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Antagonism between two intestinal parasites in humans: the importance of co-infection for infection risk and recovery dynamics
Aaron D. Blackwell, Melanie Martin, Hillard Kaplan and Michael Gurven Proc. R. Soc. B 2013 280, 20131671, published 28 August 2013

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USA Tsimane Health and Life History Project. .2 Research Cite this article: Blackwell AD. relatively little is known about the interactions between many common parasites and pathogens. Hillard Kaplan2. Melanie Martin1. Tsimane. Our results provide evidence for an antagonistic relationship between helminths and giardia.org.royalsocietypublishing. Santa Barbara. Helminths are common parasites with significant potential for interactions with other pathogens and parasites. collected during 5235 medical exams over 6 years. Finally.6]. Cross-sectionally. Helminth and giardia infections are often endemic in the same populations and both occupy the small intestine. helminths. giardia infection was less likely in helminth-infected individuals (HR: 0. Kaplan H. since co-infection may alter both treatment and susceptibility to infection [3. CA 93106-3210.2.royalsocietypublishing. Gurven M. such as severe malaria [7].2013. However.65.1098/rspb. Blackwell e-mail: blackwell@anth. lamblia (OR ¼ 0. Blackwell1. University of California. lamblia infection.royalsocietypublishing. Investigation of co-infection is important. In some cases. Bolivia 3 Department of Anthropology. Proc R Soc B 280: 20131671.1671 1 2 Department of Anthropology. Parasites were identified in faecal samples from 3275 participants. particularly with regard to antagonism between parasites sharing a host. but resulted in a marginal increase in the odds of G.1671 or via http://rspb. Infection with helminths was also less likely for individuals infected with giardia (HR: 0.3 and Michael Gurven1.org/10. Author for correspondence: Aaron D. yet few studies have examined interactions between these parasites. Received: 27 June 2013 Accepted: 1 August 2013 Subject Areas: health and disease and epidemiology.doi. Co-infection with multiple species of parasite or pathogen is likely to be the rule rather than the exception in most biological systems. and suggest that co-infection should be considered in disease transmission models and treatment decisions. Introduction A complete understanding of pathogen and parasite ecology requires knowledge about how pathogens and parasites interact with one another as they compete for limited hosts and other resources.org/10. USA Co-infection may affect transmission and recovery from infection.g. Although coinfection studies are common for diseases in which the effects of co-infection are dramatic (e. Several recent studies have demonstrated that helminth infection modifies risks for many other infections.Downloaded from rspb. Giardia lamblia (30%) and Ascaris lumbricoides (15%). NM 87131. for example affecting the balance of TH1.71). lumbricoides were negatively associated with G.4].org on November 16.doi. The most prevalent infections were hookworm (56%).2. San Borja. treatment with mebendazole reduced subsequent hookworm infections. All rights reserved. We report on helminth – giardia co-infections in a panel study of forager –horticulturalists in the Bolivian lowlands. including human populations with limited medical access. state-based modelling 1. computational biology Keywords: giardia.60. In part. OR ¼ 0. Longitudinally. Longitudinal co-infection patterns were examined using logistic mixed and multi-state Markov models. & 2013 The Author(s) Published by the Royal Society.8–11].ucsb. co-infection. HIV [1]) or where infections are similar (e. Martin M. multiple species of helminth [2]). hookworm and A. 2013 Antagonism between two intestinal parasites in humans: the importance of co-infection for infection risk and recovery dynamics.2013. respectively). University of New Mexico. Bolivia.edu Electronic supplementary material is available at http://dx. but remains an understudied element of disease ecology.org Antagonism between two intestinal parasites in humans: the importance of co-infection for infection risk and recovery dynamics Aaron D.46). 2013 rspb. these changes may protect against other conditions. Albuquerque. http://dx. ecology. the immunomodulatory effects of helminths may also limit the effectiveness of vaccination and increase susceptibility to other infectious diseases [4.1098/rspb. these interactions may arise because helminths have significant effects on host immune function. TH2 and regulatory T cells [5.g.

