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ABSTRACT |
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mixed infections have been mainly studied in animal models. Laboratory experiments have led to conflicting results (e.g., in protozoa-helminth co-infections in mice). Some studies suggested a facilitating effect of Schistosoma on Plasmodium infection,3 and others suggested a suppressive effect.4
The suppression or facilitation of one infection by another is even more difficult to evidence in humans where there is no possibility to control the burden of parasites. Observational studies in exposed populations5,6 or selected treatments of limited numbers of patients co-infected with Ascaris lumbricoides and Plasmodium falciparum7 have shown either a negative (i.e., protective effect) or a positive interaction. Recently, we showed in Senegalese children that light infections with Schistosoma haematobium were associated with lower P. falciparum densities than in children not infected with schistosomes.8
Randomized clinical trials represent a far better alternative to observational studies: significant decreases in the prevalence and the burden of a specific parasite can be obtained by means of a selective treatment, and their effects on the remaining parasites can be observed.
The island of Madagascar is a convenient setting for such trials, because a large variety of pathogens is likely to simultaneously infect human populations. Their spatial distributions differ according to environmental factors, mainly climate and altitude. For instance, malaria is hyperendemic in the hot and humid coasts, at low altitude, and scarce on the central plateaux, because of a cool and dry climate, and annual indoor spraying of Dichloro-Diphenyl-Trichloroethane (DDT) carried out from 1993 to 1998. Conversely, geohelminths such as Ascaris are particularly abundant at high altitudes.9 Schistosomes (particularly, Schistosoma mansoni) are distributed within well-delimited clusters.
We report here a randomized trial of anti-helminthic treatment in a midwestern village mainly affected by malaria and ascariasis, two of the most widely distributed parasitic diseases in the world. The main objective was to evaluate the effect of Ascaris-reduced prevalence and egg load on patients exposed to P. falciparum infection and to find evidence for antagonistic or synergistic interactions between these two parasites.
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MATERIALS AND METHODS |
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Study population and protocol. Inhabitants and families were identified by the 1995 national census. Families were randomly allocated to a treatment or control group, and inclusion of new families was stopped after the third visit. All eligible subjects were orally informed of the study protocol and, when consent was given, they were included in their family group. There was no blinding in the treatment assignment. For practical and ethical reasons, infants less than 6 months of age were excluded from the study. Because of unproven innocuousness of levamisole during pregnancy, pregnant women (N = 13) were also excluded from the study. Eligible subjects seen after the onset of the study (newcomers to the village or babies having reached 6 months of age) were included in their family group.
On inclusion, questionnaires were filled for each subject. In particular, information was obtained on age, sex, notion of ongoing pregnancy or clinical symptoms such as diarrhea or fever in the preceding days, intake of drugs, or recent migration outside the study area.
From June 1996 to October 1997, all village residents were visited by investigators every 2 months (nine visits for the whole duration of the study). The main objective of this follow-up was to combine the observation of an entire malaria transmission season and the repeated treatment of intestinal helminths at intervals adapted to the life cycle of Ascaris (8 weeks from egg ingestion until sexual maturity of adult worms). At each visit, included subjects were clinically examined and stool and blood samples were taken for malaria parasites and helminth search. Updating of questionnaires was also done on the same occasion. In addition, subjects belonging to the treated group were administered an anti-helminthic single-dose oral treatment of levamisole (3 mg/kg in children, 150 mg in adults) under control of the investigators. At each visit, a multivitamin treatment (1/2 to 3 tablets every 2 months, each tablet containing 2500 IU vitamin A, 1.0 mg thiamine, 0.5 mg riboflavine, 7.5 mg nicotinamide, and 300 IU vitamin D3) was given to subjects from the control group. All villagers infected with malaria parasites and presenting clinical symptoms (temperature > 38°C) were treated with oral chloroquine (25 mg/kg in 3 days). At the end of the study, all the population (both treated and control groups) received a curative dose of mebendazole (500 mg, single oral intake), which is an efficacious treatment of most intestinal nematodes.
The protocol was given ethical approval by the Madagascar Ministry of Health (Direction de la Lutte contre les Maladies Transmissibles).
Biologic methods. Stool samples were processed by the merthiolate iodine formalin (MIF) concentration method on a calibrated amount of stool, which allows staining, concentration, and count of helminth eggs (A. lumbricoïdes as well as S. mansoni and Necator americanus).
At each visit, finger prick thick and thin blood smears were made on all subjects. Smears were stained with Giemsa and 100 oil-immersion microscopic fields were examined for malaria parasites. Parasites and white blood cells were enumerated, and parasite density was calculated according to an assumed average of 8,000 leukocytes/mm3.
Statistical analysis.
We first performed a univariate analysis to compare the two groups on entering the study on the basis of general characteristics and prevalences and densities of the two main parasites (P. falciparum and A. lumbricoïdes). The 
2 test was used to test differences in proportions, and Student t test or non-parametric Wilcoxon test was used to test differences in means.
