TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE (2000) 94,645-651

The epidemiology of febrile malaria episodes in an area of unstable and seasonal transmission

Haider A. Giia1*2*, Susanne Rosthoj3, Daniel Dodoo ly4, Lars Hviid’, Gwiria M. H. Sat&, Thomas Scheike3, David E. ArnotS and Thor G. Theander’ ‘Centre for Medical Parasitology, Department of Infectious Diseases, Copenhagen University Hospital (Rigshospitalet), and Institute for Medical Microbiology and Immunology, University of Copenhagen, Tagensvej 20, DK 2200 Copenhagen N, Denmark; ‘Department of Biochemisty, University of Khartoum, I? 0. Box 102, Khartoum, Sudan; 3 Department OfBiostatistics, University of Copenhagen. Blegdamsvej 3, DK 2200 Copenhagen N, Denmark; 41mmunology Unit, Noguchi Memorial Institute for Medical Research, Legon, Ghana; 51nstitutefor Cell, Animal, and Population Biology, Division of Biological Sciences, University of Edinburgh, West Mains Road, Edinburgh EH9, UK

Abstract This study investigated the epidemiology of uncomplicated falciparum malaria in an area of unstable and seasonal transmission in eastern Sudan. About 90% of malaria morbiditv in this region occurs in the months of September to November, and very few malaria cases occur during the intensely&d Sudanese dry season and during years of drought. The malaria situation in the study site, the village of Daraweesh, was analysed during 3 consecutive malaria seasons in 1993-95 during which the 457 inhabitants suffered a total of 436 episodes of falciparum malaria. Using an Andersen-Gill proportional hazard model for recurrent events stratified by family, we have calculated the relative hazard for clinical malaria episodes by age, sex, haemoglobin genotype, blood type and infection in the previous season. The malaria risk was significantly lower in individuals aged 20-88 years than in the 5-19 years age-group. The relative protection due to adulthood varied between seasons (relative risk, RR, 0.34 to 0.67). Serological data were not consistent with the hypothesis that the age difference in incidence was due to differences in exposure. During the 1993 season the malaria incidence in males was lower than in females (RR = 0.75), during the 1994 season the incidences were comparable, whereas males had an increased risk of malaria in 1995 (RR = l-87). The relative risk in individuals carrying the haemoglobin AS genotype compared to homozygous AA individuals was 0.57.

Keywords: malaria, Plasmodium fulciparum, morbidity, risk factors, age, gender, haemoglobin genotype, Sudan

Introduction The development of clinical immunity to malaria in

endemic areas is governed by the transmission pattern. At one end of an epidemiological spectrum are situations of unstable transmission where there are substantial seasonal fluctuations in disease incidence. At the other extreme of the spectrum, transmission and morbidity remain stable over the seasons and years (MACDONALD, 1957). Individuals livine in areas of stable transmission acquire protection agai&t clinical attacks after several years, and the disease therefore affects mainly children (GILLES & WARREU, 1993). In areas of unstable transmission it has been generally believed that the entire population is vulnerable and that all infections are followed by clinical disease (MACDONALD, 1957). It is now recognized that this is an over-simplification, although the extent to which immunity develops remains uncertain.

We have monitored the malaria situation in a village in eastern Sudan where malaria transmission is unstable and highly seasonal. In this area clinical infections are absent during most of the year and case incidence fluctuates dramatically between years. We have pre- viously reported that a high proportion of new Plasmo- dium falciparum infections run a subclinical course and that a high percentage of village residents harbour low- level infections, detected by polymerase chain reaction (PCR) only, throughout the dry season (ELHASSAN et al., 1995; ROPER et al., 1996; ROPER et al., 1998). Thus, even though both clinical and asymptomatic infections are found in all age-groups, many individuals are appar- ently able to control some infections at subclinical levels. The purpose of this study was to investigate whether this ability to control malaria infections is a stable phenotype, or alternatively, something that develops after years and a number of infection episodes. Based on close clinical surveillance during 3 successive malaria seasons, we show that the risk of having a clinical episode of malaria

Address for correspondence: Thor G. Theander, Centre for Medical Parasitology, University of Copenhagen, Panum Build- ing 24.2, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark; e-mail theander@biobase.dk

declines markedly after the age of 20 years. This decline is most consistent with acquisition ofpartial immunity after having experienced a number of both symptomatic and asymptomatic infections.