understanding the relationship between these two pathogens has significant consequences for world health. intestinal protozoa that is estimated to infect around a billion people worldwide.9% saline solution and iodine solution. Tinidazole was given as a single 2000 mg dose.20]. that giardia and helminth co-infections may occur less frequently than would be expected (suggesting antagonism) [14]. Giardia duodenalis) is a water-borne. rice. manioc and corn. Metronidazole was prescribed at 250 or 500 mg three times a day for 7 days. larvae. respectively. particularly in individuals living with endemic infection. physicians administered vitamins. In many cases. In most cases. Material and methods (a) Subject population Tsimane are forager–horticulturalists who live along the Maniqui River and surrounding areas in lowland Bolivia.21]. Mebendazole was prescribed either as a single dose of 500 mg or as 100 mg twice daily for 3 days. methods for age estimation. symptom investigation. Beginning in 2007. multi-year. Patients seen by THLHP physicians were given routine physical exams ( patient history. height and weight). Patients were treated if they had either a positive faecal analysis or symptoms indicative of infection. At all other times mebendazole and metronidazole were prescribed. Not all patients were prescribed medications (see below). However. faecal samples were also preserved in 10% formalin solution following direct identification. 2. The few reports that exist suggest that infection with helminths may alter immune responses to giardia [13]. Proc R Soc B 280: 20131671 (c) Faecal analysis Faecal samples were analysed using two methods. lamblia infections. blood pressure and temperature. Ethnographic and epidemiological information on the Tsimane. protozoa and other parasites by direct identification on wet mounts. school performance and quality of life [12]. We use data from an on-going.royalsocietypublishing. Although G. We therefore coded individuals as positive or negative for identified species in a single dataset.edu/~tsimane). subject sampling. they subsist primarily on cultivation of plantains. infecting 57% of the population [19]. Starting in 2004. Helminth infections are highly prevalent. we considered all medications prescribed by project doctors known to be effective as anti-protozoal and anti-helminthic agents. 2013 Giardia lamblia (a. There is currently no consensus as to the value of treating helminth infections in endemically infected populations. the Percoll method identified slightly more positive cases. Given that giardia and helminths each infect around 15% of the world’s population.18].Downloaded from rspb. fishing and gathering. and the vast majority (d) Statistical analysis Logistic mixed models were used to examine odds ratios for infection status given co-infection and treatment variables. or would decline treatment. 2 rspb. we report on infections in a population of lowland Bolivian forager–horticulturalists. and examined at 100Â and 400Â [19. despite the potential for interactions due to occupation of similar intestinal substrates and the involvement of shared immune defences in fighting both types of parasites. where both infections are chronic and endemic. antibiotics. and initiatives such as frequent deworming of an entire population may need to be reconsidered. Random effects were included to control for repeated observations on the same individual and within village correlations. affecting growth. lamblia is associated with significant morbidity. All Tsimane residing in study villages are eligible to participate in the study. while also providing primary medical care. very few studies have examined interactions between giardia and helminths. then this may further alter the cost– benefit calculus for treatment. limiting the effectiveness of one-time or even periodic treatment [17. as well as hunting. the Tsimane have been participants in the on-going Tsimane Health and Life History Project (THLHP.royalsocietypublishing. Albendazole and mebendazole were prescribed as anti-helminthics but also have the potential for anti-protozoal effects [12. including growth deficits and anaemia [16]. regardless of which method was used. Since 2002. As we report elsewhere. collecting demographic. biomarker collection and physician diagnostics have been described elsewhere [19. and was used only in the later part of 2008 and 2009. THLHP researchers work extensively in Tsimane villages. for many others G. the choice of medication was made by study doctors based on availability. metronidazole and tinidazole. The data for this study were collected in 83 Tsimane villages representing a modest range of environmental (forest versus riverine) and economic (market integrated versus isolated) situations. Given the paucity of reports examining the effect of helminth