We then examined malaria parasite density in relation to the treatment group. Only P. falciparum densities, which represented 93.2% of all diagnosed parasites, were analyzed. P. malariae accounted for 4.9% and P. vivax/P. ovale for 1.9% of single malarial infections. P. falciparum densities and helminth egg loads were log-transformed [log(DP + 1)] to correct distribution asymmetry, which is usual in parasite infection data.
Because the study design was a randomized trial, we verified that subjects allocated to repeated anti-helminthic treatment and controls were similar for a number of variables (Table 1
) that we did not take into account in further analyses.
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Because there is generally a strong association between age and immunity to malaria, which in turn influences the parasite density in peripheral blood, we defined three age groups (6 months to 4 years, 5–14 years, 
15 years), in which we tested the association between P. falciparum density and treatment group by a stratified analysis.
Finally, our analysis was performed using the following model:
with ai ~ N (0, 
a2) (between-family variation); bji ~ N (0, b2) (between-subject within-family variation); eijk ~ N (0, e2) (within-subject within-family variation); and Yijk is the kth log (P. falciparum density + 1) for subject j from family i.
This model has two fixed parameters that were as follows: for ß0, the overall mean of P. falciparum density (log-transformed), and for ß1, the effect of treatment of A. lumbricoides and three random effects variances that need to be estimated.
We estimated the mixed model parameters with PROC MIXED SAS procedure (SAS Institute, Cary, NC).
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RESULTS |
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shows that, on entering the study, no significant difference between the two groups was recorded for any of these variables. However, although not significant, malaria prevalence and parasite density seemed to be slightly lower in the control group. During the follow-up, we lost contact with 9 subjects after the first visit (5 in the treated group and 4 in the control group), and we excluded from the analysis three families totaling 14 subjects that had been enrolled 1 year after the beginning of the trial and thus followed-up for a short duration (< 6 months).
At each visit, depending on the month, 0–9 subjects (mean, 3.7) in the control group and 0–11 (mean, 4.2) in the levamisole-treated group presenting with fever and parasitemia received oral chloroquine treatment. An overall proportion of 2.5% (control group) and 3.3% (treated group) received chloroquine. The maximum number of treated subjects was between February and June 1997, during the malaria transmission season.
Effect of levamisole treatment on Ascaris and other helminths. At their inclusion in the study, all subjects from the treated group received a therapeutic dose of levamisole, which was repeated on each visit. By the end of the study, each of them had received an average of 7.4 intakes.
As shown on Figure 1A, Ascaris egg loads immediately collapsed after the first visit in the treated group, whereas they persisted at a high level throughout the follow-up in the control group (all P < 0.001 for each visit between the two groups). Prevalence rates followed a similar pattern (Figure 1B). However, it is noticeable that both prevalence and egg load progressively increased after 1 year of follow-up, which could be caused by a phenomenon of resistance to the administered treatment. Because a similar tendency was observed in the control group, a seasonal relapse in the transmission of intestinal helminths during the August to October period cannot be excluded in the whole population, even if no climatological data support this hypothesis.
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Effect of anti-helminthic treatment on plasmodial infection.
Mean P. falciparum densities in the treatment groups are shown in Figure 2 for each age group (Figure 2A: 6 months to 4 years; Figure 2B: 5–14 years; Figure 2C: 15 years). There were strong seasonal fluctuations on all three graphs, as parasite densities peaked in the humid season, between March and May, and decreased in the coldest and driest months, August to October (1996 and 1997). There also seemed to be an overall decrease in the mean parasite density in the oldest age group, confirming the strong influence of age (and immune status) on malaria infection. Treatment and control groups varied in similar ways, with a tendency to higher densities in the treated group, which was particularly pronounced in the two classes of 5–14 and 15 years of age.
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) confirmed that subjects more than 5 years of age, treated with levamisole, had a significant increase in their P. falciparum densities compared with controls (P < 0.05 for age classes 5–14 and 15 years). The potential effect of treatment on parasite density was roughly equivalent in these two age groups (ratio treatment/intercept effect of 0.5 for 5–14 years, and 0.6 for 15 years). Indeed, treatment estimates, but also baseline parasite densities were higher in younger children (0.96 and 1.88 for 5–14 years and 0.34 and 0.54 for 15 years respectively). We thus pooled the two last age groups to estimate an average increase for these subjects and included an age binary variable in the model, because P. falciparum density baseline values differed between groups. In this latter model, no interaction was found between age and effect of treatment (P = 0.11). The overall estimates in this broader age class showed a higher effect of treatment on P. falciparum density (P = 0.018; Table 2). There was no effect of levamisole treatment on children 6 months to 4 years of age.