Materials and Methods Study area

The study was carried out in the village of Daraweesh, Gedaref State in eastern Sudan. Daraweesh is situated on the Sudanese Savannah, at an altitude of 603 m above sea level, 16 km from Gedaref town and 80 km from the Ethiopian border. The large-scale sorghum and sesame cultivation that supports the region depends mainly on the June-October rains. Deep wells sunk into the sub- surface water table are the source of water for human use and irrigation of small vegetable gardens around the village.

The village consists of 120 thatched mud huts and covers an area of about 40 000 m2. Anopheles arabiensis is the sole malaria vector in the area, but meaningful estimates of entomological inoculation rates cannot be calculated for individuals living in Daraweesh, since only 1 infected mosquito has been captured in the village during 5 years of surveillance (A. A. Hamad, unpub- lished data). In 1993, the monthly mean maximum temperature ranged from 33.1”C in August to 41.6”C in April, the mean minimum temperature ranged from 16.7”C in February to 25.4”C in May. The average relative humidity was 27-39% in the months from January to May, 73-79% in the months July to Septem- ber. and 44-57% in October to December. Malaria in the’area is highly seasonal, and follows the annual June- October rains. The incidence of malaria varies consider- ably from year to year, malaria being virtually absent in years of drought such as 1990 and 1991 (THEANDER, 1998).

Study population The population in Daraweesh was about 450 during

the 3 years of the study (1993-95). The villagers are descendants of a small Fulani group, originally from Burkina Faso, who settled in the area a century ago. They live in extended family compounds and can be divided

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HAIDER A. GIHA ETAL. 646

into 52 families. The haemoglobin (Hb) AS genotype is found in 2 1 families, and is carried bv 68 individuals (30 males, 38 females).- The age distribution of the popu- lation is skewed towards the younger age-groups (Fig. 1). Most villagers work as subsistence farmers, but some are lorry drivers and tradesmen in the town of Gedaref. Village children attend a local primary school. Bednets or anti-malarial chemoprophylaxis are-not used in Dara- weesh. The villaee is not endemic for schistosomiasis or leishmaniasis, b;t hehninthic and other gastrointestinal infections are common.

Blood and haemoglobin typing ABO and rhesus typing was done by routine agglutina-

tion assays. The haemoglobin typing was based on a sickling test using 2% fresh sodium metabisulphite solution and a cellulose acetate electrophoresis to con- firm the sickling test result (MARENGO-ROWE, 1965).

Clinical and parasitological surveillance A cohort of 100 individuals aged 5-30 years and

carrying the AA genotype of the a-haemoglobin gene was monitored clinically from 1990 to 1992. In 1993- 96, malaria morbidity surveillance was extended to cover all the inhabitants of the village: 415, 432, and 421 individuals were available for clinical follow-up during the malaria seasons of 1993, 1994, and 1995, respec- tively. Between September and February a health team, consisting of medical doctors, microscopists and village health workers visited the village at least every second day. The villagers were encouraged to report to the team or attend the village clinic if they had any medical complaint. If any individual had complaints or symptoms suggestive of malaria a blood smear was made and, if found positive for malaria parasites, drug treatment was initiated. The first line of treatment for uncomplicated malaria was chloroquine, sulfadoxine-pyrimetbamine treatment was given for chloroquine-resistant infections, and quinine was used as third line of treatment and also given to patients suspected to be allergic to sulfadoxine.

The team also provides free medication for the most common diseases present in the village and, owing to the longstanding collaboration between the village and the study group, it is unlikely that villagers would seek treatment for malaria elsewhere. At the beginning of September, middle of October, and beginning of January parasite surveys were done including all available villa- gers. Blood smears were collected; thick and thin smears were stained with Giemsa and examined for malaria parasites under a magnification of X1000. Films were considered negative after the examination of 200 thick- smear fields without detection of parasites. Positive films were scored semi-quantitatively as + (1- 10 parasites per 200 thick-film fields), ++ (1 l-200 per 200 fields), +++ (l-10 per field), and ++++ (>lO per field).

