Entomological vectors and inoculation rates.
Rainfall measurements identified two annual wet (April–June and September–November) and two drier (December–March and July–August) seasons (). The annual pattern was relatively consistent, but the second wet season occurred earlier in some years than others. Entomological studies conducted in 2004 resulted in the capture of 775 mosquitoes. Anopheles nili was the most abundant species that transmits malaria (45.7%), followed by Anopheles funestus (16%), An. gambiae (14.2%), and Anopheles moucheti (1.9%) (). All An. gambiae collected were identified as An. gambiae s.s. and of the M molecular form. The distribution of the malaria vectors varied throughout the year, but the change in numbers did not follow the seasons. The average monthly entomological inoculation rate (EIR) was 21.3 infectious bites/person/mon (ib/p/mon), with rates lower in the dry seasons (e.g., July [9.4 ib/p/mon] and August [14 ib/p/mon]) compared with the other months) (P = 0.046). The annual number of infective bites/person/year (ib/p/yr) for An. nili, An. funestus, An. gambiae, and An. moucheti were 144 ib/p/y, 70 ib/p/y, 38 ib/p/yr, and 5 ib/p/yr, respectively. Overall, results showed those individuals in Ngali II received ~257 P. falciparum infectious bites/p/yr or 0.7 ib/p/day throughout the year.
Figure 1. Rainfall and entomological inoculation rates in Ngali II. (A) Average rainfall (linear mm) showed annual variations between the two wet (shaded areas) and two dry periods. (B) Entomological studies conducted between May 2004 and February 2005 identified (more ...)
Prevalence of malaria in Ngali II.
The prevalence of slide-positive P. falciparum infections was determined at the end of the dry season in 1998, 2002, and 2004 and during the rainy season 2000 (). Among the four surveys, P. falciparum was detected in an average (±SEM) of 75 ± 6% of children 1–4 years of age (range 62–83%) and 68 ± 4% of those 5–14 years of age (range 52–77%). In young adults (15–19 yr), the prevalence declined to 43 ± 2% (range 40–45%) and was 26 ± 2% (range 8–38%) in adult ≥ 30 years of age. Because there was a gradual decline in prevalence between 15 and 39 years of age, adults in this age group, which includes women of child-bearing age, were still acquiring immunity to P. falciparum (). Minimal variation was found in prevalence of P. falciparum at the end of the dry season and rainy season, even though there was a difference in vector transmission rates (). The overall annual prevalence did not differ significantly during the 6-year period of study.
Figure 2. Prevalence of malaria in Ngali II. Door-to-door surveys were conducted at the end of the dry season August–September 1998 (N = 830 participants), 2002 (N = 511), and 2004 (N = 558 individuals) and in the wet season March–April 2000 (N (more ...)
The prevalence of anemia was also recorded. Hematocrits < 30% were found in 14.4 ± 3.5% for children between 1 and 4 years of age, but anemia was uncommon in older children 5–14 years of age (1.0 ± 0.18%) even though more than half of them had asymptomatic infections (). Anemia was detected in few adult males (0.8 ± 1.3%) and females (3.5 ± 1.7%). Overall, these results showed that P. falciparum
is hyperendemic (i.e., prevalence ≥ 75% in children 1–4 years of age and perennially transmitted)28
in Ngali II and that residents quickly gained immunity to disease and malaria-associated anemia after repeated malarial exposures.
Characteristics of the pregnant women enrolled in the study.
A total of 60 women were recruited early in pregnancy (median enrollment 14 wk [13.0–16.5 wk]), including 19 primigravidae and 41 multigravidae (mean 3.1 ± 1.7 prior pregnancies, range 1–8). These women had lived in Ngali II for an average of 9.6 yr. They ranged in age from 14 to 38 yr, with 41.7% being lower than 20 yr. The mean age of primigravidae and multigravidae were 18.4 ± 3.5 yr and 25.8 ± 5.4 yr, respectively. The women had an average of 6.5 yr of formal education (range 3–14 yr) and 84.5% listed farming as their primary occupation, 8.6% worked at home, and 6.9% had other employment. None of the women smoked and only 16.7% drank alcohol during pregnancy. Overall, 20.3% of the women had blood type A, 39.0% type B, 0% AB, and 40.7% type O, and 19.3% were carriers of the sickle cell trait (A/S).
Sixty-three percent (38/60) of the women were followed throughout pregnancy with a mean of 5.7 ± 1.1 prenatal visits per woman. Data from these women (12 primigravidae and 26 multigravidae) were used in longitudinal analyses. The other 22 women dropped out with a mean of 2.1 ± 0.9 prenatal visits, mostly because they moved from the village. Data from all women were used in cross-sectional analyses.
Use of malaria prevention.