infection on giardia infection and vice versa. Duplicate mounts were prepared with 0. neither is known to be effective against helminths. http://www. and often co-infect the same individuals. helminth infections are associated with significant morbidity. and that treatment for helminths may increase susceptibility to G. faecal samples were analysed for the presence of helminth eggs.org (b) Data collection and medical care Study participants were seen by the mobile THLHP biomedical team who visited Tsimane villages annually from 2006 to 2010. and later analysed using a modified Percoll (Amersham Pharmacia) technique [23]. For this study. neither medication was available. longitudinal panel study to address the following questions. Giardia intestinalis. cramping. albendazole was prescribed as a single 400 mg dose.k.org on November 16. anthropological and biomedical data. On the one hand. lamblia [15]. these symptoms can adversely affect young children. anti-protozans and anti-helminthics as warranted. This last possibility has strong implications for treatment plans in areas where both are endemic. Patients were treated with four relevant medications: albendazole. including diarrhoea. Albendazole in particular was either unavailable or prohibitively expensive during most of the study. Both giardia and helminths disproportionately affect individuals in underdeveloped areas. Metronidazole and tinidazole were prescribed as anti-protozoal agents.unm. individuals treated for helminths in highly endemic populations may become reinfected. mebendazole. In other cases.22]. On the other hand. bloating and weight loss. Following on-site analysis of participant blood and faecal samples. Owing to the difficulty of working in an isolated rural Bolivian context. Since helminth . but did not produce systematic biases in parasite identification [19]. In particular. What is the relationship between helminth and giardia infections? Does infection with one alter the odds of infection with the other? Does treatment for one alter the odds of infection with the other? of the Tsimane population chooses to do so at least once. patients either would leave the site of the mobile medical care before faecal analysis was completed. lamblia infection may appear asymptomatic. Information on treatments given and infection status during the previous examination was available for 1082 individuals at 1731 medical visits. many of which are in developing and subsistence populations [12].a. If helminth infections are antagonistic to G.

all of which can interact with covariates.4 105.org Proc R Soc B 280: 20131671 infections often show nonlinear age patterns. p ¼ 0. We began with all four treatment variables and study year in the model. obs 5 1692) mean + s. On average. model AIC was assessed. We proceeded until model AIC was minimized. lumbricoides (OR ¼ 0. we controlled for age. Twenty-three participants were age 18 or under during some examinations and over 18 during others. we ran separate models on younger (less than or equal to 16) and older (greater than or equal to 40) participants. The model allows repeated observations at arbitrary times to be used to estimate instantaneous rates of transition from one state to another. hookworm-infected (H). but not recovery rates. The transition intensity matrix from the basic model was used as the initial values matrix for subsequent models. obs 5 886) mean + s.2 150.d.2 + 4.d. age (years) height (cm) weight (kg) body mass index (kg m22) 6. a basic model was first fitted with no covariates. and in which covariates were constrained to affect only certain transition intensities. found in 56%. stercoralis (OR ¼ 1. with helminths becoming more prevalent and giardia infections declining. sex-specific effects and interactions were screened for by running separate male and female models.171 ¼ 2 2.3 + 9. respectively.7 male (n 5 766. and comparing transition intensities and hazard ratios between these models. Study year was constrained to affect only infection rates. prevalences changed significantly (figure 1). values between 0.1 162.01). In treatment analysis (§3d) we also controlled for infection status with the parasite in question at the previous visit. particularly during childhood. We therefore used a modelling strategy in which covariates were checked for significance and removed in a backward stepwise manner. Age patterns were significantly nonlinear. G. However. Direct transitions between C and U were not allowed in the model.8 + 2 adults female (n 5 868.royalsocietypublishing. t5. In treatment models.6 24.5 + 14.60. age and repeat observations. Observations from these individuals contribute to both means. See the electronic supplementary material for the data used in these analyses. lamblia and Ascaris lumbricoides. and then removed non-significant covariates one at a