Despite the reported high prevalence of polyparasitic helminth infections 
, little is known about how these contemporaneous infections interact biologically and influence morbidity. We have examined interactions, at moderate or high intensity, for concomitant infections of three pairs of helminth infections: hookworm and S. japonicum
, hookworm and trichuris, S. japonicum
and trichuris. Our results suggest the presence of synergistic interactions for the following pairs of helminths: 1) hookworm and S. japonicum
, and 2) hookworm and trichuris. Among children co-infected with hookworm and S. japonicum
, an estimated 60% of the anemia is attributable to the biologic interaction between them. Similarly, with M+ co-infection of hookworm and trichuris, an estimated 22% of the observed odds of anemia were attributable to the synergistic interaction between these species. We found no evidence of departure from additive risk model additivity for co-infection of trichuris and S. japonicum.
Also found, though not expected, was the protective association between M+ ascaris infection and anemia.
Consistent with our hypothesis, we found that individual helminth species contributed to anemia in different degrees. As in previous studies, we found that hookworm and S. japonicum
infections were independent risk factors for anemia 
. We did not find M+ intensity trichuris infection alone to be significantly associated with anemia in this study; however, there was an elevated odds of anemia among children co-infected with trichuris and hookworm.
The link between hookworm and anemia is well known and the mechanism of effect has been described 
. The mechanisms underlying S. japonicum
associated anemia are likely multi-factorial 
including, blood loss in the stool as eggs extravasate through the bowel wall, adult worm ingestion of host red blood cells 
, pro-inflammatory cytokine activity mediating anemia of inflammation whereby bio-available forms of iron are shunted to less bio-available storage forms (ferritin) 
, auto-immune hemolysis 
, and sequestration of red blood cells in an enlarged spleen 
. High intensity trichuris infection affects iron status through blood loss in the stool, particularly if dysentery syndrome is present. It is hypothesized that trichuris infection could amplify iron deficiency anemia in the presence of hookworm 
. It has been suggested that ascaris could contribute to anemia by impeding iron absorption in the lumen of the small intestine 
. This mechanism of action, however, has been investigated by one study and their findings fail to substantiate the hypothesis of iron mal-absorption with ascaris infection 
. The finding that M+ intensity ascaris infection was inversely associated with anemia was unexpected and persisted in all multivariable models. This unexpected association and detailed assessment of interactions between ascaris infection and the three helminth species: hookworm, S. japonicum
and trichuris, is the subject of a specific future investigation by our group.
That the odds of anemia was highest in the presence of hookworm and S. japonicum
infections of M+ intensity is consistent with the biology of these helminth species individually. The much higher odds of prevalent anemia observed in the presence of both helminth species is expected based on the presence of at least two pathways of anemia induction in concurrently infected individuals: active blood loss in the stool and anemia of inflammation 
. The mechanism responsible for the increased odds above additivity, however, is more complex. S. japonicum
is known to be associated with pro-inflammatory cytokine elaboration in the human host 
. While Th2 responses to hookworm antigens are well described 
, a study conducted in Brazil found elevated spontaneous cellular secretion of tumor necrosis factor alpha, a pro-inflammatory cytokine, and interleukin (IL)-10 in individuals with patent hookworm infections 
. The same study reported decreased spontaneous production of IL-5, a T-helper 2 (TH2) cytokine in these patients. Two other studies conclude that though TH2 responses predominate in the context of hookworm infection, detectable levels of the TH1 cytokines–interferon gamma 
and IL-12 
, were also observed in response to antigen stimulation of cell supernatants from hookworm infected individuals. On the basis of these data, we hypothesize that the marked increase in anemia among individuals co-infected with hookworm and S. japonicum
may be mediated by an enhanced production of pro-inflammatory cytokines. Pro-inflammatory cytokines, particularly IL-6, cause anemia of inflammation by inducing hepatocytes to produce hepcidin, an iron regulatory peptide 
A recent study from Brazil demonstrated that there was synergism in the aggregation of hookworm and some helminth species including S. mansoni
among residents in their study area; however, the impact of this phenomenon on morbidity was not explored 
. To the best of our knowledge, our report this is the first study to formally assess and report synergistic interactions between S. japonicum
and hookworm or hookworm and T. trichiura
in anemia, as a departure from additive risk profile. The demonstrated synergy between these helminths in the context of anemia have, however, been hypothesized by a few investigators 
