Giardia and growth impairment in children in high-prevalence settings: consequence or co-incidence?

Abstract

Purpose of review

Giardia is a common intestinal parasite worldwide, and infection can be associated with clear, and sometimes persistent symptomatology. However, in children in high-prevalence settings, it is most often not associated with or is perhaps even protective against acute diarrhea. Nonetheless, recent longitudinal studies in high-prevalence settings increasingly identify an association with long-term outcomes that has been difficult to discern.

Recent findings

Recent studies have made progress in disentangling this apparent paradox. First, prospective, well characterized cohort studies have repeatedly identified associations between Giardia infection, gut function, and child growth. Second, experimental animal and in-vitro models have further characterized the biological plausibility that Giardia could impair intestinal function and subsequently child development through different pathways, depending upon biological and environmental factors. Finally, new work has shed light on the potential for Giardia conspiring with specific other gut microbes, which may explain discrepant findings in the literature, help guide future higher resolution analyses of this pathogen, and inform new opportunities for intervention.

Summary

Recent prospective studies have confirmed a high, if not universal, prevalence of persistent Giardia infections in low-and-middle income countries associated with child-growth shortfalls and altered gut permeability. However, the predominance of subclinical infections limits understanding of the true clinical impact of endemic pediatric giardiasis, and global disease burdens remain uncalculated. Integrating the role of Giardia in multipathogen enteropathies and how nutritional, microbial, metabolic, and pathogen-strain variables influence Giardia infection outcomes could sharpen delineations between pathogenic and potentially beneficial attributes of this enigmatic parasite.

INTRODUCTION: WHENCE THE BLACK BOX

Giardia lamblia (also known as Giardia intestinalis/Giardia duodenalis) is one of the most common intestinal parasitic infections in both children and adults worldwide. Sporadic giardiasis has a clear-case definition characterized by malabsorptive diarrhea, abdominal bloating/cramping, and weight loss [1] that is reproducible, if strain-dependent, in human volunteers [2] and resolves with treatment. Infection can lead to intestinal inflammation [3], systemic cellular immunity [4], and lingering symptoms, even for several years [5]. Giardia has also been implicated as a cause of diarrhea in travelers from nonendemic to endemic countries [6].

The majority of the estimated 300 million annual Giardia infections worldwide occur in children living in limited-resource settings, many of whom are undernourished, and harbor a particularly large burden of Giardia infection. In the past 6 years, multisite longitudinal birth cohort studies indicate that the majority of children in these settings experience at least one Giardia infection before 2 years of age, and more than half develop infection that persists for more than 3 months [7]. In these studies, Giardia has emerged as one of a subset of gut pathogens that most strongly associates with impaired childhood growth [7–9]. These associations are identified through longitudinal surveillance studies in asymptomatic children. In contrast, assessments of pathogen-specific diarrhea in the same settings find no Giardia-attributable burden of diarrhea [10,11,12^■]. Indeed, the finding in several studies that Giardia infection is associated with a decreased likelihood of acute diarrhea [6,12^■,13] has fostered speculation that Giardia colonization is not only inconsequential but even protective in some children. Thus, despite being among the most prevalent gut pathogens in this vulnerable population, Giardia, unlike other enteropathogens (e.g. Campylobacter or Cryptosporidium) is completely absent from global burden of disease estimates [14].

Here we update our previous review with recent advances that are beginning to unravel this epidemiological conundrum, seemingly categorizing Giardia simultaneously as both harmful growth-restricting pathogen and an inconsequential bystander [15].

DOES GIARDIA CAUSE ACUTE DIARRHEA IN HIGH-PREVALENCE SETTINGS?