A survey was conducted at enrollment and a questionnaire was completed at each prenatal visit to document use of antimalarial preventive measures. At enrollment, 48% (28/59) of the women expressed concerns about having malaria. None of the women used bed nets during pregnancy, primarily because of lack of availability within the village. However, 98% (48/49) reported taking chemoprophylaxis throughout pregnancy. Both primigravid and multigravid women began taking antimalarial drugs at 18.3 ± 4.0 and 18.2 ± 4.1 wk, and continued using them until near term (33.8 ± 5.4 and 35.6 ± 5.8 wk, respectively), with 60% taking CQ, 27% using a combination of CQ and other drugs, and 13% taking other drugs. The use of prophylaxis did not differ between primigravidae and multigravidae.
Prevalence of slide positivity for malaria in women during pregnancy.
Among the 60 women enrolled, the prevalence of slide positivity was higher each month in primigravidae (54% ± 7%, mean ± SEM) than in multigravidae (26 ± 3%) throughout the course of pregnancy (P < 0.0001, type 3 LRT) (). Prevalence was also higher in primigravidae than in age-matched adults in the general population (44 ± 5%; data from four surveys, N = 174 women aged 15–22 yr) even though they took antimalarial drugs for prophylaxis and treatment. In contrast, the prevalence of malaria in multigravidae was lower than in age-matched non-pregnant women in the population (33% ± 9%; data from four surveys, N = 193 women aged 20–32 yr), most likely caused by increased antimalarial usage. Peripheral parasitemias were also significantly higher throughout pregnancy in slide-positive primigravidae (6,620 ± 1,220 parasites/μL) than multigravidae (1,210 ± 420 p/μL) (P < 0.0001, type 3 LRT) ().
Figure 3. Prevalence of slide-positive malaria during the course of pregnancy. (A) Prevalence of slide-positive malaria in primigravidae (N = 9–16 women/time point) and multigravidae (19–33 women/time point). DEL = peripheral blood at delivery. (more ...)
In the cohort followed longitudinally, primigravidae became slide-positive for P. falciparum early in pregnancy with parasites detected in 53% by the third month (14–17 wk) and all primigravidae by the fifth month (). In contrast, a gradual increase in the accumulation of slide positivity was found among multigravidae, with only 79% becoming slide-positive before delivery. Primigravidae and multigravidae were blood smear positive 3.3 ± 1.1 and 1.3 ± 1.3 times, respectively during pregnancy (P < 0.001), i.e., they were slide-positive at 60% ± 18% and 21% ± 23% of their prenatal visits. The slide-positive rate in primigravidae remained significantly higher than in multigravidae (P = 0.046) after adjusting for age, expressed concern about having malaria, and use of chemoprophylaxis in a multivariate analysis. Slide positivity rates were similar between women followed longitudinally and those in the larger group used in the cross-sectional analysis (). The finding that 92% of primigravidae were slide-positive three or more times compared with only 18.5% of multigravidae, illustrates the extent to which primigravidae were more susceptible to P. falciparum than multigravidae, even when CQ and other antimalarial drugs were used.
Figure 4. Longitudinal analysis of slide positivity, polymerase chain reaction (PCR) positivity, and submicroscopic infections during pregnancy: (A) Accumulative time until first infection detected by microscopy (N = 12 primigravidae; N = 26 multigravidae). (B (more ...)
Prevalence of PCR-detected and submicroscopic infections.
All primigravidae and 97% of multigravidae became PCR-positive for P. falciparum before the 26th week of gestation (). Longitudinal analysis estimated that primigravidae and multigravidae were PCR-positive an average of 4.6 ± 1.7 and 3.4 ± 1.7 times (i.e., at 92% and 62% of prenatal visits) before delivery, respectively, or 5.1 ± 1.8 (92%) and 4.1 ± 1.9 times (66%) including delivery. The difference was not statistically significant (P = 0.106).
Using microscopy and PCR, P. falciparum were detected in the blood of 74% ± 3% of pregnant women each month, with an average of 40% ± 3% being slide-positive and 34 ± 4% having submicroscopic infections (). The ratio of microscopic to submicroscopic infections remained relatively consistent throughout pregnancy. For comparison, 36.1% of age-matched non-pregnant women (N = 16 women, 40 blood samples) had submicroscopic infections.
Multiplicity of infectivity (MoI) during pregnancy.
The MoI were determined using 185 PCR-positive blood samples (). Results showed that MoI in samples collected < 14 wks averaged 4.8 ± 1.3 and 4.0 ± 1.6 parasite genotypes in primigravidae and multigravidae, respectively. Thereafter, the number of genotypes remained relatively constant in the peripheral blood of primigravidae (mean 3.7 ± 0.2 parasite genotypes) and multigravidae (2.7 ± 0.12 genotypes), with significantly higher numbers found in primigravidae (P = 0.0002, type 3 LTR).
Figure 5. Multiplicity of infectivity (MoI). (A) The mean number of parasite genotypes detected in the peripheral blood of primigravidae and multigravidae at each time point. Mean ± SD, average of 8 and 17 data points per bar for primigravidae and multigravidae, (more ...)
