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Keywords:

  • Giardia duodenalis ;
  • iron deficiency;
  • ferritin;
  • Rwanda

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Subjects and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Objective

Acute symptomatic infection with Giardia duodenalis impairs iron absorption, but iron deficiency may protect against infections caused by various micro-organisms including parasites. We therefore examined the association of G. duodenalis infection and iron deficiency in 575 Rwandan children under 5 years of age.

Methods

Giardia duodenalis infection was diagnosed by triplicate microscopy and PCR assays, and iron deficiency was defined as a ferritin concentration <12 ng/ml.

Results

Largely asymptomatic G. duodenalis infection was seen in 65.3% of the children and iron deficiency in 17.4%. G. duodenalis infection was less common in iron-deficient children (51%) than in non-deficient children (68%, = 0.002). In multivariate analysis, the odds of G. duodenalis infection were almost halved in iron-deficient children (adjusted odds ratio, 0.54; 95% confidence interval, 0.33–0.86).

Conclusion

In this highly endemic setting, there was no evidence that Giardia infection impairs iron status. Rather, iron deficiency appeared to protect against infection with this parasite.

Objectif

L'infection symptomatique aiguë par Giardia duodenalis altère l'absorption du fer, mais la carence en fer pourrait protéger contre les infections causées par divers micro-organismes, y compris des parasites. Nous avons donc examiné l'association entre l'infection par G. duodenalis et la carence en fer chez 575 enfants rwandais de moins de cinq ans.

Méthodes

L'infection par G. duodenalis a été diagnostiquée par microscopie en triplicat et des tests PCR, et la carence en fer a été définie comme une concentration en ferritine < 12 ng/ml.

Résultats

Une infection par G. duodenalis généralement asymptomatique a été observée chez 65,3% des enfants et la carence en fer chez 17,4% d'eux. L'infection par G. duodenalis était moins fréquente chez les enfants avec une carence en fer (51%) que chez ceux sans carence (68%, P = 0,002). En analyse multivariée, les chances pour l'infection par G. duodenalis étaient presque réduites de moitié chez les enfants avec une carence en fer (odds ratio ajusté: 0,54; intervalle de confiance de 95%: 0,33 à 0,86).

Conclusion

Dans ce contexte de forte endémicité, il n'y avait pas de preuve que l'infection par G. duodenalis altère le statut en fer. Au contraire, la carence en fer semble protège contre l'infection par ce parasite.

Objetivo

Una infección sintomática aguda con Giardia duodenalis afecta la absorción del hierro, pero la deficiencia de hierro podría proteger frente a infecciones causadas por varios microorganismos, incluídos los parásitos. Por lo tanto hemos evaluado la asociación de la infección por G. duodenalis y la deficiencia de hierro en 575 niños menores de cinco años de Ruanda.

Métodos

Se diagnosticó una infección por G. duodenalis mediante microscopía por triplicado y ensayos de PCR, y la deficiencia de hierro se definió como una concentración de ferritina <12 ng/ml.

Resultados

Se observó una infección asintomática por G. duodenalis en 65.3% de los niños y deficiencia de hierro en 17.4%. La infección por G. duodenalis era menos común en niños con deficiencia de hierro (51%) que en niños sin deficiencia (68%, = 0.002). En un análisis multivariado, la probabilidad de infección por G. duodenalis era casi la mitad entre niños con deficiencia de hierro (odds ratio ajustado, 0.54; intervalo de confianza 95%, 0.33–0.86).

