This study offered a unique opportunity to examine helminth-malaria parasite co-infection in the neglected demographic stratum of pregnant women. The principal finding was a strong association between asymptomatic infection with Plasmodium falciparum and Mansonella perstans. A weaker association was observed between hookworm and P.falciparum infections, and there was an interaction between infections of the two helminths, such that the effect of hookworm was only seen in the absence of the stronger association with M.perstans. To our knowledge, this analysis provides the first report of an association between a filarial helminth infection and malaria parasites in humans. The results have implications for understanding of the host-parasite relationship, particularly in pregnancy, and for targeting treatment of co-infections among vulnerable groups.
This study focused on the issue that the geographical distribution of parasitic infections exhibit spatial dependency over small distances [27
], and represents a step forward from studies conducted in one location that had assumed no spatial clustering, or had not measured residential location[18
]. Geographical zones were defined based on simple geography, altitude, vegetation and location of settlements, as these have been found to correlate spatially with parasite infection [27
]. The zones provide a means to analyse the different environments of, for example an exposed costal location of zone 1 compared with the very different urban environment of zone 3 as separate entities, and to adjust for these differences in analysis. However, the stratification had limitations; while the aim was to provide a detailed stratification by environment, there was a need to strike a balance between achieving homogeneity within the zones without creating zones so small that the analysis is not sufficiently powered. With probability of infections known to exhibit spatial dependency over small distances [27
], it may have been incorrect to assume homogenous parasite density within the study zones of up to four km in diameter. The use of zones in the analysis presented here enabled the description of variation between areas, but not within, areas..
Stratification by zone revealed considerable variation in the probability of co-infection with geographical location, particularly for hookworm-P.falciparum. This may be related, in part, to the observed variability in the prevalence of hookworm and P.falciparum infections. Associations were strongest in zones where the infection prevalence of both hookworm and P.falciparum were highest, consistent with a hypothesis that probability of infection with P.falciparum increases with intensity of hookworm transmission.
A considerable number of women seen at the antenatal clinic were excluded from this study; it is unlikely that the major reasons for exclusion created an important bias. The principal reason for exclusion, residence outside the study area, was appropriate to this analysis. A bias in relation to HIV status of those excluded is unlikely to have affected the results, since the principal effects showed no interaction with HIV among those analysed.
Women were included in this study only if they were well on the screening day, with no complaints (e.g. fever) and no gross evidence of severe, helminth-induced disease (such as anaemia, bloody diarrhoea, or overt liver disease). This analysis therefore addresses associations between helminth infections and asymptomatic P.falciparum
parasitaemia, which may differ from associations between helminths and symptomatic P.falciparum
]. Assuming that helminth infections are more often chronic and long-lived than infection with P.falciparum
, the observed positive association between helminths and asymptomatic P.falciparum
parasitaemia may imply an increased likelihood of P.falciparum
infection, a reduced likelihood of clearing P.falciparum
, and, or, a reduced likelihood of developing symptoms and seeking medication.
The first possibility, a helminth-P.falciparum
association due to increased likelihood of infection with P.falciparum
among women with helminth infections, could arise through behavioural or environmental factors, leading to increased exposure to both types of infection. Both M.perstans
are transmitted by flying insect vectors: M.perstans
] and P.falciparum
]. It is plausible that the distribution of these two vectors may be spatially correlated, as both require water sources for larval breeding [26
] though it is not clear whether the required conditions are exactly the same. Similarly, hookworm larvae flourish in damp soil and grass, which may be found close to stagnant water that is the breeding ground for malaria parasites. The slight reduction in the helminth-P.falciparum associations with adjustment for socio-demographic factors suggests a possible contribution of other behavioural effects; such as differences in the usage of antimalarial drugs during pregnancy, prior to enrolment in the study; however, prior consumption of antimalarials showed no statistically significant association with helminth infection and adjustment for prior consumption of antimalarials did not alter the observed effects (data not shown). On the other hand, adjustment for geographical zone strengthened the associations, suggesting that, at zone-level, common environmental factors could not explain the effect. Thus the possibility that helminths may lead to a biological increase in susceptibility to infection, or to persistence of asymptomatic infection with P.falciparum
remains plausible. This accords with previous studies suggesting associations between helminths and an increased incidence of P.falciparum
], higher parasite counts and delayed parasite clearance [17
], but a reduction in disease severity [21
]. It is not possible to say whether the higher prevalence of P.falciparum
among helminth-infected women resulted in increased incidence of disease events from the data in this cross sectional study.
Hookworm and M.perstans
were the commonest helminth infections among participants in this study. The less common infections, S.mansoni, Strongyloides, Trichuris
, showed no associations with P.falciparum
, in conflict with some previous reports [20
], but in agreement with one other study from Uganda that reported no association [17
]. For these species, a real association (if present) might not have been detected in this study because few women were infected (particularly with Ascaris
), because the intensity of helminth infections was low, or because of misclassification of low-intensity infections as negative, resulting from the examination of a single stool sample (multiple samples are required for high sensitivity of detection) [46
]. Such misclassification may also have contributed to the relatively weak observed effect of hookworm. By contrast, preliminary results suggest that the Knott’s method used for assessment of M.perstans
infection was particularly robust, with serial results in the same women showing 96% agreement for infection status and a correlation coefficient for microfilarial counts per millilitre of 0.88 (p<0.001) (AME, unpublished data).
This study does not explore potential biological mechanisms for the observed associations between helminths and P.falciparum
. However, previously proposed mechanisms include the suggestion that the immuno-regulatory effects of helminths, which allow their own long-term survival in the host [9
], “spill-over” to impair the immune response required to protect against or eliminate malaria parasites. M.perstans
is a long-lived filarial worm that inhabits serosal body cavities and reproduces through microfilaria which circulate in the blood and are transmitted through biting midges [42
]. Despite residence and migration through blood and tissues, M.perstans
infection seldom causes detectable pathology, and this attests to its particularly potent immuno-modulating properties. It is thus interesting to speculate that M.perstans
might produce a particularly strong immuno-modulating effect on the response to other pathogens, and that this might over-ride the effects of related helminths, such as hookworm, when both are present, perhaps accounting for the observed interaction between the two helminth species. This will be more comprehensively described in a future paper.
This study specifically examined co-infections in pregnant women. The unique environment that exists within the pregnant body means that one should be cautious in applying these results to the general population. This may be particularly important in relation to associations with P.falciparum
, because parasites are sequestered within the placenta during pregnancy [30
] and may be less readily detected in the peripheral blood (sampled in this study). Apparent biological associations might possibly reflect helminth effects on the sequestration of P.falciparum
parasites in the placenta, rather than effects on the prevalence of infection.
In summary, this analysis has examined helminth-P.falciparum co-infection in pregnancy and attempted to address the influence of residential location on associations between these environmentally dependent parasites. It provides evidence of an association between hookworm and P.falciparum, and the first report of an association between M.perstans and P.falciparum infection, effects not explained by measured social or geographical factors. Given the plausible hypothesis of a biological interaction between helminths and P.falciparum, and increasing advocacy for de-worming, there is a need for prospective studies of the effects of helminths and their treatment on P.falciparum and other malaria parasites, incorporating surveys of residential location, vector entomology and recording of malaria infection rates and illness events.