TRANSACTIONSOF THE ROYAL SOCIETY OFTROPICAL MEDICINE

AND HYGIENE (1993)

87, SUPPLEMENT 3,17-21

s3117

Diarrhoeal
Pathogenesis
M. J. G. Farthing UK

disease:current
of giardiasis

concepts

and future

challenges

Department of Gastroenterology, St Bartholomews

Hospital,

West Smithfield,

London,

WCIA

7BE,

Abstract

Giardiasis the mostcommon is smallintestinalprotozoa1 infection andis found worldwide. The mechanisms by which Giardia duodenalis (=G. lamblia) produces chronic diarrhoea malabsorption and have still not been clearly defined. Many infections are associated with mild to moderatemucosaldamage which, in animal modelsof infection, havefunctional correlates.Possible mechanisms include direct physicalinjury, release of parasite products such as proteinasesor lectin, and mucosalinflammation associated with T cell activation and cytokine release.Other possible mechanisms malabsorption of include associated bacterial overgrowth and bile salt deconjugation,bile salt uptake by the parasite with depletionof intraluminal bile salts,and inhibition of pancreatichydrolytic enzymes.Thus, there is no singlemechanism explain the to diarrhoeaand malabsorption causedby Giardia, which currently should be regardedas a multifactorial process.
Introduction

Giardiasis the most commonprotozoa1 is infection of the intestinal tract and is found worldwide throughout temperate and tropical locations (FARTHING, 1989; ADAM, 1991). Prevalencevariesbetween2% and 5% in the industrializedworld and up to 20-30%in the developing world. Although the clinical importance of this parasite beendebatedsincethe beginningof this cenhas tury, there is now indisputable evidence that Giardia duodenalis (=G. lamblia) can causeacute and chronic diarrhoeawith intestinal malabsorption and may be resuonsible for retarded growth and develonment in children (FARTHINGet ;Z., 1986a; SULLIVANet al., 1991).The maior clinical imuact of this narasiteis in infantsand children during the first 3 yeais of life, in the undernourished, in the immunocompromised. and Giardia cysts, which can be transmittedby direct person-to-personcontact, water and food, are the infective form of the parasite. Following excystation, which is triggered by exposureto low pH and pancreatichydrolytic enzymes,trophozoitesattach to the smallintestinal epithelium, multiply by binary fission and thus effect colonization, predominantlyin the proximal smallintestine. The parasitecompletesits life cycle by encysting before leaving the host and onceagainentering the outside environment. Encystation has been completedin vitro in the presence increased of concentrations bile of and high pH. Cysts can survive in the environment in cold moist conditionsfor weeks possiblymonths. and
manifestations of giardiasis Any hypothesisput forward to explain the pathogenesisof aiardiasis must be able to accountfor the clinical diversity of this infection, with respectto both its intestinal and extraintestinalmanifestations. shouldbe able It to take into account the broad spectrumof illnessfrom asymptomaticcarriageto severechronic diarrhoeaand malabsorption, the growth failure observed in some childrenand other well established, less but common,associationssuch as lymphoid nodular hyperplasia,protein-losingenteronathvand variousother allergicandinflammatory phenomena. Giardia accountsfor-up to 7% of cases acute diarrhoeaand ll-45% of thosewith of chronic diarrhoea(FARTHING,in press).At least50%of symptomaticpatientshave biochemicalevidenceof carbohydrate, fat and micronutrient malabsorption.A varietv of mechanisms beendescribedto explain these has phenomena, which focuson (i) the intestinalmucosa and (ii) the intestinal lumen (KATELARIS & FARTHING, Clinical ig92). Mucosal factors in the pathogenesis Morphology of diarrhoea