Conclusión

En este emplazamiento con una alta endemicidad, no se halló evidencia de que la infección por Giardia afecte los niveles de hierro. De hecho, la deficiencia de hierro parecía proteger frente a la infección contra este parásito.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Subjects and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Giardia duodenalis is one of the most common intestinal protozoa worldwide. Although a cause of gastrointestinal symptoms, notably diarrhoea, many infections in highly endemic regions are asymptomatic (Veenemans et al. 2011; Ignatius et al. 2012). However, in chronic (or recurrent) infection, malnutrition, particularly stunting, and, at later age, reduced cognitive functions may develop (Berkman et al. 2002). Malabsorption is considered the main pathogenetic determinant, but the underlying mechanisms have not been fully elucidated (Cotton et al. 2011). Giardiasis, that is, the symptomatic infection with G. duodenalis, has been associated with impaired intestinal iron absorption and reduced serum iron concentrations in children (de Vizia et al. 1985; Ertan et al. 2002; Abou-Shady et al. 2011). Accordingly, metronidazole treatment of children with giardiasis corrected iron absorption and improved iron and haematological parameters (de Vizia et al. 1985; Monajemzadeh & Monajemzadeh 2008). In contrast, in asymptomatic G. duodenalis infection, iron absorption and response to oral iron supplementation are unaffected (de Morais et al. 1996). Also, no association of G. duodenalis infection with iron deficiency was seen in a community-based study among children in Malaysia (Al-Mekhlafi et al. 2005). Iron deficiency, on its part, has been associated with protection against various infectious diseases including malaria (reviewed by Oppenheimer 2001; Sazawal et al. 2006). In line with this, iron rather stimulates the in vitro growth and survival of G. duodenalis (Gault et al. 1987), and in iron-deficient mice, reduced Giardia trophozoite counts during early infection have been observed (Duncombe et al. 1980).

In Rwandan children, we recently observed a high prevalence (60%) of largely asymptomatic G. duodenalis infection (Ignatius et al. 2012) and iron deficiency in 18% (Danquah et al. 2014). In the present analysis, we examined whether these two conditions are associated.

Subjects and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Subjects and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We conducted a cluster-sampled survey on common diseases in children under 5 years of age in Butare and its rural surroundings in 2010 (Gahutu et al. 2011). Butare (population approximately 100 000) is the capital of Huye district, southern province of Rwanda, and surrounded by densely populated farmland hills. Briefly, 25 households each were randomly chosen in 24 randomly selected villages, and one child was randomly selected per household. Paediatric health facility attendants were consecutively recruited at a rural primary health centre and at the referral district hospital, that is, the facilities serving this population. Leading primary diagnoses in the patients were respiratory tract infection, gastro-intestinal tract affection and malaria (Gahutu et al. 2011).

All children were clinically examined, medical and socio-demographic data were documented, and a venous blood sample was collected. Underweight (weight-for-age z-score <−2) and stunting (height-for-age z-score <−2) were documented. Breastfeeding was documented as any vs. none. G. duodenalis was diagnosed by triplicate light microscopy and PCR assays (Ignatius et al. 2012). Plasma concentrations of ferritin and C-reactive protein (CRP) were measured by ELISA (Assaypro, St. Charles, USA). Iron deficiency was defined using two definitions: (i) ferritin <12 ng/ml and (ii) ferritin <12 ng/ml, or ferritin <30 ng/ml if CRP >5 mg/l (WHO 2001; Thurnham et al. 2010). Children were treated according to Rwandan health authority guidelines.

Of a total of 749 children, data on G. duodenalis and iron deficiency were available for 575 children; these formed the basis of the present analysis. Data were analysed using the survey data analysis module in Stata 13.0 (Stata Corporation, College Station, TX, USA). Due to the cluster sampling and the non-normal distribution of continuous variables, weighted nonparametric tests for survey data were used to compare means (95% confidence intervals [CIs]) and proportions (95% CIs). As for factors associated with G. duodenalis, odds ratios (ORs) and 95% CIs were calculated applying logistic regression, weighting for the population size of each sampling cluster cell, that was, residence. For single villages without information on the population size, the mean population size of all villages was imputed. Due to weighting, prevalences (%) do not necessarily correspond to absolute numbers. A P-value <0.05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Subjects and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Giardia duodenalis infection was present in 65.3% (366/575) of the children (Table 1). It occurred more commonly in community children than in health facility patients, and its prevalence increased with age (Table 2; Figure 1). Overall, infection was not associated with diarrhoea, other gastrointestinal symptoms, underweight or anaemia, but among infected community children, the proportion of clinically assessed severe malnutrition was significantly increased (11.6% vs. 4.5%; Ignatius et al. 2012).

Table 1. Ferritin concentrations and iron deficiency in Rwandan children according to infection with G. duodenalis
  Giardia duodenalis P
Non-infectedInfecteda
  1. a

    by PCR.

  2. b

    ferririn <12 ng/ml.

  3. c

    for health facility attendants, conventional summary statistics were calculated, because nonparametric tests for survey data were not appropriate for the small sample size (i.e. single sampling units).