time. models were relatively insensitive to starting values. when infection prevalences are increasing .8 male (n 5 860.1 and 2. splines were refitted with natural log age.01). but not infection rates. we used a Markov multi-state model (MMSM) to examine longitudinal data in sequence. obs 5 1670) mean + s.org/) using lmer in the lme4 package. which forces the spline to fit early life patterns. sex and study year.3 105. retaining only those interactions that were significant. After each parameter change.7 63 + 8.Downloaded from rspb.0. since both transitions represented recovery from a helminth infection. to verify that effects were not due to nonlinear age patterns. which are linked together by subject to model transitions for each individual. More specifically. 3. We therefore constrained this transition intensity to be equal to the transition from a helminthonly to an uninfected state. 3275 participants age 0–90 (mean 34. Once this model was settled on. juveniles female (n 5 804. transitions between infection states over time [25]. We therefore report age as a linear parameter for these models.1 + 5.6 24 + 2. We therefore controlled for study year in all additional analyses.7 16. 51% female) were seen over the course of 5235 physician examinations (table 1). Starting transition intensities between infection states were arbitrarily set at 1.d.75. First. giardia-infected (G) and co-infected (C). age was modelled with thin-plate regression smoothing splines in generalized additive mixed models [24].8 17 + 2. t5.royalsocietypublishing.7.3 + 3. 30% and 15% of faecal samples. Sixty-five per cent of physician examinations were with adults (over age 18).46. Controlling for study year.6 + 25.1 + 11. Owing to a very low number of individuals who transitioned from a co-infected to a giardia-only state.55. Results (a) Physician examinations Between July 2006 and May 2012.org on November 16.2 + 25. Over the course of the study. 50. Significant interactions were included in the final model.8 + 4. 1 year). For all regression analyses. as we describe later. initial models had difficulty converging on this intensity.20 + 0. 6. To test for interactions between predictors and sex.0 (equivalent to a mean infection duration of approx. Because the model includes eight basic parameters.8 + 12. respectively. p ¼ 0. degrees of freedom are rapidly lost when including covariates. although only 53% of individual participants were over age 18. In these models. and more likely to be infected with S. obs 5 952) mean + s.0 produced similar final models.8 3 rspb.d. whereas adults are older than 18. which can be used to calculate the likelihood of transition over a defined period of time. Finally. and gamm in the mgcv package.2 + 23. Individuals were seen between one and five times each (mean 1. subsequent visits were just over 1 year apart (1. Transition intensities are estimated from continuous time observations.5 20. all age terms reduced to linear parameters. all eight transition intensities were unconstrained. All models and analyses were run in R v. The most common parasites found were hookworm.32 years). Our model includes four disease states: uninfected (U). (b) Helminth and protozoan infection prevalences Table 2 shows the prevalences for the principal species of helminths and giardia among juveniles and adults.4 + 14.171 ¼ 2. Hazard ratios can also be calculated for comparable transition intensities. while medication was modelled as affecting recovery rates. in this case. Sample characteristics. 2013 Table 1.6 + 0.9). we included sex-byparameter interaction terms in models one term at a time.9 55. r-project. 3. Since cross-sectional analyses are of limited usefulness in establishing causal direction. For models in which the spline reduced to a single degree of freedom. msm in the msm package.3 20. these represent the probabilities of recovery or infection at any given moment in time. males were less likely to be infected with A. instead recovery and co-infection require two transitions—first to a single infection state (H or G) and then to U or C. Juveniles are 18 and under. Models included eight intensity transitions (figure 2a).11 (http://cran.3 + 4. 48.