Data storage and akfinition ofmalaria Complaints reported by the villagers, the result of a

physical examination and of the blood film reading were

81-90 71-80

-$ 61-70 & 51-60 4 41-50 O Lb 31-40 b 21-30 2 11-20

O-10

I 0” 0

=

60 40 20 0 20 40 60 80 Number of individuals

Fig. 1. Sex and age distribution of individuals in Daraweesh (in 1994). Open bars, females; solid bars, males.

collected on forms and later entered into a Fox-Pro Database (Microsoft, Seattle, Washington, USA). A patient was defined as suffering from malaria if (a) either complaining of fever or the body temperature measured with an oral probe was > 37.5”C and (b) asexual parasites were found in the blood film. A oarasite densitv criterion was not included in the malaria definition because the percentage of slide-positive asymptomatic parasite car- riers varies considerably by season, and fever thresholds are low in many individuals. Only malaria episodes separated by 30 days from the previous episode were considered as new attacks. Thus, episodes occurring less than 30 days from the previous episode were considered to be recrudescent infections.

Blood sampling Venous blood samples were collected in heparinized

vacutainers (Becton Dickinson, Rutherford, NJ, USA). Following centrifugation, plasma was collected and stored at -20°C until use. Informed consent was always obtained prior to collection of blood samples from donors and/or their parents. The study received ethical approval from the Ethical Committee of the University of Khartoum and national clearance from the Sudan Min- istry of Health.

Measurement of malaria antibodies A crude schizont sonicate was prepared from l?

falciparum cultures. Clinical parasite isolates collected from 5 individuals in Daraweesh between 1994 and 1996, a Ghanaian isolate, and laboratory-adapted para- site FCR3 were used. Parasites were maintained in cultures of human 0 Rh+ erythrocytes in RPMI (TRA- GER & JENSEN, 1976) supplemented with Albumax II (Life Technologies, T&&up, Denmark) as previously described KRANMER et al.. 199 11. Cultures were tzrown to parasitaemia of 2-3%, with the’majority ofpara&es in the trophozoite or schizont stages. The cultures were then further synchronized by selective high-gradient maanetic sortine (MACS; Miltenvi Bio Tee. Beraisch Glabach, Germany) (PAUL et al.; 1981; ST&GE et al., 1999). In brief, culture was passed through a size C MACS column mounted with a 0.9 mm X 40 mm nee- dle. The column was then washed with 2% fetal calf serum (FCS) in phosphate-buffered saline (PBS; 2% FCS), until the effluent was colourless, thereafter the column was removed from the magnet, and the captured infected cells were released by further washing of the column with 2% FCS. Late-stage parasites were col- lected and resuspended in normal saline 0.9% (l- 2 mL). The parasite sonicate was prepared by repeated freezing (-70°C) and thawing (37°C) of the aliquoted schizonts followed by centrifugation (8000 g) for 10 min at 4°C. The supernatant containing the crude schizont sonicate of the parasites was sterilized by filtration (0.2 pm-diameter filter) and stored in the presence of enzyme inhibitors at -70°C until use.

Crude schizont sonicate (100 pL, 1.2 ug/mL) in O-1 M sodium carbonate buffer @H 9.6) was added to half of the wells in a 96-well, flat-bottomed polystyrene microtitre plate (Nunclon; Nunc, Roskilde, Denmark). To the remaining wells a control solution of ultrasoni- cated erythrocytes was added. Plates were incubated overnight at 4°C washed 3 times with 1% Tween in PBS, and blocked with 150 ~.LL of 1% bovine serum albumin in PBS for 1 h. Test plasma (100 uL diluted 1:400 in PBS with 0.37 M sodium chloride, 1% Triton X and 1% bovine serum albumin), positive control plasma pool, and negative control plasma were then added in dupli- cate to wells coated by either parasite or control antigen. After l-h incubation the plates were washed and incu- bated with 100 & per well of 1: 1000 horseradish peroxidase-conjugated goat anti-human IgG (DAKO). All incubations were done on a rotating platform at room temperature, unless otherwise mentioned. The plates were then washed again and 100 pL of citrate buffer @H

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EPIDEMIOLOGY OF UNSTABLE MALARIA

4) containing 0.16 mg/mL of o-toluidine and 4 uL/mL of 10% H202 was added. The reaction was stopped by the addition of 50 pL of 4N H2S04. The optical densities (ODs) were read at 492 nm in a Titertek multiscan plate reader. OD readings of wells coated with control antigen were subtracted from the readings of wells coated with parasite antigen. On each plate a pool of highly reactive plasma was titrated in 7 two-fold dilutions. The content of antibodies in highest dilution of the pool was arbi- trarily set to 1000 units. Using a curve-fitting programme of the OD values measured by titration of the positive pool, the OD values of the test plasma were transformed into units of antibodies. Cut-off value for positivity was determined as the mean units of antibody + 2 standard deviations measured using pooled plasma from 33 Danish volunteers never exposed to malaria.