In a recent meta-analysis of global studies of pathogen-attributable risk of acute diarrhea, among 88 079 cases and 135 755 controls in low-and-middle-income countries, Giardia detection was notable as the only pathogen demonstrating a consistent lack of association with acute diarrhea [summary odds ratio (OR) 0.4; 95% confidence interval (CI) 0.2–0.6] [12^■]. This absence of diarrhea-attributable Giardia detections is consistent across multiple age-stratified groups and is largely driven by the frequent detection of Giardia in matched asymptomatic controls. The longitudinal Etiology, Risk Factors, and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development Project (MAL-ED) Investigation provides the most comprehensive individual study analysis of Giardia infection in these settings to date. In MAL-ED, over the period of November 2009 to February 2014, Giardia was detected in more than two-thirds of 1741 children completing the study through 2 years of age, with wide variation in median time to first detection among the eight study sites [7]. Persistent infection (>3 months) was common (>56% of children, Elizabeth Rogawski McQuade, unpublished data). When adjusting for Giardia detection in nondiarrheal stools and the presence of copathogens, Giardia was not significantly associated with diarrhea, regardless of site or child age [7,11]. This strikingly high-prevalence of nondiarrheal Giardia infections [8,10,11,16] is such that Giardia detection has been negatively associated with diarrhea in several case–control and cohort studies [6,10,12^■,17, 18]. Although there is significant heterogeneity between these studies [8,12^■], these findings have led some to speculate that Giardia may have a protective effect on acute diarrhea.

Is the null, or even protective association between Giardia and acute diarrhea real, or is this an artifact of methodology? The use of quantitative nucleic acid-based diagnostics has effectively dispelled concerns of false-negative results because of inadequate detection methods in studies based on conventional microscopy. Rather, higher quantities of Giardia are associated with even greater reductions in diarrhea risk [17], an inversion of Koch’s molecular postulates and the opposite of what is seen with pathogens that are clearly diarrhea-associated [17,19]. Other factors, however, could preferentially decrease the sensitivity of Giardia detection preferentially in diarrheal stools. For example, metronidazole is commonly used as empiric therapy for diarrhea in these settings. Thus, diarrhea could be a marker of metronidazole exposure, a confounder that could reduce Giardia detection in diarrheal stools [7].

If real, a long list of potential biological mechanisms has been postulated. Giardia may induce a specific micronutrient deficiency (e.g. iron) that is protective against diarrhea [20]. In one prospective study, the protective effect of Giardia infection on acute diarrhea was not discerned in children who were randomized to receive micronutrient supplementation [13]. Giardia may more broadly modulate immune responses to other pathogens [21,22], or even directly bind enterotoxins [23]. Further work is needed to understand the specificity and potential mechanisms driving this intriguing association.

IS GIARDIA INFECTION ASSOCIATED WITH POOR GROWTH?

Child growth, rather than incident diarrhea, might be a more relevant outcome for estimating the burden of endemic pediatric giardiasis. Indeed, present together with diarrhea, Giardia infection only associates with weight reductions, whereas linear growth deficits are consistently more strongly associated with asymptomatic detections [9,24^■]. Historically, associations between Giardia and impairments in child growth have varied by study, population, and site, leaving unanswered questions regarding causal relationships between the parasite and child developmental sequelae. In MAL-ED, subclinical Giardia detection was among a restricted subset of pathogens associated with diminished height attainment at 2 years of life [9]. As the majority of first Giardia exposures occur after 1 year of age, other factors such as total pathogen burden had a greater overall effect on childhood growth at 2 years of age [25]. However, the estimate of Giardia effects on linear growth increases when considering more persistent Giardia infections [7], earlier in life Giardia exposures [8], and when Giardia is present in greater quantities (Rogawski McQuade, Bartelt, unpublished observation). Despite standardization in study protocols, however, there remains significant heterogeneity between MAL-ED sites in the presence and magnitude of estimates of Giardia effects on linear growth outcomes (Rogawski McQuade, Bartelt, unpublished observation]. These findings could suggest an early, critical window of childhood vulnerability or environment-specific cofactors, which are seldom captured in a focused manner. It is also possible that the chronic consequences of Giardia persistence into the second year of life and beyond were not yet evident at the completion of follow-up. Additionally, ongoing exposures to deworming agents with antigiardial activity, such as mebendazole for soil-transmitted helminths, which overlap with Giardia prevalence [26–28] in older children could also interrupt Giardia persistence and abrogate the impact of Giardia on growth in older children. Further challenging the distinctions between association and causality are frequent coassociations between childhood undernutrition and Giardia infection. The directionality, or possible bidirectionality, of these relationships are difficult to discern. For example, in a single site cohort in Bangladesh, although Giardia was the only pathogen to associate with reduced linear growth by age 2, most of the Giardia detections occurred at the end of the study, outside of an expected window to impact growth [29^■]. These findings raise questions about whether Giardia is driving impaired growth attainment, or if Giardia is itself a marker of poorly growing children who are undernourished and/or at increased risk of stunting from other environmental co-exposures [30], including other pathogens.

