Cortisol and Plasmodium falciparum infection in pregnant women in Kenya

TRANSACT~NS

OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE (1989) 83, 17%177

Cortisol

and Plasmodium

falciparum

infection

in pregnant

173

women

in Kenya

Michael P. H. Vleugels’, Bernard Brabin*, Wijnand M. C. Eli118 and Ruurd de Graaf4 ‘St Joseph Hospital, Department of Obstetrics and Gynuecology, Eindhoeren, The Netherlands; zInstitute of Medical Research,Madang, Papua New Guinea; ‘Department of Cell Biology& Histology, Faculty ofMedicine, Catholic University of Nijmegen, The Netherlands; 4Department of Statistical Consultation, Catholic University of Ntjmegen, The Netherlands

Abstract Women living under holoendemic conditions of malaria in Kenva exhibited an increased Drevalenceof clinical malaria during pregnancy. In a&ion parasite rate and density were higher in primigravidae compared to multigravidae. Higher serum cortisol concentrations were found in women with patent malaria during pregnancy and the levels were higher before, during and after the malaria episode. A causal relation between serum cortisol levels and suppression of malaria immunity during pregnancy is disdussed. Introduction Plaswwdiumfalciparum infections are more frequent in pregnant worn& (GILLES et al., 1969; BRUCECHWATT,1983). Darticularlv in tximieravidae. These infections duri&*pregnanci ari presbed to be due to a pregnancy-associatedalteration of immune reactivity, secondary to a reduction of cell-mediated rather than humoral responsiveness (FABRISet al., 1976; BRABIN, 1985). Previous studies revealed a linear increase of the serum cortisol level during pregnancy, which was higher in primigravidae than in multigravidae (VLEUGELSet al., 1986). In addition serum cortisol levels were related to clinical malaria in a small study group of pregnant women in Tanzania (VLEUGELSet al., 1987). Pregnancy-associated changes in serum glucocorticoid levels were responsible for loss of immunity to malaria (P. bergti> in mice (VAN ZoN et al., 1982). In the present study the relation between the concentration of total serum cortisol, gestation, parity and P. falciparum infection independent of clinical symptoms was determined in a group of women living under holoendemic conditions in west Kenya (BRABIN, 1983). Subjects and Methods The study group consisted of 289 pregnant women (118 primigravidae and 171 multigravidae), all of whom visited the antenatal clinics of the rural hospital at Nangina in Kenya between January and April 1981. Subjects were studied from the first antenatal visit onwards. Follow-up studies were made at one, 4 and about 8 weekslater. 217 women were examined twice, 117 women 3 times and 41 women were examined 4 Addressfor correspondenceand reprints: Dr W. M. C. Eling, Departmentof Cell Biology & Histology, Faculty of Medicine,Universityof Nijmegen,GeertGrootepleinNr 21, 6500 HB Nijmegen, The Netherlands.

times. At each antenatal visit 5 mg/kg chloroquine basewas given as malaria therapy, according to local hospital policy. Gestational age was calculated from the last menstrual period and checked against fundal height. Blood sampleswere collected by venous puncture. Since the serum cortisol concentration exhibits diurnal variation all samples were collected between 0900 h and 1100 h, with women sitting in the upright position. Samples were centrifuged at room temperature within one hour of collection. Serum was separatedand stored at - 12°C for less than 2 weeks and subsequently at -2O”C, transported by air in a frozen condition to Nijmegen, The Netherlands and again stored at -20°C until analysed. Parasitaemia was determined in Giemsa-stained thick blood films. Leucocyte numbers were determined and parasites were counted against 200 leucocytes to es&ate parasite density, cl&sified according to the svstem of BRUCE-CHWATT (1958). The oositive parasite density index (PPDI: ‘WHi), 1983) was calculated by multiplying the frequency of each class by the class number, adding these products and dividing the total by the number of positive slides. Total serum cortisol concentration was determined in 94% of the blood samplesby the radio-immunoassay described by VESCEI(1974) and mod&d by DE MAN et al. (1980). The sensitivity of this radioimmunoassay. was 0.01 wol/litr; at 95% B/B,, (B=actual bindinn: Bn=initial binding). The data &hibited an in& &d intra-assay z&efficient of variation of 9% and 4.5% respectively. All measurements were made with a mean maximum binding of 50.3% (standard deviation 1.6%). Results Parasitaemiain relation to ameno&oea, parity and age For the analysis of the relation between parasitaemia and amenorrhoea, parity and age, the data from only one of the antenatal visits were considered. The data of either the first or the last visit were randomly chosen for statistical reasons.Amenorrhoea was divided into classgroups: 8-16, 17-26, and 27-42 weeks. The age classesused were 13-24, 25-34, and 354 years. The age class 13-24 years contained all the primigravidae. No relation between amenorrhoea and-parity or age could be detected (x2 test; P= O-8 and 0.38 resmzctivelv). The freauencv of narasitepositive slide; decrea&d with in&as&g duiation of amenorrhoea (Table 1); x2 test of homogeneity; P-=0*006). Since parity and agewere related the analysis of the frequency of infection in relation to parity was