7 3. z ¼ 7.org on November 16. 0.0 4. lamblia (OR ¼ 2.4 868/1692 male (%) 70.001) and S.64.1 6. juveniles female (%) any helminth hookworm A. lumbricoides (OR ¼ 2. (a) Prevalences of giardia and helminths by study year. trichiura was associated with A.60. In both panels.001). z ¼ 2 7. p ¼ 0. . lamblia infection (OR ¼ 0. 0. p .) Table 2.royalsocietypublishing.33. lumbricoides T.62.0 31. 71% received mebendazole. hookworm. p . (d) Effect of treatment during the subsequent visit on risk of infection Over the course of the study. helminths and giardia were negatively associated.06. stercoralis T. Juveniles are 18 and under. These associations were not dependent upon whether a linear or nonlinear age term was used. 0. p .07. z ¼ 2 1. 2013 (a) 100 80 prevalence (%) 60 40 20 hookworm G.09). Prevalence of helminths and giardia.0 860/1670 (figure 1b). z ¼ 3.6 16. lamblia þ any helminth participants/observations 56.55.58. lumbricoides S.3 13. trichiura (b) 4 rspb. Owing to co-infection.002) and T. 0. (c) Coinfection risk Table 3 presents the results of logistic regression analyses in which the presence/absence of each individual parasite is regressed on the presence/absence of the others. lamblia G. 54% of patients infected with a helminth were given an anti-helminthic.60. Prevalence of tsimane helminth and giardia infections. having one helminth infection was predictive of having another helminth infection. 0. not stacked. 0. A lumbricoides and S. p ¼ 0.8 766/886 adults female (%) 68. p ¼ 0.94.8 29. T.63.8 16.0 60.2 13. More detailed age patterns for this population have been described elsewhere [19]. respectively). Conversely.69.74).9 3.Downloaded from rspb. p ¼ 0.001. p .60. z ¼ 2 4.0 29. p .6 12.3 5.02).9 44.99.44.org Proc R Soc B 280: 20131671 0 2006 2007 2008 2009 2010 2011 2012 year 0 20 40 age 60 80 Figure 1.42.8 4.48. lumbricoides (OR ¼ 0. stercoralis G.1 46. Both uncorrected prevalences (filled polygons) and prevalences corrected for age and village membership (dashed lines) are shown. although the converse association was not found (OR ¼ 0. (Online version in colour. we ran a series of models to test for sexspecific associations. Twenty-three participants were age 18 or under during some examinations and over 18 during others. z ¼ 2.001). Additionally.9 30. p ¼ 0.7 804/952 male (%) 52. A. 0. lamblia A. p . z ¼ 2 1.65. stercoralis was predictive of hookworm infection (OR ¼ 3. trichiura was positively associated with G. By contrast. z ¼ 2 4. No significant interactions with sex were found.25.7 4. OR ¼ 0. stercoralis (OR ¼ 0.63. p .royalsocietypublishing.26.50.001).47.001). trichiura S.1 14. z ¼ 2 7. S. Giardia lamblia infection was associated with lower odds of infection with hookworm (OR ¼ 0. z ¼ 6. z ¼ 5. stercoralis were associated with lower odds of G.3 8. (b) Total uncorrected infection prevalences for each species by 2-year age group. 25% of the helminthinfected patients also received an anti-protozoan and 26% of giardia-infected individuals received an anti-helminthic.001.0 16.80. whereas adults are older than 18. prevalences are overlapping.5 16.2 63. Of those who received an anti-helminithic.75. Hookworm infection was associated with greater odds of Strongyloides stercoralis infection (OR ¼ 37. z ¼ 2 0. A. OR ¼ 0. while 57% of patients infected with giardia were given an anti-protozoan. lumbricoides infection was associated with greater odds of Trichuris trichiura (OR ¼ 3. In general. Observations from these individuals contribute to both prevalences.

Males were more likely to become .72.36.46*** 0. The 1-year impact of mebendazole treatment was modest: 69% of individuals infected with hookworm at one visit were infected a year later without treatment.20). hookworm-infected.64*** 0. lamblia hookworm A. Models were run using only observations with complete data (participants ¼ 1082.98 0.18 0.001.43 0.55*** 0.63*** 1.96b)** Proc R Soc B 280: 20131671 The parameter given is the estimated degrees of freedom for the spline.05.02 1.07* Infected at the previous visit with the parasite listed as the dependent variable for a given column.56.71*** 0.royalsocietypublishing.83. study year and a sex-specific hazard for hookworm infection. **p  0. when males and females were analysed in separate models.94 0. ***p  0. stercoralis 0. dependent independent G. lamblia 2.10.04 0.001).61 2.Downloaded from rspb. Odds ratios for infection with one parasite given infection with another. p ¼ 0. 2013 Table 3.99 1.60*** A. z ¼ 1.33*** 1.00b) 0.001.37*** 1.50 (1. n ¼ 3275). mebendazole. dependent independent albendazole mebendazole metronidazole tinidazole sex (male) study year infected at previous visita age (decades) a # G.99 (3.57*** 0. with mebendazole reducing this only to 63%.05.08** A. After examining covariates.12 1.74. Parameter significance is indicated by asterisks.23# 1. Parameter values are odds ratios estimated in separate generalized logistic mixed model for each parasite or pathogen.91 0.98 1. albendazole treatment.10.02) # S. p ¼ 0.05 T.97 hookworm 1. This did not change when these two anti-protozoans were pooled together into a single variable.royalsocietypublishing.80 1.63*** 1. p  0.36 2. trichiura 4. *p  0.87 0.89)*** 2.53.27. controlling for nonlinear age effects with a spline term.97 T.17 0.74 1.56# 0.97 0. z ¼ 1. stercoralis sex (male) study year age (EDF)a a b 5 rspb.99** 1.001) and giardia infection (OR ¼ 2. p .91 1.10** 1.38.62* 2. p ¼ 0.25 (1. we found a significant interaction between mebendazole treatment