the smallintestinalmucosa beenreported in humans has and in animalmodelsof giardiasis.In humans,Giardia can producethe completerepertoire of abnormalities of villous architecture, ranging from entirely normal light microscopical appearances, through partial to sub-total villous atrophy (YARDLEYet al., 1964;HOSKINS al., et 1967; ALP & HISLOP, 1969; AMENT & RUBIN, 1972; WRIGHTet al., 1977; DUNCOMBE al., 1978; HARTet LONG al., 1979;ROSEKRANS al., 1981;OBERHUBER et et & STOLTE,1990).However, the majority of individuals have either normal or relatively mild villous shortening usually associated with an increasein crypt depth. Experimentalinfections in gerbils, mice and rats can produce similar abnormalitiesof mucosalarchitecture, although asin humans abnormalities often be mild the can (FAUBERT BELOSEVIC, & 1990). The gerbil provides a particularly good model to study smallintestinal structure and function, asweanlinggerbilsdevelopdiarrhoea and have significantmorphologTca1 abnormalities day by 6 of infection. with reduction in villous heirrht in the duodenum increase crypt depth in the diodenum, and in jejunum and ileum (BURETet al., 1992). In the ileum there is a smallbut significantincrease villous height. in These early changesin villus and crypt morphology occur in the absence anyinflammatory infiltrate in the of laminapropria andwithout anincrease the numbers in of intraepitheliallymphocytes. In human giardiasis,even when villous architecture appears normal bv hght microsconv, ultrastructural changes suchasshorter&g and disru$on of the microvilli are nresent(TAKANO & YARDLEY. 1965: HOSKINS et al., 1967;M~RECKI & PARKER,1967). The gerbil model has confirmed the significanceof theseobservations, demonstrating marked reduction in microvillus a membrane surfacearea in both jejunum and ileum, although theseabnormalities weretransient(BURET etal., 1992). This decrease the height of microvilli was in found uniformly and not specificilly related to sitesof trophozoite attachment (BURET et al., 1992). From humanstudies there isevidenceto suggest the extent that of the mucosal abnormality relatesto the severity of the diarrhoea (WRIGHT al., 1977; DUNCOMBE et et al., 1978).
Disaccharidase activity

A variety of structural and functional abnormalities of

In humangiardiasis, morphological abnormalities have been associated with reduction in lactase,sucraseand maltaseactivities in the microvillus membrane(DUNCOMBE et al., 1978;HARTONG et al., 1979). Similar observationshave been reported in experimentalinfection in mice, gerbilsand rats (GILLON et-al., 1982;KHANNA et al.. 1988:BELOSEVIC et al.. 1989:BURET et al.. 1990: CEV~LLOS FARTHING,1992).Reductionin disacchari: & daseactivities are maximal when diarrhoea and villous morphological abnormalities mostpronounced. are