Data presentation and statistical analysis The morbidity data were analysed using an Andersen-

Gill proportional hazard model (ANDERSEN et aZ., 1993), that was stratified by family. This model is an extension of the Cox regression model to multiple events and time- dependent covariates. It is used to describe the effect of explanatory variables on the survival time (the time from which the patient was at risk until malaria diagnosed). The dependence between events within a family was dealt with through the stratification on family. The survival time for each patient is assumed to follow its own hazard function:

hi(& Z,) = ~(t)ePl=Llw+ +Pt&(O

where i = 1, , 457 is the patient and t 2 0 is the time since the start of the study. The vector Zi = (Z,, , . , Zik) is a vector of time-dependent 0- 1 expla- natory variables and l3 = (l!$, . . . , Pk) is a vector of unknown regression parameters associated with the explanatory variables. Further, h is an arbitrary unspeci- fied baseline hazard function, which varies with the time t 2 0 and between families. The time scale considered was calendar time. The effect of age, gender, season, haemoglobin genotype, ABO blood group and rhesus blood group was tested in the model. The model was also used to test the interactions between the effects of the different parameters, i.e., whether the age effect varied between seasons. For the malaria seasons 1994 and 1995 the test was used to ascertain whether a malaria diagnosis the previous year affected the risk of getting malaria. The Andersen-Gill model was validated using a goodness-of- tit procedure, and it did give a reasonable fit of the data. To further validate the conclusions reached based on the Andersen-Gill model, we also fitted a marginal survival model (WEI et al., 1989) and got the same conclusions.

Results Malaria morbidity

Malaria in Daraweesh is seasonal and the morbidity falls around 1 to 2 months after the annual rains cease. In the 3 seasons 1993,1994, and 1995, the peak incidence fell in late October, early October and late October, respectively (Fig. 2), and 69-93% of the febrile malaria episodes appeared between 14 September and 16 No- vember. The incidence varied between 0.0018 and

647

0.0029 episodes per day of risk between the malaria seasons 1993-95, and clinical disease affected a high proportion of the inhabitants of the village (Table 1). Of the 442 episodes of malaria diagnosed in the village from September 1993 until January 1996,436 were caused by l? falciparum, 3 by l? vivax, and 3 by I? malariae. Almost all (99%) of the malaria cases were uncomplicated, and did not require admission to hospital. Although grade 1 and 2 drug resistance to chloroquine and sulfadoxine- pyrimethamine is common in the village (data not shown), all patients with uncomplicated malaria re- sponded clinically to the treatment administered. Five patients, none of whom met the criteria for cerebral malaria, had more severe disease and were admitted to Gedaref Hospital where they received parenteral treat- ment with quinine.

50

d 40 2

‘% 30

3 4 20 B

% 10 d 2 0

(cl _-

3 40 %

‘6 30 I

Fig. 2. Number of malaria cases diagnosed weekly in Daraweesh (Sudan) during the malaria seasons 1993-95.

Table 1. Study population and malaria episodes in Daraweesh (Sudan) in the period from 1993 to 1995

Malaria season Population (year) size

1993 415 1994 432 1995 421

Percent who had malariaa

34.7% 35.2% 24.7%

No. of malaria episodes

158 176 108

“Percent of the population who had at least 1 episode of malaria during the season.

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648 HAIDER A. GIHA ETAL.

Indices of malaria endemicity The seasonality of malaria in Daraweesh was reflected

in the parasite rates as judged by microscopy of Giemsa- stained thick smears obtained during cross-sectional surveys from April 1994 to September 1995 involving villagers available on the day of survey (Table 2). The parasite rates varied between 0% (June 1995) and 17% (October 1994). Spleen rates were generally low, and did not change statistically significantly with the season. The parasite and spleen rates were not statistically signifi- cantly associated with age (Table 3), although the spleen rates in October 1994 were highest among children aged 2-9 years.

