephalus; Desmonts and Couvreur, 1974). Infection later during pregnancy is more commonly transmitted, leading to ocular disease (e.g., chorioretinitis; Desmonts and Couvreur, 1974; Dunn et al., 1999), learning defects, or both, that are likely to advance with age due to recrudescence of bradyzoite cysts established in the infant (Holland, 2009; Melamed, 2009). If recognized early, transmission and the severity of infection in the child may be attenuated by treatment during pregnancy (Couvreur et al., 1984; Hohlfeld et al., 1989; Forestier et al., 1991; Cortina-Borja et al., 2010) or shortly after birth (Jones et al., 2003; Kaye, 2011). The globally patchy distribution of T. gondii, and the many complications associated with this infection, argue for epidemiological studies to shape local and regional health policies.

In an effort to assess the prevalence of T. gondii and potential public health risk of congenital toxoplasmosis in Mali, 760 sera previously collected in the context of 2 unrelated malaria case studies were tested for the presence of antibodies to this protozoan parasite. Both studies were carried out in accordance with good clinical practices; clearance to use these sera for T. gondii serotyping was obtained from the Ethical Committee of the Faculty of Medicine Pharmacy and Dentistry of the University of Bamako, Mali. This report includes all samples for which demographic and clinical data were available.

Kolle is a rural village located at 57 km south of Bamako, where residents raise millet, maize, sheep, and goats. The Kolle cohort (Djimde et al., 2007; Tekete et al., 2009) included 533 sera: 10 from 6- to 12-mo-old infants (1.9%), 124 (23.3%) from children aged 1–5 yr, 219 (41.1%) from children aged 6–10 yr, 124 (23.3%) from children aged 11–15 yr, and 56 (10.5%) from adults (>15 yr). Data were binned into age classes to enable statistical comparisons (see Supplementary Table I for detailed age information).

The Bamako cohort (from 2007 to 2008) included consenting mothers of neonates referred for inpatient care to the Unit of Reanimation and Neonatology of Hospital Gabriel Toure´ and consisted of 113 samples from adult women (>15 yr old) and 114 sera from their babies in the first month of life (including 1 pair of twins). Serum samples from mothers and babies (aged 1–30 days) were collected as reported previously (Dicko-Traore et al., 2011), and stored at –80 C until needed

Patient samples were tested for anti-T. gondii antibodies by the modified agglutination test, using formalin-fixed T. gondii tachyzoites (Desmonts and Remington, 1980; Thulliez et al., 1986; Dubey and Desmonts, 1987; Dannemann et al., 1990). In brief, sera were diluted with 0.01 M phosphate-buffered saline (pH 7.2) in round-bottomed 96-well microtiter plates (2-fold serial dilutions from 1:25 to 1:3,200; 25 µl/well). Positive and negative control sera were included on each plate. Formalin-fixed RH-strain parasites were suspended at the final concentration of 6 × 10⁵ parasites/µl in alkaline buffer (pH 8.95) containing 1% bovine serum albumin, 200 mM β-mercaptoethanol, and 40 µg/ml Evans blue dye, and 25 µl of the antigen mixture was added to each well of the microtiter plate. After overnight incubation at 37 C, agglutination (failure to precipitate) was read by eye, and sera with titers ≥1:25 were considered positive. Parasite agglutination is a well-established method for assessing exposure to T. gondii, as human sera from patients worldwide cross-react with intact parasites of any strain.

Demographic and clinical profiles were recorded and analyzed using Epi Info 6 software, with statistical significance defined as P ≤ 0.05. Confidence intervals for observed seropositivity (2 SDs from the mean) were calculated as 2 × (S [1 – S]/N)^0.5, where N is the sample size and S the observed frequency of seropositivity. Seroconversion rates were estimated by comparison with standard curves calculated according to the formula P_A(+) = 1 – (1 – S)^A, where P_A(+) represents the fraction of the population that is seropositive at age A and S is the annual seroconversion rate.

Forty-six of 169 adult Malian sera (27.2%) were positive for T. gondii antibodies (at .1:25 dilution), indicating previous parasite exposure (Table I). Differences observed between the all female Bamako cohort (31/ 113; 27.4%) and either men (5/15; 33.3%) or women in Kolle (10/41; 24.4%) were not statistically significant (P > 0.5). These levels of adult seroprevalence are well within the range reported previously at various sites around the world (Zumla et al., 1991; Dhumne et al., 2007; Jones et al., 2007; Kamani et al., 2009; Pinto et al., 2012)

TABLE I.

Seroprevalence for Toxoplasma gondii.