S3118 Functional disorders Several studies in animal models suggest that there are functional consequences of these structural abnormalities and the reduction in disaccharidase activity. In gerbils, basal transport of sodium and chloride ions was not different from that in non-infected controls, but glucosestimulated sodium absorption was significantly reduced in the ieiunum but not the ileum of infected gerbils in experiments using stripped small intestinal mucosa mounted in Ussing: chambers (BURET et al.. 1992). Perfusion studies in iivo in animals have also shown impaired water, sodium and chloride absorption in response to glucose, although basal transport was similar to that in controls (BURET et al., 1992). In a neonatal rat model of infection, basal transport of water, sodium and chloride ions was impaired, with some animals actually in a net secretory state for sodium and chloride ions. Perfusion of a lactose-containing solution enhanced these transport abnormalities (CEVALLOS & FARTHING, 1992). Studies with brush border membrane vesicles from infected mice indicated that there is impairment of glucose and amino acid transport (SAMRA et al., 1987, 1988). Mechanisms of mucosal inju y Structural and functional abnormalities in the small intestine can be detected in humans and experimentally infected animals, and it seems likely that these contribute, at least in part, to the diarrhoea and malabsorption associated with this infection. In experimental models, the abnormalities can be detected early in infection and in humans there appears to be some relationship between the extent of the structural abnormalities and the severity of diarrhoea (WRIGHT et al., 1977; DUNCOMBE et al., 1978). The mechanisms by which these abnormalities occur, however, are far from clear. Although there have been few reports of epithelial invasion (BRANDBORG et al., 1967; SAHA & GHOSH, 1977), Giardia is essentially a luminal enteropathogen and thus invasive episodes must be regarded as exceptional. Giardia trophozoites, however, do attach to the epithelium and have been shown by electron microscopy to disrupt and distort microvilli at the site where the ventral adhesive disc interfaces with the microvillus membrane (ERLANDSEN & CHASE, 1974). Ventral disc imprints are particularly marked in the murine model, although they have also been reported in human infection. It seems unlikely, however, that the localized attachment sites can account for the widespread changes in microvillus membrane surface area observed in the small intestine (BURET et al.. 1992). There is some evidence to sugiest that Giardia itself produces, and possibly releasesycytopathic substances into the intestinal lumen (SAMRA et al.. 19881. As vet no parasite product has been identified toaccount for these changes in the intestine. Giardia does, however, contain a number of thiol proteinases which might attack surface glycoproteins and disrupt microvillus membrane integrity (HARE et al., 1989; PARENTI, 1989; NORTH et al., 1990). In addition, Giardia has been shown to express a surface mannose-binding lectin (FARTHING et al., 1986b) which is cleaved from a cytoplasmic precursor molecule by trypsin (LEV et al., 1986) and can mediate attachment to enterocytes (INGE et al., 1988). Dietary plant lectins can directly damage intestinal epithelial cells and produce microvillus membrane abnormalities very similar to those seen in giardiasis (LORENZSONN & OLSEN, 1982; DOBBINS et al., 1986). It remains to be established whether any of these parasite products are injurious to host enterocytes. An alternative explanation has been put forward to explain the reduction in disaccharidase activity and the associated impairment of carbohydrate and electrolyte absorption. The increase in crypt depth seen in human infections and animal models is generally associated with increased crypt cell production rate and more rapid migration of enterocytes along the villus. In other conditions where this occurs, such as coeliac