Morbidity and age Risk factors for clinical malaria were analysed using

Andersen-Gill proportional risk hazard model. The analysis revealed that the risk of malaria varied with age, and that the distribution of risk among the age- groups varied between the 3 seasons analysed (age- season interaction, P = 0.00 13). Figure 3 shows the risk of malaria in the different age-groups relative to the risk in individuals aged 5- 19 years. In the 1993 season the relative risk (RR) of individuals aged 2-4 years was 0.37 (P = 0.003) and for those aged 20-88 years 0.61 (P = 0.011). In 1994, the O-l and the 20-88 years age-groups had reduced risks (RR = 0.12, P = 0.0034, and RR = 0.34, P < 0.001, for 0- 1 and 20-88 years, respectively). In 1995, a risk reduction was observed for the age-group 20-88 years (RR = 0.67, P = 0.077). The estimated relative risks for the age-groups younger than 5 years were also reduced, but the difference did not reach statistical significance. In the initial statistical analysis 6 age-groups were studied (O-I, 2-4, 5-19, 20-29,30-39,40-88); this analysis revealed that there were no statistically significant differences in relative risks in the over-19 years age-group (data not shown).

Gender and risk The risk in contracting malaria differed between

women and men, but the gender effect varied between the malaria seasons (gender-season interaction, P = 2.25 X 10-l’). The risk for women was set to 1. During the 1993 season the risk for males was lower than in

1994

1’5-

1 T

I I O-1 Z-4 5-19 2w38

Age-grouP (Yea@

Fig. 3. Relative malaria risk (mean f 95% confidence interval) in different age-groups in Daraweesh (Sudan) during the malaria seasons 1993-95. The risk for the 5-19 years age- group was set to 1. Calculations were adjusted for co-variant effects of haemoglobin genotype and gender.

Table 2. Point prevalence of malaria parasitaemia and splenomegaly among villagers in Daraweesh during 1994 and 1995

1994

Screening April June September

Parasite rate* 6.5% 0.7% 8.0% (14/217)” (l/151) (22/275)

Spleen rateb ND ND 0.8% (1/119)b

“Percent (slide positive/total individuals examined). bPercent (palpable spleen/total individuals examined). ND, not done.

October

17.2% (41/238)

3.4% (8/238)

January

4.1% (13/317)

1.6% (2/127)

1995

June

0.0% (O/241)

25% (6/241)

September

0.8% (3/356)

ND

Table 3. Age-specific point prevalence of malaria parasitaemia and spleno- megaly in the October 1994 survey in Daraweesh

Agwwup (yea4

Screening o-1 2-9 10-19 20-88

(n = 6) (n = 82) (n = 84) (n = 66)

Parasite rate 16.7% 19.5%

Spleen rate ‘@4$1>

(O*O-4$9)

(1166$7)

(2*7-1;3)

95% confidence intervals are given in parentheses.

13.1% 19.7% (6+;‘$2)

(0.3-8.;)

(lo.;;L3;*3)

(0.0-5.;)

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EPIDEMIOLOGY OF UNSTABLE MALARIA 649

females [RR = 0.75, 95% confidence interval (CI) = 0.54-1.06, P = 0.101, during the 1994 season there was no difference between risk of the 2 sexes, and in the 1995 season men had an increased risk of malaria (RR = 1.87, CI = 1.25-2.8, P = 0.0024).

Haemoglobin genotype, blood group and risk Of the 457 villagers included in the study, 68 carried

the AS genotype of haemoglobin. The risk of malaria in these individuals was significantly reduced as compared to individuals carrying the AA genotype (RR = 0.57, CI = 0.39-0.82, P = 0.0026). The relative protection conveyed by the AS genotype did not vary between seasons (data not shown). When the malaria risk of individuals carrying blood group 0 was set to 1, the relative risk of individuals carrying blood groups A, B, and AB were 0.82, 1.17, and 0.74, respectively. These risks did not differ statistically significantly from the risk ofblood group 0 carriers or from each other. The malaria risks of rhesus-positive and rhesus-negative individuals did not differ significantly.