WHAT IS THE PATHWAY BETWEEN GIARDIA INFECTION AND POOR GROWTH?

The current working models of subclinical entero-pathogen-associated growth shortfalls in children link nutrient deficiencies with microbial-driven intestinal inflammation, gut dysfunction, and increased intestinal permeability, termed environmental enteric dysfunction (EED) [31]. Similarly, sporadic chronic Giardia infection has been associated with altered intestinal architecture and chronic lymphocytic inflammation in humans and some experimental models [4,32,33]. In a cohort of children in Brazil, Giardia infection associated with plasma markers of epithelial cell injury that correlated with inflammatory cytokines [34]. Consistent with the parasite’s known potential to damage intestinal epithelial cells [35,36], in MAL-ED, Giardia infection was associated with an increased lactulose : mannitol (L : M) ratio, indicative of increased gut permeability. However, fecal myeloperoxidase (MPO), a marker of neutrophil inflammation, was lower in children with Giardia [37], and Giardia was not associated with increased fecal neopterin as a marker of T-cell activation or the acute phase reactant α-1-acid glycoprotein [7,38]. More directly, intestinal biopsies of stunted children in Pakistan with Giardia infection reveal diminished expression of lymphocyte genes [39^■■] despite simultaneous associations with increased gut permeability [40], an apparent contradiction to the EED paradigm of parasite-mediated gut-inflammation pathway of growth impairment. Thus, while impaired gut function represents a putative pathway for poor growth during giardiasis, the mechanisms appear to be independent of both diarrhea and inflammation.

Recent laboratory work supports these findings and suggests new lines of investigation for understanding the association between Giardia and poor growth. First, both parasite-induced and host cellular-mediated or cytokine-mediated alterations in tight junctions could participate in increased gut permeability during giardiasis. For example, organoid cell monolayers demonstrate a sequence of early changes in ion transport genes followed by a breakdown in tight junction complexes with eventual loss of barrier integrity [41]. These and other models indicate Giardia can disrupt multiple tight-junction proteins including claudin-1 and claudin-2, zonula occludens 1 (ZO-1) and may ultimately result in cytoskeleton degradation [41,42]. Changes in similar epithelial tight junctions are also measurable in vivo, but their drivers are complex and are not limited to direct parasite effects. For example, Giardia-mediated barrier injury can also lead to increased paracellular translocation of intestinal bacteria [43], and evidence of ongoing bacterial translocation and inflammation well after parasite clearance. Giardia may not only increase mucosal bacterial translocation, but elegant co-cultivation models demonstrate that the parasite may induce virulence gene expression in otherwise commensal bacteria [44,45]. Consistent with bacterial translocation-related inflammation, Giardia infections in children in low-resource settings were associated with anti-FliC IgA [46] as well as increased serum markers of oxidative stress [47], particularly in the immune-suppressed [48].

