International Journal of Gynecology and Obstetrics (2007) 97, 15–20
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / i j g o
CLINICAL ARTICLE
Malaria parasitemia and systemic cytokine bias in pregnancy E.A. Achidi a,b,⁎, T.O. Apinjoh b , V.P.K. Titanji b a b
Department of Medical Laboratory Sciences, University of Buea, Buea, Cameroon Department of Biochemistry and Microbiology, University of Buea, Buea, Cameroon
Received 3 March 2006; received in revised form 19 December 2006; accepted 20 December 2006
KEYWORDS Cytokines; Gestational age; Gravidity; Malaria parasitemia; Pregnancy trimester; T helper cell
Abstract Objectives: To investigate the effects of age, gravidity, and gestational age on peripheral malaria parasitemia and functional T helper (Th) cell heterogeneity in pregnant women. Methods: Maternal age, gravidity, and gestational age were recorded and peripheral venous blood collected from 175 women attending antenatal clinics in south western Cameroon between March and September 2002. The blood was checked for malaria parasitemia by light microscopy and plasma levels of interleukin (IL)-4 and human interferon (IFN)-γ were measured by indirect enzyme-linked immunosorbent assay. Results: Malaria parasites were detected in 45 (25.4%) of 174 women, with rates similar for different age groups, trimesters of pregnancy, and gravidity. The geometric mean parasite density was 565, and parasite density was significantly higher in younger than in older women. For all groups combined, the mean IL-4 level was significantly higher than the mean IFN-γ (P = 0.0004), irrespective of the presence and density of malaria parasites, gravidity except for women in their first trimester of pregnancy and grandmultiparas, who had similar levels of IFN-γ and IL-4. In general, the cytokine profile was biased toward Th2type of responses in 112 (84.3%) of 132 women. Conclusions: In this study the ability to control malaria parasitemia during pregnancy was found to be predominantly age dependent, suggesting naturally acquired immunity. Furthermore, the systemic cytokine profile was found to be biased towards Th2 responses, a prerequisite for a successful pregnancy. This pattern was unaffected by maternal age, gestational age, gravidity, or parasitemia. © 2007 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved.
1. Introduction ⁎ Corresponding author. Faculty of Health Sciences, University of Buea, P.O Box 63 Buea, Cameroon. Tel.: +237 332 2134; fax: +237 332 2272. E-mail address:
[email protected] (E.A. Achidi).
Pregnant women are the adult group the most at risk for malaria infestation in areas of endemic disease of Africa, with at least 50 million pregnant women infected each year [1]. In areas of high (i.e., stable) transmission, such as south
0020-7292/$ - see front matter © 2007 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijgo.2006.12.015
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E.A. Achidi et al.
western Cameroon, severe maternal anemia and low birth weight due to intrauterine growth retardation and preterm delivery are the main observable consequences [2], but malaria in pregnancy also causes significant maternal and fetal morbidity and mortality [3]. During pregnancy there is a pronounced sequestration of Plasmodium falciparum-infected erythrocytes in the intervillous spaces of the placenta. One major placental host receptor for infected erythrocytes is chondroitin sulfate, which interacts with distinct binding structures in the Duffybinding-like domain 3 (DBL3) of the P. falciparum erythrocyte membrane protein 1 (PfEMP-1) [4]. Chondroitin sulfate A (CSA)-binding parasites are not found in nonpregnant women, and probably represent variants that are distinct both antigenically and with regard to their adhesive properties [5]. Chronic plasmodial infection of the placenta is associated with thickening of the trophoblast basement membrane, and with intervillous inflammation that is especially severe in primiparas [6]. In contrast, multigravidas are less susceptible to placental malaria. This is believed to be due, in part, to the production of antibodies inhibiting placental cytoadherence, and to the response of placental mononuclear cells to malaria antigen stimulation from enhanced interferon gamma (IFN-γ) [7]. Adult women in areas of endemic malaria normally acquire, through repeated infections, substantial protective immunity against high-density parasitemia and severe disease [8], but this immunity is partially lost during pregnancy. The systemic suppression of proinflammatory responses from T helper 1 (Th1) cells, i.e., increased circulating levels of IFN-γ and tumor necrosis factor α, along with increased local expression of anti-inflammatory cytokines such as interleukin (IL)-4, IL-6, and IL-10, has been reported [9]. Placental malaria alters the cytokine balance in favor of Th1 types (i.e., proinflammatory) [10], with deleterious consequences. It is not clear whether placental immune responses to malaria antigens reflect immune responses in the peripheral blood, or whether these responses are predictive of a pregnant woman's vulnerability. Moreover, the effects of maternal age (MA), gravidity, and parasitemia, as well as gestational age (GA), particularly on systemic immunomodulation, have not been fully elucidated. The aim of this study was to examine the effect of MA, gravidity, and GA on
both malaria infestation and functional Th cell heterogeneity in pregnant women from southwestern Cameroon presenting for antenatal enrollment.