Risk of malaria in individuals who had malaria the previous season

After correcting for age, sex and Hb AS genotype, individuals who did not have malaria during the 1993 and 1994 seasons had almost the same risk of getting malaria in the following seasons (RR = 0.93, P = 0.55) as those who suffered from malaria in 1993 or 1994.

Exposure to blood-stage parasites Differences in malaria risk between mouvs could be

due to variations in exposure to new infectious bites, or to differences in the ability to control new infections. Since it is impossible to estimate exposure to infections using vector-based measurements in Daraweesh owing to the very low density of infectious mosquitoes, we attempted to measure exposure by comparing antibody levels to blood-stage antigens in samples collected before and

(4

after the 1995 malaria season. Individuals were defined to have acquired responses to the parasites if(i) the level was undetectable in the September sample and detect- able in the January sample, or (ii) the level in the January sample was over twice as high as in the September sample. Figure 4 compares the proportion of individuals who acquired antibodies in different age-groups (panel a), between males and females (panel b), and between individuals carrying the AA or the AS haemoglobin genotype (panel c). No differences could be detected between any of these groups, suggesting that the differ- ence in malaria risk was not due to differences in exposure to the parasite.

Discussion Daraweesh is a classical example of seasonal and

unstable malaria transmission in the Sahel Sudan Sa- vannah transition zone. Under such conditions malaria is known to affect all age-groups (IMACDONALD, 1957), but the detailed epidemiology of febrile malaria episodes has not been closely investigated. We have monitored the malaria situation in Daraweesh since 1988, and this paper presents morbidity data collected during the 3 vears 1993. 1994 and 1995. In the vears vrior to this Study, there were very few cases of malaria in-tie village in 1990 and 199 1, while about 30% of the population had an attack of malaria in 1992. It has not been possible to establish precisely when transmission occurs using vec- tor-based measurements (ARNOT, 1998). However, the fact that malaria morbidity is concentrated in the 2 months following the rains strongly indicates that most transmission takes place in this period and that little, if any, transmission occurs the rest of the year.

During the prolonged dry season malaria is almost absent from the village and very few sporadic cases are detected between February and August. This transmis- sion pattern is also reflected in fluctuations of antibody directed against malaria antigens during the year. The IgG levels to several erythrocyte-stage antigens such as

r s 4 60.

3 .e $ 40. 8

‘2 E e 20. c

o._ .jfi

Z-4 5-9 lo-14 15-19 20-24 25-34 3544 45-68

Age-group (years)

(b) (cl

Male Female AS donors AA donors

Fig. 4. Percentage and 95 % confidence interval ofindividuals acquiring antibodies to crude schizont sonicates during the malaria season 1995. Panel (a), acquisition according to age; panel (b), acquisition according to gender; panel (c), acquisition according to haemoglobin genotype.

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HAIDER A. GIHA ETAL. 650

RESA (ELHASSAN et al., 1995), PfEMPl (GIHA et al., 1998) and MSPl antigens (CAVANAGH et al.. 19981 are low in September and-high in January. ’ ’

During the 3 years of study the individuals in Dar- aweesh were therefore considered to be at risk of malaria infection for 1792 10 person-days, and 442 malaria episodes were recorded. Using an Andersen-Gill hazard model for survival data we analysed how age, gender, ABO and rhesus blood grouping, haemoglobin genotype and previous disease history affected the risk of having episodes of malaria during each season, and the extent to which these factors influenced each other.

Althoueh febrile malarial euisodes were observed in all age-groups, the incidence varied considerably between groups. The most noticeable observation was the con- sistently reduced risk (RR = 0.34-0.67) of having clin- ical malaria after the age of 20 years compared to older children and teenagers (5 19 years of age). Infants and smaller children (O-4 years) also had a lower risk of disease than the 5- 19 years age-group, but the differ- ences were not always significant. As estimated from the acquisition of antibodies to malaria antigens during the season (Fig. 4a), there was no age difference in the exposure to malaria parasites during the season of 1995. It is thus unlikelv that the reduced risk of malaria in the individuals over age 20 years was due to less exposure to new infectious bites.