In contrast, the absence of an association between Giardia and markers of inflammation in MAL-ED and other studies [37,39^■■] are consistent with the frequent lack of acute inflammation on histopathology during Giardia infection, decreased fecal neutrophil chemotactic markers such as IL-8 during more prolonged Giardia infections [49], and an association between Giardia and lower systemic C-reactive protein levels [13]. In experimental models, Giardia modestly diminishes intestinal inflammatory markers and macrophage responses to LPS stimulation during co-infection with enteroaggregative Escherichia coli [50,51]. Indeed, Giardia contains an impressive repertoire of immunoevasive products [52] that are upregulated upon sensing secreted host factors even prior to parasite attachment [53] and could have implications for modulating host responses to co-pathogens or resident microbiota. For example, cathepsin B-producing Giardia strains are capable of cleaving IL-8 and attenuating neutrophil chemotaxis to inflammatory stimuli [22,54]. Interestingly, inhibition of cathepsin B prevents Giardia-induced epithelial cell villin protein degradation, potentially linking a single virulence factor that could lead to divergent gut permeability and inflammation findings [42]. Alternatively, Giardia may stimulate the production of antimicrobial peptides in epithelial cells that result in enhanced innate defenses and consequently diminished burden of co-infecting pathogens [55,56].

The finding that persistent Giardia infections have the greatest influence on childhood growth [7] suggest that a subset of children fail to make an appropriate immune response. IL-17A-producing CD4+ T cells are increasingly recognized as the predominate memory cell phenotype [57] in those who cleared parasites more rapidly, consistent with murine models invoking the role of a IL17 axis and Th17 cells in Giardia clearance [58,59]. The specific relevance of the Th17 axis during Giardia infection has not been elucidated, but as IL17 has been shown to enhance intestinal epithelial barrier function [60], determining whether children with persistent Giardia infection have impairments in IL17-mediated immunity and, consequently, barrier dysfunction even in the absence of increased inflammation may reveal additional mechanisms driving increased intestinal permeability in this population. For example, recent findings that resident microbiota enhance a Giardia-specific Th17 cellular immune [61^■] response invoke the possibility that a disrupted microbiota underlies both persistent Giardia infection and attenuated mucosal inflammation in a subset of children.

Recent advances in Giardia pathogenesis models are leading the field to examine longstanding questions surrounding the role of persistent Giardia colonization within the broader context of the complex community of intestinal microbiota [45,62–64]. In undernourished children, Giardia co-localizes not only with EED but also with small intestinal bacterial overgrowth (SIBO) [65]. In both experimental models and real-world studies, Giardia associates with a change in the community structure of the intestinal resident microbiota across multiple mammalian hosts [66–68,69^■]. Using dietary interventions like protein deprivation or high-fat diets in experimental models has revealed that the range of Giardia pathogenesis is highly diet-dependent and likely to be mediated by diet-induced changes in resident intestinal microbiota [50,70]. For example, in a model of multipathogen enteropathy, Giardia-induced growth restriction required exposure to a protein-deficient diet, but depletion of intestinal microbiota with antibiotics prevented this outcome. In the same model, Giardia led to robust changes in more than 20 bacteria-derived urinary metabolites [50]. Technological advances in metabolomics and microbiomics profiling are, therefore, opening new paradigms that Giardia-mediated growth shortfalls may result from unique biological properties of the parasite, such as utilization of bile constituents, and the consequences of metabolic dysregulation induced by Giardia-disrupted bacterial communities [69^■].

Future work into better and more pathogen-specific and EED-specific noninvasive biomarkers may also help resolve the relationships between Giardia and childhood growthviaEED, SIBO, or otherunique pathways. For example, increases in urinary phenolic acids, like para-cresol, seen in experimental Giardia infection also predict growth shortfalls in children [71] and ongoing work is identifying certainphenolic acids as uniquely associated with Giardia infection in MAL-ED (Giallourou, Bartelt, unpublished observation). It will be important to resolve whether such changes are because of direct effects of Giardia, or more general alterations in the metabonome mediated by Giardia-associated microbial interactions and/or immune modulation.