174 performed in the age class of the primigravidae, i.e. lL24 years. Onlv the data for one visit ner woman were included in ihe analysis as described &we. The frequency of a parasite-positive slide was significantly higher in primigravidae than in multigravidae (Table 2: Fisher’s exact test; P=O+O7). Further analysis of the data of the multigravidae revealed a significant decreasein the rate of infection with an increasing number of pregnancies{Table 3: x2, P=OW). Again, the data of only one visit were included in the analysis (see above). In the same group of multigravidae no sign&ant decrease in the rate of infection with increasing age was found (x2 test, P=O*21; data not shown). Parasite density expressedas PPDI was analysed in relation to amenorrhoea, parity and age. Although the rate of infection altered during gestation (Table l), the PPDI was not significantly different. (one-way analysis of variance.;P=O*37). Comparison of primigravidae and mulugravidae in the age class 13-24 years not only revealed a higher frequency of infection (Table 2), but also a signiiicantly higher PPDI in primigravidae (Table 2: Student’s t test; P=O*OOl). On the other hand, the PPDI was not significantly different in relation to the number of gravidities, or the age classesof multigravidae (one-way analysis of variance; P=O-3).

In holoendemic areas asymptomatic parasitaemias frequently occur in adults and clinical symptoms depend to someextent on parasite density. Following previous observations (TRAPE et al., 1985; VLEUGELS, 1984), a PPDI of class4 or higher was taken to select the group of more severely infected women. Again the data of only one visit per woman were included in the analysis. A PPDI of 4 or higher was more frequently found in primigravidae than in multigravidae in the ageclass 13-24 years (Table 4; Fisher’s exact test; P
v

Table 1. Frequency of parasite-positive visits ia relation to amenorrhea Duration of -orrhoea (weeks)

NO. examined

No. parasite-positive

18-16 17-26 27-42

35 123 131

20 (57%) 62 (50%) 44 (34%)

Table 2. Frenquency of parasitaemia aad the positive parasite density iadea (PPDI) in primigravidae aad multigravidae ia the age class 13-24 yesrs

Primigravidae Multigravidae

NO. examined

NO. parasite-positive

Mm PPDI (*SD)

112 10s

66 (59%) 42 (40%)

5.6 (2.2) 4.0 (2.1)

1 I 50-I

. . .

B .

Fig. 1. A. Serum concentration of total cortisol in 287 pregnant women plotted against duration of amenorrhoea. Only one visit per woman was included; see materials and methods. B. Comparison of the serum concentrations of total cortisol in primigravidae (n=112; V and ----), and multigravidae aged 1>24 yeprs (n= 103; 0 and -), plotted against duration of amenorrhoea. The individual data are shown, together with the correspond-

Table 3. Frequency of parasitaemia in relation to the gravidity number in multlgravidae GlWidity

No. examined

No. parasite-positive

46 39 29 57

16 (35%) 17 (44%) 14 (48%) 12 (21%)

2nd 3rd 4th 5th or more

Table 4. Frequency of infection and positive parasite density index (PPDI) (class 4 or higher) ia primigavidae aad multigravidae io the ageclaasn-24years Mean PPDI (*SD) No. examined PPDia4 Primigravidae Multiaravidae

112 105

57 (51%) 27 (26%)

6.3 (15) 5.4 (1.4)

Fig. 2. Serum concentration of total cortisol in pregnant women detemtined during a parasite-positive visit, plotted against duration of amenorrhoea. Comparison of primigravidae (n=64; V and ----) and multigravidae aged E-24 years (n=42; 0 and -). Individual data are plotted together with the corresponding regression lines.

175 Table 5. Serum cortisol conceotration in primigravidae and mnkigravidae aged 11-24 years with and without malaria (positive parasite density index of class 4 or higher)

No. examined

Mean cortisol concentration (nnroklitre)

Primigravidae non-malarious malarious

15 60

0.67 0.81

Mnltigravidae non-malatious malarious

31 23

0.65 0.61

‘Concentrations recalcnlated for amenorrhoea of 24.5 weeks duration, the mean value for all visits.