		Female			Male			Total
	Age (yr)	N	Positive	%	N	Positive	%	N	Positive	%	SD (%)*
Bamako (urban)	<15	113	31	27.4				113	31	27.4	8.3
	Neonate†	35	11	31.4	79	20	25.3	114	31	27.2	8.4
Kolle (rural)	0.5–1	4	0	0.0	6	0	0.0	10	0	0.0	0.0
	1–4	58	1	1.7	66	0	0.0	124	1	0.8	1.4
	5–9	128	3	2.3	91	3	3.3	219	6	2.7	5.7
	10–14	63	6	9.5	61	8	13.1	124	14	11.3	5.7
	≥15	41	10	24.4	15	5	33.3	56	15	26.8	5.8

^*Two SDs from the mean, based on observed sample size (N) and seropositivity (S): 2 ⨉ 3 (S [1 – S]/N)^0.5.

^†All seropositive neonate samples were from seropositive mothers.

Considering younger samples in the Kolle cohort reveals a progressive increase in seroprevalence with age, rising from 0% in infants, to 0.8 % in the 1- to 5-yr age group, 2.7% in the 6- to 10-yr age group, 11.3 % in the 11- to 15-yr age group, and 26.8% in adults. Differences in seroprevalence were highly significant between children in the 11–15-yr age group and those <10 yr (P < 0.002) or adults >15 yr (P = 0.01). Modeling these data (as described above) suggests an overall seroconversion rate of approximately 1% (gray lines in Fig. 1).

FIGURE 1.

Toxoplasma gondii seroconversion rates in Mali. Data collected from Kolle (Table I and Supplementary Table I) were binned by age (1–4, 5–9, 10–14, and 15–64 yr) to provide sample sizes of ≥50 and plotted to estimate seroconversion rates. Error bars indicate SD in age and seropositivity (see text); the broad bar for adults (broken line) is a consequence of pooling all available adult samples. Comparison with standard curves calculated for various rates of seroconversion (gray) suggests an overall rate of ~1%/yr.

Toxoplasma gondii–positive serotiters also seem to increase with age, as antibody levels were highest among subjects aged .>10 yr (P < 0.02; Table II). 71.4% (5/7) of positive sera from children ,10 yr in the displayed antibody titers !1:100, whereas 85.8% (12/14) of positive sera from children 11–15 yr and 84.8% (39/46) of adults presented anti–T. gondii serotiters ≥1:200. Serotiters in babies from the Bamako cohort were uninformative for this study, as all reflected the serum status of their mothers, presumably due to residual immunoglobulin G transmitted across the placenta before birth.

TABLE II.

Anti–Toxoplasma gondii serum titers.^*

				Low titer			Moderate titer			High titer
Age (yr)	N	Positive	%	1:25	1:50	1:100	1:200	1:400	1:800	1:1,600	1:3,200	Median (log)
0.5–1†	10	0	0.0	0	0	0	0	0	0	0	0	0
1–4	124	1	0.8	1	0	0	0	0	0	0	0	1:25
5–9	219	6	2.7	0	2	2	0	0	0	0	2	1:200
10–14	124	14	11.3	0	2	0	2	0	1	0	9	1:800
≥15‡	169	46	27.2	3	3	1	4	8	8	4	15	1:800

^*Pooled data from males and females, as no sex-specific differences in seroprevalence or titer were observed (see Table I).

^†All seropositive neonate samples were from seropositive mothers.

^‡Pools data from Kolle and Bamako, as no differences were observed in anti–T. gondii seropositivity (see Table I).

Previous reports from Bamako and surrounding areas have described T. gondii seroprevalence in adults of ~60% in 1974 (Quilici et al., 1976), 34% in 1984 (Maiga et al., 1984), and 21% in 2001(Maiga et al., 2001). It is possible that exposure may be declining, but longitudinal studies are required to determine whether this is indeed the case. Although age data are not available for individual samples in these earlier studies, pooled data in the 1974 study suggest a seroconversion rate of ~3–4%/yr. More recent studies from elsewhere in West Africa report T. gondii seropositivity in the range of 20–25% (Julvez et al., 1996; Faye et al., 1998; Simpore et al., 2006; Ndiaye et al., 2007; Ayi et al., 2009; Kamani et al., 2009; Akinbami et al., 2010) consistent with seroconversion rates in the range of 0.5–1.0%/yr. All reports agree that seroprevalence is similar in urban and rural communities and that it is similar in men and women.

It is clear that T. gondii is widespread in Mali, and elsewhere in West Africa, in both urban and rural areas, and that rising seroprevalence in women of child-bearing age indicates a significant risk of congenital disease. The current population (>15 million) and birth rate (4.5%) in Mali suggests that several thousand pregnancies are at risk for transplacental toxoplasmosis annually, leading to fetal abortion, mild or severe congenital neurological defects, chorioretinitis, learning disabilities, or a combination. The availability of effective treatments minimizing the adverse effects of congenital toxoplasmosis suggests that the public health impact of routine screening during pregnancy should be considered.