and sex on Ascaris infection in which treatment was associated for greater odds of infection in males.72. # p  0.69*** 0. These age terms were fitted to log age.02). trichiura 2.03).75 0.14. lumbricoides T.12.36 1.01.10).72* 1. obs ¼ 1731).02 1. p ¼ 0.06 1.00*** 1.71* 0. ORfemales ¼ 0.12 0.28. controlling for repeat observations and within community correlation with random effects. Odds ratios for infection based on receipt of anti-helminthic or anti-protozoal agents and infection status at the previous medical visit.06 1.27# 1. lamblia infections 1 year later (OR ¼ 1. and lower odds in females (ORmales ¼ 1.org G. Individuals who received mebendazole were also marginally more likely to have G. strongyloides 0. the final model included mebendazole treatment.90 0.78# 1.61 1.99** 2.47 1. ***p  0.75# 0. The most common helminth treatment.23 1.65*** 0. trichiura S.10 1. p ¼ 0.27 3. By contrast.03 any helminth 1.82 0. giardia-infected and co-infected (figure 2).45. lumbricoides 0. neither of these odds ratios was significant (ORmales ¼ 1. 0. and controlling for repeat observations and within community correlation with random effects terms (obs ¼ 5171.01 (7. z ¼ 3.13 7. When we tested for interactions by sex. (e) Changes in infection status over time An MMSM was used to examine transitions between four disease states over time: uninfected.2 1. ORfemales ¼ 0. trichiura (OR ¼ 4.99b)*** 1. lumbricoides 0. We examined the effect of treatment on infection at the subsequent visit (table 4). Table 4. However.10.61*** 1.21.20 3.50# 37.51 0. albendazole was associated with increased odds of subsequent T.66.65*** 0. *p  0. Odds ratios were calculated in generalized additive logistic mixed models with all independent variables in one model. Metronidazole and tinidazole treatments were not significantly associated with changes in the odds of infection in the subsequent year for any helminth or giardia.86 # S. interaction: z ¼ 2. z ¼ 2 1. lamblia hookworm 0.10# 2. was associated with a decreased likelihood of hookworm infection at the subsequent visit (OR ¼ 0.60*** 0.01. p ¼ 0.01 1. z ¼ 3.77** (2.14*** 1. z ¼ 2 2. **p  0.org on November 16.

95% CI (0. ¼ 789.02.70 H 3. 95% CI (0.57. 0. Although both types of analysis suggest mutual antagonism. recovery from helminth was much less likely when co-infected with giardia (B ! G/H ! U.18. HR: 1. Apart from a sex difference in hookworm infection.67)).86. giardia-infected (G) and co-infected with both (C). for individuals sampled two (r ¼ 2 0.79.99 (c) U 2.Downloaded from rspb.d ) Examples of simulated 10-year infection histories for hookworm (lower. or to host differences in genetic or phenotypic susceptibility. suggesting that differences are due not to persistent host characteristics but to short-term dynamics. The model confirms that individuals infected with hookworm are significantly less likely to become infected with giardia (H ! C/U ! G.88. 1.f. we find that co-infected individuals are much more likely to clear giardia rather than (f ) Individual differences or parasite interaction Negative associations between helminths and giardia might be due to antagonism between parasites. (c.57. 1. lamblia infections in only 24% of those infected with helminths. light bars) and giardia (upper. p .c) Arrows and values indicate the estimated instantaneous transition intensities between the possible states of uninfected (U).04 1.19.) infected with hookworm (HR: 1. 1.88.26)). we would expect giardia infections to cluster in some hosts and helminth infections in others. hookworm-infected (H).36. no other interactions by sex were found. out.31. d.f.71.03 G 1. 0. 95% CI (1. (a.10 (b) 6 rspb. but did not affect the giardia recovery rate (HR: 1. To put the odds ratios we report into perspective.90)). some associations may be mediated by third variables. the model predicts that over a 10-year period the average Tsimane will be infected with helminths for 6.52)). 95% CI (1. in longitudinal analysis.81. Models run separately by age cohort also did not differ significantly in model parameters. To parse this .43). we find G. but end up with similar infection frequencies given sufficient time. so were not included in final models. p ¼ 0.royalsocietypublishing. the estimation of odds ratios from crosssectional data is of somewhat limited value.98 C 0 2 4 6 8 10 Figure 2. 1.65)). 95% CI: (1. However. 1.74 1. (a.00. although 42% of those without helminth infection were infected with G. 6.08)) and with giardia for 2. HR: 0. 0.51 G (d ) C 0 2 4 6 8 10 Proc R Soc B 280: 20131671 0.63 0. if negative associations are due to parasite interactions. p ¼ 0.30. 50% were infected with at least one helminth. 