2. Patients and methods 2.1. Study region The study was carried out in maternity centers located in Mutengene and Buea, Fako Division, southwestern Cameroon. These 2 towns are situated on the lower and upper windward slope of Mount Cameroon, at about 300 m (Mutengene) and 1000 m (Buea) above sea level. There are 2 seasons in this region, the dry (November–February) and the rainy (March– October) seasons. Malaria parasite transmission is perennial, mesoendemic from November to February and hyperendemic during the rainy season [11], with P. falciparum accounting for up 32.8% of malaria infections in pregnant women [11,12]. Anopheles gambiae is the dominant mosquito vector.
2.2. Study population and sampling A total of 175 consecutive pregnant women attending antenatal clinics at 2 maternity centers, Mutengene Integrated Health Center and Buea Integrated Health Center, were enrolled from March to September 2002 after they gave informed consent. Information relating to maternal age, last menstrual period, gravidity, and GA (calculated from last menstrual period) were recorded. Then, 4-mL blood samples were collected by venipuncture into heparinized vacutainers (BD Vacutainer systems, Plymouth, UK). The samples were centrifuged and plasma supernatants stored in a freezer (Sanyo Electric Co. Ltd, Tokyo, Japan) at − 86 °C until analyzed.
2.3. Parasitologic assessment Thick blood films were prepared, air dried, Giemsa stained (Sigma, St. Louis, MO, USA), and examined for malaria parasites by light microscopy for all participants. Parasites were counted against 200 white blood cells to determine parasite density. A count of 5000 or fewer parasites per microliter of blood was considered low, and a count of more
Table 1 Malaria parasite rates and geometric mean ± SEM of parasitemia densities in pregnant women of different age groups, gestational age, and gravidity status Variable Maternal age ≤ 20 y > 20 y Pregnancy duration 1st trimester 2nd trimester 3rd trimester Gravidity Primigravidae Multigravidae a b
Malaria parasite rate, % a
P value
Geometric mean ± SEM of parasite density b
P value
35.7 (15/42) 22.6 (30/132)
P = 0.09
1008 ± 588 444 ± 117
P = 0.049
33.3 (2/6) 27.3 (33/121) 17.1 (7/41) 23.3 (14/60) 27.0 (31/115)
P = 0.56
P = 0.60
517 ± 127 595 ± 135 475 ± 161 941 ± 418 456 ± 138
P > 0.05
P = 0.06
Malaria Parasite Rate means percentage of women with asexual Plasmodium species parasites detected by thick blood film method. Parasite density refers to the number of asexual-stage parasites per a minimum of 200 leucocytes detected in thick blood film.
Malaria parasitemia and systemic cytokine bias in pregnancy
Figure 1 Frequency distribution of OD405 values for plasma interferon γ and interleukin 4 in pregnant women from southwest Cameroon. Absorbance represents midpoints for a range of sample optical density values.
than 5000 parasites was considered high. Fifty high-power fields were scanned to confirm malaria parasite-negative slides.
17
Figure 2 Correlation between malaria parasite density and plasma levels of interferon γ in pregnant women from southwest Cameroon. Values indicated are log10 transformations of the respective parasite densities and interferon γ OD405 values. reader. The detection limit for the assay was 10 pg/mL. The IFN-γ to IL-4 ratio was used to determine whether the functional Th cell response was Th1- or Th2-oriented [13,14].