GIARDIA BY ANY OTHER NAME…

G. lamblia is conventionally categorized into eight genotypically distinct assemblages, each with a restricted host range, with the majority of human Giardia infections because of assemblage A or B. Unlike the characteristics that distinguish the pathogenicity of some other enteric infections, no single set of markers ascribes virulence potential to clinical Giardia isolates. The extent to which putative virulence genes such as cysteine proteases [72], tenascins, and certain variant surface proteins [73] vary between strains and contribute to phenotypic variability in laboratory [74] or natural infection is poorly understood. Despite clear strain-dependent differences in experimental models and in human volunteers, there remains no consistent delineation of assemblage infection type by symptoms or outcomes [75], even in recent case–control study designs with improved molecular diagnostics.

The two human-infecting Giardia assemblages are genetically and phenotypically distinguishable [76], sharing less than 70% sequence identity across syntenic regions [77]. Within assemblage B, there is a consistently greater degree of genetic diversity than assemblage A [78,79]. Additionally, there is ongoing population expansion of Giardia haplotype diversity across all continents [80]. Even within a single isolate, Giardia displays a high degree of allelic sequence heterozygosity [81]. Proteomics studies reveal that even within the less genetically heterogeneous assemblage A, specific differences in proteins correspond with variable gene families. Notably, differences in variant surface proteins among eight separate isolates aligned with two different profiles that were independent of host origin, sub-assemblage designation, or geographic region [82–84]. With an increasing reference database of different Giardia laboratory strains, use of optimized techniques to perform whole genome sequencing from clinical isolates may help identify strain-specific correlates of disease [85,86]. Applied to field studies, this additional resolution may help separate out the pathogenic signal from commensal noise.

IMPLICATIONS FOR MANAGEMENT

Although deliberation surrounding the harmful versus potential beneficial effects of endemic pediatric Giardia carriage proceeds, the high-prevalence of Giardia acquisition in the first 2 years of life continues. Unfortunately, attempts to use existing tools to limit spread of Giardia in limited resource settings have been disappointing. Neither water sanitation and hygiene interventions nor mass chemotherapy campaigns consistently nor substantially diminish Giardia prevalence in the community [28,87,88]. Episodic treatment seems impractical given a very high rate of reinfection after targeted treatment with metronidazole [89] and the increasing concerns of emerging nitroimidazole resistance [90]. Finally, there is little known about the potential effects of repeated exposures to nitroimidazoles that have broad activity against intestinal anaerobes in addition to protozoa.

CONCLUSION

In conclusion, Giardia has distinct population-dependent epidemiological patterns. The parasite can cause malabsorptive diarrhea as well as chronic intestinal sequelae when exposures are infrequent and occur later in life. However, with frequent exposures in early childhood, Giardia is seemingly inconsequential or even beneficial for diarrhea incidence, while simultaneously contributing to impaired intestinal permeability and growth attainment. To consider Giardia a commensal would, therefore, be inappropriate, though a paucity of clearly defined factors to distinguish virulence strains limits our ability to confidently ascribe pathogenicity. In the meantime, the direct influence of host-immune, nutritional, and microbial factors on experimental giardiasis outcomes position Giardia as a eukaryotic pathobiont in high-prevalence settings. Giardia’s pathogenic potential varies across unique isolates, interacts with and may in turn be influenced by the resident intestinal ecology. As such, defining and addressing the elusive burden of one of the most common intestinal parasitic infections in humans continues to require broad thinking about complex microbial–host interactions, and careful examination and characterization of long-term sequelae.

KEY POINTS.

• New field studies have helped clarify associations between Giardia and poor growth in children living in high-prevalence settings.

• Recent work in Giardia pathogenesis is helping reconcile new paradigms through which Giardia could protect against acute diarrhea but simultaneously impair growth attainment independent of intestinal inflammation in these children.

• Advances in comparative genomics and multiomic studies are needed to identify novel characteristics that better delineate more pathogenic Giardia strains from those that are less consequential, which in turn could help refine burden of disease estimates for this complex organism.