2.51.60. infection with hookworm is less likely for individuals with giardia (G ! C/U ! H.07)).54 0.60)).22. compared with 70% of those without G.57.12 years out of 10 years (95% CI: (5.d) significantly increased the recovery rate from hookworm-infected to uninfected (H ! U and B ! G. frequencies of hookworm and giardia infections were negatively correlated. and for these differences to persist over time. albendazole was not significantly associated with likelihood of giardia recovery (HR: 0.96 1.51 U 2.org 0. Additionally.02. for individuals sampled four or five times there was no correlation between frequencies (r ¼ 2 0.04. Similarly. we examined correlations between individual frequency of hookworm infection and frequency of giardia infection. In a model assuming annual mebendazole treatment. 4.47)). lamblia infection. Indeed.d ) with annual mebendazole treatment. not significantly less time than without treatment.07.63 years (95% CI (6.org on November 16. Multi-state Markov models.002) times.46. Treatment with mebendazole (figure 2c. HR: 1. 95% CI (0. Overall. but generally cannot illuminate causality. 2013 (a) 1. 95% CI (0. Discussion We report on helminth and giardia infections in an on-going longitudinal study of a lowland Bolivian population.27. (b. 1. We find significant evidence for an interaction between helminth and giardia infections. Additionally. HR: 0. time infected with helminths was 6. Similarly. Taken alone.royalsocietypublishing.76)). d.14 years (95% CI (1. 95% CI (1. However.15)) or hookworm recovery (HR: 0. ¼ 245. ¼ 189. 2.24 years. 95% CI (0.b) With no treatment. (Online version in colour. Anti-protozoans were not significant in any model and did not improve model fit (by AIC).10 1. of those with G. recovery from giardia is more likely when coinfected with helminths (C ! H/G ! U. 0. By contrast. 7. dark bars). Although the inclusion of albendazole improved model fit.001) or three (r ¼ 2 0.74)). 95% CI (0.70 H 3. Time infected with giardia was also not significantly changed (2. lamblia.f. HR: 0. Odds ratios may uncover associations between parasites.54 0.28)).64 0. individuals should cycle between infections.06. If negative associations are due to host differences. lamblia. d. We therefore analysed the time course of infections using longitudinal data in a state-based model.

since we would expect this to result in persistent negative correlations between individual infection frequencies. or which induce similar immune responses. 7 rspb. but more frequently they become infected with one parasite when not infected with the other. which reside on microvilli in the small intestine. This work was supported by grants from the National Institutes of Health/National Institute on Aging (R01AG024119 and R56AG024119) and the National Science Foundation (BCS-0422690). individuals alternate between infections. rather than systemic immune interaction between the two species [14]. but depended on logistical and clinical factors. trichiura (with adults located further down in the large intestine) was not. consistent with the only other similar study we are aware of [15]. Rather. Finally. S. these results illustrate the importance of considering co-infection in epidemiological models and treatment strategies. This is particularly apparent in the mebendazole treatment condition. The apparent antagonism may reflect competitive inhibition or cross-immunity. lamblia. our study provides strong support for an interaction between helminths and giardia. which may present a problem due to patient compliance. were inhibited by Trichinella spiralis when these helminths inhabited the small intestine but not at later stages when they moved to muscular tissue. were negatively associated with G. A. should treatment be provided? Only a careful analysis of the impact of each species on quality of life can answer this question. However. it is possible that helminth-induced TH2 activity may provide some cross-immunity against G. Second. hookworm. Additionally. lamblia presents a conundrum for treatment decisions in regions in which infection with these species is common. lamblia. This can be seen in simulated infection histories (figure 2b. Typical albendazole treatment recommendations for these species require multiple days of treatment. Our results also suggest that in the neotropics. we caution against drawing strong conclusions since albendazole was used primarily in only one study year. treatment in our study was not truly randomized. suggesting a physical or localized. all of which inhabit the small intestine. Tsimane consent procedures were approved by the IRBs at the University of California. Not only do we see fewer G. G. the many THLHP staff and students who contributed to this project and two anonymous reviewers for their helpful comments.royalsocietypublishing. Our results nonetheless show that treatment for either parasite is unlikely to be effective without changes in sanitation and access to clean water. lamblia. A. the effect of albendazole was