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15), which probably indicates the progressive build-up of an adequate immune control of parasitemia in older children. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We performed the first controlled clinical trial of anti-helminthic treatment, and we showed a clear negative interaction between Ascaris infection and malaria parasite density in children more than 5 years of age. Because these results seem to be quite convincing of a protective effect of Ascaris on malaria, we are in complete disagreement, for these two particular parasites at least, with a recent paper that dealt with the consequences on malaria control of a positive interaction (i.e., a facilitating effect), between Plasmodium and all kinds of worms considered as systematic in endemic areas.13
Because the study design was a randomized trial and not an observation study and the data were analyzed as "intention to treat," we are quite confident that no major bias could interfere with our results and that it is indeed the effect of anti-helminthic treatment that led to an increase in malaria parasite density. It is noteworthy that the slight difference (although not significant) in malaria parasite densities observed between the groups at entry (before anti-helminthic treatment) vanished in the age classes (5–14 and 15 years), where a protective effect was shown.
It was rather logical that no effect was found in children 0–4 years of age, because in areas such as the Madagascar highlands where malaria is mesoendemic, it takes a few years of exposition to malaria to establish a stable immune response and an equilibrium between the two parasites.
In our study, P. falciparum and A. lumbricoides were the predominant, but not the only, parasites found in the followed up patients. We cannot categorically exclude that other helminths, such as S. mansoni or N. americanus, may have interfered with the two main parasites. However, because the two treatment groups were randomized, one can expect (and it can be verified in Table 1) that these two parasites were evenly distributed in each arm.
To our knowledge, levamisole has no efficacy on the treatment of trematodes. There is no particular reason for a selective interaction of S. mansoni with either of the groups. The situation is somewhat different with N. americanus, which is sensitive to the same drugs as A. lumbricoides, but at much higher doses. Nevertheless, there was no difference in either prevalence or egg loads between treatment groups for any of these parasites at each visit after inclusion. Therefore, we do not think that these infrequent helminths interfered in our study population with P. falciparum–A. lumbricoides interaction.
We do not think that the intakes of drugs, other than anti-helminthics, had any effect on our results. Chloroquine was given in a similar way to both groups (2.5% of subjects in the control group versus 3.3% in the levamisole-treated group, P = 0.21) and concerned only a limited number of individuals. As for multivitamins, doses were very low and certainly unable to boost the immune system of control subjects.
From a pathogenic point of view, an explanation to such an interaction may be found in the immunomodulation generated by both parasites, particularly the effects of cytokines controlling the orientation toward the Th1 or the Th2 arm of the immune response. Schistosoma and Plasmodium are known to induce a Th2-like response, and a strong correlation has been recently shown between malaria- and schistosome-specific IgG3 responses in individuals from Kenya, Uganda, and the Sudan, indicating a cross-reactivity between helminths and plasmodia that could confer a protection to schistosome-infected subjects exposed to malaria.14 A similar phenomenon could be involved in A. lumbricoides and P. falciparum co-infections, as suggested by the demonstration of a reactivity of patients with A. lumbricoides to schistosome antigens.15 Other immunologic mechanisms, such as the inhibition of dendritic cell maturation by P. falciparum–infected erythrocytes,16 have also been put forward. Last, because such interactions between parasites are probably the outcome of several complex mechanisms, genetic susceptibility/resistance of the host may play a role, as suggested by the location in the same genetic region of loci controlling the levels of S. mansoni and P. falciparum infections.17,18
There have been millions of years of host–parasite co-evolution, and it is not surprising that parasites have developed mechanisms to facilitate or to prevent host invasion by other infectious agents, even if such phenomena are difficult to show. From a public health point of view, positive or negative interactions may have crucial importance. Each pathogen, considered separately, contributes to the health weakening of exposed populations, especially in children. In the case of multiple infections in the same individuals, positive or negative interactions between parasites may have unexpected consequences on the efficacy of systematic treatments of parasites like helminths, such as an increase of malaria attacks after mass treatment of ascariasis.7 The advantage of using a single community therapy program to deliver treatments against several parasitic infections should be assessed after taking into account these considerations.
Received August 26, 2005. Accepted for publication December 21, 2005.
* Address correspondence to Michel Cot, IRD UR 010, Faculté de Pharmacie, 4 Avenue de l’Observatoire, 75270 Paris Cedex 06, France. E-mail: michel.cot{at}ird.fr 
Authors’ addresses: Laurent Brutus, IRD UR 010, CP 9214, La Paz, Bolivia. Laurence Watier, Inserm U 780, 16 Avenue Paul Vaillant-Couturier, 94807, Villejuif Cedex, France. Valérie Briand and Michel Cot, Institut de Recherche pour le Développement, UR 010, Faculté de Pharmacie, 4 Avenue de l’Observatoire, 75270 Paris Cedex 06, France. Virginie Hanitrasoamampionona, Hélène Razanatsoarilala, Direction de la Lutte contre les Maladies Transmissibles (DLMT), Ministère de la Santé, BP 460, 101 Antananarivo, Madagascar.
Reprint requests: Michel Cot, IRD UR 010, Faculté de Pharmacie, 4 Avenue de l’Observatoire, 75270 Paris Cedex 06, France. E-mail: michel.cot{at}ird.fr.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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