disease, it results in the villus becoming populated by relatively immature enterocytes with reduced digestive and absorptive capacities. Increased proliferation has been confirmed in the gerbil model (BURET et al., 1992) but, using thymidine kinase activity as a marker of maturity, there was no evidence in the jejunum or ileum that the cells repopulating the villus were less mature than those in non-infected control animals. It seems likely therefore that the structural and functional abnormalities observed in the microvillus membrane relate to direct injury rather than another secondary mechanism which is causing crypt cell proliferation. In human giardiasis there is a variable immune response within the mucosa, although infection is often associated with an increase in the numbers of lamina propria lymphocytes and intraepithelial lymphocytes (WRIGHT & TOMKINS, 1977; DUNCOMBE et al., 1978; ROSEKRANS et al., 1981; GILLON, 1985). These inflammatory changes have been more difficult to reproduce in experimental models. However, there is compelling evidence that T cell activation alone can produce villous atrophy. The enteropathy occurring in intestinal graftversus-host disease and rejection of transplanted intestinal allografts is characterized by villous atrophy, crypt cell hyperplasia and a lymphocytic infiltrate. Using human foetal small intestinal explants it has been possible partly to characterize the mechanisms involved, by activating T cells with either pokeweed mitogen or anti-CD3 antibody; both approaches produced villous atrophy, crypt cell hyperplasia and increased interleukin 2 production, confirming T cell activation (MACDONALD & SPENCER, 1988). Further support for this hypothesis has been obtained from studies of experimental G. murk infection in athymic nuinu mice (ROBERTS-THOMSON & MITCHELL, 1978). Despite prolonged infection! the alteration of villusicrypt cell ratio is less severe in nuinu mice than in immunocompetent controls. When Iymphocytes from the spleens of immunologically intact mice were iniected into athvmic infected mice. histoloaical abnormalities in the small intestine became m&e pronounced. However, reduction in the villusicrypt cell ratio did occur in the immunocompromised mice before reconstitution, and thus it seems likely that T cell-independent mechanisms are also involved. In addition, immunosuppression in mice results in more profound effects on disaccharidase activities in conventional animals, indicating that epithelial damage is not solely dependent on immune function (KHANNA et al., 1988). Furthermore, although intraepithelial lymphocytes are frequently increased in number in giardiasis, and have been incriminated in contributing to immune mediated damage in experimental G. muris infection., intraepithelial lymphocyte numbers increased after villus shortening and the decrease in brush border disaccharidases had already occurred (GILLON et al., 1982). Whether Giardia lectin can act as a mitogen and directly activate T cells remains to be established. However, challenge of mice (previously infected with G. muris) with G. muris trophozoite extract resulted in a rapid decrease in disaccharidase activity, which might have been immune mediated (DANIELS & BELOSEVIC, 1992). The effect was most marked in the genetically susceptible C3H/HeN mice. Luminal factors in the pathogenesis of diarrhoea Bacterial overgrowth There is some evidence to suggest that symptomatic giardiasis is associated with increased numbers of aerobic and/or anaerobic bacteria in the proximal small intestine. In one studv from India. 8 of 17 natients 147%) with steatorrhoea had more than lo4 aerobic bacteria cultured from duodenal fluid, whereas none from a giardiasis control group without steatorrhoea had bacterial overgrowth (TANDON et al., 1977). Three of the natients with steatorrhoea also had anaerobes present: TOMKINS et al. (1978) found increased numbers of aerobic bacteria in 9 of 14symptomatic overland travellers (64%) with giar-