not significant in the state-based model. We would like to thank the Tsimane who participated in this study. Overall. Although mebendazole was associated with reduced odds of being infected with hookworm 1 year later. Individuals may be co-infected. an open question is whether treatment for helminths increases susceptibility to giardia infection in the weeks or months following treatment. in which hookworm infections are disrupted (figure 2d ). lamblia. limiting our ability to examine short-term dynamics of reinfection or changing infection status. we do not have a clear explanation for the apparent increases.royalsocietypublishing. We suspect a shorter sampling period might reveal stronger effects. Thus. consistent with either a physical interaction or the effect of a localized immune response. Therefore. frequency of hookworm infection is uncorrelated with frequency of giardia infection. IgG. treatment did not significantly affect time spent with either infection. Our analyses suggest that this is not a direct effect of the drug administered. infectious status among the Tsimane was diagnosed only on the basis of a single faecal sample. lamblia. consent forms were signed for literate participants and verbal approval with fingerprint signature given for non-literate participants. we cannot rule out the possibility that some medications may have been obtained from other sources between study visits.org Proc R Soc B 280: 20131671 Acknowledgements. Instead. In a murine model. as well as TH2 antibody responses (IgA. which may underestimate the true prevalence of both single and multiple species infections in this population. Funding statement. Studies have also shown that G. stercoralis and hookworm. Given the highly significant negative associations between G. particularly for infectious agents that directly compete for substrate or resources. lamblia/helminth co-infections than would be expected by random assortment. After explanation of a formal protocol by bilingual Tsimane assistants. suggesting that the increased likelihood of male hookworm infection may drive males’ lower odds of giardia infection. lamblia and hookworm. Santa Barbara and the University of New Mexico. although most Tsimane have limited access to medications other than those provided by our medical team.2 years apart.d). but that the removal of helminths increases giardia susceptibility. Co-infection affects both recovery and reinfection. If occasional treatment for helminths increases susceptibility to G. over a longer time period. The apparent interaction between hookworm. study visits were on average 1. lamblia clearance and protective immunity are mediated by mixed TH1 and TH2 cytokine production (characterized by both INF-g and IL-4). the lack of significant changes in time spent infected in the longitudinal model probably reflects the time between samples and the overall rapid reinfection rates for both giardia and hookworm. and (iii) individual consent during medical visits and before each procedure. we found no lasting effect of anti-protozoan treatment. IgE) [26 –28]. Informed consent was obtained at three levels: from (i) the Gran Consejo Tsimane.Downloaded from rspb. the reduction was minimal. mebendazole may be more effective at treating the predominant helminth. antiprotozoans may provide short-term benefits. and the inhibition of giardia infection by hookworm is more pronounced than the converse. Additionally. Owing to its large size and longitudinal analysis. It is not the case that some individuals suffer from giardia and others from hookworm. However. In our study. 2013 hookworm. In our study. Finally.org on November 16. but our analyses suggest that treatment with albendazole or mebendazole may increase the odds of giardia infection. whereas T. . there are several limitations that should be noted. lumbricoides. although males in cross-sectional analysis were more likely to have hookworm and less likely to have giardia. First. The details of this interaction will need to be investigated in future studies. trichiura and G. in the state-based model the only significant sex difference was an increased hazard of hookworm infection for males. The negative association between hookworm and giardia does not appear to be driven by differential host sensitivity to these two parasites. (ii) community officials and participants in village meetings. While this may explain the lack of efficacy. than is albendazole. since this is the time period in which we would most expect to see an effect. but do not appear to have lasting effects due to constant reinfection. the local Tsimane government organization that represents Tsimane interests and oversees all projects. Additionally. However. Albendazole was associated with an increase in T. lumbricoides and G.

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