Infection with multiple parasitic species is a common observation in parasitological surveys of communities in tropical and sub-tropical countries [1–3], but attempts to understand their combined health impact on affected individuals and communities have been very limited in both scope and number. An inherent problem is the lack of specificity of many signs and symptoms associated with parasitic infection, which can complicate attempts at attributing morbidity to a particular cause [4].

Hepatomegaly and splenomegaly fall into this category as common signs amongst school-aged children in Africa with multiple aetiologies. Two of the most commonly attributed causes are infections with Schistosoma mansoni and Plasmodium falciparum. It has long been suggested that the presence of one species may confound or modify the effects of the other on the liver and spleen in endemic situations [5–7], but it has also been suggested that hepatosplenomegaly in infants is likely attributable to malaria infection, whereas amongst school-aged children it is more likely attributable to S. mansoni infection [8]. This paradigm has prevailed in the absence of systematic research, and the relative contribution of the two infections in the aetiology of chronic hepatosplenomegaly of school-aged children remains essentially unknown.

Recent insights have been gained from a series of epidemiological and immunological studies in Kenya. In a cross-sectional study of children, morbidity profiles were compared in two communities. After testing for the effect of several other infections, only the prevalence of malaria was higher in the worst affected community, an ecological observation that led to the suggestion that co-infection with malaria may exacerbate hepatosplenic morbidity associated with schistosome infection [9]. This hypothesis received support after retrospective assays and analysis of the blood samples from the same cohort indicated that Ab istotypes, in particular IgG3 to P. falciparum schizont antigen, were elevated in hepatosplenic patients compared to controls [10].

Small-area variation in exposure to malaria infection estimated from mosquito densities have been associated with variation in the prevalence of splenomegaly in children up to the age of 5 years [11]. The degree of splenomegaly has also been correlated with S. mansoni egg counts in school-aged children [9, 12]. However, until now, the level of joint exposure to these infections on risk or magnitude of splenomegaly has not been investigated. Here, we have undertaken a retrospective study of splenomegaly amongst a cohort of children selected for a longitudinal study on the basis of ultrasound detectable hepatomegaly [12]. Exposure to malaria was assessed by measuring IgG3 responses to P falciparum schizont antigen (anti-Pfs IgG3) whereas exposure to schistosomiasis was assessed by egg counts. These measurements were supplemented with geographical data to identify areas of low and high transmission of each infection.

The study area and cohort have been described in detail elsewhere [12]. Briefly, 96 children were enrolled in October 1999 on the basis of having ultrasound detectable hepatomegaly. The majority of these children also presented with splenomegaly. The children were living in Mbeetwani community, situated close to the River Kambu in the district of Makueni, Kenya and attending Mbeetwani school. They were examined clinically by four clinicians at baseline to record hepatomegaly below the mid sternal line (left lobe) and mid clavicular line (right lobe), and spleen enlargement below the mid axillary line (MAL) and mid clavicular line (MCL). Enlargement was measured in cm extension below the rib cage. Also recorded was the consistency of the organ if palpable. Organs were recorded as being either soft, firm or hard as described previously [12]. An ultrasonograpic examination of the liver was conducted by experienced ultrasonographers to identify any cases of peri-portal fibrosis.

Egg counts were estimated using the Kato-Katz technique [13] from 3 stool samples (2 readings per stool), submitted before treatment. The IgG3-response to schizont antigen was measured by ELISA, as previously described [14], from blood samples collected before praziquantel treatment, as well as one and two years after praziquantel treatment. Preliminary repeated-measures analysis of the IgG3 data indicated no significant difference in the responses over time (F = 0.123, p = 0.884); however not all children contributed blood samples at all surveys. Data from the first year follow-up were available for more children than any other survey, and these data were therefore used to map the distribution of IgG3 across the study area.

Local variation in the level of exposure is being increasingly recognised as an important determinant of the risk of morbidity attributable to parasitic infections. Most research efforts have previously focussed on factors affecting the micro-epidemiology of malaria [11, 15–18], although it has been also observed that exposure to S. mansoni infection can vary over time within small areas due to environmental variables such as distance to the transmission site [19, 20] and micro-climatic effects on the ecology of snail intermediate hosts [21]. Here, we have demonstrated for the first time that small area variation in exposure to both malaria and schistosomiasis can be exploited in efforts to understand their combined effects on the health of school-aged children living in areas with endemic infections.