. Despite differences in assessment methods that preclude evaluation of the extent to which the adverse health effect of polyparasitic infections departed from additivity of risks, two studies provide some support for our findings. One study reported lower mean hemoglobin among children co-infected with hookworm and trichuris relative to the reference group consisting of children without either infection or children with single infections of either species only 
. Another study indirectly corroborates our findings by reporting evidence of altered immune function in multiply infected individuals 
. Specifically, altered immune responses to antigenic preparations were reported for co-infections including S. mansoni
, hookworm and ascaris. These altered immune responses, the investigators suggest, could impede immune responses to infection 
Strengths and Limitations
Of the few studies that have attempted to link polyparasitic helminth infections to morbidity, all have defined concomitant infections as present or absent for pairs of species 
. Infection categories defined as such–even when single and combined infection categories are present, often lack a fourth constant reference group without both pairs of infections - make any inferences of biologically and public health relevant interactions difficult 
. In addition, the majority of these studies were conducted in the context of malaria 
, many did not assess the intensity of co-infecting species, and none of them examined interactions as departure from additivity of risks. We have examined interactions between helminth species in a naturally occurring context for an important morbidity free of possible confounding from malaria while incorporating information on infection intensity. To ensure the most de-confounded estimates of effect possible, important confounders have been adjusted for and the lack of observation independence within household units was analytically addressed. All assessments were made within in a causally relevant framework, allowing some inferences regarding the public health impact of concomitant infections to be made for the sub-group of individuals exposed to multiple helminth species at moderate or higher intensity.
Despite these strengths, we cannot exclude the possibility that anemia predisposes children to multiple infections based on the cross-sectional design of this study, or that unmeasured factors or residual confounding could still bias our results despite careful control for known confounders. Evidence from prospective 
and randomized clinical studies 
that helminth infections cause anemia suggests that reverse causality is an unlikely alternative explanation for our findings. Yet, the evaluation of interactions at M+ intensity and the over-sampling of those with S
infection as a function of the sampling scheme in the main study, may limit the generalizability of these findings to children co-infected at low intensity, and to representative community samples with different joint distributions of helminth species. Further, the fact that a smaller number of individuals were co-exposed at M+ intensity in this study is likely to have limited the power of this study - potentially making it difficult to achieve statistical significance where one existed for some pairs of helminth species.
Our study suggests that for children in helminth endemic regions, contemporaneous infections with hookworm and either S. japonicum
or trichuris may result in higher levels of anemia than would be expected if the effects of these species on anemia were simply additive. The results provide support for the hypothesis that co-infecting parasites, even living in separate anatomic locations, can interact synergistically to modify anemia, likely via the hosts' immune response to concomitant infections. The presence of synergistic interactions between some helminth species as demonstrated here has implications for treatment given the high burden of anemia associated with concomitant infections of hookworm and S. japonicum
or trichuris. Specifically, our results support the notion that concomitant treatment for S. japonicum
and some geohelminths may provide an even greater public health benefit than that based on the assumption of additive morbidity. This is a particularly appealing option given joint treatment with albendazole and praziquantel has been shown to significantly improve hemoglobin levels, without greater side-effects than treatment with praziquantel alone 
. We note however, that in light of the limited number of studies on the morbidity consequence of polyparasitic helminth infections in general, and of the biologic interactions between concurrent infections in the mediation of helminth-associated morbidities in particular, more epidemiologic studies of this phenomenon are necessary to better predict the impact of any public health intervention. Specifically, epidemiologic studies that explore the full range of biologic interactions between co-infecting species - including the possibility of antagonism, in their joint effects on anemia and other helminth-associated morbidities are necessary to fully evaluate the possible risks and benefits of combined mass therapy in helminth endemic regions.