It is generally considered that naturally acquired immunity fails to develop where malaria endemicity is low (~&CD• NALD, 1957), or once developed is lost without continuous exposure. Based on serological evidence (ELHASSAN et al.. 1995: GIHA et al.. 1998) and PCRresults (ROPER et a?., 1998), we have previously concluded that a substantial percentage of infectious bites gives rise to blood-stage infections, which are controlled at subclinical levels. It is tempting to speculate that the reduced malaria risk in adults reflects an acquired capacity to control a higher percentage of new infectious challenges than children and teenagers. In the adults protection was not absolute, and, on an individual basis, protection or susceptibility one year could not predict the risk ofgetting a malaria episode the next year. This indicates that an acquired immunity affords protec- tion to only a proportion of challenges, and also suggests that the protective immune responses are directed against antigens that are either variable (GIHA et al., 1999a, 1999b) or polymorphic (CAVANAGH et al., 1998). In this context it is interesting that we have found a significant inverse association between pre-malaria season IgG reactivity to PfEMPl-like protein expressed on infected erythrocytes of a parasite isolated in Ghana and risk of getting malaria during the subsequent season (GIHA et al., 2000).

The acquisition of immunity by age may be due either to a gradual build up of immunological memory covering larger and larger parts of the parasite’s antigenic reper- toire (h&RSH & HOWARD 1986; MARSH et al., 1989; BULL et al., 1998) or to a physiological effect of age which makes adults more effective in combating the parasite. The latter suggestion is supported by studies of non- immune immigrants to highly malaria endemic areas of Irian Jaya, showing that the malaria risk diminished more rapidly in adults than in children in the years after arrival in the area (BAIRD et al., 1991; 1993). However, these studies did not consider the possible confounding effect of easier access to anti-malarial drugs of adults (MOLI- NEAUX, 1996). Our findings do not rule out an intrinsic role for age in development of natural immunity, and the roles of cumulative exposure and of age per se in the acquisition of immunity against I? falciparum malaria are not mutually exclusive.

The effect of gender on malaria risk varied between the years. The first year males had lower risk than females (RR = 0.75), but the difference was not statistically significant; in the second year there were no differences between the sexes, while in the third year males were at a

higher risk than females. We do not have a clear-cut explanation for this observation. However, the absence of malaria in the area due to drought for 2 consecutive years prior to the study might have compromised im- munity to malaria more among the women, who largely remained in the village, than among the men, many of whom travelled to areas less affected by the drought. Following a rather speculative line of argument, the gradually increasing relative risk of males could be due to the fact that an immunological advantage of males faded out, and that women inherently are better in controlling parasitaemia than men as suggested from the results of the Garki project (MOLINBAUX & GRA- MICCIA, 1980).

It is well established that individuals carrying the AS genotype of haemoglobin are partly protected against severe forms of malaria (ALLISON, 1954; INGRAM, 1957). In this study we show that the risk of getting febrile malaria attacks was reduced to about half that of individuals carrying the normal Hb AA genotype. In contrast to the effects of age and gender, the reduced risk attributed to the Hb AS genotype did not vary with season.

During the 10 years we have worked in the village there have been very few cases of severe malaria. This could be because severe malaria is uncommon under the condi- tions prevailing in Daraweesh, but more likely it reflects the prompt diagnosis and treatment provided by the team in the villaae. The incidence of malaria is hiah in Daraweesh and&i a community level comparable to areas with much more intense transmission (TRAPE etal., 1994; LEMNGE et al., 1997; DODOO et al., 1999). Our data support the notion that even very significant reduc- tion in transmission will not necessarily reduce the burden of malaria in the long term (SNOW et al., 1997; SNOW & MARSH, 1998). The situation in Daraweesh could represent the end-point for malaria control under conditions where transmission is reduced by vector control and use of insecticide-treated materials, but where interventions are not sufficient to completely block transmission. Despite very low and seasonal transmission levels, malaria remains one of the most serious health problems in northern and eastern Sudan.

Acknowledgement We gratefully acknowledge the continued support and col-

laboration of the people in Daraweesh. Mustafa Hamid, Fail Omer, Elhadi Abd Elmagid, Adil Ameen, the late Eltayib Hayatti, Abdalla A. Nasr, and Marjan Yousefi are thanked for excellent assistance in the field and laboratory. This work was supported financially by the Danish International Development Agency (DANIDA), the Wellcome Trust, and the INCO-DC Programme of the Commission of the European Union (ERBIC 18CT970238).

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Received 1 June 1999; revised 26 April 2000; accepted for publication 9 May 2000

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