2.4. Enzyme-linked immunosorbent assay (ELISA) for cytokines
2.5. Statistical analyses
Plasma cytokine levels were measured by sandwich ELISA using commercially available kits (Mabtech AB, Naaka, Sweden). Duplicate plasma samples (100 μL per well) were applied to microtiter plates for the measurement of IFN-γ and IL-4 using matched pairs of monoclonal antibodies for the capture and detection of IFN-γ (mAb1-D1K and mAb7-B6-1, biotin) and IL-4 (mAbIL4-1 and mAbIL4-2, biotin). The assays were performed according to the manufacturer's instructions and developed using streptavidin-ALP and para-nitrophenyl phosphate (Sigma, Poole, UK). The reaction was stopped with 10 mM EDTA and the absorbance read at 405 nm using a Dentley Welscan ELISA plate
Data were entered in a Microsoft Excel 2002 database (Microsoft Corporation Inc., Redmond, WA, USA) and analyzed using SPSS (SPSS Inc., Chicago, IL, USA). Values for parasite density and plasma cytokine optical density were log10-transformed before statistical analyses. The Pearson χ2 test was used to evaluate group differences in parasite rates and the t test to evaluate differences in group means. Correlation between variables was determined using the Pearson correlation. Statistical significance was set at P < 0.05. The influence of MA was assessed by comparing values for women aged 20 years or younger with values for women older than 20 years; the influence of GA by
Table 2 Geometric mean of OD405 values for plasma interferon γ and interleukin 4 in pregnant women of different age groups, trimester of pregnancy, gravidity, and malarial status from southwestern Cameroon Variable
No. of patients
Maternal age ≤20 y 26 > 20 y 104 P value Pregnancy duration 1st trimester 4 2nd trimester 94 3rd trimester 30 P value Gravidity Primigravidae 40 Multigravidae 90 P value Malaria parasitemia Yes 96 No 34 P value
Interferon γ, mean ± SEM
No. of patients
Interleukin 4, mean ± SEM
P value
0.083 ± 0.022 0.184 ± 0.041 P = 0.04
28 108
0.214 ± 0.046 0.383 ± 0.064 P = 0.13
P = 0.005 P = 0.002
0.073 ± 0.010 0.168 ± 0.042 0.173 ± 0.060 P = 0.737
6 96 32
0.244 ± 0.136 0.385 ± 0.071 0.275 ± 0.062 P = 0.923
P = 0.126 P = 0.002 P = 0.078
0.113 ± 0.043 0.186 ± 0.045 P = 0.20
42 94
0.341 ± 0.093 0.352 ± 0.064 P = 0.95
P = 0.013 P = 0.008
0.169 ± 0.038 0.148 ± 0.073 P = 0.59
101 35
0.391 ± 0.065 0.226 ± 0.074 P = 0.09
P = 0.001 P = 0.118
18 comparing values for the first, second, and third trimesters of pregnancy; and the influence of gravidity by comparing values for primigravidae with values for multigravidae.
3. Results Samples from 175 pregnant women were analyzed. Seven women could not remember the date of their last menstrual period and GA could not be determined in their cases. Most of the women (133 of 175) were older than 20 years, multigravidae (115 of 175), and in the second trimester of pregnancy (121 of 168) when their samples were analyzed.
3.1. Malaria parasitemia Malaria parasites were found in 45 (25.4%) of 174 of the pregnant women. Although rates of malaria infestation were higher among younger than older women, the difference was not statistically significant (P = 0.09). Rates of infestation were similar for women grouped by pregnancy trimester (P = 0.56) or gravidity (P = 0.60) (Table 1). The geometric mean of parasite density for the pregnant women was 565, and the density was higher in women younger than 20 years (P < 0.05). Furthermore, there was no significant difference in geometric mean parasite density among women grouped by trimester of pregnancy (P > 0.05) or gravidity (P = 0.06).
3.2. Plasma cytokine levels are biased towards Th2 responses Wide-ranging values for plasma concentration of IFN-γ and IL-4 were obtained for the 145 samples analyzed. In some cases, the volume of plasma was insufficient to determine levels for both cytokines. The geometric mean OD405 nm value was 0.058 for plasma IFN-γ level (range, 0.01–0.52) and 0.130 for IL-4 (range, 0.01–0.53). In general, values were significantly higher for IL-4 than for IFN-γ (P = 0.0004) (Fig. 1). The IFN-γ to IL-4 ratio was determined to compare functional T-cell heterogeneity. A cytokine ratio greater than 1 denoted a shift towards Th1 immune responses whereas a
Figure 3 Correlation between parasite density and plasma levels of interleukin 4 in pregnant women from southwest Cameroon. Values indicated are log10 transformations of the respective parasite densities and interleukin 4 OD405 values.
E.A. Achidi et al. ratio lower than 1, represented a shift towards Th2 immune responses. In 112 of 132 women, the cytokine profile of the study participants was biased towards Th2 responses. The frequency of women with Th1 or Th2 cytokine responses was similar for women with or without peripheral malaria parasitemia (P > 0.10). Furthermore, cytokine levels were not influenced by MA, GA, or gravidity (P > 0.10). In fact, all women in the first trimester (5 of 139) were Th2 responders.