Total number of parasite genotypes detected during pregnancy.
The total number of different msp1 and msp2 genotypes detected in the peripheral blood of each woman during pregnancy was determined (). It was assumed that if a genotype was detected early in pregnancy and again later, it resulted from the same infection. The total number of parasite genotypes detected by msp1 and msp2 alleles was similar (). Overall, the total number of different parasite genotypes infecting primigravidae and multigravidae during pregnancy was similar, 8.9 ± 3.2 and 7.0 ± 2.9, respectively.
Total number of parasite genotypes detected in the peripheral blood of women during pregnancy
The number of MSP1-19 variants present in the peripheral blood of 20 pregnant women was determined by DNA sequencing of 75 samples (). Nine variants were identified, with 43% being EKSNGL (FUP type), 35% QKSNGL (FVO type), and only 2.7% ETSSRL (3D7 type). The remaining six variants were detected in less than 4% of the samples. A difference in the distribution of the variants was found between primigravidae and multigravidae. Both the FUP and FVO types were detected in the blood of all primigravidae and these were the only variants found. On the other hand, this combination of variants was detected in only 18% of multigravidae, whereas 10 of the 11 multigravidae were infected with one or more of the rare variants. Although parasites were sequenced from only 20 women during the course of pregnancy, the distribution of MSP1-19 genotypes was clearly different between the two groups.
An attempt was made to determine how long women had been infected with parasite genotypes present in the IVS at term. This was possible because most msp1 and msp2 genotypes were detected in < 15% of the women. Because most genotypes were rare in the population, it is likely their presence in the same woman over time was because of persistence and not to new infections. A total of 31 parasites present in blood collected from the IVS could be “traced backward” through pregnancy by msp1 and 38 parasites by msp2 genotyping (). Overall, 62% of the parasites in the IVS had not been detected during pregnancy, but 13.2–19.4% had persisted since the fifth month of pregnancy ().
Clinical manifestations of P. falciparum infections.
Because symptoms associated with malaria are nonspecific, it is difficult to assess how often they occur in women with mild infections. To investigate the frequency of clinical symptoms, each woman was asked at her monthly prenatal visits if she had had any of the following symptoms within the last 2 days and last 2 wk: fever, chills, headache, vomiting, nausea, diarrhea, lethargy, cough, abdominal pain, myalgia, or puritis. On the basis of 299 responses, 39.2% of the women who were slide-positive and 40.6% who were slide-negative reported having one or more of the symptoms. Only three of the 38 (7.9%) women followed longitudinally were diagnosed as having one or more episodes of malaria, i.e., they were slide-positive, had fever, plus one or more of the above symptoms, for an overall prevalence rate of 2% (5 episodes/299 visits). Thus, there was little evidence that women developed malaria-associated symptoms when they were slide-positive, or that slide-positive and submicroscopic infections ultimately developed into clinical episodes.
With the exception of anemia and hypertension, the clinical parameters studied remained within normal ranges (). Among the cohort followed longitudinally, 50% of primigravidae were anemic during the second and third trimesters compared with only 16.2% and 7.7% of multigravidae (P < 0.01). Five of the 38 women showed signs of hypertension in the second and third trimester, including three primigravidae and two secundigravidae. Fever, severe anemia, and high or low WBC counts were uncommon.
Percentage (%) of women who had the following conditions*
At each visit, maternal weight was recorded and BMI determined. Between the 14th and 38th wk, multigravidae gained more weight (6.5 ± 3.4 kg) than primigravidae (1.9 ± 2.5 kg) (P = 0.009 t test). The BMI rose from an average of 21.5 to 23.0 in primigravidae and 20.9 to 22.9 in multigravidae (P = 0.008 t test) during this period. The effect of malaria on weight gain has received little attention. Unfortunately, the effect could not be assessed in primigravidae because they were blood smear positive on multiple occasions, however, multigravidae who were slide-positive only 0–1 times during pregnancy gained 7 ± 6 kg compared with those who were slide-positive ≥ 2 times who gained 5 ± 3 kg. Although the difference was not significant because of wide variation and small sample size, results suggest that malaria may have an impact on maternal weight gain.
At delivery, 57% of primigravidae were peripheral blood smear positive, 83% were peripheral blood PCR-positive, and 100% had placental malaria (PM) with evidence of active (50%) and chronic (50%) infections (). In comparison, only 17% of multigravidae were peripheral blood smear positive, 68% were PCR-positive, and 38% had PM with 19% having active and 19% having chronic infections. Overall, 52% of the placentas of multigravidae showed no evidence of past infections even though 97% of the multigravidae were PCR-positive at least once during pregnancy. Babies born to primigravidae had a lower birth weight than those born to multigravidae (P = 0.05). The impact of PM on birthweight could not be assessed, because all primigravidae were infected at the time of delivery. Most infants were born full term and all survived.
Summary at delivery and pregnancy outcomes*