Table 6. Serum cortisol concentration of primigravidae mnkigravidae aged between lM4 years

and

No. examined

Mean cortisol concentration (mnol/litre)

Primigravidae non-malarious malarious

15 13

0.62 0.74

Mnltigravidae non-malarious

31

0.60 0.58

IlldtiOUS

4

“Data from parasite-negative visits preceding a parasite positive visit (positive parasite density index of class 4 or nigher) of malarious women compared to the date from the first visit of non-malarious women. Data recalcnlated for amenorrhwa of 23.3 weeks duration, the mean value for these visits.

Table 7. Serum cortisol concentration multigravidae aged 13-24 years

No. examined

of primigravidae

MCitl cortisol level (~01 per line)

Primigravidae non-malarious malarious

26

0.80 0.86

Multigravidae non-malarious malarious

31 12

0.66

15

and

0.71

‘Data recalculated for amenorrhoeaof 26.6 weeks duration, the mean value for these visits. Comparison of data for the first parasitenegative visit (positive parasite density index [PPDI]=O) of malarious women after their first parasite-positive visit (PPDI of class4 or higher) with the data for the last visit of non-malarious women.

Serum cortisol in relation to amenowhoea, parity and age

Total serum cortisol concentrations were plotted against duration of amenorrhoea (Fig. 1). Only the data of one visit were included (the first or the last visit was randomly chosen). A linear relationship with function y=O.l6+0*021x (y=mean serum cortisol concentration in umol/litre, and x=amenorrhoea in weeks; Fig. la) was obtained. Analysis of the relationship between total serum cortisol and amenorrhoea for primigravidae and multigravidae, for ages 13-24 years only, yielded the functions y=O.13+0*024 x and y=O*20+0.017 x respectively (Fig. lb). Assuming parallelism of these regression lines (parallelism test: P=O.19), the corti-

sol levels were signnicantly higher in primigravidae than in multigravidae (one-way analysis of covariance: P=ONl7). The difference was estimated to be 0.09 pmoMitre. In the multigravidae neither age nor parity number significantly affected the total serum cortisol value during pregnancy (one-way analysis of covarimce: P=O40 and P=O*31 respectively). Analysis of data from women without infection at the randomly chosen times gave regression lines with function y=O.26+0*016x for primigravidae and y=O.32+0*012x for multigravidae in the age class 13-24 years. Assuming parallelism, the difference between primigravidae and multigravidae was not significant (parallelism test: P=O*52; one-way analysis of covariance: P=O*22). Analysis of cortisol data in primigravidae and multigravidae (ageclass 13-24 years) with a record of infection on the day of sampling revealed cortisol regression lines y=O.O16+0*032x and y=O*OO8+0*026xrespectively (Fig. 2). A significant difference of 0.13 urnol/litre was found (parallelism test: P=O.40; one-way analysis of covariance: P=O.Ol). To study the relation between PPDI and cortisol, the data of randomly chosen visits with a record of infection were used. A sign&cant correlation between PPDI and cortisol was found in the primigravidae (Spearman correlation test: R=0.35; P=O*OOS)but not in multigravidae (R=0*12; P=O.35). We also analysed changes in cortisol levels during pregnancy and in relation to development of infection. Data of primigravidae or multigravidae with a more severe infection (class 4 or higher according to BRUCE-CHWATT, 1958) were compared with those of women with no record of infection. All data from visits at least 2 weeks apart were included. In these groups, again, primigravidae, malarious and non-malarious, exhibited higher cortisol values than the corresponding muhigravidae (Table 5; twoway analysis of covariance with amenorrhoea as covariable: P=O.O4). The difference betrween malarious and non-malarious women averaged over both parity groups was not significant (P=O.35), but an indication of interaction between gravidity and malaria was found (P=O.O9). This might indicate that the effect of parity on the cortisol level was stronger than the effect of infection. Two-way analysis of covariance and the linearity hypothesis (t tests) of the above randomized groups revealed significantly higher cortisol values in malarious primigravidae than in malarious multigravidae (P=O=OO2),but not in the non-malarious women (P=O.78). In addition, malarious primigravidae had higher cortisol values than non-malarious primigravidae (P=O*O7), but this was not the casefor multigravidae (P=O.59). An important question is whether cortisol levels rise before and/or during a malaria infection. The cortisol data from the first visits of women without a record of malaria during pregnancy were compared with the cortisol data from the last parasite-negative visit before a parasite-positive visit for women with an infection of class 4 or higher. On the basis of a two-way analysis of covariance with gravidity and malaria as factors, and amenorrhoea as a covariable, separatet tests were carried out (linear hypothesis) for primigravidae and multigravi-