Here, we have focused on whether or not variation in exposure to malaria and schistosome infections over a small area was correlated with variation in the degree of splenomegaly, recorded during an examination of children with ultrasound detectable hepatomegaly. The cohort was formed as part of a longitudinal study of the benefits of praziquantel treatment [12]. Although the selection process meant that the cohort was not necessarily representative of the community, the lack of geographical variation in hepatomegaly indicates that our observations concerning splenomegaly are not simply a proxy for hepatomegaly. The primary observation was that the extent of splenomegaly, irrespective of whether it was measured below the mid axillary line or mid clavicular line, was greatest amongst children living in a small area where both S. mansoni egg counts and IgG3 responses to malaria schizont antigen were significantly elevated. There was no difference in the extent of splenomegaly amongst children from elsewhere in the study area.

Together, these observations suggest that children with relatively high levels of exposure to both parasites were measurably worse affected in terms of spleen enlargement. An important question, though, is whether or not elevated IgG3 responses to P. falciparum schizont antigen are an appropriate proxy for exposure to malaria infection. As all examinations were conducted towards the end of the dry season, very few cohort members were expected to present with either clinical malaria or blood-smear detectable parasitaemia. We therefore required a proxy measure of exposure to malaria that was sensitive enough to reveal small area variation. In the present study, the highest IgG3 responses to schizont antigen were clustered along the northern border of the study area formed by the course of the river Kambu, and there was a rapid decline in this response with increasing distance from the river. This observation is consistent with the idea of IgG3 as a proxy measure of exposure, and this response may be expected to fulfil that role for a number of other reasons. It is a dominant IgG sub-class response to malaria antigens and toxins [22, 23], but has a short half-life, and it has been hypothesised that the response wanes rapidly in areas of seasonal transmission [24]. Furthermore, IgG3 responses to blood stage antigen have been correlated with the level of exposure to infection during a bednet intervention in western Kenya [25] – a further indication that reducing exposure to malaria infection results in a measurable difference in this antibody isotype response. Finally, the distance over which the decline in IgG3 responses was measured is consistent with observations from other areas concerning the limited flight range of mosquitoes [26], and personal observations have made it clear that during the dry season there is no other surface water in the area that could harbour mosquito larvae. It is therefore reasonable to conclude that children with elevated responses had been recently exposed, or were most heavily exposed during the transmission season.

The use of egg counts as a measure of exposure to S. mansoni infection is less equivocal, especially amongst children. Children with the highest egg counts lived at the western end of the study area, which was concomitant with unpublished observations concerning the distribution of snails prior to the study, as well as the availability of surface water at different times of year. At the western end of the river, the surface water is less transient than at the east and there were more snails before treatment (HC. Kariuki, personal observation). Local residents situated at the western end of the river therefore are therefore likely to have experienced higher cumulative exposure than residents at the eastern end of the river, where surface water is often unavailable and wells are dug into the dry riverbed to extract subterranean water.

Hardened spleens were associated with relatively high egg count and a greater degree of splenomegaly, but the geographical clustering of hard spleens extended beyond the sector with highest IgG3 titres and high egg counts into the sector with high IgG3 levels and low egg counts. These observations alone would suggest a dominant role for malaria in the aetiology of hardened spleens, but we have recently reported elsewhere [27] that the prevalence of hard spleens reduced significantly after treatment of S. mansoni infection with praziquantel, and only a few children presented with hard spleens three years after treatment. Further analysis of these data will indicate whether or not the presence of hardened spleens was associated with exposure to malaria only, or attributable to exposure to both infections.

The strong negative correlation between anti-Pfs IgG3 level and distance to the river offers insights into other observations concerning this isotype. A low level of IgG3 antibodies against P. falciparum variant surface antigen (VSA) was recently associated with an increased risk of severe malaria in children from Gabon [28]. The authors suggested this association was due to the inability of affected individuals to produce a sustained anti-VSA response. Their hypothesis is supported by observations of genetic restriction in the production of IgG3 responses against malaria antigens [29, 30]. However, it is equally plausible that a low IgG3 response is simply a marker for reduced exposure, and that concomitantly reduced exposure to VSAs is a risk factor for severe malaria upon infection.

Although the potential for malaria infections to affect the health of school-aged children in Kenya has been recently highlighted [31], our results stress the importance of considering the joint impact of multiple-species infections when addressing the health of children in this age group. This study has also demonstrated the value of a multidisciplinary approach to investigating the impact of schistosome and malaria infections. Whereas we have focused on splenomegaly in the present study, it is possible that enlargement of the spleen is not the only form of morbidity that is exacerbated by relatively high exposure to malaria and schistosomiasis. Further analysis of the present cohort will therefore focus on whether or not the outcome of praziquantel treatment for schistosomiasis on both hepatomegaly and splenomegaly was associated with exposure to malaria infection.