3.3. Effect of studied variables on cytokine levels Plasma IFN-γ levels were significantly lower than IL-4 levels, and this was not affected neither by MA, GA, or gravidity (Table 2). The levels of both cytokines were, however, similar among women grouped by GA (P = 0.126) or by malaria infestation status (P = 0.118). Furthermore, the levels of both cytokines were similar among groups based on MA, GA, and gravidity. An insignificant negative correlation was observed between malaria parasitemia density and plasma levels of IFN-γ (P > 0.05) (Fig. 2) and IL-4 (P > 0.05) (Fig. 3).
4. Discussion This study investigated rates of malaria infestation and parasite density in infested pregnant women during the rainy season in south western Cameroon according to MA, GA, and gravidity, and determined which, if any, of the 3 variables measured provided information that could be exploited for the control of malaria in this endemic area. The results suggest that young pregnant women are at risk for malaria in this area, an observation in agreement with the intense, perennial dynamics of transmission in the area. Young age [15] and low gravidity [13,16] have been previously reported as important risk factors for malaria infection and density in pregnancy. Although these factors are interrelated, in that primigravidae tend to be younger, the age dependence has also been attributed to intrinsic immune factors [17]. It is also believed that nonsterile natural immunity to malaria develops with age and after repeated exposure, sometimes resulting in asymptomatic parasitemia. Both humoral- and cell-mediated immune mechanisms have been implicated in the observed age dependence [18]. The increased vulnerability of primigravidae may reflect the absence of specific antibodies to variant surface antigen, delayed onset of antiadhesion antibodies [19] and, possibly, lower MSPI-19 antibody titers [20]. The similar parasite rates found across age and gravidity groups, as well as the insignificant differences in parasite density found across the different gravidity groups in this study are in disagreement with previous reports, perhaps because this study was based on a smaller sample size. It may also be that most of the primigravidae were more compliant with taking sulfadoxine/pyrimethamine, an antimalarial chemoprophylactic agent given to pregnant women during antenatal enrollment. In a previous study in the study environment, these groups tended to be more regular at antenatal follow-up clinics and better complied with healthrelated advice given by the midwives [13]. Interferon γ and IL-4, respectively, are the chief cytokines that determine Th1 and Th2 immune responses. In this study,
Malaria parasitemia and systemic cytokine bias in pregnancy IL-4 levels were found to be significantly higher than IFN-γ levels, thus biasing the immune response towards Th2, which was consistent with previous reports [21]. The anti-inflammatory cytokine environment is thought to be maintained, in part, by the high progesterone levels in pregnancy, which induces both Th0 to Th2 conversion [22] and the ability of CD56+ T cells to secrete IL-4 [23]. The enhanced IL-4 expression, perhaps in concert with other anti-inflammatory immunomodulatory cytokines, curtails the potentially hazardous effects of IFN-γ and other Th1-related cytokine production on systemic immunity during pregnancy, thus ensuring the retention of the fetal allograft [24]. It may be that IL-4 blocks NK activity and DNA synthesis in the CD16– CD56-bright NK cells of the decidua by inhibiting IL-2 receptor and IL-2-induced killer activity—an activity potentially deleterious to the fetus because it may cause thrombosis, inflammation, and abortion [25]. The fact that the IFN-γ to IL-4 ratio did not change throughout pregnancy is consistent with previous reports [24], which suggests that a strong Th2 placental environment is compatible with successful pregnancy. Previous reports have suggested that the systemic cytokine bias that occurs upon infestation with malaria parasites does parallel placental infestation [10]. In this study, the lack of conversion of Th2 responses to Th1 responses in the presence of peripheral malaria parasitemia could partly be explained by the predominantly low (subthreshold) parasite densities recorded. Furthermore, peripheral malaria may not be as relevant as placental malaria in the induction of Th1 immune responses. In this case, malaria parasitemia may alter the preponderant Th2 bias at high densities or when sequestration of infected red blood cells occurs in the placenta. Consistent with previous results, the similar levels of IFN-γ and IL-4 found in malaria-infested pregnant women suggest diminished type 2 cytokine responses [10]. It is expected that multigravidae with previous feto-maternal interactions, maybe due to malaria infection, should portray a cytokine picture different from that of primigravidae. This difference would be a result of both immune cell priming, especially against placental malaria parasites, and the development of antibodies against adhesion molecules responsible for placental sequestration of parasites. Further work is needed to fully elucidate the cytokine profile that prevails in pregnant women of different gravidity. Taken together, the study's results suggest a bias towards Th2 cytokine responses in the pregnant women investigated, and this pattern was unaffected by the age or gravidity status of study participants.
Acknowledgements This study received financial support from the Malaria Immunology and Pathogenesis Network and the UNDP/ World Bank/WHO Special Program for training in Tropical Disease Research (Grant 970973).
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