All children
No.209366 
Age (months) (mean, 95% CI)26.6 (24.4–28.9)34.8 (33.2–36.4)<0.0001
Ferritin (ng/ml) (geometric mean, 95% CI)13.5 (8.7–20.4)33.1 (25.7–42.7)<0.0001
Iron deficiency (%, 95% CI)b24.4 (18.8–30.9)13.7 (10.4–17.7)0.002
Community children
No166326 
Age (months) (mean, 95% CI)26.1 (23.6–28.6)34.8 (33.1–36.5)<0.0001
Ferritin (ng/ml) (geometric mean, 95% CI)13.8 (8.7–21.9)30.9 (23.4–40.7)0.004
Iron deficiency (%, 95% CI)b22.5 (16.6–29.7)14.5 (11.0–18.9)0.03
Health facility attendantsc
No4340 
Age (months) (mean, 95% CI)28.8 (23.5–34.1)34.4 (28.9–39.8)0.15
Ferritin (ng/ml) (geometric mean, 95% CI)13.2 (5.2–33.1)55.0 (33.9–91.2)0.009
Iron deficiency (%, 95% CI)b30.2 (18.2–45.8)7.5 (2.4–21.4)0.02
Table 2. Univariate and multivariate analysis of factors associated with Giardia duodenalis
Parameter n G. duodenalis positive (%)aOR95% CI P aOR95% CI P
  1. Adding inflammation (CRP >5 mg/l) to the model did not change the estimate of iron deficiency (aOR, 0.53; 95% CI, 0.33–0.84). The same was true when adding clinically assessed severe malnutrition (aOR, 0.55; 95% CI, 0.35–0.88) or underweight (aOR, 0.55; 95% CI, 0.35–0.87).

  2. OR, odds ratio; aOR, odds ratio adjusted for all univariately associated parameters; CI, confidence interval.

  3. a

    by PCR.

  4. b

    ferritin <12 ng/ml.

Subgroup
Community49268.01  1  
Health facility8350.10.470.29–0.760.0020.480.29–0.800.005
Age (years)
<15941.91  1  
1 < 214053.91.620.84–3.110.151.450.73–2.860.29
2 < 314069.33.131.62–6.070.0012.221.08–4.560.03
3 < 412472.83.721.88–7.37<0.00012.190.98–4.890.06
4 < 511278.35.012.45–10.27<0.00013.041.25–7.370.01
Breastfed
No28176.41  1  
Yes29354.70.370.26–0.54<0.00010.610.36–1.030.06
No. of siblings
None13058.61  1  
1–340265.21.330.88–2.010.180.990.63–1.550.97
≥44283.33.541.47–8.520.0052.390.91–6.300.08
Iron deficiencyb
Absent47468.21  1  
Present10151.40.490.31–0.770.0020.540.33–0.860.01
image

Figure 1. Prevalence of iron deficiency and Giardia duodenalis infection among 475 children from southern Rwanda. Crude prevalences (unweighted) shown as percentages.

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Iron deficiency was present in 17.4% (101/575) of the children, regardless of the definition applied (i.e. adjusting for inflammation did not change the estimate). Of note, G. duodenalis infection was significantly less common in iron-deficient children (51.4%, 51/101) than in non-deficient children (68.2%, 315/474, = 0.002). Vice versa, iron deficiency was seen in 14% of G. duodenalis infected and in 24% of non-infected children, and correspondingly, ferritin concentrations were higher in infected than in non-infected children. These associations were present in both, community children and health facility attendants (Table 1). The proportion of submicroscopic infections among all G. duodenalis infections was not influenced by iron deficiency: it was 70.0% (36/51) in deficient children and 68.9% (216/315) in non-deficient children (= 0.89).

Iron deficiency was not associated with age (Figure 1), study subgroup (community, health facility), sex, residence, socio-economic indicators, underweight, stunting, clinically assessed severe malnutrition, CRP levels or infection with soil-transmitted helminths (data not shown), but it was less common in breastfed children (6.8% (41/281) vs. 10.6% (60/293), = 0.03).

In multivariate analysis including previously identified predictors of G. duodenalis infection in this group, that is, study subgroup, age, breastfeeding and number of siblings (Ignatius et al. 2012), iron deficiency proved to be associated with 46% reduced odds of G. duodenalis (Table 2).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Subjects and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

In highly endemic southern Rwanda, iron deficiency was associated with reduced odds of G. duodenalis infection. In this population, infection was linked with malnutrition, but not with diarrhoea (Ignatius et al. 2012). This predominance of largely asymptomatic and likely chronic infections accords with the notion that the production of acute symptoms caused by Giardia infection may be more common in less endemic areas (reviewed by Cotton et al. 2011). In fact, at high endemicity, there even is evidence for protection against acute diarrhoea in infected individuals (Veenemans et al. 2011).