176 dae in the age group 13-24 years. The cortisol values in sera from parasite-negative visits before a positive visit were higher than those of negative visits in women without a record of malaria during pregnancy. There was a good indication for significance in primigravidae, but not in multigravidae (Table 6; P=O*O8 and P=O*84 respectively). Similarly, on the basis of a two-way analysis of covariance of data from the first parasite-negative visit following the hrst parasite-positive visit, higher cortisol values were found compared to the data from the last visits of women with no record of infection in primigravidae, but the difference was not significant (Table 7: P=O-39). However, parasite-positive multigravidae exhibited even lower cortisol levels after a parasite positive visit than multigravidae without a record of infection. Discussion This study confirmed a higher prevalence of malaria (GILLES et al., 1969; CAMPBELL et al., 1980) and of patent malaria infections during pregnancy (KORTMANN, 1972; VLEUCELS 1984) in women living under holoendemic conditions. In addition, parasite rate and density were higher in primigravidae than in multigravidae controlled for gestational age, as was observed in a study of Tanzanian pregnant women (VLEUGELS et al., 1987).

As a consequence, primigravidae apparently lose their malaria immunitv more frequently during infection than multigravidae, which is in line with-results of MCGREGOR (1984) and with observations on clinical malaria during pregnancy (VLEUGELS, 1984;

VLEUGELSet al., 1987). This has also been shown in studies of the loss of P. berg/k immunity in pregnant mice (VAN ZON 81 ELING, 1980a, 1980b).

The parasite rate decreasedwith increasing parity number but not with increasing age. This indicates a reinforcement or improvement of malarial immunity after reseated pregnancies, a phenomenon already observed in the mouse malaria model (VAN ZON et al., 1985) and in the Tarzanian studv (VLEUGELS, 1984). The lower frequency of malaria d&g the secondand third trimester might be related to a reinforcement of immune reactivitv (BRABIN. 1983). No relation was found between parasite density ‘and amenorrhoea, suggesting that loss of immunity and the associated parasite density are determined by independent factors. Total serum cortisol increased linearly during gestation, and was significantly higher in primigravidae than in multigravidae with a patent infection, as observed in the Tanzanian study (VLEUGELS et al., 1986). Studies on malaria immunity during pregnancy in endemic areasare hampered by the fact that people in endemic areasare premune, i.e. harbour parasites in their peripheral blood without clinical symptoms. Previous studies have indicated that par&e densities of class 4 or higher (BRUCE-CH~ATT, 1958) in endemic areas can be considered to indicate patent and even clinical infection (TRAPE et al., 1985; VLEUGELS,1984). Indeed, parasite densities of class4 or higher were found more frequently in primigravidae than in multigravidae. In contrast to previous studies, cortisol concentrations were not significantly different in primigravidae and multigravidae without patent infection

(VLEUGELS,1984; VLEUGELSet al., 1986). In this study women were grouped according to gravida and not parity number. Nulliparae may therefore complicate the group of multigravidae, a problem that has been indicated in experimental studies with P. bergti (VAN ZON et al., 1985). Different cortisol concentrations in nulliparous women with or without patent infection (VLEUGELS, 1984) might support this notion. More studies are

needed for the analysis of differences in the cortisol levels of primigravidae and nulliparae in relation to a possible effect of abortion on the cortisol levels in a subsequent pregnancy. A higher cortisol concentration in primigravidae with patent infection is taken to indicate a cortisol-related loss of immunity during pregnancy. Studies in the P. berg/k-mouse model showed that the serum glucocorticoid level could be responsible for loss of immunity during pregnancy (VAN ZON et al:, 1982, 1985) and this was confirmed in the Tanzaman study (VLEUGELS et al., 1987). In this study, not only were higher cortisol concentrations found in relation to patent infection in primigravidae, but these women exhibited higher serum values before, during and after the patent episode. The increase of total serum cortisol before patent infection is taken to indicate a causal relation between cortisol and infection (loss of immunity) during pregnancy. A similar relation was not observed among multigravidae, in contrast to observations on multiparae with clinical malaria in the Tanzanian study (VLEUGELS, 1984). The presence of nulliparae in the multigravidae group, and the choice of a parasite density of class 4-or higher in this study instead of clinical infection as used in the Tanzanian study, may have been important factors involved in this difference. Thus, the results of this study support the observations, made in the P. berghi-mouse model, that serum glucocorticoid regulates malaria immunity (VAN ZON ec al., 1982? 1985). With regard to the mechanism of malaria Immunity, it is important to note that suppression associated with pregnancy is considered to represent a reduction in cell mediated, rather than humoral, immunity (FABFUS et al., 1976). Whether reduction of the malaria prevalence in multigravidae is related to quantitative or qualitative changes in malaria immunity remains to be determined. Acknowledgements

Mr C. C. Hermsen and Mrs J. Peters are gratefully acknowledged for their skilled technical assistance,and Mrs M. Scholten for typing the manuscript. References

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