s3/19 diasis returning to the UK, 2 of whom also had anaerobes present. Bacterial overgrowth can produce architectural abnormalities in the small intestine similar to those seen in giardiasis, and thus it may have a role in producing mucosal injury. Bile salt deconjugation The removal of the glycine or taurine conjugate from bile salts reduces their solubility in aqueous solution and thus reduces their efficacy in micelle formation within the intestinal lumen. In addition, free bile salts are membranotoxic and can cause intestinal secretion, thus potentially contributing to the pathogenesis of diarrhoea: TANDON et al. (1977) found evidence of bile salt deconiugation in allof their Indian patients with bacterial overgrowth and in 40% of giardiasis controls without malabsorption, but other studies have not confirmed this (HALLIDAY et al., 1988). Giardia does not itself have the ability to deconjugate bile sale (SMITH et al., 1981; HALLIDAY et aZ., 1988). Bile salt uptake by Giardia Bile and bile salts appear to make important contributions to the life cycle of the parasite. Mammalian bile at low concentration has been shown to stimulate parasite growth and reduce generation time, and thus may be an rmportant colonization factor for this parasite (FARTHING et al.. 1983. 1985: KEISTER. 1983). The effect can be reproduced iartially by theaddition of conjugated bile salts alone, which appear to increase uptake of cholesterol and membrane phospholipid that the parasite is unable to svnthesise de nova (FARTHING et al., 1985). More recently-it has been shown that parasites grownin bile are larger than those grown in a bile free medium, and that the presence of bile alters antigenic expression by the parasite (KATELARIS et al., 1991a). High concentrations of bile salts trigger parasite encystation. During the course of bile stimulation experiments it became apparent that the parasite was consuming conjugated bile salts (FARTHING et al., 1985). Further studies showed that this was relatively specific for Giardia (HALLIDAY et al., 1988) and that uptake appeared to be occurring by a carrier-mediated., active transport process. The metabolic advantage of bile salt uptake for the parasite has not been defined, although bile salts do appear to be fully internalized into the cytoplasm and not sequestered in the surface membrane. Theoretically, consumption of host bile salts during chronic diarrhoea could deplete the bile salt pool and thus contribute to fat malabsorption by impairing micellar solubilization of ingested fats and decreasing the effectiveness of pancreatic lipase, the action of which is bile salt-dependent. Inhibition of hydrolytic enzymes Intraluminal concentrations of trypsin, chymotrypsin and lipase have been shown to be reduced in symptomatic patients with giardiasis (GUPTA & MEHTA, 1973; CHAWLA et al., 1975; OKADA et al., 1983). There is no evidence that this is due to a failure of pancreatic exocrine secretion but it could be related to the more recent observation that live Giardia trophozoites and trophozoite sonicates inhibit trvnsin activitv and linolvsis in vitro (SMITH et al., 1981; KATELARIS et hZ., 199ib;SEow etpl:,l993). The mechanism by which the parasite inhibits these enzyme activities has not been established but could be related to the direct interaction between a parasite product, such as its own proteinases, and the host enzymes. However, the pancreas has a large functional reserve and the magnitude of the reduction observed in clinical studies is itself unlikely to account for malabsorption; however, it could contribute to the cascade of abnormalities that together impair the absorptive mechanisms of the gut and contribute to diarrhoea and malabsorption. Phenotypic and genotypic variation in isolates Host susceptibility to Giardia appears to vary and experiments with animal models indicate that this is at least in part genetically determined. However, there is increasing evidence to suggest that Giardia isolates differ in genotype and phenotype (NASH et al., 1985; ANDREWS et al., 1989; MELONI et al., 1989; CARNABY et al., 1991; KORMAN et al., 1992). There is also evidence to indicate that Giardia isolates may interact differently with their host with respect to their ability to colonize the host and nroduce clinical disease (AGGARWAL & NASH. 1987: DASH et al., 1987; CEVAL~OS & FARTHING, 199i; UDEZULU et al., 1992). Sequelae of giardiasis Nutritional insufficiency and growth failure have been associated with giardiasis, although the mechanisms have not been studied in detail. In the early stages of infection reduced food intake is likely to be a major contributor, although as the illness progresses and malabsorption and steatorrhoea become more apparent loss of energy substrates through the gut will compound the problem. However, these mechanisms remain speculative since energy balance studies have not been performed in chronic giardiasis. Protein losing enteropathy has been described in several case reports (KORMAN et al., 1990; SHERMAN & LIEBMAN, 1980) but from a recent survey of children in The Gambia it appears that this is uncommon and rarely of a degree that has a major clinical impact (SULLIVAN et al., 1992). Loss of protein through the gut can occur as a result of a breach in mucosal integrity following inflammation and/or direct damage associated with enterocyte loss. The precise mechanisms of protein losing enteropathy in giardiasis have not been defined. Occasionally allergic and other inflammatory phenomena have been described in criardiasis. Although immediate-type hypersensitivity is a relatively common association with helminthic infections. it is rare with protozoa. However, urticaria, arthralgia and other allergic phenomena have been described. Increased serum immunoglobulin (Ig) E concentrations have been reported, but in one study where this was investigated in more detail there was no evidence that the IgE was Giardia-specific, suggesting that the intestinal damage induced by the parasite merely facilitated parenteral sensitization with food or other luminal antigens (FARTHING et al., 1984). Lymphoid nodular hyperplasia has been associated with both chronic giardiasis and immune deficiency. Several studies have examined the prevalence of giardiasis in patients with hypogammaglobulinaemia and found it to occur in 29-71% of cases (AJDUKIEWICZ et al., 1972; WEBSTER et al., 1977; NAGURA et al., 1979). However, in one study from India 25 patients were described, all of whom had giardiasis and lymphoid nodular hyperplasia but none had immunoglobulin deficiency (WARD et al., 1983). Thus, the relationship between lymphoid nodular hyperplasia, hypogammaglobulinaemia and giardiasis remains unclear, although it appears that any 2 can occur in combination without any direct implication for pathogenesis. There is no clear indication as to the pathogenesis of lymphoid nodular hyperplasia, although several studies have shown a predominance of B cells producing IgM within the mucosa and lymphoid noduies, suggesting that there might be immune overactivity against a luminal antigen, possibly with failure to switch from IgM to IgA production within the intestine.
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