Although not specifically assessed, our results argue against reduced iron uptake in children with essentially asymptomatic G. duodenalis infections. This corroborates unaffected iron absorption found in asymptomatically infected Brazilian children (de Morais et al. 1996). In contrast, reduced iron absorption and reduced iron levels have been seen in children with acute symptomatic giardiasis in Italy, Turkey and Egypt (de Vizia et al. 1985; Ertan et al. 2002; Abou-Shady et al. 2011).

As a matter of fact, the present study was not a priori designed to assess an association between iron deficiency and G. duodenalis infection. Considering the given prevalences, the study had a power of 85% to detect a 50% reduction in infection in iron-deficient children. Confounders may have affected our finding, but in multivariate analysis including known predictors of infection, the reduced odds in iron-deficient children were confirmed. Moreover, association does not mean causality, and the direction of association could be both ways. Nevertheless, we suggest that iron deficiency in this highly endemic setting may provide some degree of protection against G. duodenalis infection. A weakness of this study is that iron deficiency was defined by reduced ferritin levels only; however, serum ferritin is the most specific marker of body iron stores and recommended by WHO as the best indicator of iron deficiency. But it is also an acute-phase protein that is elevated in the presence of infection or inflammation. Increasing the ferritin threshold from <12 ng/ml in the presence of inflammation (here defined as CRP >5 mg/l) to, for example, <30 ng/ml is therefore recommended (World Health Organization 2001; Thurnham et al. 2010). In the present study, the prevalence of iron deficiency was identical with both definitions, and CRP levels did not differ significantly between children with or without iron deficiency. Also, when including raised CRP levels into the logistic regression model of factors associated with G. duodenalis, the association with iron deficiency did not substantially change. We therefore conclude that inflammation did not confound our finding. Nevertheless, studies on Giardia infection and iron deficiency using more than on iron biomarker are required.

Despite mild immune-suppressive effects, iron deficiency has been associated with protection against several infectious diseases including malaria, and iron supplementation may enhance pathogens as well as associated morbidity and mortality (reviewed by Oppenheimer 2001; Sazawal et al. 2006). Considering the virtual absence of hookworms in the present study group (Ignatius et al. 2012), iron deficiency likely reflects low nutritional intake of iron. For the intestinal and extracellular parasite G. duodenalis, a low iron status of the host may create an unfavourable environment and interfere with the parasite's fitness and replication (Duncombe et al. 1980; Gault et al. 1987). Additional mechanisms could contribute to reduced susceptibility in iron-deficient individuals and/or limit parasite persistence: Nitric oxide produced by epithelial cells or subepithelial macrophages has cytostatic effects on G. duodenalis in vitro (Eckmann et al. 2000), and in iron deficiency, nitric oxide production is increased (reviewed by Koskenkorva-Frank et al. 2013). Enhanced host defence via nitric oxide could thus be one explanation for our finding. Alternatively, iron-related modification of lactoferrin activity might be involved. This main iron-binding protein of mucosal surfaces, present in neutrophi1 granules, bile and mucosal secretions, has demonstrated giardicidal activity. In vitro, iron reduces the binding of lactoferrin to trophozoites and protects from subsequent killing (Turchany et al. 1997). Low iron levels could thus contribute to improved clearance of G. duodenalis.

Irrespective of the actual mechanisms involved, the observation of the present study of reduced odds of G. duodenalis infection in iron-deficient children may have implications for the management of both iron deficiency and the parasitic infection. Studies able to infer causality, for example following-up Giardia infection during iron supplementation, are needed.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Subjects and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We thank the children and their families for participation in this study, the staff at Sovu Health Centre, Kabutare District Hospital, and University Teaching Hospital of Butare central laboratory for assistance and cooperation. The active assistance by the Rwanda office of the German Development Cooperation (GIZ) is gratefully acknowledged. This study was supported by the German Federal Ministry for Economic Cooperation and Development via the ESTHER programme and by the German Federal Ministry of Education and Research (grant 01DG13006A, MOPACUR).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Subjects and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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